transforming-growth-factor-beta and Hypertension--Pulmonary

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disorder, wherein mean systemic arterial pressure (mPAP) becomes abnormally high because of aberrant changes in various proliferative and inflammatory signalling pathways of pulmonary arterial cells. Currently used anti-PAH drugs chiefly target the vasodilatory and vasoconstrictive pathways. However, an imbalance between bone morphogenetic protein receptor type II (BMPRII) and transforming growth factor beta (TGF-β) pathways is also implicated in PAH predisposition and pathogenesis. Compared to currently used PAH drugs, various biologics have shown promise as PAH therapeutics that elicit their therapeutic actions akin to endogenous proteins. Biologics that have thus far been explored as PAH therapeutics include monoclonal antibodies, recombinant proteins, engineered cells, and nucleic acids. Because of their similarity with naturally occurring proteins and high binding affinity, biologics are more potent and effective and produce fewer side effects when compared with small molecule drugs. However, biologics also suffer from the limitations of producing immunogenic adverse effects. This review describes various emerging and promising biologics targeting the proliferative/apoptotic and vasodilatory pathways involved in PAH pathogenesis. Here, we have discussed sotatercept, a TGF-β ligand trap, which is reported to reverse vascular remodelling and reduce PVR with an improved 6-minute walk distance (6-MWDT). We also elaborated on other biologics including BMP9 ligand and anti-gremlin1 antibody, anti-OPG antibody, and getagozumab monoclonal antibody and cell-based therapies. Overall, recent literature suggests that biologics hold excellent promise as a safe and effective alternative to currently used PAH therapeutics.

Topics: Biological Products; Humans; Hypertension, Pulmonary; Ligands; Pulmonary Arterial Hypertension; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a rare cardiovascular disorder leading to pulmonary hypertension and, often fatal, right heart failure. Sex differences in PAH are evident, which primarily presents with a female predominance and increased male severity. Disturbed signalling of the transforming growth factor-β (TGFβ) family and gene mutations in the bone morphogenetic protein receptor 2 (BMPR2) are risk factors for PAH development, but how sex-specific cues affect the TGFβ family signalling in PAH remains poorly understood. In this review, we aim to explore the sex bias in PAH by examining sex differences in the TGFβ signalling family through mechanistical and translational evidence. Sex hormones including oestrogens, progestogens, and androgens, can determine the expression of receptors (including BMPR2), ligands, and soluble antagonists within the TGFβ family in a tissue-specific manner. Furthermore, sex-related genetic processes, i.e. Y-chromosome expression and X-chromosome inactivation, can influence the TGFβ signalling family at multiple levels. Given the clinical and mechanistical similarities, we expect that the conclusions arising from this review may apply also to hereditary haemorrhagic telangiectasia (HHT), a rare vascular disorder affecting the TGFβ signalling family pathway. In summary, we anticipate that investigating the TGFβ signalling family in a sex-specific manner will contribute to further understand the underlying processes leading to PAH and likely HHT.

Topics: Bone Morphogenetic Protein Receptors, Type II; Familial Primary Pulmonary Hypertension; Female; Humans; Hypertension, Pulmonary; Male; Pulmonary Arterial Hypertension; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a complex multifactorial disease with poor prognosis characterized by functional and structural alterations of the pulmonary circulation causing marked increase in pulmonary vascular resistance, ultimately leading to right heart failure and death. Mutations in the gene encoding BMPRII-a receptor for the TGF-β (transforming growth factor-beta) superfamily-account for over 70% of families with PAH and ≈20% of sporadic cases. In recent years, however, less common or rare mutations in other genes have been identified. This review will consider how these newly discovered PAH genes could help to provide a better understanding of the molecular and cellular bases of the maintenance of the pulmonary vascular integrity, as well as their role in the PAH pathogenesis underlying occlusion of arterioles in the lung. We will also discuss how insights into the genetic contributions of these new PAH-related genes may open up new therapeutic targets for this, currently incurable, cardiopulmonary disorder.

Topics: Humans; Hypertension, Pulmonary; Mutation; Pulmonary Arterial Hypertension; Transforming Growth Factor beta; Vascular Resistance

Pulmonary arterial hypertension (PAH) is a severe complication of connective tissue disease (CTD), causing death in systemic sclerosis (SSc). The past decade has yielded many scientific insights into microRNA (miRNAs) in PAH and SSc. This growth of knowledge has well-illustrated the complexity of microRNA (miRNA)-based regulation of gene expression in PAH. However, few miRNA-related SSc-PAH were elucidated. This review firstly discusses the role of transforming growth factor-beta (TGF-β) signaling and bone morphogenetic protein receptor type II (BMPR2) in PAH and SSc. Secondly, the miRNAs relating to TGF-β and BMPR2 signaling pathways in PAH and SSc or merely PAH were subsequently summarized. Finally, future studies might develop early diagnostic biomarkers and target-oriented therapeutic strategies for SSc-PAH and PAH treatment.

Topics: Bone Morphogenetic Protein Receptors, Type II; Humans; Hypertension, Pulmonary; MicroRNAs; Pulmonary Arterial Hypertension; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) superfamily includes several groups of multifunctional proteins that form two major branches, namely the TGF-β-activin-nodal branch and the bone morphogenetic protein (BMP)-growth differentiation factor (GDF) branch. The response to the activation of these two branches, acting through canonical (small mothers against decapentaplegic (Smad) 2/3 and Smad 1/5/8, respectively) and noncanonical signalling pathways, are diverse and vary for different environmental conditions and cell types. An extensive body of data gathered in recent years has demonstrated a central role for the cross-talk between these two branches in a number of cellular processes, which include the regulation of cell proliferation and differentiation, as well as the transduction of signalling cascades for the development and maintenance of different tissues and organs. Importantly, alterations in these pathways, which include heterozygous germline mutations and/or alterations in the expression of several constitutive members, have been identified in patients with familial/heritable pulmonary arterial hypertension (PAH) or idiopathic PAH (IPAH). Consequently, loss or dysfunction in the delicate, finely-tuned balance between the TGF-β-activin-nodal branch and the BMP-GDF branch are currently viewed as the major molecular defect playing a critical role in PAH predisposition and disease progression. Here we review the role of the TGF-β-activin-nodal branch in PAH and illustrate how this knowledge has not only provided insight into understanding its pathogenesis, but has also paved the way for possible novel therapeutic approaches.

Topics: Bone Morphogenetic Proteins; Humans; Hypertension, Pulmonary; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factors

In this review, we discuss the role of transforming growth factor-beta (TGF-β) in the development of pulmonary vascular disease (PVD), both pulmonary arteriovenous malformations (AVM) and pulmonary hypertension (PH), in hereditary hemorrhagic telangiectasia (HHT). HHT or Rendu-Osler-Weber disease is an autosomal dominant genetic disorder with an estimated prevalence of 1 in 5000 persons and characterized by epistaxis, telangiectasia and AVMs in more than 80% of cases, HHT is caused by a mutation in the ENG gene on chromosome 9 encoding for the protein endoglin or activin receptor-like kinase 1 (ACVRL1) gene on chromosome 12 encoding for the protein ALK-1, resulting in HHT type 1 or HHT type 2, respectively. A third disease-causing mutation has been found in the SMAD-4 gene, causing a combination of HHT and juvenile polyposis coli. All three genes play a role in the TGF-β signaling pathway that is essential in angiogenesis where it plays a pivotal role in neoangiogenesis, vessel maturation and stabilization. PH is characterized by elevated mean pulmonary arterial pressure caused by a variety of different underlying pathologies. HHT carries an additional increased risk of PH because of high cardiac output as a result of anemia and shunting through hepatic AVMs, or development of pulmonary arterial hypertension due to interference of the TGF-β pathway. HHT in combination with PH is associated with a worse prognosis due to right-sided cardiac failure. The treatment of PVD in HHT includes medical or interventional therapy.

Topics: Activin Receptors, Type II; Animals; Arteriovenous Malformations; Endoglin; Humans; Hypertension, Pulmonary; Lung Diseases; Mutation; Risk; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta; Vascular Diseases

Pulmonary arterial hypertension (PAH) is a severe condition characterised by remodelling of precapillary pulmonary arteries sometimes associated with mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene. Even in the absence of BMPR2 mutations, increased transforming growth factor (TGF)β receptor signalling and decreased BMPRII signalling have been shown to contribute to PAH pathogenesis. In this Keynote, we review the potential mechanisms by which the imbalance of BMP/TGFβ signalling contributes to endothelial dysfunction, vascular remodelling, inflammation and disordered angiogenesis in PAH. Additionally, we highlight how currently used drugs can influence BMP/TGFβ signalling. Finally, we browse the newly developed therapeutic approaches targeting BMPRII and TGFβ signalling pathways by focusing on preclinical studies and clinical trials and put them into perspectives.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Humans; Hypertension, Pulmonary; Inflammation; Neovascularization, Physiologic; Signal Transduction; Transforming Growth Factor beta

Approximately 5% of individuals chronically infected with

Topics: Animals; Biomarkers; Diagnostic Imaging; Disease Susceptibility; Heart Function Tests; Humans; Hypertension, Pulmonary; Schistosoma; Schistosomiasis; Th2 Cells; Thrombospondin 1; Transforming Growth Factor beta; Vascular Remodeling

Genetic studies in animals and humans indicate that gene mutations that functionally perturb transforming growth factor β (TGF-β) signaling are linked to specific hereditary vascular syndromes, including Osler-Rendu-Weber disease or hereditary hemorrhagic telangiectasia and Marfan syndrome. Disturbed TGF-β signaling can also cause nonhereditary disorders like atherosclerosis and cardiac fibrosis. Accordingly, cell culture studies using endothelial cells or smooth muscle cells (SMCs), cultured alone or together in two- or three-dimensional cell culture assays, on plastic or embedded in matrix, have shown that TGF-β has a pivotal effect on endothelial and SMC proliferation, differentiation, migration, tube formation, and sprouting. Moreover, TGF-β can stimulate endothelial-to-mesenchymal transition, a process shown to be of key importance in heart valve cushion formation and in various pathological vascular processes. Here, we discuss the roles of TGF-β in vasculogenesis, angiogenesis, and lymphangiogenesis and the deregulation of TGF-β signaling in cardiovascular diseases.

Topics: Activin Receptors, Type II; Animals; Aortic Aneurysm; Atherosclerosis; Cardiovascular Diseases; Cell Communication; Endothelial Cells; Fibrosis; Humans; Hypertension, Pulmonary; Lymphangiogenesis; Myocardium; Neovascularization, Physiologic; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Pulmonary artery hypertension (PAH) is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis)-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia), caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Caveolin 1; Child; Endoglin; Humans; Hypertension, Pulmonary; Kv1.5 Potassium Channel; Mutation; Transforming Growth Factor beta

Knowledge pertaining to the involvement of transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling in pulmonary arterial hypertension (PAH) is continuously increasing. There is a growing understanding of the function of individual components involved in the pathway, but a clear synthesis of how these interact in PAH is currently lacking. Most of the focus has been on signaling downstream of BMPR2, but it is imperative to include the role of TGF-β signaling in PAH. This review gives a state of the art overview of disturbed signaling through the receptors of the TGF-β family with respect to vascular remodeling and cardiac effects as observed in PAH. Recent (pre)-clinical studies in which these two pathways were targeted will be discussed with an extended view on cardiovascular research fields outside of PAH, indicating novel future perspectives.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Epithelial-Mesenchymal Transition; Humans; Hypertension, Pulmonary; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a progressive and lethal pulmonary vascular disease (PVD). Although in recent years outcome has improved by new treatments that delay disease progression, a cure has not yet been achieved. In PAH associated with congenital heart disease (CHD), remodeling of the pulmonary vasculature reaches an irreversible phenotype similar to all forms of end-stage PAH. In PAH-CHD, however, also an early stage is recognised, which can be completely reversible. This reversible phase has never been recognised in other forms of PAH, most likely because these patients are only diagnosed once advanced disease has developed. We propose that the clinical model of PAH-CHD, with an early reversible and advanced irreversible stage, offers unique opportunities to study pathophysiological and molecular mechanisms that orchestrate the transition from reversible medial hypertrophy into irreversible plexiform lesions. Comprehension of these mechanisms is not only pivotal in clinical assessment of disease progression and operability of patients with PAH-CHD; specific targeting of these mechanisms may also lead to pharmacological interventions that transform 'irreversible' plexiform lesions into a reversible PVD: one that is amenable for a cure. In recent years, significant steps have been made in the strive to 'reverse the irreversible'. This review provides an overview of current clinical and experimental knowledge on the reversibility of PAH, focussing on flow-associated mechanisms, and the near-future potential to advance this field.

Topics: Adult; Animals; Antihypertensive Agents; Apoptosis; Bone Morphogenetic Proteins; Child; Disease Models, Animal; Disease Progression; Heart Defects, Congenital; Humans; Hypertension, Pulmonary; Pulmonary Circulation; Signal Transduction; Transforming Growth Factor beta; Vascular Remodeling; Vasculitis

Pulmonary arterial hypertension (PAH) is a group of vascular diseases that produce right ventricular dysfunction, heart failure syndrome, and death. Although the majority of patients appear idiopathic, accumulated research work combined with current sequencing technology show that many gene variants could be an important component of the disease. However, current guidelines, clinical practices, and available gene panels focus the diagnosis of PAH on a relatively low number of genes and variants associated with the bone morphogenic proteins and transforming Growth Factor-β pathways, such as the BMPR2, ACVRL1, CAV1, ENG, and SMAD9.. To provide an expanded view of the genes and variants associated with PAH, we performed a systematic literature review. Facilitated by a web tool, we classified, curated, and annotated most of the genes and PubMed abstracts related to PAH, in which many of the mutations and variants were not annotated in public databases such as ClinVar from NCBI. The gene list generated was compared with other available tests.. Our results reveal that there is genetic evidence for at least 30 genes, of which 21 genes shown specific mutations. Most of the genes are not covered by current available genetic panels. Many of these variants were not annotated in the ClinVar database and a mapping of these mutations suggest that next generation sequencing is needed to cover all mutations found in PAH or related diseases. A pathway analysis of these genes indicated that, in addition to the BMP and TGFβ pathways, there was connections with the nitric oxide, prostaglandin, and calcium homeostasis signalling, which may be important components in PAH.. Our systematic review proposes an expanded gene panel for more accurate characterization of the genetic incidence and risk in PAH. Their usage would increase the knowledge of PAH in terms of genetic counseling, early diagnosis, and potential prognosis of the disease.

Topics: Bone Morphogenetic Proteins; Databases, Genetic; Humans; Hypertension, Pulmonary; Mutation; Risk; Signal Transduction; Transforming Growth Factor beta

Hereditary haemorrhagic telangiectasia is a rare systemic autosomal dominantly inherited disorder of the fibrovascular tissue with a wide variety of clinical manifestations. Diagnosis is based on the clinical Curaçao criteria or molecular genetic testing. Dilated vessels can develop into telangiectases or larger vascular malformations in various organs, calling for an interdisciplinary approach. Epistaxis and gastrointestinal bleeding can result from these vascular defects. Various conservative and interventional treatments have been described for these conditions. However, no optimal therapy exists. Treatment can become especially difficult due to progressive anaemia or when anticoagulant or anti-thrombotic therapy becomes necessary. Screening for pulmonary arteriovenous malformations (PAVM) should be performed in all confirmed and suspected patients. Treatment by percutaneous transcatheter embolotherapy and antibiotic prophylaxis is normally effective for PAVM. Cerebral or hepatic vascular malformations and rare manifestations need to be evaluated on a case-by-case basis to determine the best course of action for treatment.

Topics: Anemia, Iron-Deficiency; Antibiotic Prophylaxis; Anticoagulants; Arteriovenous Malformations; Disease Management; Embolization, Therapeutic; Epistaxis; Fibrinolytic Agents; Gastrointestinal Hemorrhage; Hemostatics; Humans; Hypertension, Pulmonary; Intracranial Arteriovenous Malformations; Liver; Lung; Neovascularization, Pathologic; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Thrombophilia; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II-like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next-generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus-specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Disease Models, Animal; Genetic Association Studies; Genetic Counseling; Genetic Predisposition to Disease; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Hypertension, Pulmonary; Multigene Family; Mutation; Signal Transduction; Transforming Growth Factor beta

Fibrotic lung diseases carry a significant mortality burden worldwide. A large proportion of these deaths are due to right heart failure and pulmonary hypertension. Underlying contributory factors which appear to play a role in the mechanism of progression of right heart dysfunction include chronic hypoxia, defective calcium handling, hyperaldosteronism, pulmonary vascular alterations, cyclic strain of pressure and volume changes, elevation of circulating TGF-β, and elevated systemic NO levels. Specific therapies targeting pulmonary hypertension include calcium channel blockers, endothelin (ET-1) receptor antagonists, prostacyclin analogs, phosphodiesterase type 5 (PDE5) inhibitors, and rho-kinase (ROCK) inhibitors. Newer antifibrotic and anti-inflammatory agents may exert beneficial effects on heart failure in idiopathic pulmonary fibrosis. Furthermore, right ventricle-targeted therapies, aimed at mitigating the effects of functional right ventricular failure, include β-adrenoceptor (β-AR) blockers, angiotensin-converting enzyme (ACE) inhibitors, antioxidants, modulators of metabolism, and 5-hydroxytryptamine-2B (5-HT2B) receptor antagonists. Newer nonpharmacologic modalities for right ventricular support are increasingly being implemented. Early, effective, and individualized therapy may prevent overt right heart failure in fibrotic lung disease leading to improved outcomes and quality of life.

Topics: Adrenergic beta-Antagonists; Angiotensin-Converting Enzyme Inhibitors; Fibrosis; Heart Failure; Humans; Hypertension, Pulmonary; Molecular Targeted Therapy; Nitric Oxide; rho-Associated Kinases; Transforming Growth Factor beta; Ventricular Dysfunction, Right

Pulmonary arterial hypertension (PAH) is a rare disease characterized by distinctive changes in pulmonary arterioles that lead to progressive elevation of pulmonary artery pressure, pulmonary vascular resistance, right ventricular failure, and a high mortality rate. The etiology of PAH is heterogeneous and incompletely understood. Based on clinical classification, WHO Group 1 PAH includes sporadic disease (idiopathic PAH), inherited PAH (heritable PAH), and association with certain medical conditions (associated PAH). Genes play an important role in idiopathic and heritable PAH. Mutations in bone morphogenetic protein receptor 2 (BMPR2), a member of the transforming growth factor β (TGFβ) superfamily of receptors, have been identified in 70 % of cases of familial PAH, as well as in 10-40 % of cases of idiopathic PAH. Mutations in ALK-1, ENG, SMAD4 and SMAD8, other TGFβ family members, are additional rare causes of PAH. CAV1 regulates SMAD2/3 phosphorylation, and mutations in CAV1 are a rare cause of PAH. KCNK3 is a member of the two-pore domain potassium channels expressed in pulmonary artery smooth muscle cells, and mutations in KCNK3 are a rare cause of both familial and IPAH. The genetics of PAH are complex due to incomplete penetrance and genetic heterogeneity. In addition to rare mutations as a monogenic cause of HPAH, common variants in cerebellin 2 (CBLN2) increase the risk of PAH by approximately twofold. PAH in children is much more heterogeneous than in adults and can be associated with several genetic syndromes, specifically syndromes with congenital heart disease, vascular disease, and hepatic disease. Clinical genetic testing is available for PAH and should be considered in families to allow for more definitive risk stratification and allow for reproductive planning.

Topics: Adult; Bone Morphogenetic Protein Receptors, Type II; Child; Genes, Recessive; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Mutation; Nerve Tissue Proteins; Potassium Channels, Tandem Pore Domain; Transforming Growth Factor beta

Germ-line mutations in the bone morphogenetic protein type II receptor (BMPR2; BMPR-II) gene, a transforming growth factor-β (TGFβ) receptor superfamily member, cause the majority of cases of heritable pulmonary arterial hypertension (PAH). Pulmonary arterial hypertension is a subset of pulmonary hypertension (PH) disorders, which also encompass hypoxia-related lung diseases. Bone morphogenetic proteins (BMPs), via BMPR-II, activate the canonical Smad1/5/9 pathway, whereas TGFβs (TGFβ1-3) activate the Smad2/3 pathway via the ALK5 receptor. Dysregulated TGFβ1 signalling is pathogenic in fibrotic diseases. We compared two rat PH models, monocrotaline-induced PAH (MCT-PAH) and chronic normobaric hypoxia (fractional inspired O2 10%), to address whether BMPR-II loss is common to PH and permits pathogenic TGFβ1 signalling. Both models exhibited reduced lung BMPR-II expression, but increased TGFβ1 signalling and decreased BMP signalling were observed only in MCT-PAH. Furthermore, a pharmacological ALK5 inhibitor prevented disease progression in the MCT-PAH model, but not in hypoxia. In vitro studies using human pulmonary artery smooth muscle cells showed that TGFβ1 directly inhibits BMP-Smad signalling. In conclusion, BMPR-II loss is common to the hypoxic and MCT-PAH models, but systemic ALK5 inhibition is effective only in the MCT model, highlighting a specific role for TGFβ1 in vascular remodelling in MCT-PAH, potentially via direct inhibition of BMP signalling.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Familial Primary Pulmonary Hypertension; Homeostasis; Humans; Hypertension, Pulmonary; Hypoxia; Pulmonary Artery; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a rare disorder that may be hereditary (HPAH), idiopathic (IPAH), or associated with either drug-toxin exposures or other medical conditions. Familial cases have long been recognised and are usually due to mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2), or, much less commonly, two other members of the transforming growth factor-β superfamily, activin-like kinase-type 1 (ALK1), and endoglin (ENG), which are associated with hereditary hemorrhagic telangiectasia. In addition, approximately 20% of patients with IPAH carry mutations in BMPR2. Clinical testing for BMPR2 mutations is available and may be offered to HPAH and IPAH patients but should be preceded by genetic counselling, since lifetime penetrance is only 10% to 20%, and there are currently no known effective preventative measures. Identification of a familial mutation can be valuable in reproductive planning and identifying family members who are not mutation carriers and thus will not require lifelong surveillance. With advances in genomic technology and with international collaborative efforts, genome-wide association studies will be conducted to identify additional genes for HPAH, genetic modifiers for BMPR2 penetrance, and genetic susceptibility to IPAH. In addition, collaborative studies of BMPR2 mutation carriers should enable identification of environmental modifiers, biomarkers for disease development and progression, and surrogate markers for efficacy end points in clinical drug development, thereby providing an invaluable resource for trials of PAH prevention.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Familial Primary Pulmonary Hypertension; Genetic Predisposition to Disease; Genetic Testing; Genetic Therapy; Humans; Hypertension, Pulmonary; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta family members are multifunctional cytokines that elicit their effects on cells, including endothelial and mural cells, via specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. Knock-out mouse models for TGF-beta family signaling pathway components have revealed their critical importance in proper yolk sac angiogenesis. Genetic studies in humans have linked mutations in these signaling components to specific cardiovascular syndromes such as hereditary hemorrhagic telangiectasia, primary pulmonary hypertension and Marfan syndrome. In this review, we present recent advances in our understanding of the role of TGF-beta receptor signaling in vascular biology and disease, and discuss how this may be applied for therapy.

