semaxinib and Familial-Primary-Pulmonary-Hypertension

Topics: Animals; Disease Models, Animal; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Hypoxia; Indoles; Mineralocorticoid Receptor Antagonists; Pulmonary Arterial Hypertension; Pyrroles; Rats; Ventricular Dysfunction, Right

Pulmonary arterial hypertension (PAH) is a devastating disease with poor prognosis and limited therapeutic options. We screened for pathways that may be responsible for the abnormal phenotype of pulmonary arterial smooth muscle cells (PASMCs), a major contributor of PAH pathobiology, and identified cyclin-dependent kinases (CDKs) as overactivated kinases in specimens derived from patients with idiopathic PAH. This increased CDK activity is confirmed at the level of mRNA and protein expression in human and experimental PAH, respectively. Specific CDK inhibition by dinaciclib and palbociclib decreases PASMC proliferation via cell cycle arrest and interference with the downstream CDK-Rb (retinoblastoma protein)-E2F signaling pathway. In two experimental models of PAH (i.e., monocrotaline and Su5416/hypoxia treated rats) palbociclib reverses the elevated right ventricular systolic pressure, reduces right heart hypertrophy, restores the cardiac index, and reduces pulmonary vascular remodeling. These results demonstrate that inhibition of CDKs by palbociclib may be a therapeutic strategy in PAH.

Topics: Animals; Cell Line; Cyclin-Dependent Kinases; Disease Models, Animal; Familial Primary Pulmonary Hypertension; Humans; Indoles; Lung; Male; Mice; Mice, Inbred C57BL; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Piperazines; Protein Kinase Inhibitors; Pulmonary Artery; Pyridines; Pyrroles; Rats; Rats, Inbred WKY; Rats, Sprague-Dawley; Treatment Outcome

We have investigated the temporal relationship between the hemodynamic and histological/morphological progression in a rat model of pulmonary arterial hypertension that develops pulmonary arterial lesions morphologically indistinguishable from those in human pulmonary arterial hypertension. Adult male rats were injected with Sugen5416 and exposed to hypoxia for 3 wk followed by a return to normoxia for various additional weeks. At 1, 3, 5, 8, and 13 wk after the Sugen5416 injection, hemodynamic and histological examinations were performed. Right ventricular systolic pressure reached its maximum 5 wk after Sugen5416 injection and plateaued thereafter. Cardiac index decreased at the 3∼5-wk time point, and tended to further decline at later time points. Reflecting these changes, calculated total pulmonary resistance showed a pattern of progressive worsening. Acute intravenous fasudil markedly reduced the elevated pressure and resistance at all time points tested. The percentage of severely occluded small pulmonary arteries showed a similar pattern of progression to that of right ventricular systolic pressure. These small vessels were occluded predominantly with nonplexiform-type neointimal formation except for the 13-wk time point. There was no severe occlusion in larger arteries until the 13-wk time point, when significant numbers of vessels were occluded with plexiform-type neointima. The Sugen5416/hypoxia/normoxia-exposed rat shows a pattern of chronic hemodynamic progression similar to that observed in pulmonary arterial hypertension patients. In addition to vasoconstriction, nonplexiform-type neointimal occlusion of small arteries appears to contribute significantly to the early phase of pulmonary arterial hypertension development, and plexiform-type larger vessel occlusion may play a role in the late deterioration.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Angiogenesis Inhibitors; Animals; Familial Primary Pulmonary Hypertension; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Indoles; Male; Pulmonary Artery; Pyrroles; Rats; Rats, Sprague-Dawley; Vasodilator Agents

NF-κB and IL-6, a NF-κB downstream mediator, play a central role in the inflammatory response of tissues. We aimed to determine the role of the classical NF-κB pathway in severe pulmonary arterial hypertension (PAH) induced by SU5416 and chronic hypoxia (SuHx) in rats. Tissue samples from patients with idiopathic PAH (iPAH) and control subjects were investigated. SuHx rats were treated from Days 1 to 3, 1 to 21, and 29 to 42 with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and/or from Days 1 to 21 with anti-IL-6 antibody. Nuclear staining for NF-κB, an indicator of the activation of the classical NF-κB pathway, was detected in pulmonary arterial lesions of patients with iPAH and SuHx rats. NF-κB inhibition with PDTC prevented and reduced pulmonary arterial obliteration without reducing muscularization. However, the elevated lung levels of IL-6 were not reduced in PDTC-treated SuHx animals. PDTC treatment prevented or reduced apoptosis of pulmonary artery wall cells and pulmonary arterial obliteration. IL-6 inhibition had only a partial effect on apoptosis and obliteration. Pulmonary arterial media wall thickness was not affected by any of these treatments. Preventive and therapeutic PDTC treatment promoted immune regulation by increasing the number of perivascular CD4(+) T cells, in particular regulatory T cells (early treatment), and by reducing the number of perivascular CD8(+) T lymphocytes and CD45RA(+) B lymphocytes. Therapeutic PDTC treatment further preserved right ventricular function in SuHx animals. Inhibition of NF-κB may represent a therapeutic option for pulmonary arterial obliteration via reduced vessel wall cell apoptosis and improved regulation of the immune system.