Topics: Animals; Bone Morphogenetic Proteins; Hypertension, Pulmonary; Mice; Mice, Knockout; Neovascularization, Pathologic; Neovascularization, Physiologic; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Genetic mutations in members of the transforming growth factor-beta receptor superfamily are associated with two diseases characterised by changes in the structure of the lung vasculature, pulmonary arterial hypertension and hereditary haemorrhagic telangiectasia. Pulmonary arterial hypertension is characterised by increased vessel muscularisation, sharply contrasting with the reduction in smooth muscle that occurs in hereditary haemorrhagic telangiectasia. Intriguingly, both pathologies can exist in some patients with combined disease. In this review, we discuss the contributions of convergent and divergent ligand response profiles and differing tissue expression patterns of the affected receptors to the pathologies of these diseases. We address the possible contribution of inflammation in disease progression and focus on potential emerging therapeutic targets.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Disease Progression; Drug Delivery Systems; Humans; Hypertension, Pulmonary; Ligands; Protein Binding; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a rare disorder that may be hereditable (HPAH), idiopathic (IPAH), or associated with either drug-toxin exposures or other medical conditions. Familial cases have long been recognized and are usually due to mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2), or, much less commonly, 2 other members of the transforming growth factor-beta superfamily, activin-like kinase-type 1 (ALK1) and endoglin (ENG), which are associated with hereditary hemorrhagic telangiectasia. In addition, approximately 20% of patients with IPAH carry mutations in BMPR2. We provide a summary of BMPR2 mutations associated with HPAH, most of which are unique to each family and are presumed to result in loss of function. We review the finding of missense variants and variants of unknown significance in BMPR2 in IPAH/HPAH, fenfluramine exposure, and PAH associated with congenital heart disease. Clinical testing for BMPR2 mutations is available and may be offered to HPAH and IPAH patients but should be preceded by genetic counseling, since lifetime penetrance is only 10% to 20%, and there are currently no known effective preventative measures. Identification of a familial mutation can be valuable in reproductive planning and identifying family members who are not mutation carriers and thus will not require lifelong surveillance. With advances in genomic technology and with international collaborative efforts, genome-wide association studies will be conducted to identify additional genes for HPAH, genetic modifiers for BMPR2 penetrance and genetic susceptibility to IPAH. In addition, collaborative studies of BMPR2 mutation carriers should enable identification of environmental modifiers, biomarkers for disease development and progression, and surrogate markers for efficacy end points in clinical drug development, thereby providing an invaluable resource for trials of PAH prevention.

Topics: Bone Morphogenetic Protein Receptors, Type II; Frameshift Mutation; Genetic Counseling; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Mutation, Missense; Open Reading Frames; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a complex and heterogeneous chronic illness characterized by substantial patient to patient variability in clinical manifestations, internal organ involvement, and outcome. Genetic factors contribute to disease susceptibility, but environmental influences also play a significant role. The pathogenesis of SSc encompasses vascular, immunological, and fibrotic processes, which contribute to clinical manifestations and morbidity and must be addressed in the treatment plan. Although vascular interventions appear to reduce the frequency and severity of complications, such as scleroderma renal crisis and pulmonary hypertension, current therapies generally target the immune component of SSc in a non-selective fashion and have largely failed as diseases-modifying interventions. Newer insights into the mechanisms underlying autoimmunity, vascular injury and destruction, and particularly tissue fibrosis provide novel potential targets for therapy. Transforming growth factor-ss is a ubiquitous cytokine that appears to contribute to fibroblast activation, collagen overproduction, and pathological tissue fibrosis. Neutralizing antibodies and small molecules that block TGF-beta activation or function are effective in shutting down TGF-beta signaling and selectively inhibit the progression of fibrosis and may be entering clinical trials for the treatment of SSc.

Topics: Blood Vessels; Cardiovascular Agents; Fibrosis; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Immunosuppressive Agents; Inflammation; Kidney Diseases; Lung Diseases, Interstitial; Raynaud Disease; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Treatment Outcome

Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of small, peripheral resistance vessels in the lung involving proliferation and migration of vascular smooth muscle, endothelial cell and fibroblasts. The increase in pulmonary vascular resistance leads to right heart failure, and, without treatment, death occurs within 3 years of diagnosis. The etiology of PAH is multifactorial. In some patients, there is a major genetic predisposition in the form of heterozygous germline mutations in a transforming growth factor-beta superfamily receptor, the bone morphogenetic type II receptor (BMPR-II). In addition, it is likely that additional factors, such as inflammation, are important to manifest disease. The currently available treatments for PAH were developed mainly as vasodilators, and although they improve symptoms they have limited impact on survival. This review examines the role of the BMPR-II signaling pathway in the process of pulmonary vascular remodeling. We discuss the ways in which manipulation of BMPR-II signaling might be targeted with the aim of preventing or reversing vascular remodeling and improving survival in patients with PAH.

Topics: Bone Morphogenetic Protein Receptors, Type II; Humans; Hypertension, Pulmonary; Mutation; Signal Transduction; Transforming Growth Factor beta; Vascular Resistance

Growth factors of the transforming growth factor (TGF) beta superfamily have emerged as important regulators of normal cardiovascular development, as well as modulators of the onset or progression of vascular diseases. Recently, familial and idiopathic pulmonary arterial hypertension (IPAH) has been causally linked to somatic and genetic perturbations to the TGF-beta/bone morphogenic protein (BMP) system, particularly because heterogeneous germline mutations in bmpr2 (encoding the type II BMP receptor) have been detected in IPAH patients. Transgenic animal models and functional genomic studies have begun investigating TGF-beta/BMP-induced effects in the pulmonary vasculature, as well as the cellular effects of bmpr2 mutations on vascular cell phenotypes. While these studies have significantly increased our knowledge about the biologic effects of TGF-beta/BMP signaling in the lung vasculature, the molecular mechanisms leading to pulmonary vasculopathy in the context of bmpr2 mutations in IPAH remain largely unknown.

Topics: Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Disease Progression; Endothelium; Humans; Hypertension, Pulmonary; Muscle, Smooth; Mutation; Risk Factors; Signal Transduction; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) control the development and homeostasis of multiple tissue types in many organisms, from humans to invertebrates. These morphogens are expressed in a tissue-specific manner and they signal by binding to serine-threonine kinase receptors, resulting in coordinated changes in gene expression that regulate the differentiation and development of multiple tissue types. In addition, these proteins are regulated post-transcriptionally through binding to several soluble proteins. In this review we focus on a subset of BMPs and GDFs that have been implicated in the pathophysiology of type 2 diabetes and cardiovascular disease.

Topics: Animals; Atherosclerosis; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Cardiovascular Agents; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Growth Differentiation Factor 3; Humans; Hypertension, Pulmonary; Hypoglycemic Agents; Intercellular Signaling Peptides and Proteins; Kidney Diseases; Metabolic Diseases; Signal Transduction; Transforming Growth Factor beta

Genetic studies in familial pulmonary arterial hypertension (FPAH) have revealed heterozygous germline mutations in the bone morphogenetic protein type II receptor (BMPR-II), a receptor for the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) superfamily. PAH is characterized by intense remodeling of small pulmonary arteries by myofibroblast and smooth muscle proliferation. BMPR-II mutation in pulmonary artery smooth muscle cells contributes to abnormal growth responses to BMPs and TGF-beta. Reduced expression or function of BMPR-II signaling leads to exaggerated TGF-beta signaling and altered cellular responses to TGF-beta. The likely mechanism involves an interaction between BMP and TGF-beta-regulated Smad pathways. In endothelial cells, BMPR-II mutation increases the susceptibility of cells to apoptosis. The combination of increased endothelial apoptosis and failure of growth suppression in pulmonary artery smooth muscle cells provides important clues to the cellular pathogenesis of PAH. The reciprocal regulation of TGF-beta and BMP signaling in models of tissue repair may provide new approaches to our understanding of lung disease.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Mutation, Missense; Signal Transduction; Transforming Growth Factor beta

Novel discoveries of the genetic basis of pulmonary hypertension have recently inspired a whole new area of research in pulmonary medicine. The finding that germ-line mutations in the BMPR2 locus, encoding a transmembrane receptor of the TGF beta superfamily, are the cause for familial and partly idiopathic pulmonary arterial hypertension has provided new clues for our understanding of this complex, yet highly localized disease. The TGF beta superfamily comprises more than 40 different growth and differentiation factors essential in the control of proliferation and differentiation of most cell types. In this review, we summarize the novel genetic and functional genomic findings to provide an outlook for the understanding of this disease.

Topics: Bone Morphogenetic Protein Receptors, Type II; DNA Mutational Analysis; Genetic Predisposition to Disease; Genetic Testing; Genomics; Humans; Hypertension, Pulmonary; Incidence; Polymorphism, Genetic; Protein Serine-Threonine Kinases; Risk Assessment; Risk Factors; Transforming Growth Factor beta

Idiopathic pulmonary arterial hypertension (formerly primary pulmonary hypertension) can affect more than one member of the same family. In the past 25 years scientists have exposed the inheritance pattern and a major element of the molecular basis for inherited pulmonary arterial hypertension. Familial pulmonary arterial hypertension is inherited as an autosomal dominant trait with incomplete penetrance (i.e., several individuals inherit a predisposition to the disease, but never express it). Mutations in the gene that codes for bone morphogenetic protein receptor type II (BMPR-II) are a major predisposition for the development of pulmonary arterial hypertension. These mutations are present in at least half of familial cases of pulmonary arterial hypertension and 10 to 25% of idiopathic pulmonary arterial hypertension patients. Mutations in the gene that codes for activin receptor-like kinase (ALK 1), another transforming growth factor beta (TGF-beta) cell surface receptor, appear responsible for the rare occurrence of pulmonary arterial hypertension in patients with hereditary hemorrhagic telangiectasia. These discoveries coupled with other basic investigations offer opportunities for advances in the management of pulmonary arterial hypertension.

Topics: Activin Receptors; Bone Morphogenetic Protein Receptors, Type II; Forecasting; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Mutation; Pedigree; Polymorphism, Genetic; Risk Factors; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

In agreement with previous data in the literature, our results indicate that serotonin, a monoamine neurotransmitter, can also regulate cell proliferation, cell movements and cell differentiation. We have recently shown that serotonin is required for embryonic heart development. Genetic ablation of the 5-HT2B receptor leads to partial embryonic and postnatal lethality with abnormal heart development. Similar molecular mechanisms seem to be involved in adult cardiomyocytes since mutant mice surviving to adulthood display a dilated cardiomyopathy. Furthermore this receptor appears to be involved in survival of cardiomyocytes. The 5-HT2B receptor is also implicated in systemic hypertension. Furthermore, mice with pharmacological or genetic ablation of 5-HT2B receptor are totally resistant to hypoxia-induced pulmonary hypertension, indicating that this receptor is regulating the pathologic vascular proliferation leading to this disease. Underlying mechanisms are still to be discovered.

Topics: Adult; Animals; Cardiomyopathy, Dilated; Cell Survival; Fenfluramine; Fetal Heart; Genes, Lethal; Genetic Predisposition to Disease; Heart Defects, Congenital; Humans; Hypertension; Hypertension, Pulmonary; Hypoxia; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Smooth, Vascular; Myocytes, Cardiac; Organ Specificity; Pancreatic Elastase; Pulmonary Artery; Rats; Receptor, Serotonin, 5-HT2B; Serotonin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Bone Morphogenetic Protein Receptors, Type II; Humans; Hypertension, Pulmonary; Mutation; Protein Serine-Threonine Kinases; Transforming Growth Factor beta

Pulmonary hypertension (PH) is a progressive disease of the pulmonary vasculature characterized by increased vascular resistance and pressure overload of the right ventricle. Histologically, PH lungs demonstrate medial hypertrophy of small pulmonary arteries and proliferation of endothelial cells resulting in plexiform lesions. Recent studies have identified mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene and the activin-receptor-like kinase 1 (ALK1) gene, that affect the transforming growth factor beta (TGF-beta) receptor superfamily, a group of transmembrane signaling molecules with serine-threonine kinase activity that are involved in the regulation of cell growth. Several lines of evidence indicate that the development of PH is a multi-hit process, where one of the events is having a gene mutation and another might be a circumstantial condition or other disease-modifying genes. It is unknown which mechanism that is critical in rheumatic diseases causes pulmonary vascular disease. PH is most frequently associated with systemic sclerosis (SS), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), however, it is still a rare manifestation of these disorders. For example, approximately 10% of SS cases manifest pulmonary vascular disease. In recent years symptomatic vasodilator therapies have been employed and have been able to improve exercise capacity and survival in these patients.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Anticoagulants; Bone Morphogenetic Protein Receptors, Type II; Cell Division; Endothelium, Vascular; Humans; Hypertension, Pulmonary; Protein Serine-Threonine Kinases; Rheumatic Diseases; Signal Transduction; Transforming Growth Factor beta; Vascular Resistance; Vasodilator Agents

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder in humans which has been mapped to two genes, endoglin and activin receptor-like kinase-1 (ALK-1) both of which mediate signaling by transforming growth factor beta ligands in vascular endothelial cells. Animal models have shown that these receptors are not only important for maintaining vascular integrity but also for angiogenesis both during embryonic development and during tumor growth. Here, we review the current status of reported mutations in the context of the clinical manifestations and the effects on the vessel wall both in patients and in animal models of the disease.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Animals; Antigens, CD; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 9; Endoglin; Humans; Hypertension, Pulmonary; Mice; Mice, Mutant Strains; Mutation; Neovascularization, Pathologic; Receptors, Cell Surface; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1; Zebrafish

Topics: Animals; Cell Division; DNA-Binding Proteins; Humans; Hypertension, Pulmonary; Neoplasms; Signal Transduction; Smad Proteins; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

Primary pulmonary hypertension has been described as either sporadic or clustered in families. Familial primary pulmonary hypertension segregates as an autosomal dominant trait with markedly reduced disease gene penetrance. Defects within bone morphogenetic protein receptor type II gene, coding for a receptor member of the transforming growth factor-beta family, underlie familial primary pulmonary hypertension. Several lines of evidence point to the potential requirement of additional factors, either environmental or genetic, in the pathogenesis of the disease. In addition, a proportion of so-called sporadic primary pulmonary hypertension turns out to have an inherited basis, as demonstrated by germline bone morphogenetic protein receptor type II gene mutations. Analysis of cases in association with hereditary haemorrhagic telangiectasia led to the demonstration that pulmonary arterial hypertension can involve activin-receptor-like kinase 1 mutations, a type I transforming growth factor-beta receptor. These findings emphasise the critical role of the transforming growth factor-beta signalling pathway in pulmonary arterial hypertension. While this achievement has generated extreme interest, the pathobiology of severe pulmonary arterial hypertension remains unclear and genomic approaches to pulmonary hypertension research may identify additional molecular determinants for this disorder. Finally, there is an urgent need to develop relevant guidelines for genetic counselling to assist patients, their relatives and pulmonary vascular specialists to utilise these recent observations.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Bone Morphogenetic Protein Receptors, Type II; Genetic Testing; Germ-Line Mutation; Glycogen Storage Disease Type I; Humans; Hypertension, Pulmonary; Mutation; Protein Serine-Threonine Kinases; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Members of the transforming growth factor (TGF)-beta superfamily play critical roles in regulation of various cellular functions. Dysregulation of the signaling mechanisms of the TGF-beta superfamily proteins is associated with clinical diseases such as cancer, fibrotic diseases, and vascular disorders. Therefore, understanding these signaling mechanisms may provide us with novel ways to develop strategies for treating clinical diseases induced by these cytokines.. This review discusses our current understanding of the mechanisms of TGF-beta signaling, focusing on the roles of TGF-beta in regulation of vascular wall cells and on the regulation of TGF-beta superfamily signals by inhibitory Smads.

Topics: Bone Morphogenetic Proteins; DNA-Binding Proteins; Humans; Hypertension, Pulmonary; Ligases; Neovascularization, Pathologic; Signal Transduction; Smad Proteins; Telangiectasia, Hereditary Hemorrhagic; Trans-Activators; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

There have been remarkable advances in our understanding of the pathobiology of pulmonary hypertension. A region on chromosome 2 encoding bone morphogenetic receptor type 2 has been identified to underlie familial and many cases of sporadic primary pulmonary arterial hypertension. The vasoactive mediators, discovered and defined by vascular biologists, have been translated into promising treatments of human disease. Prostacyclin, endothelin receptor blockers, sildenafil, and nitric oxide have been applied therapeutically to limit, and occasionally reverse, the inexorable damage to the pulmonary circulation initiated by recently identified genetic and environmental triggers of pulmonary arterial hypertension.

Topics: Antihypertensive Agents; Bone Morphogenetic Protein Receptors, Type II; Bronchodilator Agents; Child; Chromosomes, Human, Pair 2; Eisenmenger Complex; Epoprostenol; Humans; Hypertension, Pulmonary; Mutation; Nitric Oxide; Piperazines; Protein Serine-Threonine Kinases; Pulmonary Artery; Purines; Receptors, Endothelin; Sildenafil Citrate; Sulfones; Transforming Growth Factor beta; Vasodilator Agents

Topics: Activin Receptors, Type I; Animals; Cell Division; Humans; Hypertension, Pulmonary; Osteochondrodysplasias; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Trans-Activators; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.

Topics: Animals; Bone Morphogenetic Proteins; DNA-Binding Proteins; Humans; Hypertension, Pulmonary; Ligands; Mice; Multigene Family; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Vascular Diseases

Topics: Bone Morphogenetic Protein Receptors, Type II; Carrier Proteins; Humans; Hypertension, Pulmonary; Membrane Glycoproteins; Membrane Transport Proteins; Models, Biological; Muscle, Smooth, Vascular; Nerve Tissue Proteins; Polymorphism, Genetic; Protein Serine-Threonine Kinases; Serotonin; Serotonin Plasma Membrane Transport Proteins; Signal Transduction; Transforming Growth Factor beta

This paper concentrates on the genetic aspects of pulmonary arterial hypertension (PAH), a diagnostically based subclass of pulmonary hypertension that includes primary pulmonary hypertension (PPH). During the past year, patients with familial and sporadic PPH were found to have germline heterozygous missense, nonsense and frameshift mutations in bone morphogenetic protein receptor II (BMPR2). Mutations in BMPR2, a member of the transforming growth factor-beta (TGF-beta) receptor superfamily, are predicted to interrupt the bone morphogenetic protein (BMP) signalling pathway, resulting in proliferation, rather than apoptosis of cells within small arterioles. Mechanistically, haploinsufficiency was found by using in vitro gene expression experiments, but a dominant-negative mechanism has not been excluded. The failure to find BMPR2 mutations in all families with familial PPH and in all patients with sporadic PPH suggests that other genes remain to be identified. Mutations in ALK1, a TGF-beta type 1 receptor, previously known to cause type 2 hereditary haemorrhagic telangiectasia (HHT), have also been reported in a few HHT families with clinical and histological features of PPH. The clinical development of PPH, as in neoplasia, appears to require 'two hits' The two hits can be provided either by genetic or environmental factors.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Frameshift Mutation; Humans; Hypertension, Pulmonary; Mutation, Missense; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Topics: Animals; Humans; Hypertension, Pulmonary; Mice; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Topics: Animals; Arachidonate 5-Lipoxygenase; Endothelial Growth Factors; Endothelins; Humans; Hypertension, Pulmonary; Inflammation; Insulin-Like Growth Factor I; Lymphokines; Platelet Activating Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Skeletal muscle dysfunction is a clinically important complication of pulmonary arterial hypertension (PAH). Growth/differentiation factor 15 (GDF-15), a prognostic marker in PAH, has been associated with muscle loss in other conditions. We aimed to define the associations of GDF-15 and muscle wasting in PAH, to assess its utility as a biomarker of muscle loss and to investigate its downstream signalling pathway as a therapeutic target.. GDF-15 levels and measures of muscle size and strength were analysed in the monocrotaline (MCT) rat, Sugen/hypoxia mouse and in 30 patients with PAH. In C2C12 myotubes the downstream targets of GDF-15 were identified. The pathway elucidated was then antagonised in vivo.. Circulating GDF-15 levels correlated with tibialis anterior (TA) muscle fibre diameter in the MCT rat (Pearson r=-0.61, p=0.003). In patients with PAH, plasma GDF-15 levels of <564 pg/L predicted those with preserved muscle strength with a sensitivity and specificity of ≥80%. In vitro GDF-15 stimulated an increase in phosphorylation of TGFβ-activated kinase 1 (TAK1). Antagonising TAK1, with 5(Z)-7-oxozeaenol, in vitro and in vivo led to an increase in fibre diameter and a reduction in mRNA expression of atrogin-1 in both C2C12 cells and in the TA of animals who continued to grow. Circulating GDF-15 levels were also reduced in those animals which responded to treatment.. Circulating GDF-15 is a biomarker of muscle loss in PAH that is responsive to treatment. TAK1 inhibition shows promise as a method by which muscle atrophy may be directly prevented in PAH.. NCT01847716; Results.

Topics: Adult; Animals; Biomarkers; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Female; Growth Differentiation Factor 15; Humans; Hypertension, Pulmonary; Immunohistochemistry; Male; MAP Kinase Kinase Kinases; Mice; Middle Aged; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Vitamin D deficiency is common and linked to poor prognosis in pulmonary arterial hypertension (PAH). We investigated the differential effect of basal vitamin D levels in monocrotaline (MCT) induced PAH in normal and vitamin D deficient (VDD) rats. Rats were fed a VDD diet and exposed to filtered fluorescent light to deplete vitamin D. Normal rats were pretreated with vitamin D 100 IU/d and treated with vitamin D 100 and 200 IU/d, while VDD rats received vitamin D 100 IU/d. Vitamin D receptor (VDR) silencing was done in human umbilical vein endothelial cells (HUVECs) using VDR siRNA. Calcitriol (50 nM/mL) was added to human pulmonary artery smooth muscle cells (HPASMCs) and HUVECs before and after the exposure to TGF-β (10 ng/mL). Vitamin D 100 IU/d pretreatment in normal rats up-regulated the expression of eNOS and inhibited endothelial to mesenchymal transition significantly and maximally. Vitamin D 100 IU/d treatment in VDD rats was comparable to vitamin D 200 IU/d treated normal rats. These effects were significantly attenuated by L-NAME (20 mg/kg), a potent eNOS inhibitor. Exposure to TGF- β significantly reduced the expression of eNOS and increased the mesenchymal marker expression in normal and VDR-silenced HUVECs and HPASMCs, which were averted by treatment and maximally inhibited by pretreatment with calcitriol (50 nM). To conclude, this study provided novel evidence suggesting the beneficial role of higher basal vitamin D levels, which are inversely linked with PAH severity.

Topics: Animals; Calcitriol; Human Umbilical Vein Endothelial Cells; Humans; Hypertension, Pulmonary; Monocrotaline; Pulmonary Arterial Hypertension; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta; Vitamin D; Vitamin D Deficiency; Vitamins

Cannabidiol (CBD) is a non-intoxicating compound of Cannabis with anti-fibrotic properties. Pulmonary hypertension (PH) is a disease that can lead to right ventricular (RV) failure and premature death. There is evidence that CBD reduces monocrotaline (MCT)-induced PH, including reducing right ventricular systolic pressure (RVSP), vasorelaxant effect on pulmonary arteries, and decreasing expression of profibrotic markers in the lungs. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on profibrotic parameters in the RVs of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters and parameters related to RV dysfunction, i.e. plasma pro-B-type natriuretic peptide (NT-proBNP), cardiomyocyte width, interstitial and perivascular fibrosis area, amount of fibroblasts and fibronectin, as well as overexpression of the transforming growth of factor β1 (TGF-β1), galectin-3 (Gal-3), suppressor of mothers against decapentaplegic 2 (SMAD2), phosphorylated SMAD2 (pSMAD2) and alpha-smooth muscle actin (α-SMA). In contrast, vascular endothelial cadherin (VE-cadherin) levels were decreased in the RVs of MCT-induced PH rats. Administration of CBD reduced the amount of plasma NT-proBNP, the width of cardiomyocytes, the amount of fibrosis area, fibronectin and fibroblast expression, as well as decreased the expression of TGF-β1, Gal-3, SMAD2, pSMAD2, and increased the level of VE-cadherin. Overall, CBD has been found to have the anti-fibrotic potential in MCT-induced PH. As such, CBD may act as an adjuvant therapy for PH, however, further detailed investigations are recommended to confirm our promising results.

Topics: Animals; Cannabidiol; Fibronectins; Fibrosis; Heart Failure; Hypertension, Pulmonary; Monocrotaline; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

In pulmonary hypertension (PH), pulmonary arterial remodeling is often accompanied by perivascular inflammation. The inflammation is characterized by the accumulation of activated macrophages and lymphocytes within the adventitial stroma, which is comprised primarily of fibroblasts. The well-known ability of fibroblasts to secrete interleukins and chemokines has previously been implicated as contributing to this tissue-specific inflammation in PH vessels. We were interested if pulmonary fibroblasts from PH arteries contribute to microenvironmental changes that could activate and polarize T-cells in PH.. We used single-cell RNA sequencing of intact bovine distal pulmonary arteries (dPAs) from PH and control animals and flow cytometry, mRNA expression analysis, and respirometry analysis of blood-derived bovine/human T-cells exposed to conditioned media obtained from pulmonary fibroblasts of PH/control animals and IPAH/control patients (CM-(h)PH Fibs vs CM-(h)CO Fibs).. Single-cell RNA sequencing of intact bovine dPAs from PH and control animals revealed a pro-inflammatory phenotype of CD4+ T-cells and simultaneous absence of regulatory T-cells (FoxP3+ Tregs). By exposing T-cells to CM-(h)PH Fibs we stimulated their proinflammatory differentiation documented by increased IFNγ and decreased IL4, IL10, and TGFβ mRNA and protein expression. Interestingly, we demonstrated a reduction in the number of suppressive T-cell subsets, i.e., human/bovine Tregs and bovine γδ T-cells treated with CM-(h)PH-Fibs. We also noted inhibition of anti-inflammatory cytokine expression (IL10, TGFβ, IL4). Pro-inflammatory polarization of bovine T-cells exposed to CM-PH Fibs correlated with metabolic shift to glycolysis and lactate production with increased prooxidant intracellular status as well as increased proliferation of T-cells. To determine whether metabolic reprogramming of PH-Fibs was directly contributing to the effects of PH-Fibs conditioned media on T-cell polarization, we treated PH-Fibs with the HDAC inhibitor SAHA, which was previously shown to normalize metabolic status and examined the effects of the conditioned media. We observed significant suppression of inflammatory polarization associated with decreased T-cell proliferation and recovery of mitochondrial energy metabolism.. This study demonstrates how the pulmonary fibroblast-derived microenvironment can activate and differentiate T-cells to trigger local inflammation, which is part of the vascular wall remodeling process in PH.

Topics: Animals; Cattle; Culture Media, Conditioned; Humans; Hypertension, Pulmonary; Inflammation; Interleukin-10; Interleukin-4; T-Lymphocyte Subsets; Transforming Growth Factor beta

Dysregulated BMP (bone morphogenetic protein) or TGF-β (transforming growth factor beta) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. MED1 (mediator complex subunit 1) is a key transcriptional co-activator and KLF4 (Krüppel-like factor 4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-β axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive. We investigate the MED1/KLF4 co-regulation of the BMP/TGF-β axes in endothelium by studying the epigenetic regulation of BMPR2 (BMP receptor type II), ETS-related gene (. High-throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, ATAC-seq, and high-throughput chromosome conformation capture together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells (ECs) and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic PAH, animals with experimental PH, and mice with endothelial ablation of MED1 (EC-. Levels of MED1 were decreased in lung tissue or pulmonary arterial endothelial cells from idiopathic PAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-. A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for the normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-β signaling is implicated in the disease progression of PAH in humans and PH in rodent models.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Endothelial Cells; Endothelium, Vascular; Epigenesis, Genetic; Humans; Hypertension, Pulmonary; Mediator Complex Subunit 1; Mice; Pulmonary Arterial Hypertension; Pulmonary Artery; Receptor, Transforming Growth Factor-beta Type II; Transcription Factors; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Topics: Animals; Disease Models, Animal; Endothelial Cells; Humans; Hypertension, Pulmonary; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Pulmonary Artery; Rats; Transforming Growth Factor beta; Vascular Remodeling

We recently identified oncologic miR-182 as a new regulator of pulmonary artery hypertension (PAH) that targets myeloid-associated differentiation marker (Myadm), which is expressed in bone marrow stem cells and multipotent progenitors. Both miR-182 and Myadm are expressed in the cardiopulmonary system and correlated with the balance between the bone morphogenetic protein (BMP) and the transforming growth factor (TGF)-β signalling pathways, which are disturbed in PAH. We hypothesize that miR-182/Myadm are involved in BMP-TGF-β-signalling way in PAH. Hypoxia triggered pathological progression in cardiopulmonary PAH in vivo and in vitro; these changes were accompanied by strongly dowregulated BMP/SMAD1/5/8 expression and enhanced TGF-β/SMAD2/3 signalling pathway, favouring SMAD4/SMAD2 transcript formation and inhibiting the PAH negative regulator Id1 expression. miR-182 gain-of-function significantly inhibited the pathological progression in hypoxia-induced PAH (HPH) in vivo and in vitro, with a restoration of the balance in BMP-TGF-β signalling pathway. This recovery was abrogated by overexpression of Myadm. Conversely, loss-of-function of miR-182 increased the pathological progression of HPH followed by severe disturbance of BMP and TGF-β signal transduction and reduced Id1 expression, which was restored by Myadm knockdown. We also showed that the miR-182/Myadm relate BMP-TGF-β pathway is associated with NOS3/NO/cGMP via the crosstalk between endothelial cells and smooth muscle cells. Our findings further support the therapeutic significance of miR-182/Myadm in PAH via the balance of BMP- and TGF-β-associated mechanisms.

Topics: Bone Morphogenetic Proteins; Endothelial Cells; Humans; Hypertension, Pulmonary; Hypoxia; MicroRNAs; Myelin and Lymphocyte-Associated Proteolipid Proteins; Pulmonary Artery; Transforming Growth Factor beta

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFβ-signaling both in vitro and in vivo. Endothelial-specific TGFβR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFβR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Topics: Animals; Gene Deletion; Gene Expression Regulation; Genotype; Hindlimb; Human Umbilical Vein Endothelial Cells; Humans; Hypertension, Pulmonary; Ischemia; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinase 3; Mice; Receptors, Transforming Growth Factor beta; Selective Estrogen Receptor Modulators; Signal Transduction; Tamoxifen; Transforming Growth Factor beta; Vascular Remodeling

Recent translational studies highlighted the inhibition of transforming growth factor (TGF)-β signaling as a promising target to treat pulmonary arterial hypertension (PAH). However, it remains unclear whether alterations in TGF-β signaling are consistent between PAH patients and animal models. Therefore, we compared TGF-β signaling in the lungs of PAH patients and rats with experimental PAH induced by monocrotaline (MCT) or SU5416+hypoxia (SuHx). In hereditary PAH (hPAH) patients, there was a moderate increase in both TGFβR2 and pSMAD2/3 protein levels, while these were unaltered in idiopathic PAH (iPAH) patients. Protein levels of TGFβR2 and pSMAD2/3 were locally increased in the pulmonary vasculature of PAH rats under both experimental conditions. Conversely, the protein levels of TGFβR2 and pSMAD2/3 were reduced in SuHx while slightly increased in MCT. mRNA levels of plasminogen activator inhibitor (PAI)-1 were increased only in MCT animals and such an increase was not observed in SuHx rats or in iPAH and hPAH patients. In conclusion, our data demonstrate considerable discrepancies in TGFβ-SMAD signaling between iPAH and hPAH patients, as well as between patients and rats with experimental PAH.

Topics: Animals; Blood Pressure; Disease Models, Animal; Humans; Hypertension, Pulmonary; Phosphorylation; Plasminogen Activator Inhibitor 1; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; RNA, Messenger; Signal Transduction; Smad Proteins; Systole; Transforming Growth Factor beta

Clinical outcomes of pulmonary arterial hypertension (PAH) may be improved using targeted delivery system. We investigated the efficacy of ONO1301 (prostacyclin agonist) nanospheres (ONONS) in Sugen5416/hypoxia rat models of PAH. The rats were injected with saline (control) or ONONS (n = 10, each) on days 21 and 28, respectively. Hepatocyte growth factor (HGF)-expressing fibroblasts and inflammatory cytokines were measured. Cardiac performance was assessed and targeted delivery was monitored in vivo, using Texas red-labeled nanoparticles. Compared with control, HGF-expressing fibroblasts and HGF expression levels were significantly higher in the ONONS group, while the levels of interleukin-6, interleukin-1β, transforming growth factor-β, and platelet-derived growth factor were lower. Histological assessment revealed significant amelioration of the percent medial wall thickness in pulmonary vasculature of rats in the ONONS group. Rats in the ONONS group showed decreased proliferating cell nuclear antigen-positive smooth muscle cells and improved right ventricle pressure/left ventricle pressure. No difference was seen in the accumulation of Texas red-labeled nanoparticles in the brain, heart, liver, and spleen between PAH and normal rats. However, a significant area of nanoparticles was detected in the lungs of PAH rats. ONONS effectively ameliorated PAH, with selective delivery to the damaged lung.

Topics: Animals; Antihypertensive Agents; Cells, Cultured; Epoprostenol; Fibroblasts; Hepatocyte Growth Factor; Hypertension, Pulmonary; Interleukins; Male; Myocytes, Smooth Muscle; Nanocapsules; Platelet-Derived Growth Factor; Pulmonary Artery; Pyridines; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFβ (transforming growth factor-β) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFβ in the pathophysiology of CTEPH is unknown.. To determine whether defective TGFβ signaling in endothelial cells contributes to thrombus nonresolution and fibrosis.. Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFβ1 in platelets (Plt.TGFβ-KO) or TGFβ type II receptors in endothelial cells (End.TGFβRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFβ1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFβRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFβ1 levels, endothelial TGFβRI/ALK1 (activin receptor-like kinase), and TGFβRI/ALK5 expression were detected in End.TGFβRII-KO mice, and activated TGFβ signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFβRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFβ1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFβRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFβ1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFβRII-KO mice.. Endothelial TGFβ1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Topics: Activin Receptors, Type II; Aged; Aged, 80 and over; Animals; Blood Platelets; Endothelin-1; Female; Human Umbilical Vein Endothelial Cells; Humans; Hypertension, Pulmonary; Male; Mice; Mutation; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Transforming Growth Factor beta; Venae Cavae; Venous Thrombosis

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-β treatment would phenocopy these metabolic changes. We used

Topics: Adolescent; Adult; Case-Control Studies; Cell Proliferation; Cells, Cultured; Citric Acid Cycle; Endothelium, Vascular; Female; Glycolysis; Humans; Hypertension, Pulmonary; Isotope Labeling; Male; Metabolome; Middle Aged; Muscle, Smooth, Vascular; Pentose Phosphate Pathway; Pulmonary Artery; Signal Transduction; Transforming Growth Factor beta

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells (ECs) contribute to severe pulmonary arterial hypertension (PAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). The remodelled pulmonary arteries of PAH patients harboured CD117

Topics: Animals; Apoptosis; Arterial Occlusive Diseases; Bone Morphogenetic Proteins; Cell Lineage; Cell Separation; Cells, Cultured; Chronic Disease; Clone Cells; Endothelial Cells; Flow Cytometry; Humans; Hypertension, Pulmonary; Hypoxia; Male; Mesoderm; Proto-Oncogene Proteins c-kit; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Transcriptome; Transforming Growth Factor beta

Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied.. Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension.. Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-β1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression.. MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

Topics: Animals; Cells, Cultured; DNA (Cytosine-5-)-Methyltransferase 1; Epigenesis, Genetic; Fibrosis; Heart Ventricles; Hypertension, Pulmonary; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mitochondria, Heart; Mitochondrial Dynamics; Monocrotaline; Myofibroblasts; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

ProT is a cell survival gene, which modulates oxidative stress and transforming growth factor (TGF)-β signaling. We hypothesized that the delivery of the ProT cDNA gene in rats could protect against right heart dysfunction secondary to pulmonary hypertension (PH) induced by left-to-right shunt.. A 2-hit rat model of flow-induced PH was used, and a single intravenous injection of adenoviral vectors (2 billion plaque-forming unit) carrying ProT or Luc gene was administered. The animals were euthanized 21 days after gene delivery to assess cardiopulmonary function, serum biochemistry, pulmonary artery (PA), and vasomotor reactivity. Immunohistology and immunoblotting of PA tissues were also performed.. ProT transduction significantly reduced PA pressure, right ventricle muscle mass, and wall stress, thereby improving the overall survival of the treated rat. Increased production of ProT through gene therapy preserved both the smooth muscle myosin heavy chain-II and α-smooth muscle actin while counteracting the abundance of TGF-β in PA. Protein abundances of phosphorylated p47-phox, heme oxygenase-1, caspase-3, inducible nitric oxide synthase, cyclo-oxygenase 2, and monocyte chemoattractant protein-1 in PA tissues were reduced. ProT also preserved microRNA-223, thereby suppressing the abundance of PARP-1, which is independent of hypoxia-inducible factor-1α signaling.. ProT gene transduction improved PA function by reducing oxidative stress, attenuating inflammation, and preserving the contractile phenotype of vascular smooth muscle cells. The modification of microRNA-223-associated downstream signaling through ProT transduction may play an important role in mitigating cardiopulmonary remodeling in flow-induced PH.

Topics: Animals; Disease Models, Animal; Gene Transfer Techniques; Hypertension, Pulmonary; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Survival Rate; Transforming Growth Factor beta

Primary myelofibrosis (PMF) is a Ph-negative myeloproliferative neoplasm (MPN), characterized by advanced bone marrow fibrosis and extramedullary haematopoiesis. The bone marrow fibrosis results from excessive proliferation of fibroblasts that are influenced by several cytokines in the microenvironment, of which transforming growth factor-β (TGF-β) is the most important. Micromechanics related to the niche has not yet been elucidated. In this study, we hypothesized that mechanical stress modulates TGF-β signalling leading to further activation and subsequent proliferation and invasion of bone marrow fibroblasts, thus showing the important role of micromechanics in the development and progression of PMF, both in the bone marrow and in extramedullary sites. Using three PMF-derived fibroblast cell lines and transforming growth factor-β receptor (TGFBR) 1 and 2 knock-down PMF-derived fibroblasts, we showed that mechanical stress does stimulate the collagen synthesis by the fibroblasts in patients with myelofibrosis, through the TGFBR1, which however seems to be activated through alternative pathways, other than TGFBR2.

Topics: Animals; Biomechanical Phenomena; Disease Progression; Fibroblasts; Humans; Hypertension, Pulmonary; Mice, Nude; Models, Biological; Primary Myelofibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Stress, Mechanical; Transforming Growth Factor beta

Heart failure with reduced ejection fraction (HFrEF) causes lung remodelling with myofibroblasts proliferation and fibrosis leading to a restrictive lung syndrome with pulmonary hypertension (PH) and right ventricular (RV) dysfunction. PBI-4050 is a first-in-class anti-fibrotic, anti-inflammatory, and anti-proliferative compound. The present study evaluated the therapeutic impact of PBI-4050 on PH in an HFrEF model.. HFrEF was induced after myocardial infarction (MI) in rats. Two weeks later, sham-operated and MI groups received PBI-4050 (200 mg/kg/day by gavage) or saline for 3 weeks. Animals were analysed according to infarct size as large (≥30% left ventricle) or medium MI (<30%). Large MI caused PH and RV hypertrophy (RVH) with a restrictive lung syndrome. PBI-4050 did not adversely affect left ventricular (LV) function but markedly reduced PH and RVH and improved RV dysfunction. PBI-4050 reduced lung remodelling and improved respiratory compliance with decreased lung fibrosis, alveolar wall cellular proliferation and α-smooth muscle actin expression. The increased expression of endothelin-1 (ET-1), transforming growth factor beta (TGF-β), interleukin-6 (IL-6) and of tissue inhibitor of metalloprotease-1 in the lungs from HFrEF were reduced with PBI-4050 therapy. Activation of isolated human lung fibroblasts (HLFs) to a myofibroblastic pro-fibrogenic phenotype was markedly reduced by PBI-4050. The fatty acid receptor GPR84 was increased in HFrEF lungs and in activated HLFs, and reduced by PBI-4050. GPR84 agonists activated fibrogenesis in HLFs and finally, PBI-4050 reduced ERK1/2 phosphorylation.. PBI-4050 reduces PH and RVH in HFrEF by decreasing lung fibrosis and remodelling. This novel agent decreases the associated restrictive lung syndrome and recovers RV function. A contributing mechanism involves reducing the activation of lung fibroblasts by IL-6, TGF-β, and ET-1 by antagonism of GPR84 and reduced ERK1/2 phosphorylation. PBI-4050 is a novel promising therapy for targeting lung remodelling in group II PH.

Topics: Acetates; Animals; Cells, Cultured; Disease Models, Animal; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibrosis; Heart Failure; Heart Ventricles; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Interleukin-6; Lung; Male; Phosphorylation; Pulmonary Fibrosis; Rats, Wistar; Receptors, G-Protein-Coupled; Signal Transduction; Transforming Growth Factor beta; Ventricular Dysfunction, Right; Ventricular Function, Right; Ventricular Remodeling

Transforming growth factor beta (TGF-β) has been shown to play a critical role in pathogenesis of pulmonary arterial hypertension (PAH) although the precise role of TGF-β signaling remains uncertain. A recent report has shown that periostin (Pn) is one of the most upregulated proteins in human PAH lung compared with healthy lungs. We established type I TGF-β receptor knockout mice specifically with Pn expressing cell (Pn-Cre/Tgfb1fl/fl mice). Increases in PA pressure and pulmonary artery muscularization were induced by hypoxia of 10% oxygen for 4 weeks. Lung Pn expression was markedly induced by 4 week-hypoxia. Pn-Cre/Tgfb1fl/fl mice showed lower right ventricular pressure elevation, inhibition of PA medial thickening. Fluorescent co-immunostaining showed that Smad3 activation in Pn expressing cell is attenuated. These results suggest that TGF-β signaling in Pn expressing cell may have an important role in the pathogenesis of PAH by controlling medial thickening.

Topics: Animals; Cell Adhesion Molecules; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Pulmonary Arterial Hypertension; Pulmonary Artery; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factors

Excessive TGF-β signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their contents and significance have not yet been studied. Here, we aimed to analyse the contents and biological relevance of HPASMC-EVs and their transport to human pulmonary arterial endothelial cells (HPAECs), as well as the potential alteration of these under pathological conditions.. We used low-input RNA-Seq to analyse the RNA cargoes sorted into released HPASMC-EVs under basal conditions. We additionally analysed the effects of excessive TGF-β signalling, using TGF-β1 and BMP4, in the transcriptome of HPASMCs and their EVs. We then, for the first time, optimised Cre-loxP technology for its use with primary cells in vitro, directly visualising HPASMC-to-HPAEC communication and protein markers on cells taking up EVs. Furthermore we could analyse alteration of this transport with excessive TGF-β signalling, as well as by other cytokines involved in PAH: IL-1β, TNF-α and VEGFA.. We were able to detect transcripts from 2417 genes in HPASMC-EVs. Surprisingly, among the 759 enriched in HPASMC-EVs compared to their donor cells, we found Zeb1 and 2 TGF-β superfamily ligands, GDF11 and TGF-β3. Moreover, we identified 90 genes differentially expressed in EVs from cells treated with TGF-β1 compared to EVs in basal conditions, including a subset involved in actin and ECM remodelling, among which were bHLHE40 and palladin. Finally, using Cre-loxP technology we showed cell-to-cell transfer and translation of HPASMC-EV Cre mRNA from HPASMC to HPAECs, effectively evidencing communication via EVs. Furthermore, we found increased number of smooth-muscle actin positive cells on HPAECs that took up HPASMC-EVs. The uptake and translation of mRNA was also higher in activated HPAECs, when stimulated with TGF-β1 or IL-1β.. HPASMC-EVs are enriched in RNA transcripts that encode genes that could contribute to vascular remodelling and EndoMT during development and PAH, and TGF-β1 up-regulates some that could enhance this effects. These EVs are functionally transported, increasingly taken up by activated HPAECs and contribute to EndoMT, suggesting a potential effect of HPASMC-EVs in TGF-β signalling and other related processes during PAH development.