Topics: Animals; Apoptosis; CD4-Positive T-Lymphocytes; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Hypoxia; Indoles; Inflammation; Interleukin-6; Leukocyte Common Antigens; Lung; NF-kappa B; Pulmonary Artery; Pyrroles; Pyrrolidines; Rats; Signal Transduction; Thiocarbamates; Time Factors

Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH.. Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression.. These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Topics: Adventitia; Animals; Antihypertensive Agents; Cell Movement; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Matrix; Familial Primary Pulmonary Hypertension; Fibroblasts; HEK293 Cells; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Indoles; Male; Mice; Mice, Inbred C57BL; Monocrotaline; NADPH Oxidase 4; NADPH Oxidases; Pulmonary Artery; Pyrroles; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Time Factors; Transfection; Up-Regulation

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator-activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end-products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH-PASMC proliferation and resistance to apoptosis.. In vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH-PASMC (6-fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6-fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline- and Sugen-induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%).. We have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.

Topics: Adult; Aged; Animals; Apoptosis; Arterial Pressure; Bone Morphogenetic Protein Receptors, Type II; Case-Control Studies; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Familial Primary Pulmonary Hypertension; Female; Glycation End Products, Advanced; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Indoles; Male; Middle Aged; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; PPAR gamma; Pulmonary Artery; Pyrroles; Rats; Rats, Sprague-Dawley; Receptor for Advanced Glycation End Products; Receptors, Immunologic; RNA Interference; S100 Proteins; Signal Transduction; STAT3 Transcription Factor; Transfection; Up-Regulation

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

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

Topics: Animals; Antibodies, Monoclonal; Bone Morphogenetic Proteins; Chronic Disease; Familial Primary Pulmonary Hypertension; Heart Ventricles; HEK293 Cells; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Indoles; Intercellular Signaling Peptides and Proteins; Lung; Mice; Pyrroles; Signal Transduction

Conduit pulmonary artery (PA) stiffening is characteristic of pulmonary arterial hypertension (PAH) and is an excellent predictor of mortality due to right ventricular (RV) overload. To better understand the impact of conduit PA stiffening on RV afterload, it is critical to examine the arterial viscoelastic properties, which require measurements of elasticity (energy storage behavior) and viscosity (energy dissipation behavior). Here we hypothesize that PAH leads to frequency-dependent changes in arterial stiffness (related to elasticity) and damping ratio (related to viscosity) in large PAs. To test our hypothesis, PAH was induced by the combination of chronic hypoxia and an antiangiogenic compound (SU5416) treatment in mice. Static and sinusoidal pressure-inflation tests were performed on isolated conduit PAs at various frequencies (0.01-20 Hz) to obtain the mechanical properties in the absence of smooth muscle contraction. Static mechanical tests showed significant stiffening of large PAs with PAH, as expected. In dynamic mechanical tests, structural stiffness (κ) increased and damping ratio (D) decreased at a physiologically relevant frequency (10 Hz) in hypertensive PAs. The dynamic elastic modulus (E), a material stiffness, did not increase significantly with PAH. All dynamic mechanical properties were strong functions of frequency. In particular, κ, E and D increased with increasing frequency in control PAs. While this behavior remained for D in hypertensive PAs, it reversed for κ and E. Since these novel dynamic mechanical property changes were found in the absence of changes in smooth muscle cell content or contraction, changes in collagen and proteoglycans and their interactions are likely critical to arterial viscoelasticity in a way that has not been previously described. The impact of these changes in PA viscoelasticity on RV afterload in PAH awaits further investigation.

Topics: Angiogenesis Inhibitors; Animals; Blood Pressure; Chronic Disease; Collagen; Elastic Modulus; Familial Primary Pulmonary Hypertension; Hypertension, Pulmonary; Hypoxia; Indoles; Male; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Proteoglycans; Pyrroles; Stress, Mechanical; Vascular Stiffness; Ventricular Dysfunction, Right; Viscosity