Topics: Bone Morphogenetic Proteins; Endothelium, Vascular; Extracellular Vesicles; Growth Differentiation Factors; Humans; Hypertension, Pulmonary; Interleukin-1beta; Myocytes, Smooth Muscle; Phenotype; Pulmonary Artery; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta3; Vascular Remodeling; Zinc Finger E-box-Binding Homeobox 1

Topics: Growth Differentiation Factor 15; Humans; Hypertension, Pulmonary; Muscle, Skeletal; Muscular Atrophy; Transforming Growth Factor beta

Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, C─C motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Epithelium; Fibronectins; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Inflammation; Lung; Monocrotaline; Phosphodiesterase 5 Inhibitors; Rats, Wistar; Transforming Growth Factor beta

Pulmonary hypertension (PH) is a devastating pulmonary vascular disease characterized by aberrant muscularization of the normally nonmuscularized distal pulmonary arterioles. The expression of the transcription factor, Twist1, increases in the lungs of patients with pulmonary arterial hypertension. However, the mechanisms by which Twist1 controls the pathogenesis of PH remain unclear. It is becoming clear that endothelial-to-mesenchymal transition (EndMT) contributes to various vascular pathologies, including PH; Twist1 is known to mediate EndMT. In this report, we demonstrate that Twist1 overexpression increases transforming growth factor (TGF) β receptor2 (TGF-βR2) expression and Smad2 phosphorylation, and induces EndMT in cultured human pulmonary arterial endothelial (HPAE) cells, whereas a mutant construct of Twist1 at the serine 42 residue (Twist1S42A) fails to induce EndMT. We also implanted fibrin gel supplemented with HPAE cells on the mouse lung, and found that these HPAE cells form vascular structures and that Twist1-overexpressing HPAE cells undergo EndMT in the gel, whereas Twist1S42A-overexpressing cells do not. Furthermore, hypoxia-induced EndMT is inhibited in endothelial cells overexpressing Twist1S42A mutant construct in vitro. Hypoxia-induced accumulation of α-smooth muscle actin-positive cells in the pulmonary arterioles is attenuated in Tie2-specific Twist1 conditional knockout mice in vivo. These findings suggest that Twist1 serine 42 phosphorylation plays a key role in EndMT through TGF-β signaling and that modulation of Twist1 phosphorylation could be an effective strategy for managing PH.

Topics: Animals; Cell Hypoxia; Cells, Cultured; Endothelial Cells; Humans; Hypertension, Pulmonary; Lung; Mice; Mice, Knockout; Nuclear Proteins; Pulmonary Artery; Receptors, Transforming Growth Factor beta; Smad2 Protein; Transforming Growth Factor beta; Twist-Related Protein 1

Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-β (transforming growth factor β) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH.. We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression.. SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats.. SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-β treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-β-treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-β-induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats.. Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and-via MRTF disinhibition-hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype.

Topics: Animals; Cell Movement; Cell Proliferation; Disease Models, Animal; Down-Regulation; Humans; Hypertension, Pulmonary; Male; Muscle Cells; Random Allocation; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Smad3 Protein; Transcription Factors; Transfection; Transforming Growth Factor beta; Vascular Remodeling

Topics: Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Smad3 Protein; Transforming Growth Factor beta; Vascular Remodeling

Hypoxic pulmonary hypertension is characterized by the remodeling of pulmonary artery. Previously we showed that tanshinone IIA, one lipid-soluble component from the Chinese herb Danshen, ameliorated hypoxic pulmonary hypertension by inhibiting pulmonary artery remodeling. Here we explored the effects of danshensu, one water-soluble component of Danshen, on hypoxic pulmonary hypertension and its mechanism. Rats were exposed to hypobaric hypoxia for 4 weeks to develop hypoxic pulmonary hypertension along with administration of danshensu. Hemodynamics and pulmonary arterial remodeling index were measured. The effects of danshensu on the proliferation of primary pulmonary artery smooth muscle cells and transforming growth factor-β-smad3 pathway were assessed in vitro. Danshensu significantly decreased the right ventricle systolic pressure, the right ventricle hypertrophy and pulmonary vascular remodeling index in hypoxic pulmonary hypertension rats. Danshensu also reduced the increased expression of transforming growth factor-β and phosphorylation of smad3 in pulmonary arteries in hypoxic pulmonary hypertension rats. In vitro, danshensu inhibited the hypoxia- or transforming growth factor-β-induced proliferation of primary pulmonary artery smooth muscle cells. Moreover, danshensu decreased the hypoxia-induced expression and secretion of transforming growth factor in primary pulmonary adventitial fibroblasts and NR8383 cell line, inhibited the hypoxia or transforming growth factor-β-induced phosphorylation of smad3 in rat primary pulmonary artery smooth muscle cells. These results demonstrate that danshensu ameliorates hypoxic pulmonary hypertension in rats by inhibiting the hypoxia-induced proliferation of pulmonary artery smooth muscle cells, and the inhibition effects is associated with transforming growth factor-β-smad3 pathway. Therefore danshensu may be a potential treatment for hypoxic pulmonary hypertension.

Topics: Animals; Cell Hypoxia; Cell Line; Cell Proliferation; Fibroblasts; Hypertension, Pulmonary; Lactates; Male; Myocytes, Smooth Muscle; Phosphorylation; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Right ventricular (RV) hypertrophy is characterized by cardiac fibrosis due to endothelial-mesenchymal transition (EndMT) and increased collagen production in pulmonary arterial hypertension (PAH) patients, but the mechanisms for restoring RV function are unclear. Prostanoid agonists are effective vasodilators for PAH treatment that bind selective prostanoid receptors to modulate vascular dilation. The importance of prostanoid signaling in the RV is not clear. We investigated the effects of the EP4-specific agonist L-902,688 on cardiac fibrosis and TGF-β-induced EndMT. EP4-specific agonist treatment reduced right ventricle fibrosis in the monocrotaline (MCT)-induced PAH rat model. L-902,688 (1 µM) attenuated TGF-β-induced Twist and α-smooth muscle actin (α-SMA) expression, but these effects were reversed by AH23848 (an EP4 antagonist), highlighting the crucial role of EP4 in suppressing TGF-β-induced EndMT. These data indicate that the selective EP4 agonist L-902,688 attenuates RV fibrosis and suggest a potential approach to reducing RV fibrosis in patients with PAH.

Topics: Animals; Epithelial-Mesenchymal Transition; Fibrosis; Heart Ventricles; Human Umbilical Vein Endothelial Cells; Humans; Hypertension, Pulmonary; Male; Monocrotaline; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin E, EP4 Subtype; Tetrazoles; Transforming Growth Factor beta

Pulmonary vascular remodeling due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is the hallmark feature of pulmonary arterial hypertension (PAH). Recent evidence suggests that miR-125a-5p plays a role in a rat model of monocrotaline-induced PAH (MCT-PAH); however, the underlying mechanism is currently unknown. Here, we examined the expression profile of miR-125a-5p in MCT-PAH rats and investigated the putative therapeutic effect of miR-125a-5p using the miR-125a-5p agomir. In addition, the miR-125a-5p agomir or antagomir was transfected into rat PASMCs, and proliferation and apoptosis were measured. Activity of the miR-125a-5p target STAT3 was measured using a luciferase reporter assay, and the expression of downstream molecules was measured using RT-qPCR and/or western blot analysis. Importantly, inducing miR-125a-5p expression in vivo slowed the progression of MCT-PAH by reducing systolic pulmonary arterial pressure, the Fulton index, and pulmonary vascular remodeling. Moreover, overexpressing miR-125a-5p inhibited the proliferation and promoted the apoptosis of PASMCs. In addition, stimulating PASMCs with TGF-β1 or IL-6 upregulated miR-125a-5p expression, whereas overexpressing miR-125a-5p reduced TGF-β1 and IL-6 production, as well as the expression of their downstream targets STAT3 and Smad2/3; in contrast, downregulating miR-125a-5p increased TGF-β1 and IL-6 production. Finally, a dual-luciferase reporter assay revealed that miR-125a-5p targets the 3'-UTR of STAT3, suppressing the downstream molecules PCNA, Bcl-2, and Survivin. Taken together, these findings suggest that miR-125a-5p ameliorates MCT-PAH in rats, has a negative feedback regulation with TGF-β1 and IL-6, and regulates the proliferation and apoptosis of PASMCs by directly targeting STAT3.

Topics: Animals; Apoptosis; Cells, Cultured; Down-Regulation; Gene Expression Regulation; Hypertension, Pulmonary; Interleukin-6; Male; MicroRNAs; Monocrotaline; Pulmonary Artery; Rats; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta; Vascular Remodeling

Pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) is suggested as the consequence of emphysematous destruction of vascular bed and hypoxia of pulmonary microenvironment, mechanisms underpinning its pathogenesis however remain elusive. The dysregulated expression of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases and superoxide generation by pulmonary vasculatures have significant implications in the hypoxia-induced PH.. In this study, the involvement of NADPH oxidase subunit 4 (NOX4) in pulmonary arteriolar remodeling of PH in COPD was investigated by ascertaining the morphological alteration of pulmonary arteries and pulmonary blood flow using cardiac magnetic resonance imaging (cMRI), and the expression and correlation of NOX4 with pulmonary vascular remodeling and pulmonary functions in COPD lungs.. Results demonstrated that an augmented expression of NOX4 was correlated with the increased volume of pulmonary vascular wall in COPD lung. While the volume of distal pulmonary arteries was inversely correlated with pulmonary functions, despite it was positively associated with the main pulmonary artery distensibility, right ventricular myocardial mass end-systolic and right ventricular myocardial mass end-diastolic in COPD. In addition, an increased malondialdehyde and a decreased superoxide dismutase were observed in sera of COPD patients. Mechanistically, the abundance of NOX4 and production of reactive oxygen species (ROS) in pulmonary artery smooth muscle cells could be dynamically induced by transforming growth factor-beta (TGF-β), which in turn led pulmonary arteriolar remodeling in COPD lungs.. These results suggest that the NOX4-derived ROS production may play a key role in the development of PH in COPD by promoting distal pulmonary vascular remodeling.

Topics: Adult; Aged; Cells, Cultured; Female; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Magnetic Resonance Imaging; Male; Middle Aged; Myocytes, Smooth Muscle; NADPH Oxidase 4; Pulmonary Disease, Chronic Obstructive; Reactive Oxygen Species; Transforming Growth Factor beta; Vascular Remodeling

Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known.. We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O. We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling.. Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Proliferation; Cells, Cultured; Contractile Proteins; Disease Models, Animal; Female; Forkhead Box Protein M1; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pulmonary Artery; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Vascular Remodeling

Topics: Activins; Adult; Antibodies; Cell Proliferation; Culture Media, Conditioned; Female; Humans; Hypertension, Pulmonary; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Pulmonary Artery; Signal Transduction; Smad2 Protein; Smad3 Protein; Solubility; Transforming Growth Factor beta; Up-Regulation; Young Adult

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-β signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-β blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-β is regulated at the level of activation, but how TGF-β is activated in this disease is unknown. Here we show TGF-β activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-β activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-β could thus be a therapeutic approach in TGF-β-dependent vascular diseases.

Topics: Animals; Antigens, Ly; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Cells; Cattle; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Mice, Inbred C57BL; Monocytes; RNA, Messenger; Schistosoma; Signal Transduction; Th2 Cells; Thrombospondin 1; Transforming Growth Factor beta

We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly.. Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with. Disrupting BMPR2 impairs TGFβ1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Elastic Tissue; Elastin; Familial Primary Pulmonary Hypertension; Fibrillin-1; Fibroblasts; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mutation; Myocytes, Smooth Muscle; Phenotype; Pulmonary Artery; RNA Interference; Transfection; Transforming Growth Factor beta; Vascular Remodeling

Topics: Bone Morphogenetic Protein 2; Cells, Cultured; Humans; Hypertension, Pulmonary; MicroRNAs; PPAR gamma; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Bone Morphogenetic Protein 2; Cells, Cultured; Humans; Hypertension, Pulmonary; MicroRNAs; PPAR gamma; Transforming Growth Factor beta; Transforming Growth Factor beta1

Death and morbidity in pulmonary arterial hypertension (PAH) are often due to right ventricular (RV) failure and associated left ventricular (LV) dysfunction. We investigated regional myocardial remodeling and function as the basis for adverse ventricular-ventricular interactions in experimental chronic RV pressure overload. Two distinct animal models were studied: A rabbit model of increased RV pressure-load through progressive pulmonary artery banding A rat model of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Regional myocardial function was assessed by speckle-tracking strain echocardiography and ventricular pressures measured by catheterization before termination. Regional RV and LV myocardium was analyzed for collagen content, apoptosis and pro-fibrotic signaling gene and protein expression. Although the RV developed more fibrosis than the LV; in both models the LV was substantially affected. In both ventricles, particularly the LV, fibrosis developed predominantly at the septal hinge-point regions in association with decreased regional and global circumferential strain, reduced global RV and LV function and up-regulation of regional transforming growth factor-

Topics: Animals; Apoptosis; Collagen; Echocardiography; Fibrosis; Heart Septum; Heart Ventricles; Hypertension, Pulmonary; Male; Rabbits; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Ventricular Dysfunction, Left; Ventricular Dysfunction, Right; Ventricular Remodeling

To evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).. IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF.. We demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements.

Topics: Animals; Benzothiazoles; Bleomycin; Cell Proliferation; Disease Models, Animal; Fibroblasts; Fos-Related Antigen-2; Hypertension, Pulmonary; Mice; Mice, Transgenic; Myofibroblasts; Pulmonary Artery; Pulmonary Fibrosis; Severity of Illness Index; Sulfonamides; Transforming Growth Factor beta; Treatment Outcome; Vascular Remodeling

Idiopathic, familial and secondary pulmonary arterial hypertension (PAH) are associated with reduced bone morphogenetic protein receptor type 2 (BMPR2) expression, and in some contexts, TGF-β upregulation. Our aims were to assess BMPR2 gene therapy in a PAH mouse model and to assess the impact on TGF-β signalling.. Using a targeted in vivo gene delivery approach, we assessed the impact of BMPR2 gene delivery in a transgenic mouse model in which PAH was first induced by doxycycline driven expression of a dominant negative BMPR2 mutant (R899X). We also assessed the impact of BMPR2 gene delivery on TGF-β-induced changes in cell signalling in human pulmonary vascular endothelial and smooth muscle cells.. In the mouse model, changes in TGF-β levels were not detected, but BMPR2 gene delivery reversed the increase in right ventricle systolic pressure (RVSP) and Fulton Index (FI), associated with a trend to increased pulmonary endothelial nitric oxide synthase (eNOS) gene expression. In vitro, BMPR2 gene transfer reduced TGF-β effects on Smad2, Smad1/5/8 and Erk1/2 phosphorylation in human pulmonary arterial smooth muscle cells (HPASMC). BMPR2 was also found to upregulate nitric oxide (NO) production in lung derived human microvascular endothelial cells (HMVEC-L).. This study provides further evidence that BMPR2 modulation may have therapeutic potential. See Editorial, page 406.

Topics: Animals; Blotting, Western; Bone Morphogenetic Protein Receptors, Type II; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Gene Transfer Techniques; Humans; Hypertension, Pulmonary; Mice; Mice, Transgenic; Mutation; Pulmonary Artery; Real-Time Polymerase Chain Reaction; RNA; Transforming Growth Factor beta

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor-β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Animals; Antihypertensive Agents; Epithelial Cells; Gene Expression; Genes, Reporter; Genetic Vectors; High-Throughput Screening Assays; Humans; Hypertension, Pulmonary; Lentivirus; LIM Domain Proteins; Luciferases; Lung; Mink; Paclitaxel; Phosphorylation; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) continues to be a fatal disease and is associated with downregulation of bone morphogenetic protein receptor type-2 (BMPR2). Our approach is to upregulate BMPR2 in the pulmonary vasculature allowing us to examine the changes in endothelial cell signalling and better understand what pathways are altered when disease is attenuated using this treatment approach.. We used gene delivery of BMPR2 to human pulmonary endothelial cells to investigate downstream signalling, then assessed the impact of this approach on downstream signalling in vivo in rats with PAH using the monocrotaline (MCT) model.. Gene delivery of BMPR2 leads to an increase in BMPR2 protein expression, and this is associated with increased Smad1/5/8 and reduced Smad2/3 signalling. Additionally, we have found that BMPR2 modulation has effects on non-Smad signalling with increases found in phosphoinositide-3 kinase (PI3K) and a decrease in phosphorylated-p38-mitogen activated protein kinase (p38-MAPK) in vivo. These findings are associated with amelioration of PAH (reduced right ventricular, mean pulmonary artery pressures and Fulton Index).. These results indicate that the therapeutic effect of BMPR2 gene delivery on PAH is associated with a switch between TGF-β-Smad2/3 signalling to BMPR2-Smad1/5/8 signalling. This supports the further development of this treatment approach.

Topics: Animals; Arterial Pressure; Bone Morphogenetic Protein Receptors, Type II; Cell Line; Endothelial Cells; Genetic Therapy; Humans; Hypertension, Pulmonary; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Pulmonary Artery; Rats; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-β (TGF-β) ligands signal via type I and type II serine-threonine kinase receptors to regulate broad transcriptional programs. Excessive TGF-β-mediated signaling is implicated in the pathogenesis of pulmonary arterial hypertension, based in part on the ability of broad inhibition of activin-like kinase (ALK) receptors 4/5/7 recognizing TGF-β, activin, growth and differentiation factor, and nodal ligands to attenuate experimental pulmonary hypertension (PH). These broad inhibition strategies do not delineate the specific contribution of TGF-β versus a multitude of other ligands, and their translation is limited by cardiovascular and systemic toxicity.. We tested the impact of a soluble TGF-β type II receptor extracellular domain expressed as an immunoglobulin-Fc fusion protein (TGFBRII-Fc), serving as a selective TGF-β1/3 ligand trap, in several experimental PH models.. Signaling studies used cultured human pulmonary artery smooth muscle cells. PH was studied in monocrotaline-treated Sprague-Dawley rats, SU5416/hypoxia-treated Sprague-Dawley rats, and SU5416/hypoxia-treated C57BL/6 mice. PH, cardiac function, vascular remodeling, and valve structure were assessed by ultrasound, invasive hemodynamic measurements, and histomorphometry.. TGFBRII-Fc is an inhibitor of TGF-β1 and TGF-β3, but not TGF-β2, signaling. In vivo treatment with TGFBRII-Fc attenuated Smad2 phosphorylation, normalized expression of plasminogen activator inhibitor-1, and mitigated PH and pulmonary vascular remodeling in monocrotaline-treated rats, SU5416/hypoxia-treated rats, and SU5416/hypoxia-treated mice. Administration of TGFBRII-Fc to monocrotaline-treated or SU5416/hypoxia-treated rats with established PH improved right ventricular systolic pressures, right ventricular function, and survival. No cardiac structural or valvular abnormalities were observed after treatment with TGFBRII-Fc.. Our findings are consistent with a pathogenetic role of TGF-β1/3, demonstrating the efficacy and tolerability of selective TGF-β ligand blockade for improving hemodynamics, remodeling, and survival in multiple experimental PH models.

Topics: Animals; Disease Models, Animal; Heart; Hemodynamics; Hypertension, Pulmonary; Immunoglobulin Fc Fragments; Ligands; Male; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta; Vascular Remodeling

Chronic obstructive sleep apnea syndrome (OSAS) is considered to be associated with pulmonary diseases. However, the roles and mechanisms of OSA in pulmonary remodeling remain ambiguous. Thus, this study was aimed to elucidate the morphological and mechanical action of OSA in lung remodeling. In the present study, we employed a novel OSA model to mimic the OSA patient and investigate the role of OSA in pulmonary remodeling. We showed that pulmonary artery pressure of OSA group has no significant increased compared with the sham group. Nevertheless, we found that fibrotic tissue was predominantly located around the bronchi and vascular in the lung. Additionally, inflammatory cell infiltration was also detected in the peribonchial and perivascular space. The morphological change in OSA canines was ascertained by ultrastructure variation characterized by mitochondrial swelling, lamellar bodies degeneration and vascular smooth muscle incrassation. Moreover, sympathetic nerve sprouting was markedly increased in OSA group. Mechanistically, we showed that several pivotal proteins including collagen type I(CoLA1), GAP-43, TH and NGF were highly expressed in OSA groups. Furthermore, we found OSA could activated the expression of TGF-β, which subsequently suppressed miR-185 and promoted CoL A1 expression. This signaling cascade leads to pulmonary remodeling. In conclusion, Our data demonstrates that OSA can accelerate the progression of pulmonary remodeling through TGF-β/miR-185/CoLA1 signaling, which would potentially provide therapeutic strategies for chronic OSAS.

Topics: Animals; Bronchi; Cell Line; Chronic Disease; Collagen Type I; Disease Models, Animal; Dogs; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Inflammation; Lung; Male; MicroRNAs; Muscle, Smooth, Vascular; Sleep Apnea, Obstructive; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a leading cause of heart failure. Although pulmonary endothelial dysfunction plays a crucial role in the progression of the PAH, the underlying mechanisms are poorly understood. The HIF-α hydroxylase system is a key player in the regulation of vascular remodeling. Knockout of HIF-2α has been reported to cause pulmonary hypertension. The present study examined the role of endothelial cell specific prolyl hydroxylase-2 (PHD2) in the development of PAH and pulmonary vascular remodeling. The PHD2f/f mouse was crossbred with VE-Cadherin-Cre promoter mouse to generate an endothelial specific PHD2 knockout (Cdh5-Cre-PHD2ECKO) mouse. Pulmonary arterial pressure and the size of the right ventricle was significantly elevated in the PHD2ECKO mice relative to the PHD2f/f controls. Knockout of PHD2 in EC was associated with vascular remodeling, as evidenced by an increase in pulmonary arterial media to lumen ratio and number of muscularized arterioles. The pericyte coverage and vascular smooth muscle cells were also significantly increased in the PA. The increase in vascular pericytes was associated with elevated expression of fibroblast specific protein-1 (FSP-1). Moreover, perivascular interstitial fibrosis of pulmonary arteries was significantly increased in the PHD2ECKO mice. Mechanistically, knockout of PHD2 in EC increased the expression of Notch3 and transforming growth factor (TGF-β) in the lung tissue. We conclude that the expression of PHD2 in endothelial cells plays a critical role in preventing pulmonary arterial remodeling in mice. Increased Notch3/TGF-β signaling and excessive pericyte coverage may be contributing to the development of PAH following deletion of endothelial PHD2.

Topics: Animals; Cardiomegaly; Echocardiography; Endothelium, Vascular; Fibrosis; Hypertension, Pulmonary; Hypoxia-Inducible Factor-Proline Dioxygenases; Mice; Mice, Knockout; Pericytes; Pulmonary Artery; Receptor, Notch3; Transforming Growth Factor beta; Tunica Media; Vascular Remodeling

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-β significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

Topics: Actins; Airway Remodeling; Angiotensin II; Animals; Biomarkers; Cell Differentiation; Collagen Type I; Diabetes Mellitus, Type 1; Fibroblasts; Heart Failure; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Lung; Male; MicroRNAs; Myocardial Contraction; Myocardial Infarction; Nestin; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Streptozocin; Transforming Growth Factor beta

Pulmonary hypertension (PH) is characterized by increased pressure in the pulmonary artery and right ventricular hypertrophy (RVH). Recently, angiotensin-converting enzyme 2 (ACE2), which converts angiotensin (Ang) II into Ang-(1-7), was shown to inhibit experimental PH. Here we identified a novel ACE2 activator and investigated how the compound reduced monocrotaline (MCT)-induced PH.. To induce PH, Sprague-Dawley rats were injected subcutaneously with MCT, followed by the continuous administration of NCP-2454, an ACE2 activator, using osmotic pumps. Pulmonary arterial compliance was monitored every week until 4 weeks post-injection (wpi). RVH and lung remodeling was evaluated using lung tissue at 4 wpi.. NCP-2454 upregulated the production of Ang-(1-7) when incubated with ACE2 and Ang II. Notably, a continuous infusion of NCP-2454 significantly improved pulmonary arterial compliance, right ventricular systolic pressure, and RVH in MCT-treated rats. Interestingly, NCP-2454 increased the relative expression of ACE2 and MAS mRNA in lung tissue, especially in MCT-treated rats. In addition, the compound inhibited the MCT-induced overexpression of transforming growth factor β, phosphorylation of signal transducer and activator of transcription-3 (STAT3), and interleukin-6 production. The compound also restored the expression of caveolin-1 (Cav-1), which negatively regulates the Janus kinase-STAT signaling cascade.. NCP-2454 prevented MCT-induced PH by suppressing intracellular inflammatory cascades, an upstream molecular change of which is the disruption of Cav-1 expression.

Topics: Angiotensin-Converting Enzyme 2; Animals; Caveolin 1; Drug Evaluation, Preclinical; Enzyme Activation; Feedback, Physiological; Heart Ventricles; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Janus Kinases; Male; Monocrotaline; Oxazoles; Peptidyl-Dipeptidase A; Pyrimidines; Rats, Sprague-Dawley; STAT Transcription Factors; Transforming Growth Factor beta

Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH.. To explore BMPRII expression and function in a mouse model of SSc (TβRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung.. BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TβRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts.. Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-β-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-β activity.. We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

Topics: Animals; Blotting, Western; Bone Morphogenetic Protein Receptors, Type II; Disease Models, Animal; Fibroblasts; Humans; Hypertension, Pulmonary; Lung; Mice; Mice, Transgenic; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca(2+) entry (VDCE) and store-operated Ca(2+) entry (SOCE) are important mechanisms for controlling [Ca(2+)]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca(2+) regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Signaling; Cell Dedifferentiation; Cell Proliferation; Cells, Cultured; Hypertension, Pulmonary; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nifedipine; ORAI2 Protein; Pulmonary Artery; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Stromal Interaction Molecule 2; Transforming Growth Factor beta; TRPC Cation Channels; TRPC6 Cation Channel; Vascular Remodeling; Vascular Resistance; Vasoconstriction

Pulmonary arterial hypertension (PAH) can be idiopathic or secondary to autoimmune diseases, and it represents one of the most threatening complications of systemic sclerosis (SSc). Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with proinflammatory functions that appears to be involved in the pathogenesis of hypoxia-induced PH. In SSc patients, high serum levels of MIF have been associated with the development of ulcers and PAH. Stem cell growth factor β (SCGF β) is a human growth factor that, together with MIF, is involved in the pathogenesis of chronic spinal cord injury. The aim of our study was to measure serum levels of MIF in patients with idiopathic and SSc-associated PAH. We enrolled 13 patients with idiopathic PAH and 15 with SSc-associated PAH. We also selected 14 SSc patients without PAH and 12 normal healthy controls, matched for sex and age. PAH was confirmed by right hearth catheterism (mPAP>25 mmHg). MIF and SCGF β levels were measured by ELISA. We found significantly higher circulating levels of MIF and of SCGF β in patients with idiopathic PAH (P=0.03 and P=0.004) and with PAH secondary to SSc (P=0.018 and P=0.023) compared to SSc patients without PAH. Higher levels of MIF were found in those patients with an higher New York Heart Association (NYHA) class (P=0.03). We can hypothesize that MIF and SCGF β are able to play a role in PAH, both idiopathic or secondary, and in the future they may be evaluated as useful biomarkers and prognostic factors for this serious vascular disease.

Topics: Aged; Biomarkers; Case-Control Studies; Female; Humans; Hypertension, Pulmonary; Macrophage Migration-Inhibitory Factors; Middle Aged; Predictive Value of Tests; Prognosis; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Sensitivity and Specificity; Transforming Growth Factor beta

The etiology of schistosomiasis-associated pulmonary arterial hypertension (PAH), a major cause of PAH worldwide, is poorly understood. Schistosoma mansoni exposure results in prototypical type-2 inflammation. Furthermore, transforming growth factor (TGF)-β signaling is required for experimental pulmonary hypertension (PH) caused by Schistosoma exposure.. We hypothesized type-2 inflammation driven by IL-4 and IL-13 is necessary for Schistosoma-induced TGF-β-dependent vascular remodeling.. Wild-type, IL-4(-/-), IL-13(-/-), and IL-4(-/-)IL-13(-/-) mice (C57BL6/J background) were intraperitoneally sensitized and intravenously challenged with S. mansoni eggs to induce experimental PH. Right ventricular catheterization was then performed, followed by quantitative analysis of the lung tissue. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH was also systematically analyzed.. Mice with experimental Schistosoma-induced PH had evidence of increased IL-4 and IL-13 signaling. IL-4(-/-)IL-13(-/-) mice, but not single knockout IL-4(-/-) or IL-13(-/-) mice, were protected from Schistosoma-induced PH, with decreased right ventricular pressures, pulmonary vascular remodeling, and right ventricular hypertrophy. IL-4(-/-)IL-13(-/-) mice had less pulmonary vascular phospho-signal transducer and activator of transcription 6 (STAT6) and phospho-Smad2/3 activity, potentially caused by decreased TGF-β activation by macrophages. In vivo treatment with a STAT6 inhibitor and IL-4(-/-)IL-13(-/-) bone marrow transplantation also protected against Schistosoma-PH. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH had evidence of type-2 inflammation.. Combined IL-4 and IL-13 deficiency is required for protection against TGF-β-induced pulmonary vascular disease after Schistosoma exposure, and targeted inhibition of this pathway is a potential novel therapeutic approach for patients with schistosomiasis-associated PAH.

Topics: Animals; Bone Marrow Transplantation; Cell Adhesion Molecules; Humans; Hypertension, Pulmonary; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-13; Interleukin-4; Interleukin-4 Receptor alpha Subunit; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Schistosoma mansoni; Schistosomiasis mansoni; Smad2 Protein; Smad3 Protein; STAT6 Transcription Factor; Th1 Cells; Th17 Cells; Transforming Growth Factor beta; Vascular Remodeling

PS-341, a proteasome inhibitor, is suggested to prevent the vascular remodeling induced by high-flow pulmonary artery hypertension (PAH), but the mechanism remains unclear. The aim of the current study was to investigate the effects and possible mechanism of PS-341 on hypertension-induced vascular remodeling. Male Sprague-Dawley rats were subjected to surgical methods to produce a shunt model of PAH. Three days after the surgical procedure, the animals randomly assigned to four groups (n = 10 in each group): I: sham group; II: shunt group; III: vehicle; IV: treated group. Eight weeks postoperative, the hemodynamics data were measured through Swan-Ganz catheter; the protein expression level of proliferating cell nuclear antigen, nuclear factor-κB (NF-κB), inhibitor of nuclear factor-κB (I-κBα), transforming growth factor beta-β (TGF-β), drosophila mothers against decapentaplegic protein (Smad) and vascular endothelia growth factor (VEGF) were investigated by immunohistochemical and Western blotting; the mRNA expression level of Ubiquitin (Ub), Smad3, TGF-β1and Smad2 in lung were performed to detect by real-time reverse transcription-polymerase chain reaction analysis. The results showed that hemodynamic data and right ventricular hypertrophy were significantly improved (P < 0.05), the expression level of Ub, NF-κB, TGF-β1, Smad2 and VEGF were decreased (P < 0.05), but the level of I-κBα was increased in PS-341 treated group as compared with the shunt and vehicle groups (P < 0.05). In conclusion, the present study indicated that PS-341 could significantly improve the lung damage, attenuate pulmonary vascular remodeling induced by high blood PAH model. The mechanism may be mediated by inhibition of NF-κB and TGF-β/Smad signaling pathway and modulation the effect of VEGF.

Topics: Animals; Blotting, Western; Boronic Acids; Bortezomib; Disease Models, Animal; Gene Expression Profiling; Hypertension, Pulmonary; Immunohistochemistry; Lung; Male; NF-kappa B; Proliferating Cell Nuclear Antigen; Proteasome Inhibitors; Pyrazines; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Remodeling

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary arterial pressure and vascular resistance. Despite advances in therapy for PAH, its treatment and prognosis remain poor. We aimed to investigate whether the transplantation of bone marrow mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF), alone or in combination with granulocyte colony-stimulating factor (G-CSF), attenuates the development of experimental monocrotaline (MCT)-induced PAH. Three weeks after MCT administration, rats were divided into the following groups: (1) untreated (PAH); (2) HGF treated; (3) MSCs administered; (4) HGF-MSCs treated; and (5) HGF-MSCs plus G-CSF treated. After 3 weeks, hemodynamic changes, histomorphology, and angiogenesis were evaluated. To elucidate the molecular mechanisms of vascular remodeling and angiogenesis, serum levels of transforming growth factor (TGF)-β and endothelin-1 (ET-1) were measured, and the gene and protein expression levels of vascular cell adhesion molecule-1 (VCAM-1) and matrix metalloproteinase-9 (MMP-9) were determined. Compared with the PAH, MSC, and G-CSF groups, the HGF and HGF+G-CSF groups exhibited significantly reduced right ventricular hypertrophy and mean pulmonary arterial pressure (P < 0.05). Histologically, vessel muscularization or thickening and collagen deposition were also significantly decreased (P < 0.05). The number of vessels in the HGF+G-CSF group was higher than that in the other groups (P < 0.05). The TGF-β and ET-1 concentrations in the plasma of pulmonary hypertensive rats were markedly lower in the HGF and HGF+G-CSF groups (P < 0.05). Furthermore, HGF induced the expression of VCAM-1, and HGF treatment together with G-CSF synergistically stimulated MMP-9 expression. Transplanted HGF-MSCs combined with G-CSF potentially offer synergistic therapeutic benefit for the treatment of PAH.

Topics: Animals; Arterial Pressure; Cells, Cultured; Disease Models, Animal; Endothelin-1; Genetic Therapy; Granulocyte Colony-Stimulating Factor; Hepatocyte Growth Factor; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Male; Matrix Metalloproteinase 9; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Neovascularization, Physiologic; Pulmonary Artery; Rats, Sprague-Dawley; Recovery of Function; Time Factors; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1; Vascular Remodeling

Vascular remodelling is a hallmark of pulmonary hypertension (PH) and is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates a crucial role of transcription factors in the vascular remodelling processes. Here, we characterize the involvement of meprin β, a novel activator protein-1 (AP-1) effector molecule, in PH. Fra-2 transgenic (TG) mice exhibited increased right ventricular systolic pressure (RVSP), accompanied by vascular remodelling and activation of the pro-proliferative and pro-fibrotic AKT pathway. Microarray studies revealed the collagen-processing metalloprotease meprin β as the most up-regulated gene in Fra-2 TG mice. Its expression, increased at all investigated time points, preceded the decreased expression of MMPs and increased TGFβ, followed by collagen deposition. Correspondingly, remodelled pulmonary arteries from explanted idiopathic pulmonary arterial hypertension (IPAH) patients' lungs exhibited pronounced expression of meprin β. Fra-2 and meprin β expression in human PASMCs was regulated by PDGF-BB and TGFβ in a complementary fashion. Importantly, PDGF-BB-dependent proliferation was attenuated by silencing AP-1 expression or by meprin β inhibition. This study delineates a novel molecular mechanism underlying PASMCs proliferation and extracellular matrix (ECM) deposition by identifying meprin β as an important mediator in regulating vascular remodelling processes. Thus, meprin β may represent a new molecule that can be targeted in pulmonary hypertension.

Topics: Animals; Becaplermin; Cell Proliferation; Cells, Cultured; Collagen; Disease Models, Animal; Familial Primary Pulmonary Hypertension; Fibrosis; Fos-Related Antigen-2; Humans; Hypertension, Pulmonary; Matrix Metalloproteinases; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proto-Oncogene Proteins c-sis; Pulmonary Artery; RNA Interference; Signal Transduction; Time Factors; Transcription Factor AP-1; Transfection; Transforming Growth Factor beta; Up-Regulation; Ventricular Function, Right; Ventricular Pressure

Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder.. Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro.. Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFβ-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients.. These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.

Topics: Animals; Atrial Natriuretic Factor; Bleomycin; Cell Differentiation; Dose-Response Relationship, Drug; Humans; Hypertension, Pulmonary; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Natriuretic Peptide, C-Type; Natriuretic Peptides; Protein Precursors; Pulmonary Fibrosis; Structure-Activity Relationship; Transforming Growth Factor beta

It is suggested that interleukin (IL)-13 and transforming growth factor (TGF)-beta play a role in the pulmonary vascular changes found in animal models of schistosomiasis. The aim of this study was to assess and compare the serum levels of total TGF-beta and IL-13 of patients with schistosomiasis with pulmonary arterial hypertension (PAH) and patients with schistosomiasis without PAH.. 34 patients from the schistosomiasis outpatient clinic of the Hospital das Clinicas, Recife, Pernambuco, Brazil, without PAH assessed by echocardiography and 34 patients from the Reference Centre of Pulmonary Hypertension of Pronto Socorro Cardiológico de Pernambuco, Recife, Brazil with PAH, confirmed by right heart catheterization, were enrolled on the study. Both groups presented with schistosomal periportal fibrosis after abdominal ultrasound. Serum levels of TGF-beta1 and IL-13 were determined by ELISA. Student t test to independent samples, Mann-Whitney test to nonparametric variables, Pearson correlation test for correlation analyses and Fisher Chi-squared test to compare categorical analyses were used.. The median value of TGF-beta1 was significantly higher in patients with PAH (22496.9 pg/ml, interquartile range [IR] 15936.7 - 32087.8) than in patients without PAH (13629.9 pg/ml, IR: 10192.2- 22193.8) (p = 0.006). There was no difference in the median value of IL-13 in the group with Sch-PAH compared to patients without Sch-PAH (p > 0.05).. Our results suggest that TGF-beta possibly plays a role in the pathogenesis of schistosomiasis-associated PAH.

Topics: Adult; Animals; Brazil; Female; Humans; Hypertension, Pulmonary; Interleukin-13; Male; Middle Aged; Schistosomiasis; Schistosomiasis mansoni; Transforming Growth Factor beta; Transforming Growth Factor beta1

Mutations affecting transforming growth factor-beta (TGF-β) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/-) and wild-type (Eng+/+) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/- mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

Topics: Activin Receptors, Type II; Animals; Blotting, Western; Case-Control Studies; Cell Proliferation; Cells, Cultured; Endoglin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Familial Primary Pulmonary Hypertension; Female; Follow-Up Studies; Humans; Hypertension, Pulmonary; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Prognosis; Pulmonary Artery; Real-Time Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH. Its effect on disease development, however, is unknown. We aimed to uncover a novel role for Egr-1 as a molecular inductor for disease development in PAH.. In experimental flow-associated PAH in rats, we investigated the effects of Egr-1 down-regulation on pulmonary vascular remodelling, including neointimal development, and disease progression. Intravenous administration of catalytic oligodeoxynucleotides (DNA enzymes, DNAzymes) resulted in down-regulation of pulmonary vascular Egr-1 expression. Compared with vehicle or scrambled DNAzymes, DNAzymes attenuated pulmonary vascular remodelling, including the development of occlusive neointimal lesions. Selective down-regulation of Egr-1 in vivo led to reduced expression of vascular PDGF-B, TGF-β, IL-6, and p53, resulting in a reduction of vascular proliferation and increased apoptosis. DNAzyme treatment further attenuated pulmonary vascular resistance, RV systolic pressure, and RV hypertrophy. In contrast, in non-neointimal PH rodents, DNAzyme treatment had no effect on pulmonary vascular and RV remodelling. Finally, pharmacological inhibition of Egr-1 with pioglitazone, a peroxisome proliferator activated receptor-γ ligand, attenuated vascular remodelling including the development of neointimal lesions.. These results indicate that Egr-1 governs pulmonary vascular remodelling and the development of characteristic vascular neointimal lesions in flow-associated PAH. Egr-1 is therefore a potential target for future PAH treatment.

Topics: Animals; Down-Regulation; Early Growth Response Protein 1; Hemodynamics; Hypertension, Pulmonary; Interleukin-6; Male; Muscle, Smooth, Vascular; Oligodeoxyribonucleotides; Pulmonary Artery; Rats, Wistar; Transforming Growth Factor beta; Vascular Remodeling

Pulmonary hypertension is a progressive disease of various origins that is associated with right ventricular dysfunction. In the present study, the protective effect of diosgenin was investigated in monocrotaline-induced pulmonary hypertension in rats. Pulmonary hypertension was induced by a single subcutaneous injection of monocrotaline (60 mg/kg). Diosgenin (100 mg/kg) was given by oral administration once daily for 3 weeks. At the end of the experiment, mean arterial blood pressure, electrocardiography and echocardiography were recorded. Rats were then sacrificed and serum was separated for determination of total nitrate/nitrite level. Right ventricles and lungs were isolated for estimation of oxidative stress markers, tumor necrosis factor-alpha, total nitrate/nitrite and transforming growth factor-beta contents. Myeloperoxidase and caspase-3 activities in addition to endothelial and inducible nitric oxide synthase protein expression were also determined. Moreover, histological analysis of pulmonary arteries and cardiomyocyte cross-sectional area was performed. Diosgenin treatment provided a significant improvement toward preserving hemodynamic changes and alleviating oxidative stress, inflammatory and apoptotic markers induced by monocrotaline in rats. Furthermore, diosgenin therapy prevented monocrotaline-induced changes in nitric oxide production, endothelial and inducible nitric oxide synthase protein expression as well as histological analysis. These findings support the beneficial effect of diosgenin in pulmonary hypertension induced by monocrotaline in rats.

Topics: Animals; Caspase 3; Diosgenin; Glutathione; Heart Ventricles; Hypertension, Pulmonary; Inflammation; Lung; Male; Monocrotaline; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxidase; Protective Agents; Pulmonary Artery; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Although hypoxia is detrimental to most cell types, it aids survival of progenitor cells and is associated with diseases like cancer and pulmonary hypertension in humans. Therefore, understanding the underlying mechanisms that promote survival of progenitor cells in hypoxia and then developing novel therapies to stop their growth in hypoxia-associated human diseases is important. Here we demonstrate that the proliferation and growth of human CD133(+) progenitor cells, which contribute to tumorigenesis and the development of pulmonary hypertension, are increased when cultured under hypoxic conditions. Furthermore, glucose-6-phosphate dehydrogenase (G6PD) activity was increased threefold in hypoxic CD133(+) cells. The increased G6PD activity was required for CD133(+) cell proliferation, and their growth was arrested by G6PD inhibition or knockdown. G6PD activity upregulated expression of HIF1α, cyclin A, and phospho-histone H3, thereby promoting CD133(+) cell dedifferentiation and self-renewal and altering cell cycle regulation. When CD133(+) cells were cocultured across a porous membrane from pulmonary artery smooth muscle cells (PASMCs), G6PD-dependent H2O2 production and release by PASMCs recruited CD133(+) cells to the membrane, where they attached and expressed smooth muscle markers (α-actin and SM22α). Inhibition of G6PD reduced smooth muscle marker expression in CD133(+) cells under normoxia but not hypoxia. In vivo, CD133(+) cells colocalized with G6PD(+) cells in the perivascular region of lungs from rats with hypoxia-induced pulmonary hypertension. Finally, inhibition of G6PD by dehydroepiandrosterone in pulmonary arterial hypertensive rats nearly abolished CD133(+) cell accumulation around pulmonary arteries and the formation of occlusive lesions. These observations suggest G6PD plays a key role in increasing hypoxia-induced CD133(+) cell survival in hypertensive lungs that differentiate to smooth muscle cells and contribute to pulmonary arterial remodeling during development of pulmonary hypertension.

Topics: AC133 Antigen; Administration, Oral; Animals; Antigens, CD; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Coculture Techniques; Dehydroepiandrosterone; Glucosephosphate Dehydrogenase; Glycoproteins; Humans; Hypertension, Pulmonary; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Male; Peptides; Protein Transport; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5) and is considered first-line therapy for erectile dysfunction. Nowadays, Sildenafil is used extensively throughout the world on patients with pulmonary hypertension. However, few studies have evaluated the possible side effects of chronic Sildenafil treatment on the male reproductive system, specifically in the prostate. In the present study, it was demonstrated via morphological and ultrastructural analysis that chronic treatment with Sildenafil induced an enhancement of the glandular activity of the prostate. In addition, mice treated with Sildenafil showed a significant increase in testosterone serum levels. However, no statistically significant differences were observed in nitric oxide serum levels, or in sGC, eNOS, PSA and TGF-β prostatic expression. In conclusion, the present study suggests that chronic use of Sildenafil does not cause evident prostatic damage, and therefore, can be used pharmacologically to treat a variety of disorders.

Topics: Animals; Erectile Dysfunction; Humans; Hypertension, Pulmonary; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type III; Piperazines; Prostate; Prostate-Specific Antigen; Purines; Sildenafil Citrate; Sulfonamides; Testosterone; Transforming Growth Factor beta

Pulmonary hypertension (PH) represents an important complication of idiopathic pulmonary fibrosis (IPF) with a negative impact on patient survival. Herpes viruses are thought to play an etiological role in the development and/or progression of IPF. The influence of viruses on PH associated with IPF is unknown. We aimed to investigate the influence of viruses in IPF patients focusing on aspects related to PH. A laboratory mouse model of gamma-herpesvirus (MHV-68) induced pulmonary fibrosis was also assessed.. Lung tissue samples from 55 IPF patients and 41 controls were studied by molecular analysis to detect various viral genomes. Viral molecular data obtained were correlated with mean pulmonary arterial pressure (mPAP) and arterial remodelling. Different clinical and morphological variables were studied by univariate and multivariate analyses at time of transplant and in the early post-transplant period. The same lung tissue analyses were performed in MHV-68 infected mice.. A higher frequency of virus positive cases was found in IPF patients than in controls (p = 0.0003) and only herpes virus genomes were detected. Viral cases showed higher mPAP (p = 0.01), poorer performance in the six minute walking test (6MWT; p = 0.002) and higher frequency of primary graft (PGD) dysfunction after lung transplant (p = 0.02). Increased arterial thickening, particularly of the intimal layer (p = 0.002 and p = 0.004) and higher TGF-β expression (p = 0.002) were demonstrated in viral cases. The remodelled vessels showed increased vessel cell proliferation (Ki-67 positive cells) in the proximity to metaplastic epithelial cells and macrophages. Viral infection was associated with higher mPAP (p = 0.03), poorer performance in the 6MWT (p = 0.008) and PGD (p = 0.02) after adjusting for other covariates/intermediate factors. In MHV-68 infected mice, morphological features were similar to those of patients.. Herpesviral infections may contribute to the development of PH in IPF patients.

Topics: Alveolar Epithelial Cells; Animals; Arterial Pressure; Blood Vessels; Disease Models, Animal; Epithelial Cells; Female; Genome, Viral; Herpesviridae; Herpesviridae Infections; Humans; Hypertension, Pulmonary; Idiopathic Pulmonary Fibrosis; Lung; Lung Transplantation; Male; Mice; Middle Aged; Pulmonary Artery; Transforming Growth Factor beta

To delineate the constitutive pulmonary vascular phenotype of the TβRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH).. The TβRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor β (TGFβ) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFβ signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ and VEGF signaling axes in tissue sections and explanted smooth muscle cells.. In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFβ and VEGF ligand and receptor was consistent with a scleroderma phenotype.. In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFβ and VEGF, which provides insight into the pathogenesis of this disease.

Topics: Angiogenesis Inhibitors; Animals; Disease Models, Animal; Endothelium, Vascular; Familial Primary Pulmonary Hypertension; Female; Hypertension, Pulmonary; Hypoxia; Indoles; Lac Operon; Male; Mice; Mice, Transgenic; Phenotype; Protein Serine-Threonine Kinases; Pulmonary Circulation; Pyrroles; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Receptors, Vascular Endothelial Growth Factor; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

This study tested the hypothesis that combined therapy with nicorandil and colchicine is superior to either alone in attenuating monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH). Adult male Sprague-Dawley rats (n=50) were equally randomized into group 1 (sham control), group 2 [MCT (60 mg/kg i.p.)], group 3 [MCT-Nicorandil (5.0 mg/kg/day)], group 4 [MCT-Colchicine (1.0 mg/kg/day)], and group 5 (MCT-Nicorandil-Colchicine). Drugs were given on day 5. All animals were sacrificed on day 90 after MCT administration. Right ventricular systolic blood pressure (RVSBP) and RV weight were increased in group 2 compared to group 1, reduced in groups 3 and 4 compared to group 2, and further reduced in group 5, whereas arterial-oxygen saturation showed an opposite pattern (all p<0.001). Pulmonary damage severity (thickened alveolar septum and pulmonary arteriolar wall, decreased alveolar-sac numbers), number of CD3+ cells, and protein expressions of inflammatory (MMP-9, NF-κB, VCAM-1, angiotensin II-receptor), apoptotic (Bax, caspase 3, cleaved PARP), and fibrotic (TGF-β, Smad3) biomarkers showed an identical pattern compared to that of RVSBP, whereas pulmonary expressions of anti-apoptotic (Bcl-2) and anti-fibrotic (BMP-2, Smad1/5) biomarkers displayed a reverse pattern (all p<0.01). The protein expressions of RV damage markers (BNP, caspase 3) were increased, whereas expression of biomarker for RV functional preservation (Cx43) was reduced in group 2 compared with group 1, elevated in groups 3 and 4 compared to group 2, and further increased in group 5 (all p<0.01). Combined therapy with nicorandil and colchicine is superior to either alone in attenuating MCT-induced PAH in rats.

Topics: Animals; Antihypertensive Agents; Aorta, Thoracic; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line; Colchicine; Connexin 43; Drug Therapy, Combination; Familial Primary Pulmonary Hypertension; Heart Ventricles; Hypertension, Pulmonary; Lung; Male; Matrix Metalloproteinase 9; Monocrotaline; Myocytes, Smooth Muscle; NF-kappa B; Nicorandil; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Smad3 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Topics: Animals; Humans; Hypertension, Pulmonary; Schistosoma mansoni; Schistosomiasis mansoni; Signal Transduction; Transforming Growth Factor beta

The pathogenic mechanisms underlying pulmonary arterial hypertension resulting from schistosomiasis, one of the most common causes of pulmonary hypertension worldwide, remain unknown. We hypothesized that transforming growth factor-β (TGF-β) signaling as a consequence of Th2 inflammation is critical for the pathogenesis of this disease.. Mice sensitized and subsequently challenged with Schistosoma mansoni eggs developed pulmonary hypertension associated with an increase in right ventricular systolic pressure, thickening of the pulmonary artery media, and right ventricular hypertrophy. Rho-kinase-dependent vasoconstriction accounted for ≈60% of the increase in right ventricular systolic pressure. The pulmonary vascular remodeling and pulmonary hypertension were dependent on increased TGF-β signaling, as pharmacological blockade of the TGF-β ligand and receptor, and mice lacking Smad3 were significantly protected from Schistosoma-induced pulmonary hypertension. Blockade of TGF-β signaling also led to a decrease in interleukin-4 and interleukin-13 concentrations, which drive the Th2 responses characteristic of schistosomiasis lung pathology. Lungs of patients with schistosomiasis-associated pulmonary arterial hypertension have evidence of TGF-β signaling in their remodeled pulmonary arteries.. Experimental S mansoni-induced pulmonary vascular disease relies on canonical TGF-β signaling.

Topics: Animals; Disease Models, Animal; Humans; Hypertension, Pulmonary; Mice; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Pulmonary Circulation; Schistosoma mansoni; Schistosomiasis mansoni; Signal Transduction; Transforming Growth Factor beta; Vasoconstriction

The idiopathic pulmonary arterial hypertension is a complex disease that mainly affects pulmonary arterial circulation. This undergoes a remodeling with subsequent reduction of flow in the small pulmonary arteries. Because of this damage an increased vascular resistance gradually develops, and over time it carries out in heart failure. The inflammatory process is a key element in this condition, mediated by various cytokines. The inflammatory signal induces activation of NF-κB, and prompts TGF-β-related signaling pathway. Clinical evolution leads to progressive debilitation, greatly affecting the patient quality of life. The actual therapeutic approaches, are few and expensive, and include systemic drugs such as prostanoids, phosphodiesterase inhibitors and antagonists of endothelin-1 (ERBs). Some researchers have long investigated the anti-inflammatory effects of curcumin. It shows a role for inactivation of NF-κB-mediated inflammation. On the basis of these findings we propose a potential role of curcumin and its pharmacologically fit derivatives for treatment of idiopathic pulmonary arterial hypertension.

Topics: Curcumin; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Models, Biological; NF-kappa B; Signal Transduction; Transforming Growth Factor beta; Vascular Resistance

Transforming growth factor beta (TGF β ) is a family of genes that play a key role in mediating tissue remodeling in various forms of acute and chronic lung disease. In order to assess their role on pulmonary hypertension in broilers, we determined mRNA expression of genes of the TGF β family and endothelin 1 in lung samples from 4-week-old chickens raised either under normal or cold temperature conditions. Both in control and cold-treated groups of broilers, endothelin 1 mRNA expression levels in lungs from ascitic chickens were higher than levels from healthy birds (P < 0.05), whereas levels in animals with cardiac failure were intermediate. Conversely, TGF β 2 and TGF β 3 gene expression in lungs were higher in healthy animals than in ascitic animals in both groups (P < 0.05). TGF β 1, T β RI, and T β RII mRNA gene expression among healthy, ascitic, and chickens with cardiac failure showed no differences (P > 0.05). BAMBI mRNA gene expression was lowest in birds with ascites only in the control group as compared with the values from healthy birds (P < 0.05).

Topics: Animals; Chickens; Endothelin-1; Gene Expression Regulation; Hypertension, Pulmonary; Ligands; Lung; Male; Poultry Diseases; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene (BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-β and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene (THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-β regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-β. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.

Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Binding Sites; Cell Growth Processes; Cells, Cultured; Cohort Studies; Conserved Sequence; DNA-Binding Proteins; Endothelial Cells; Familial Primary Pulmonary Hypertension; Female; Humans; Hypertension, Pulmonary; Introns; Male; Mutation, Missense; Myocytes, Smooth Muscle; Polymorphism, Genetic; Protein Binding; Pulmonary Artery; RNA Splicing; Sp1 Transcription Factor; Transcription Factors; Transforming Growth Factor beta

To observe the effect of adrenomedullin (ADM) on the pulmonary vascular collagen metabolism in hypoxic rats in order to study the effect of ADM on chronic hypoxic pulmonary vascular structural remodeling and its possible mechanism.. Nineteen male Wistar rats were randomly divided into three groups: normal control (n=6), hypoxia (n=7) and ADM-treated hypoxia (n=6). ADM was subcutaneously administered into rats of the ADM-treated hypoxia group by mini-osmotic pump (300 ng/h) for two weeks. After two weeks of hypoxic challenge, mean pulmonary arterial pressure (mPAP) was evaluated using a right cardiac catheterization procedure. The ratio of right ventricular mass to left ventricular plus septal mass[RV/ (LV+S)] was measured. The changes of pulmonary vascular microstructure were observed. Meanwhile, the expression levels of collagen I, collagen III and transforming growth factor (TGF)-β in pulmonary arteries were detected by immunohistochemical assay.. mPAP and RV/(LV+S) increased significantly in the hypoxia group compared with normal controls (P<0.01). The muscularization of small pulmonary vessels and the relative medial thickness of pulmonary arteries increased obviously in the hypoxia group compared with those in the normal control group (P<0.01). Meanwhile, the expression levels of collagen I, collagen III and TGF-β of pulmonary arteries in the hypoxia group increased markedly compared with those in the normal control group. However, mPAP and RV/(LV+S) were significantly reduced in the ADM-treated hypoxia group compared with those in the hypoxia group (P<0.01). ADM ameliorated pulmonary vascular structural remodeling of hypoxic rats, with a decrease in the expression of collagen I, collagen III and TGF-β of pulmonary arteries.. ADM might play a regulatory role in the development of hypoxic pulmonary hypertension and hypoxic pulmonary vascular remodeling, through inhibiting the expression of TGF-β and alleviating the collagen accumulation of pulmonary arteries.

Topics: Adrenomedullin; Animals; Collagen; Hypertension, Pulmonary; Hypoxia; Male; Pulmonary Artery; Rats; Rats, Wistar; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a cardiovascular disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR2) underlie the majority of the inherited and familial forms of PAH. The transforming growth factor β (TGFβ) pathway is activated in both human and experimental models of PAH. However, how these factors exert pro-proliferative and anti-apoptotic responses in PAH remains unclear. Using mouse primary PASMCs derived from knock-in mice, we demonstrated that BMPR-II dysfunction promotes the activation of small mothers against decapentaplegia-independent mitogen-activated protein kinase (MAPK) pathways via TGFβ-associated kinase 1 (TAK1), resulting in a pro-proliferative and anti-apoptotic response. Inhibition of the TAK1-MAPK axis rescues abnormal proliferation and apoptosis in these cells. In both hypoxia and monocrotaline-induced PAH rat models, which display reduced levels of bmpr2 transcripts, this study further indicates that the TGFβ-MAPK axis is activated in lungs following elevation of both expression and phosphorylation of the TAK1 protein. In ex vivo cell-based assays, TAK1 inhibits BMP-responsive reporter activity and interacts with BMPR-II receptor. In the presence of pathogenic BMPR2 mutations observed in PAH patients, this interaction is greatly reduced. Taken together, these data suggest dysfunctional BMPR-II responsiveness intensifies TGFβ-TAK1-MAPK signalling and thus alters the ratio of apoptosis to proliferation. This axis may be a potential therapeutic target in PAH.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein Receptors, Type II; Cell Proliferation; Cells, Cultured; Familial Primary Pulmonary Hypertension; Hypertension, Pulmonary; MAP Kinase Kinase Kinases; Mice; Pulmonary Artery; Rats; Signal Transduction; Transforming Growth Factor beta

It is believed that growth factors play an important role in vascular remodeling that is evident in pulmonary hypertension (PH) pathogenesis. In the present study, the vascular endothelial growth factor (VEGF) levels in serum and pulmonary artery samples of rats have been analyzed with monocrotaline (MCT)-induced PH after treatments with iloprost, bosentan, and sildenafil. Serum VEGF and pulmonary artery VEGF levels were found to be significantly lower in MCT groups compared with control groups and significantly higher in treatment groups compared with MCT groups. In conclusion, treatment strategies directed at increasing VEGF levels may be reasonable in PH management.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Bosentan; Female; Hypertension, Pulmonary; Iloprost; Monocrotaline; Piperazines; Pulmonary Artery; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfonamides; Sulfones; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vasodilator Agents

Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type 2 pulmonary hypertension. Understanding the molecular mechanisms for type 2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, as well as type 2 pulmonary hypertension, in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure are associated with a ≤5.3-fold increase in lung wet weight and dry weight, pulmonary hypertension, and right ventricular hypertrophy. Interestingly, the aortic constriction-induced increase in lung weight was not associated with pulmonary edema but resulted from profound pulmonary remodeling with a dramatic increase in the percentage of fully muscularized lung vessels, marked vascular and lung fibrosis, myofibroblast proliferation, and leukocyte infiltration. The aortic constriction-induced left ventricular dysfunction was also associated with right ventricular hypertrophy, increased right ventricular end-diastolic pressure, and right atrial hypertrophy. The massive lung fibrosis, leukocyte infiltration, and pulmonary hypertension in mice after transverse aortic constriction clearly indicate that congestive heart failure also causes severe lung disease. The lung fibrosis and leukocyte infiltration may be important mechanisms in the poor clinical outcome in patients with end-stage heart failure. Thus, the effective treatment of left ventricular failure may require additional efforts to reduce lung fibrosis and the inflammatory response.

Topics: Animals; Aorta; Blotting, Western; Collagen; Constriction, Pathologic; Fibrosis; Heart Failure; Hypertension, Pulmonary; Hypertrophy, Left Ventricular; Hypoxia; Leukocytes; Lung; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Organ Size; Pulmonary Edema; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Ventricular Dysfunction, Left; Water

Chronic right ventricular (RV) pressure overload results in pathologic RV hypertrophy and diminished RV function. Although aortic constriction has been shown to improve systolic function in acute RV failure, its effect on RV responses to chronic pressure overload is unknown.. Adjustable vascular banding devices were placed on the main pulmonary artery and descending aorta. In 5 animals (sham group), neither band was inflated. In 9 animals (PAB group), only the pulmonary arterial band was inflated, with adjustments on a weekly basis to generate systemic or suprasystemic RV pressure at 28 days. In 9 animals, both pulmonary arterial and aortic devices were inflated (PAB + AO group), the pulmonary arterial band as for the PAB group and the aortic band adjusted to increase proximal systolic blood pressure by approximately 20 mm Hg. Effects on the functional performance were assessed 5 weeks after surgery by conductance catheters, followed by histologic and molecular assessment.. Contractile performance was significantly improved in the PAB + AO group versus the PAB group for both ventricles. Relative to sham-operated animals, both banding groups showed significant differences in myocardial histologic and molecular responses. Relative to the PAB group, the PAB + AO group showed significantly decreased RV cardiomyocyte diameter, decreased RV collagen content, and reduced RV expression of endothelin receptor type B, matrix metalloproteinase 9, and transforming growth factor β genes.. Aortic constriction in an experimental model of chronic RV pressure overload not only resulted in improved biventricular systolic function but also improved myocardial remodeling. These data suggest that chronically increased left ventricular afterload leads to a more physiologically hypertrophic response in the pressure-overloaded RV.

Topics: Animals; Aorta; Arterial Pressure; Chronic Disease; Collagen; Collagenases; Connective Tissue Growth Factor; Constriction; Disease Models, Animal; Endothelin-1; Familial Primary Pulmonary Hypertension; Heart Failure; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Myocardial Contraction; Myocardium; Pulmonary Artery; Rabbits; Receptors, Endothelin; Recovery of Function; Time Factors; Transforming Growth Factor beta; Ventricular Function, Left; Ventricular Function, Right; Ventricular Pressure; Ventricular Remodeling

The drugs that are currently used to treat pulmonary hypertension (PH) lack the ability to inhibit or reverse the pulmonary vascular remodeling that occurs during the course of the disease. We propose a novel method that combines the therapeutic powers of the potassium channel opener pinacidil and the statin drug simvastatin. These two drugs do not share similar mechanisms of treating PH. We used rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) as a model and examined the combined effects of pinacidil and simvastatin on pulmonary vascular remodeling. A series of indicators, including those for pulmonary vascular obstruction, proliferation, and cell phenotype, pulmonary vascular matrix and pulmonary vascular smooth muscle cell phenotype were used to monitor changes in pulmonary structure over the course of disease and treatment in normal controls, untreated PAH rats, pinacidil-treated subjects, simvastatin-treated subjects, and combination-treated subjects. We found that levels of mPAP, right ventricle Fulton index, pulmonary arteriolar wall thickness and muscularization, cell growth rate, transforming growth factor beta (TGF-beta), lung tissue matrix metalloproteinase-2 (MMP-2), MMP-9 and lung tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), vascular smooth muscle cell (VSMC) contractile protein SM-alpha-actin, and SM-alpha-actin mRNA of these different groups were all significantly lower in the combination-treated group than in the untreated group. Subjects in the combination-treated group also showed lower levels than those in either the pinacidil-treated or simvastatin-treated group. These results support our hypothesis and provide basis for a new, more effective therapeutic methods of treating PAH in human patients.

Topics: Actins; Animals; Arterioles; Blood Vessels; Cell Proliferation; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertension, Pulmonary; Immunochemistry; Immunohistochemistry; KATP Channels; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pinacidil; Poisons; Polymerase Chain Reaction; Pulmonary Circulation; Rats; Simvastatin; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Vasodilator Agents

Beyond their role as innate immune effectors, natural killer (NK) cells are emerging as important regulators of angiogenesis and vascular remodeling. Pulmonary arterial hypertension (PAH) is characterized by severe pulmonary vascular remodeling and has long been associated with immune dysfunction. Despite this association, a role for NK cells in disease pathology has not yet been described.. Analysis of whole blood lymphocytes and isolated NK cells from PAH patients revealed an expansion of the functionally defective CD56(-)/CD16(+) NK subset that was not observed in patients with chronic thromboembolic pulmonary hypertension. NK cells from PAH patients also displayed decreased levels of the activating receptor NKp46 and the killer immunoglobulin-like receptors 2DL1/S1 and 3DL1, reduced secretion of the cytokine macrophage inflammatory protein-1β, and a significant impairment in cytolytic function associated with decreased killer immunoglobulin-like receptor 3DL1 expression. Genotyping patients (n=222) and controls (n=191) for killer immunoglobulin-like receptor gene polymorphisms did not explain these observations. Rather, we show that NK cells from PAH patients exhibit increased responsiveness to transforming growth factor-β, which specifically downregulates disease-associated killer immunoglobulin-like receptors. NK cell number and cytotoxicity were similarly decreased in the monocrotaline rat and chronic hypoxia mouse models of PAH, accompanied by reduced production of interferon-γ in NK cells from hypoxic mice. NK cells from PAH patients also produced elevated quantities of matrix metalloproteinase 9, consistent with a capacity to influence vascular remodeling.. Our work is the first to identify an impairment of NK cells in PAH and suggests a novel and substantive role for innate immunity in the pathobiology of this disease.

Topics: Adult; Aged; Animals; CD56 Antigen; Chemokine CCL4; Cytotoxicity, Immunologic; Extracellular Matrix; Female; Genotype; GPI-Linked Proteins; Humans; Hypertension, Pulmonary; Immunophenotyping; Killer Cells, Natural; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Middle Aged; Natural Cytotoxicity Triggering Receptor 1; Pulmonary Embolism; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, IgG; Receptors, KIR2DL1; Receptors, KIR3DL1; Receptors, KIR3DS1; Transforming Growth Factor beta

Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.

Topics: Animals; Bleomycin; Cell Proliferation; Early Growth Response Protein 1; Extracellular Space; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide Synthase Type III; Nitrosation; Oxidation-Reduction; Pulmonary Artery; Pulmonary Fibrosis; Stress, Physiological; Superoxide Dismutase; Transforming Growth Factor beta; Tyrosine

Loss-of-function mutations in genes coding for transforming growth factor-beta/bone morphogenetic protein receptors and changes in nitric oxide(*) (NO(*)) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-beta coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO(*) synthase (eNOS)-derived NO(*) production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng(+/-) mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension.. Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng(+/-) mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng(+/-) mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide ((*)O(2)(-)) production in a BH(4)-independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-beta/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol.. Adult Eng(+/-) mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increased (*)O(2)(-) production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension.

Topics: Animals; Antioxidants; Bone Morphogenetic Protein Receptors; Cyclic N-Oxides; Disease Models, Animal; Endoglin; Endothelium, Vascular; Hypertension, Pulmonary; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mutation; Nitric Oxide Synthase Type III; Oxidative Stress; Reactive Oxygen Species; Spin Labels; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-β/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bone Morphogenetic Protein Receptors, Type II; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Endothelial Cells; Gene Transfer Techniques; Genetic Therapy; Hemodynamics; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Interleukin-10; Interleukin-6; Monocrotaline; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Survival Analysis; Tissue Extracts; Transforming Growth Factor beta

Our study aimed to investigate the relationship between exercise-induced pulmonary arterial hypertension and genetic changes related to the transforming growth factor-β (TGF-β) signalling pathway in patients with cardiac septal defects.. In a population-based group of 44 patients (age 13-25 years) with either isolated ventricular septal defect (n=27) or isolated atrial septal defect (n=17), right ventricular systolic pressure response to submaximal exercise was studied by echocardiography and classified as normal (≤45 mmHg), borderline (45-50 mmHg) or abnormal (>50 mmHg). Three genes related to TGF-β, bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1) and endoglin (ENG), were analyzed by DNA sequencing (only BMPR2) and multiplex ligand-dependent probe amplification (BMPR2, ALK1 and ENG).. Pressure response was borderline in five and abnormal in nine patients. Five patients showed mutations in exon 12 of the bone morphogenetic protein receptor type 2 gene. The previously described polymorphism S775N (c. 2324, G > A) was found in three patients with normal pressure response. The mutation Y589C (c. 1766, A > G), which has not been described previously, was found in two of 14 patients with borderline/abnormal pressure response.. Genetic changes in the BMPR2 gene may be overrepresented in patients with cardiac septal defects and exercise-induced pulmonary hypertension.

Topics: Activin Receptors, Type II; Adolescent; Adult; Antigens, CD; Base Sequence; Bone Morphogenetic Protein Receptors, Type II; Endoglin; Exercise; Female; Heart Septal Defects, Atrial; Heart Septal Defects, Ventricular; Humans; Hypertension, Pulmonary; Male; Mutation; Receptors, Cell Surface; Risk Assessment; Signal Transduction; Transforming Growth Factor beta; Ultrasonography, Doppler; Ventricular Function, Left; Ventricular Function, Right; Young Adult

Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6-overexpressing transgenic mice (Tg(+)) and compared to wild-type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-beta and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

Topics: Animals; Apoptosis; Arterioles; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blood Pressure; Cell Proliferation; Chronic Disease; Endothelial Cells; Hyperplasia; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Inhibitor of Apoptosis Proteins; Interleukin-6; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Pulmonary Artery; Repressor Proteins; Survivin; Time Factors; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Resistance; Ventricular Function, Right; Ventricular Pressure

Recent genetic studies have highlighted the role of the bone morphogenetic protein (BMP)/transforming growth factor (TGF)-beta signaling pathways in the pathogenesis of familial pulmonary arterial hypertension (PAH). It remains unclear whether alterations in these pathways contribute to other forms of pulmonary hypertension and to what extent these changes can be exploited for therapeutic intervention.. We studied BMP/TGF-beta signaling in 2 rat models of PAH due to chronic hypoxia and monocrotaline. In both models, there was a significant reduction in lung BMP type IA receptor and BMP type II receptor mRNA expression, although these changes were more pronounced in the monocrotaline model. This was accompanied by a reduction in lung levels of phospho-Smad1/5 and Id (inhibitor of DNA binding) gene expression in the monocrotaline model. In contrast, we observed increased TGF-beta activity, again more marked in the monocrotaline model, as evidenced by increased phospho-Smad2/3 and increased expression of TGF-beta-regulated genes. Immunohistochemistry revealed increased TGF-beta(1) expression in pulmonary artery smooth muscle cells and macrophages surrounding remodeled pulmonary arteries in monocrotaline rats. Inhibition of activin receptor-like kinase-5 signaling in vivo with the selective small-molecule inhibitor IN-1233 prevented PAH, right ventricular hypertrophy, and vascular remodeling after monocrotaline injection and inhibited the progression of established PAH in this model. No significant effect was observed in hypoxic PAH. In vitro studies confirmed that TGF-beta stimulated migration of distal rat pulmonary artery smooth muscle cells and that this effect was inhibited by IN-1233.. Disruption of BMP/TGF-beta signaling is more pronounced in the monocrotaline model of PAH than in the chronic hypoxia model. Increased TGF-beta activity is associated with greater macrophage recruitment with monocrotaline treatment. Inhibition of TGF-beta signaling via activin receptor-like kinase-5 prevents development and progression of PAH in the monocrotaline model and may involve inhibition of pulmonary artery smooth muscle cell migration.

Topics: Animals; Benzamides; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Movement; Chronic Disease; Disease Models, Animal; Disease Progression; Hypertension, Pulmonary; Hypoxia; Male; Monocrotaline; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Quinolines; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Topics: Epithelium; Extracellular Matrix; Fibroblasts; Humans; Hyaluronic Acid; Hypertension, Pulmonary; Idiopathic Interstitial Pneumonias; Prognosis; Pulmonary Fibrosis; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Bone Marrow Transplantation; Clinical Trials as Topic; Fibrosis; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Kidney Diseases; Oligonucleotide Array Sequence Analysis; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Stem Cell Transplantation; Transforming Growth Factor beta

Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-beta receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-beta family signaling in PH and pulmonary vascular remodeling remains unclear.. To determine whether inhibition of TGF-beta signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH).. We have used an orally active small-molecule TGF-beta receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH.. The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-beta signaling as documented by psmad2 expression. Inhibition of TGF-beta signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-beta signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling.. These findings provide evidence that increased TGF-beta signaling participates in the pathogenesis of experimental severe PH.

Topics: Animals; Disease Models, Animal; Endothelial Cells; Hepatocytes; Hypertension, Pulmonary; Male; Monocrotaline; Plant Extracts; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

Topics: AC133 Antigen; Adult; Animals; Antigens, CD; Antigens, CD34; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Female; Glycoproteins; Hematopoietic Stem Cells; Humans; Hypertension, Pulmonary; Male; Mice; Mice, Inbred NOD; Middle Aged; Peptides; Stem Cells; Transforming Growth Factor beta

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.

Topics: Activin Receptors; Animals; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Disease Models, Animal; Endothelium, Vascular; Hypertension, Pulmonary; Injections, Intraperitoneal; Lung; Male; Monocrotaline; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARgamma and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB-induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARgamma that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARgamma agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient-derived BMP-RII mutant PASMCs, a PPARgamma antagonist, and PASMCs isolated from PPARgamma- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARgamma, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARgamma in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARgamma-mediated events could protect against PAH, and PPARgamma agonists may reverse PAH in patients with or without BMP-RII dysfunction.

Topics: Animals; Apolipoproteins E; Becaplermin; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoglycemic Agents; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Platelet-Derived Growth Factor; PPAR gamma; Proto-Oncogene Proteins c-sis; RNA Interference; Rosiglitazone; Signal Transduction; Thiazolidinediones; Transforming Growth Factor beta

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a "second hit." There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38(MAPK) inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Cells, Cultured; Chemokine CCL2; Humans; Hypertension, Pulmonary; Inhibitor of Differentiation Protein 1; Interleukin-6; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Pulmonary Artery; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

Mutations in genes encoding members of the transforming growth factor (TGF)-beta superfamily have been identified in idiopathic forms of pulmonary arterial hypertension (PAH). The current study examined whether perturbations to the TGF-beta/Smad2,3 signalling axis occurred in a monocrotaline (MCT) rodent model of experimental PAH. Expression of the TGF-beta signalling machinery was assessed in the lungs and kidneys of MCT-treated rodents with severe PAH by semi-quantitative reverse-transcription (RT)-PCR, real-time RT-PCR and immunoblotting. TGF-beta signalling was assessed in the lungs and in pulmonary artery smooth muscle cells (PASMC) from MCT-treated rodents by Smad2 phosphorylation, expression of the connective tissue growth factor gene, activation of the serpine promoter in a luciferase reporter system and by the induction of apoptosis. The expression of type1 TGF-beta receptor (TGFBR) activin-A receptor-like kinase1, TGFBR-2, TGFBR-3 (endoglin), Smad3 and Smad4; as well as TGF-beta signalling and TGF-beta-induced apoptosis, were dramatically reduced in the lungs and PASMC, but not the kidneys, of MCT-treated rodents that developed severe PAH. The current data indicate that the transforming growth factor-beta/Smad2,3 signalling axis is functionally impaired in monocrotaline-treated rodents with severe pulmonary arterial hypertension, underscoring the potential importance of transforming growth factor-beta/Smad2,3 signalling in the onset or development of pulmonary arterial hypertension.

Topics: Animals; Apoptosis; Disease Models, Animal; Hypertension, Pulmonary; Lung; Models, Biological; Monocrotaline; Mutation; Pulmonary Artery; Rats; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Germ-line mutations in bone morphogenic protein type II receptor (Bmpr2) confer susceptibility to pulmonary arterial hypertension (PAH), which is characterized by obstructive vascular lesions in small arteries. The molecular and cellular mechanisms that account for the etiology of this disorder remain elusive, as does the role of Bmpr2 in postnatal tissue homeostasis. Here we show that in adult mice, stably silencing Bmpr2 expression by RNA interference does not increase pulmonary arterial resistance but results in severe mucosal hemorrhage, incomplete mural cell coverage on vessel walls, and gastrointestinal hyperplasia. We present evidence that BMP receptor signaling regulates vascular remodeling during angiogenesis by maintaining the expression of endothelial guidance molecules that promote vessel patterning and maturation and by counteracting growth factor-induced AKT activation. Attenuation of this function may cause vascular dysmorphogenesis and predisposition to angioproliferative diseases. Our findings provide a mechanistic link between PAH and other diseases associated with the BMP/TGF-beta pathways, such as hereditary hemorrhagic telangiectasia and juvenile polyposis syndrome.

Topics: Adenomatous Polyposis Coli; Animals; Bone Morphogenetic Protein Receptors, Type II; Gene Dosage; Germ-Line Mutation; Hemorrhage; Homeostasis; Hypertension, Pulmonary; Mice; Mice, Knockout; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; RNA Interference; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFbeta and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle alpha-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases.

Topics: Actins; Active Transport, Cell Nucleus; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line; Cell Movement; Cell Nucleus; DNA-Binding Proteins; Elafin; Humans; Hypertension, Pulmonary; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oncogene Proteins, Fusion; Phenotype; Pluripotent Stem Cells; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Signal Transduction; Trans-Activators; Transforming Growth Factor beta

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor alpha, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 microg of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Topics: Adult; Aged; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Survival; Cells, Cultured; Endothelial Cells; Female; Humans; Hypertension, Pulmonary; Male; Middle Aged; Mutation; Pulmonary Artery; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR-II) and dysfunction of BMPR-II have been implicated in patients with primary pulmonary hypertension (PH). To clarify the possible involvement of BMP and BMPR-II in the development of hypoxic PH, the expression of BMP-2, BMPR-II, and their downstream signals were investigated in rat lung under normal and hypoxic conditions by RT-PCR, immunoblot, and immunohistochemical methods. In rats under normal conditions, BMP-2 is localized in the endothelium of the pulmonary artery, whereas BMPR-II is abundantly expressed in the endothelium, smooth muscle cells, and adventitial fibroblasts. After 0.5 and 3 days of exposure to hypoxia, upregulation of BMP-2 was observed in the intrapulmonary arteries. The change was accompanied by activation of its downstream signaling, p38 MAPK, and Erk1/2 MAPK, and the apoptotic process, measured by caspase-3 activity and TdT-mediated dUTP nick end labeling-positive cells. In contrast, a significant decrease in the expression of BMPR-II and inactivation of p38 MAPK and caspase-3 were observed in the pulmonary vasculature after 7-21 days of hypoxia exposure. Because BMP-2 is known to inhibit proliferation of vascular smooth muscle cells and promote cellular apoptosis, disruption of BMP signaling pathway through downregulation of BMPR-II in chronic hypoxia may result in pulmonary vascular remodeling due to the failure of critical antiproliferative/differentiation programs in the pulmonary vasculature. These results suggest abrogation of BMP signaling may be a common molecular pathogenesis in the development of PH with various pathophysiological events, including primary and hypoxic PH.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Caspases; Down-Regulation; Endothelium, Vascular; Fibroblasts; Hypertension, Pulmonary; Hypoxia; Immunoblotting; In Situ Nick-End Labeling; Lung; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; p38 Mitogen-Activated Protein Kinases; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II (BMPR-II) receptor underlie the majority (>70%) of cases of familial pulmonary arterial hypertension (FPAH), and dysfunction of BMP signaling has been implicated in other forms of PAH. The reduced disease gene penetrance in FPAH indicates that other genetic and/or environmental factors may also be required for the clinical manifestation of disease. Of these, the serotonin pathway has been implicated as a major factor in PAH pathogenesis. We investigated the pulmonary circulation of mice deficient in BMPR-II (BMPR2(+/-) mice) and show that pulmonary hemodynamics and vascular morphometry of BMPR2(+/-) mice were similar to wild-type littermate controls under normoxic or chronic hypoxic (2- to 3-week) conditions. However, chronic infusion of serotonin caused increased pulmonary artery systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling in BMPR2(+/-) mice compared with wild-type littermates, an effect that was exaggerated under hypoxic conditions. In addition, pulmonary, but not systemic, resistance arteries from BMPR2(+/-) mice exhibited increased contractile responses to serotonin mediated by both 5-HT2 and 5-HT1 receptors. Furthermore, pulmonary artery smooth muscle cells from BMPR2(+/-) mice exhibited a heightened DNA synthesis and activation of extracellular signal-regulated kinase 1/2 in response to serotonin compared with wild-type cells. In vitro and in vivo experiments suggested that serotonin inhibits BMP signaling via Smad proteins and the expression of BMP responsive genes. These findings provide the first evidence for an interaction between BMPR-II-mediated signaling and the serotonin pathway, perturbation of which may be critical to the pathogenesis of PAH.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Proliferation; Cells, Cultured; Disease Susceptibility; Extracellular Signal-Regulated MAP Kinases; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Artery; Receptors, Serotonin; RNA, Messenger; Serotonin; Serotonin Plasma Membrane Transport Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Vasoconstriction

Activity of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on K(V) channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent gamma-subunits that form heterotetramers with functional K(V) channel alpha-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell K(V) currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K(+) currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional K(V) channels. These results suggest that BMP-2 divergently regulates mRNA expression of various K(V) channel alpha-, beta-, and gamma-subunits and significantly increases whole cell K(V) currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in K(V) channel activity and regulation of K(V) channel expression. The increased K(V) channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.

Topics: Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cells, Cultured; Gene Expression; Humans; Hypertension, Pulmonary; Membrane Potentials; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Potassium; Potassium Channels, Voltage-Gated; Protein Subunits; Proto-Oncogene Proteins c-myc; Pulmonary Artery; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cells, Cultured; Curcuma; Cytochromes c; Down-Regulation; Enzyme Activation; Gene Deletion; Humans; Hypertension, Pulmonary; Mice; Muscle, Smooth, Vascular; Proto-Oncogene Proteins c-bcl-2; Pulmonary Artery; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a potentially fatal vasculopathy that can develop at any age. Adult-onset disease has previously been associated with mutations in BMPR2 and ALK-1. Presentation in early life may be associated with congenital heart disease but frequently is idiopathic.. We performed mutation analysis in genes encoding receptor members of the transforming growth factor-beta cell-signaling pathway in 18 children (age at presentation <6 years) with PAH. Sixteen children were initially diagnosed with idiopathic PAH and 2 with PAH in association with congenital heart defects. Germ-line mutations were observed in 4 patients (22%) (age at disease onset, 1 month to 6 years), all of whom presented with idiopathic PAH. The BMPR2 mutations (n=2, 11%) included a partial gene deletion and a nonsense mutation, both arising de novo in the proband. Importantly, a missense mutation of ALK-1 and a branch-site mutation of endoglin were also detected. Presenting clinical features or progression of pulmonary hypertension did not distinguish between patients with mutations in the different genes or between those without mutations.. The cause of PAH presenting in childhood is heterogeneous in nature, with genetic defects of transforming growth factor-beta receptors playing a critical role.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Amino Acid Motifs; Amino Acid Substitution; Antigens, CD; Bone Morphogenetic Protein Receptors, Type II; Child; Child, Preschool; Codon, Nonsense; DNA Mutational Analysis; Endoglin; Exons; Female; Genetic Predisposition to Disease; Genotype; Germ-Line Mutation; Heart Defects, Congenital; Humans; Hypertension, Pulmonary; Infant; Infant, Newborn; Male; Mutation, Missense; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA Splicing; Sequence Deletion; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Asymmetric dimethylarginine (ADMA) is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear.. We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix). Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K), SMA-related protein 5 (Smad5), bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3), were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 microM). We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo.. Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and thereby indicates possible targets for therapies.

Topics: Arginine; Atherosclerosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Cardiovascular Diseases; Cell Culture Techniques; Endothelial Cells; Gene Expression Profiling; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Oligonucleotide Array Sequence Analysis; Protein-Arginine N-Methyltransferases; Smad5 Protein; Transforming Growth Factor beta

We established a rat chronic alveolar hypoxia in vivo model to evaluate the efficacy against hypoxic pulmonary hypertension of a new angiotensin II-receptor I blocker, olmesartan medoxomil. Three groups of rats were established: rats exposed for 2-6 weeks to 10% oxygen atmosphere in a normobaric chamber; hypoxic rats treated with olmesartan medoxomil oral administration (5 mg/day) every day; and control rats fed in a normoxic condition. After hypoxia treatment, the presence, etiology and severity of pulmonary hypertension, was echocardiographically evaluated, and expressions of brain natriuretic peptide (BNP), transforming growth factor (TGF-beta) and endothelin-1 genes measured by both immunohistochemical assay and real-time polymerase chain reaction. Olmesartan medoxomil significantly reduced the induction of hypoxic cor pulmonale not only on echocardiographical observations but also in BNP, TGF-beta and endothelin gene expressions in molecular studies. However, systolic blood pressure was independent of olmesartan medoxomil. The present study clearly indicates that the angiotensin II-type I-receptor blocker olmesartan medoxomil has significant efficacy for hypoxic cor pulmonale.

Topics: Administration, Oral; Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Collagen; Disease Models, Animal; Echocardiography; Endothelins; Heart; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Imidazoles; Lung; Male; Myocardium; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Pulmonary Artery; Pulmonary Heart Disease; Rats; Rats, Wistar; RNA, Messenger; Tetrazoles; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in pulmonary artery smooth muscle cells (PASMCs) from normal subjects. Dysfunction of BMP signaling due to mutations in and/or down-regulation of BMP receptors has been implicated in idiopathic pulmonary arterial hypertension (IPAH). The authors examined whether BMP differentially regulates gene expression in PASMCs from normal subjects and IPAH patients using the Affymetrix microarray analysis. BMP-2 treatment (200 nM for 24 hours) altered expression levels of 6206 genes in normal and IPAH PASMCs. Of these genes, 1063 were regulated oppositely by BMP-2: 523 genes were down-regulated by BMP-2 in normal PASMCs but up-regulated in IPAH PASMCs, whereas 540 genes were up-regulated by BMP-2 in normal PASMCs but down-regulated in IPAH PASMCs. The divergent effects of BMP-2 on gene expression profiles indicate that PASMCs may undergo significant phenotypic changes in IPAH patients during development of the disease. The transition of the antiproliferative effect of BMP-2 in normal PASMCs to its proliferative effect in IPAH patients is attributed potentially to its differential effect on expression patterns of various genes that are involved in cell proliferation and apoptosis. Among the 6206 BMP-2-sensitive genes, there are more than 1800 genes whose expression levels were negatively (correlation coefficient, r, <-0.9) or positively (with r >+ 0.9) correlated with the pulmonary arterial pressure. These results suggest that BMP-mediated gene regulation is significantly altered in PASMCs from IPAH patients and mRNA expression changes in BMP-regulated genes may be involved in the development of IPAH.

Topics: Adult; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Proliferation; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Male; Middle Aged; Muscle, Smooth, Vascular; Pulmonary Artery; Pulmonary Wedge Pressure; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Up-Regulation

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Humans; Hypertension, Pulmonary; Mutation, Missense; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Disease Models, Animal; Genes, Synthetic; Humans; Hypertension, Pulmonary; Mice; Mice, Transgenic; Microfilament Proteins; Muscle Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Potassium Channels; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta; Transgenes

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. The transforming growth factor (TGF)-beta superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-beta1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38(MAPK) or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-beta1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-beta1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-beta superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.

Topics: Antibodies; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cells, Cultured; Cyclooxygenase 2; DNA-Binding Proteins; Gene Expression Regulation, Enzymologic; Humans; Hypertension, Pulmonary; Hypoxia; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Mutations in transforming growth factor-beta family receptor-II, bone morphogenetic protein receptor-2, and activin-like kinase-1 have been associated with pulmonary hypertension. In the present study, we determined that pulmonary arteries in normal lungs and in lungs of patients with emphysema and idiopathic pulmonary arterial hypertension comparably expressed transforming growth factor-beta receptors I and II, Smad(1, 5, 8), Smad2, Smad3, Smad4, phosphorylated Smad(1, 5, 8), and phosphorylated Smad2 (the latter two both indicative of active in vivo signaling) in endothelial cells, as assessed by immunohistochemistry and quantitative morphometry. Medial or intimal smooth muscle cells had weak or absent expression of these molecules. In clear contrast to endothelial cell expression in pulmonary arteries and in endothelial cells lining incipient vessels within plexiform lesions of hypertensive lungs, endothelial cells present in the core of the lesions lacked expression of all examined members of the signaling molecules. These findings were made irrespective of the mutation status of bone morphogenetic protein receptor-2 in hypertensive patients. Our findings suggest that pulmonary artery endothelial cells in both normal and severely hypertensive lungs have active transforming growth factor-beta family signaling, and that loss of signaling might contribute to the abnormal growth of endothelial cells in plexiform lesions in idiopathic pulmonary arterial hypertension.

Topics: Arteries; Humans; Hypertension, Pulmonary; Transforming Growth Factor beta

Pulmonary hypertension (PH) is a progressive disease characterized by raised pulmonary vascular resistance, thought to be curable only through lung transplantation. Pathophysiologically, proliferation of pulmonary artery smooth muscle cells triggers pulmonary arterial stenosis and/or regurgitation, especially in advanced PH.. Using a rat model of advanced pulmonary vascular disease produced by injecting monocrotaline, we show that hepatocyte growth factor (HGF) targets pulmonary arterioles and blocks the progression of PH. In these rats, endogenous HGF production was dramatically downregulated during developing experimental PH, but c-Met/HGF receptor was abundant in the medial layers of pulmonary arterioles. HGF gene transfection 2 weeks after the monocrotaline injection resulted in milder medial hyperplasia in lung arterioles and inhibited overgrowth of pulmonary artery smooth muscle cells. Notably, exogenous HGF reduced lung expression levels of endothelin-1 and transforming growth factor-beta, which are critically involved in PH-linked fibrogenic events. Overall, medial wall thickening of pulmonary arteries was almost completely prevented by HGF, and the total collagen deposition in the lung decreased; both effects contributed to the suppression of pulmonary artery hypertension.. Our results suggest that the loss of endogenous HGF may be a feature of the pathogenesis of PH and that HGF supplementation may minimize pathological lung conditions, even advanced PH.

Topics: Animals; Arterioles; Collagen; Endothelin-1; Extracellular Matrix; Fibrosis; Genetic Therapy; Hepatocyte Growth Factor; Humans; Hyperplasia; Hypertension, Pulmonary; Male; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proto-Oncogene Proteins c-met; Rats; Rats, Wistar; Transfection; Transforming Growth Factor beta; Tunica Media

The pathogenesis of primary pulmonary hypertension (PPH) remains poorly understood. Molecular genetic studies have identified that mutations within the gene BMPR2 on the long arm of chromosome 2 underlie familial PPH. This review explores the significance of the PPH gene identification and examines additional genetic determinants, emphasizing the immediate implications for assessment and management of patients and their relatives.

Topics: Bone Morphogenetic Protein Receptors, Type II; Genetic Testing; Humans; Hypertension, Pulmonary; Mutation; Protein Serine-Threonine Kinases; Telangiectasis; Transforming Growth Factor beta

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.

Topics: Activin Receptors, Type I; Animals; Female; Hypertension, Pulmonary; Lung; Neovascularization, Pathologic; Pregnancy; Protein Serine-Threonine Kinases; Pulmonary Artery; Pulmonary Circulation; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sheep; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.

Topics: Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Cells, Cultured; DNA-Binding Proteins; Down-Regulation; Fas Ligand Protein; Gene Expression; Humans; Hypertension, Pulmonary; Membrane Glycoproteins; Muscle, Smooth, Vascular; Proto-Oncogene Proteins c-bcl-2; Pulmonary Artery; Receptors, Growth Factor; Smad Proteins; Smad1 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Germline mutations in the BMPR2 gene encoding bone morphogenetic protein (BMP) type II receptor (BMPR-II) have been reported in patients with primary pulmonary hypertension (PPH), but the contribution of various types of mutations found in PPH to the pathogenesis of clinical phenotypes has not been elucidated. To determine the biological activities of these mutants, we performed functional assays testing their abilities to transduce BMP signals. We found that the reported missense mutations within the extracellular and kinase domains of BMPR-II abrogated their signal-transducing abilities. BMPR-II proteins containing mutations at the conserved cysteine residues in the extracellular and kinase domains were detected in the cytoplasm, suggesting that the loss of signaling ability of certain BMPR-II mutants is due at least in part to their altered subcellular localization. In contrast, BMPR-II mutants with truncation of the cytoplasmic tail retained the ability to transduce BMP signals. The differences in biological activities among the BMPR-II mutants observed thus suggest that additional genetic and/or environmental factors may play critical roles in the pathogenesis of PPH.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; COS Cells; Cytoplasm; DNA-Binding Proteins; DNA, Complementary; Germ-Line Mutation; Humans; Hypertension, Pulmonary; Ligands; Lung; Microscopy, Fluorescence; Mutation; Mutation, Missense; Phosphoproteins; Phosphorylation; Plasmids; Precipitin Tests; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad5 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

Prostanoids are major regulators of smooth muscle function that are generated by cyclooxygenase (COX). Here we hypothesized that cytokines and mediators that regulate the pulmonary circulation would alter COX expression and prostanoid generation in pulmonary artery smooth muscle cells. Bradykinin, transforming growth factor-beta1 (TGF-beta1), and interleukin-1beta (IL-1beta) increased inducible COX-2 expression and prostaglandin E(2) (PGE(2)) release. Transfection studies using a COX-2 promoter construct demonstrated that all three agents acted transcriptionally. Constitutive COX-1 protein expression was unchanged. The COX inhibitor indomethacin, the COX-2 inhibitor NS-398, the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone abrogated the increased PGE(2) levels. Dexamethasone and cycloheximide prevented COX-2 induction. Hypoxia (3% O(2)-5% CO(2)-92% N(2)) for 24 h selectively augmented TGF-beta1-stimulated PGE(2) production and COX-2 induction but had no effect alone. Prolonged hypoxic culture alone for 48 and 72 h enhanced COX-2 induction and increased PGE(2). These studies show that a number of stimuli are capable of inducing COX-2 in pulmonary artery smooth muscle cells. The interaction between hypoxia and TGF-beta1 may be particularly relevant to pulmonary hypertension.

Topics: Adult; Bradykinin; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Humans; Hypertension, Pulmonary; Hypoxia; Interleukin-1; Isoenzymes; Male; Membrane Proteins; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Female; Gene Expression; Genetic Linkage; Humans; Hypertension, Pulmonary; Lung; Male; Microsatellite Repeats; Middle Aged; Mutation; Oligonucleotide Array Sequence Analysis; Pedigree; Protein Serine-Threonine Kinases; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Dexfenfluramine; Fenfluramine; Gaucher Disease; Humans; Hypertension, Pulmonary; Mutation; Protein Serine-Threonine Kinases; Risk Factors; Transforming Growth Factor beta

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Chromosome Mapping; Chromosomes, Human, Pair 2; Echocardiography, Doppler; Echocardiography, Stress; Genetic Heterogeneity; Genetic Linkage; Genotype; Heterozygote; Humans; Hypertension, Pulmonary; Lod Score; Mutation; Pedigree; Protein Serine-Threonine Kinases; Transforming Growth Factor beta

Topics: Base Pair Mismatch; bcl-2-Associated X Protein; Child; DNA-Binding Proteins; Endothelium, Vascular; Frameshift Mutation; Humans; Hypertension, Pulmonary; Microsatellite Repeats; Mutation; MutS Homolog 2 Protein; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Line; Exons; Humans; Hypertension, Pulmonary; Mice; Mutation; Phenotype; Protein Serine-Threonine Kinases; Transforming Growth Factor beta

A wide range of mutations in the type II receptor for bone morphogenetic protein (BMPR-II) have been shown to underlie primary pulmonary hypertension. To determine the mechanism of altered BMPR-II function, we employed transient transfection studies in cell lines and primary cultures of pulmonary vascular smooth muscle cells using green fluorescent protein (GFP)-tagged wild-type and mutant BMPR2 constructs and confocal microscopy to localize receptors. Substitution of cysteine residues in the ligand binding or kinase domain prevented trafficking of BMPR-II to the cell surface, and reduced binding of (125)I-BMP4. In addition, transfection of cysteine-substituted BMPR-II markedly reduced basal and BMP4-stimulated transcriptional activity of a BMP/Smad responsive luciferase reporter gene (3GC2wt-Lux), compared with wild-type BMPR-II, suggesting a dominant-negative effect of these mutants on Smad signalling. In contrast, BMPR-II containing non-cysteine substitutions in the kinase domain were localized to the cell membrane, although these also suppressed the activity of 3GC2wt-Lux. Interestingly, BMPR-II mutations within the cytoplasmic tail trafficked to the cell surface, but retained the ability to activate 3GC2wt-Lux. Transfection of mutant, but not wild-type, constructs into a mouse epithelial cell line (NMuMG cells) led to activation of p38(MAPK) and increased serum-induced proliferation compared with the wild-type receptor, which was partly p38(MAPK)-dependent. We conclude that mutations in BMPR-II heterogeneously inhibit BMP/Smad-mediated signalling by diverse molecular mechanisms. However, all mutants studied demonstrate a gain of function involving upregulation of p38(MAPK)-dependent proproliferative pathways.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Line; Cells, Cultured; Genes, Reporter; HeLa Cells; Humans; Hypertension, Pulmonary; Ligands; Mice; Mitogen-Activated Protein Kinases; Mutation; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Recombinant Proteins; Transforming Growth Factor beta

Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients.. We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations.. We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries.. Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension.

Topics: Activin Receptors; Adult; Bone Morphogenetic Protein Receptors, Type II; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 12; Female; Humans; Hypertension, Pulmonary; Lung; Male; Microsatellite Repeats; Middle Aged; Mutation; Mutation, Missense; Pedigree; Protein Serine-Threonine Kinases; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Topics: Activin Receptors; Bone Morphogenetic Protein Receptors, Type II; Genetic Markers; Humans; Hypertension, Pulmonary; Mutation; Protein Serine-Threonine Kinases; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-beta (TGF-beta) superfamily, underlie many cases of familial and sporadic primary pulmonary hypertension (PPH). We postulated that pulmonary artery smooth muscle cells (PASMCs) from patients with PPH might demonstrate abnormal growth responses to TGF-beta superfamily members.. For studies of (3)H-thymidine incorporation or cell proliferation, PASMCs (passages 4 to 8) were derived from main pulmonary arteries. In control cells, 24-hour incubation with TGF-beta(1) (10 ng/mL) or bone morphogenetic protein (BMP)-2, -4, and -7 (100 ng/mL) inhibited basal and serum-stimulated (3)H-thymidine incorporation, and TGF-beta(1) and BMPs inhibited the proliferation of serum-stimulated PASMCs. In contrast, TGF-beta(1) stimulated (3)H-thymidine incorporation (200%; P<0.001) and cell proliferation in PASMCs from PPH but not from patients with secondary pulmonary hypertension. In addition, BMPs failed to suppress DNA synthesis and proliferation in PASMCs from PPH patients. Reverse transcription-polymerase chain reaction of PASMC mRNA detected transcripts for type I (TGF-betaRI, Alk-1, ActRI, and BMPRIB) and type II (TGF-betaRII, BMPR-II, ActRII, ActRIIB) receptors. Receptor binding and cross-linking studies with (125)I-TGF-beta(1) confirmed that the abnormal responses in PPH cells were not due to differences in TGF-beta receptor binding. Mutation analysis of PASMC DNA failed to detect mutations in TGF-betaRII and Alk-1 but confirmed the presence of a mutation in BMPR-II in 1 of 5 PPH isolates.. We conclude that PASMCs from patients with PPH exhibit abnormal growth responses to TGF-beta(1) and BMPs and that altered integration of TGF-beta superfamily growth signals may contribute to the pathogenesis of PPH.

Topics: Activin Receptors; Adult; Binding, Competitive; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Division; Cells, Cultured; Cross-Linking Reagents; DNA; DNA Mutational Analysis; Female; Gene Expression Profiling; Humans; Hypertension, Pulmonary; Male; Middle Aged; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Pulmonary Artery; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Thymidine; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Anticipation, Genetic; Bone Morphogenetic Protein Receptors, Type II; Female; Genetic Linkage; Humans; Hypertension, Pulmonary; Male; Models, Biological; Mutation; Penetrance; Protein Isoforms; Protein Serine-Threonine Kinases; Sex Factors; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Diterpenes; Epoxy Compounds; Humans; Hypertension, Pulmonary; Immunosuppressive Agents; Phenanthrenes; Rats; Transforming Growth Factor beta

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

Topics: Animals; Cells, Cultured; DNA, Complementary; Endothelin-1; Endothelins; Gene Expression; Hemodynamics; Hypertension, Pulmonary; In Situ Hybridization; Lung; Lymph; Muscle, Smooth; Polymerase Chain Reaction; Protein Precursors; Pulmonary Artery; RNA, Messenger; Sheep; Transcription, Genetic; Transforming Growth Factor beta

The high mortality rate for patients with congenital diaphragmatic hernia (CDH) has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PPH). The factors that cause vasoconstriction and vascular remodeling in PPH are not fully understood. Immunohistochemistry was performed on lung tissue obtained from postmortem CDH patients with pulmonary hypoplasia and PPH (n = 21) using the following antibodies: alpha smooth muscle-actin (ASMA), transforming growth factor-beta (TGF-beta), isoform specific (TGF-beta 1, -beta 2, -beta 3), and M-57. Normal lung tissues from age-matched sudden infant death syndrome patients (SIDS, n = 8) were obtained as controls. TGF-beta 3 immunoreactivity was observed in the adventitia but not in the media of pulmonary muscular arteries in patients with CDH. TGF-beta 1, -beta 2 immunoreactivity was either absent or faintly expressed in pulmonary arteries in CDH patients. No TGF-beta staining was observed in the pulmonary vasculature of SIDS patients. Newly synthesized procollagen (M-57) was easily detected in the media and adventitia in a large number of pulmonary arteries in all patients with CDH and in the neointima in two patients with long standing PPH. No M-57 staining was seen in the media of pulmonary arteries of the lungs of SIDS patients. These observations suggest a potential role of TGF-beta 3 but not TGF beta 1 or TGF beta 2 in pulmonary vascular remodeling and that smooth muscle cells in muscular pulmonary arteries are actively synthesizing collagen in patients with CDH complicated by PPH.

Topics: Antibodies, Monoclonal; Cadaver; Extracellular Matrix; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Humans; Hypertension, Pulmonary; Immunohistochemistry; Infant, Newborn; Muscle, Smooth; Procollagen; Pulmonary Artery; Transforming Growth Factor beta; Tunica Media

Monocrotaline (MCT)-induced pulmonary vascular injury was used to begin studying the mechanism(s) of vascular remodeling in Fischer 344 rats, using extracellular matrix (ECM) gene expression to define areas of remodeling. By day 28 after injection, pulmonary artery pressures were increased and right ventricular hypertrophy had developed compared with normal controls. Tropoelastin, fibronectin, and alpha 1(I) procollagen mRNA levels increased at least 2-fold by day 28. In situ hybridization demonstrated tropoelastin gene expression by cells adjacent to the lumen and procollagen gene expression at the medial-adventitial border in both small muscular and large elastic pulmonary arteries. This pattern of gene expression was observed as early as 1 wk after MCT injury. These observations indicated two distinct areas of remodeling, one along the vascular lumen at the site of monocrotaline-induced injury and the other at a second distinct site. To determine whether other differences may be involved at these two sites, the presence of transforming growth factor-beta (TGF-beta) was studied. Total TGF-beta protein was 4-fold higher in remodeling lungs compared with normal lungs. Gene expression for all three isoforms of TGF-beta colocalized with tropoelastin gene expression along the vascular lumen but not with alpha 1(I) procollagen gene expression. These results suggest a complex site-specific response to injury mediated by two distinct pathways in this model of pulmonary vascular remodeling.

Topics: Animals; Blotting, Northern; Endothelium, Vascular; Extracellular Matrix Proteins; Gene Expression; Hemodynamics; Hypertension, Pulmonary; In Situ Hybridization; Lung; Male; Monocrotaline; Poisons; Procollagen; Pulmonary Artery; Rats; Rats, Inbred F344; RNA, Messenger; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Tropoelastin

Transforming growth factor-beta (TGF-beta) has been suggested as one of the mediators of vascular remodeling in chronic pulmonary hypertension. We have previously shown a transient early increase in TGF-beta levels in lung lymph during the development of sustained pulmonary hypertension in a sheep model (12 days of air embolization). The present study examines expression and cellular localization of mRNA and protein of the three mammalian isoforms of TGF-beta in lung biopsy tissue taken during the development of pulmonary hypertension (0, 1, 4, 8, and 12 days of embolization). In control tissue, immunohistochemical techniques localized each of the TGF-beta proteins in an identical pattern in large preacinar airways--bronchial epithelium and subepithelial cells--and in the medial wall of muscular vessels; no protein was detected in intraacinar regions. Following air embolization, immunoreactivity appeared in peripheral lung. At day 1, immunoreactivity for TGF-beta 1 and TGF-beta 3 proteins was seen in edema fluid, in perivascular cells associated with nonmuscular intraacinar arteries, and in alveolar walls; no increased immunoreactivity was detected for TGF-beta 2. After 4, 8, and 12 days of embolization, immunoreactivity for all three TGF-beta proteins was associated with newly muscularized intraacinar arteries. With in situ hybridization, the three TGF-beta mRNAs co-localized in lung tissue from both control and air-embolized animals. In control tissue, hybridization was seen around preacinar airways and muscular vessels; no hybridization seen in intraacinar regions of the lung. After 1 day of embolization, the pattern of hybridization was similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Disease Models, Animal; Embolism, Air; Gene Expression; Hypertension, Pulmonary; Immunohistochemistry; In Situ Hybridization; Lung; RNA, Messenger; Sheep; Tissue Distribution; Transforming Growth Factor beta

Active exogenous transforming growth factor-beta s (TGF-beta s) are potent modulators of extracellular matrix synthesis in cell culture and stimulate matrix synthesis in wounds and other remodeling tissues. The role of endogenous TGF-beta s in remodeling tissues is less well defined. Vascular remodeling in the pulmonary arteries of patients with primary pulmonary hypertension is characterized, in part, by abnormal deposition of immunohistochemically detectable procollagen, thereby identifying actively remodeling vessels. We used this marker of active matrix synthesis to begin defining the in vivo role of TGF-beta in the complex milieu of actively remodeling tissues. Immunohistochemistry using isoform-specific anti-TGF-beta antibodies was performed to determine whether TGF-beta was present in actively remodeling hypertensive pulmonary arteries 20 to 500 microns in diameter. Intense, cell-associated TGF-beta 3 immunoreactivity was observed in the media and neointima of these hypertensive muscular arteries. Immunostaining was present, but less intense, in normal arteries of comparable size. TGF-beta 2 immunoreactivity was observed in normal vessels and was increased slightly in hypertensive vessels, in a pattern resembling TGF-beta 3 immunoreactivity. No staining was associated with the adventitia. TGF-beta 1 immunostaining was either faint or absent in both normal and hypertensive vessels. Comparison of procollagen and TGF-beta localization demonstrated that TGF-beta 2 and TGF-beta 3 colocalized at all sites of procollagen synthesis. However, TGF-beta was observed in vessels, or vascular compartments, where there was no procollagen synthesis. Procollagen immunoreactivity was not present in normal vessels that showed immunoreactivity for TGF-beta 2 and TGF-beta 3. These observations suggest: a) the stimulation of procollagen synthesis by TGF-beta in vivo is more complex than suggested by in vitro studies and b) a potential role for TGF-beta 2 or TGF-beta 3, but not TGF-beta 1, in hypertensive pulmonary vascular remodeling.

Topics: Extracellular Matrix; Humans; Hypertension, Pulmonary; Immunoenzyme Techniques; Lung; Muscle, Smooth, Vascular; Procollagen; Pulmonary Artery; Transforming Growth Factor beta

The development of pulmonary hypertension in hypoxic newborn calves is associated with a complex pattern of increased tropoelastin and type I procollagen synthesis and deposition by smooth muscle cells in large elastic pulmonary arteries compared to normoxic controls. We examined the possibility that transforming growth factor-beta 1 (TGF-beta 1) may be associated with the production of extracellular matrix protein in this model of pulmonary hypertension. Medial smooth muscle cells in both normotensive and hypertensive vessels, as assessed by immunohistochemistry, were the major source of TGF-beta 1. Staining was confined to foci of smooth muscle cells in the outer media and appeared greater in normotensive than hypertensive vessels. Consistent with the immunohistochemistry, a progressive, age-dependent increase in normotensive pulmonary artery TGF-beta 1 mRNA was observed after birth, whereas TGF-beta 1 mRNA remained at low, basal levels in hypertensive, remodeling pulmonary arteries. These observations suggest that local expression of TGF-beta 1 is not associated with increased extracellular matrix protein synthesis in this model of hypoxic pulmonary hypertension.

Topics: Animals; Base Sequence; Cattle; Gene Expression; Hypertension, Pulmonary; Immunohistochemistry; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Pulmonary Artery; RNA, Messenger; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, we demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, we now demonstrate the presence of transforming growth factor beta 1 (TGF-beta 1), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-beta 1 may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-beta 1 is the alveolar macrophage, and we demonstrate the expression of abundant TGF-beta 1 mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.

Topics: Fibronectins; Gene Expression; Humans; Hypertension, Pulmonary; Lung; Nucleic Acid Hybridization; Procollagen; Pulmonary Fibrosis; Reference Values; RNA Probes; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Animals, Newborn; Cattle; Hypertension, Pulmonary; Hypoxia; Polymerase Chain Reaction; Pulmonary Artery; RNA, Messenger; Transforming Growth Factor beta

Chronic pulmonary hypertension is associated with extensive structural remodeling of the pulmonary arterial bed. The structural changes in the arterial walls include increased production of extracellular matrix components and smooth muscle cell hypertrophy, changes that have been similarly induced by transforming growth factor-beta (TGF-beta) in culture. In the present study, experiments were performed to determine whether TGF-beta is present in sheep lung lymph, and whether TGF-beta levels were altered in an animal model of chronic pulmonary hypertension induced by continuous air embolization. Several standard biological assays for TGF-beta activity were used for these determinations including soft agar assays, inhibition of epithelial cell proliferation, and a TGF-beta-specific radioreceptor assay. In each case, control lung lymph contained high concentrations of TGF-beta (100 ng/ml) which required transient acidification for detection. Samples of lung lymph from hypertensive sheep showed a transient and early two- to threefold increase in concentrations of latent TGF-beta. This activity could be partially blocked by TGF-beta antibodies. These studies indicate that sheep lung lymph contains TGF-beta and that the level of TGF-beta increases early during the development of pulmonary hypertension. Thus, TGF-beta may contribute to the development of the structural changes in the pulmonary arteries that occur during the onset of chronic pulmonary hypertension.

Topics: Animals; Biological Assay; Cell Division; Hypertension, Pulmonary; Lung; Lymph; Pulmonary Embolism; Radioligand Assay; Sheep; Thymidine; Transforming Growth Factor beta