transforming-growth-factor-beta and Heart-Defects--Congenital

Dilatation of the aorta is a constantly evolving condition that can lead to the ultimate life-threatening event, acute aortic dissection. Recent research has tried to identify quantifiable biomarkers, with both diagnostic and prognostic roles in different aortopathies. Most studies have focused on the bicuspid aortic valve, the most frequent congenital heart disease (CHD), and majorly evolved around matrix metalloproteinases (MMPs). Other candidate biomarkers, such as asymmetric dimethylarginine, soluble receptor for advanced glycation end-products or transforming growth factor beta have also gained a lot of attention recently. Most of the aortic anomalies and dilatation-related studies have reported expression variation of tissular biomarkers. The ultimate goal remains, though, the identification of biomarkers among the serum plasma, with the upregulation of circulating MMP-1, MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), asymmetric dimethylarginine (ADMA), soluble receptor for advanced glycation end-products (sRAGE) and transforming growth factor beta (TGF-β) being reported in association to several aortopathies and related complications in recent research. These molecules are apparently quantifiable from the early ages and have been linked to several CHDs and hereditary aortopathies. Pediatric data on the matter is still limited, and further studies are warranted to elucidate the role of plasmatic biomarkers in the long term follow-up of potentially evolving congenital aortopathies.

Topics: Aorta; Aortic Valve; Biomarkers; Child; Dilatation, Pathologic; Heart Defects, Congenital; Heart Valve Diseases; Humans; Matrix Metalloproteinases; Receptor for Advanced Glycation End Products; Tissue Inhibitor of Metalloproteinase-1; 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

Marfan Syndrome (MFS) and Loeys-Dietz Syndrome (LDS) represent heritable connective tissue disorders that cosegregate with a similar pattern of cardiovascular defects (thoracic aortic aneurysm, mitral valve prolapse/regurgitation, and aortic root dilatation with regurgitation). This pattern of cardiovascular defects appears to be expressed along a spectrum of severity in many heritable connective tissue disorders and raises suspicion of a relationship between the normal development of connective tissues and the cardiovascular system. Given the evidence of increased transforming growth factor-beta (TGF-β) signaling in MFS and LDS, this signaling pathway may represent the common link in this relationship. To further explore this hypothetical link, this chapter will review the TGF-β signaling pathway, heritable connective tissue syndromes related to TGF-β receptor (TGFBR) mutations, and discuss the pathogenic contribution of TGF-β to these syndromes with a primary focus on the cardiovascular system.

Topics: Adrenergic beta-Antagonists; Angiotensin II Type 1 Receptor Blockers; Antibodies, Neutralizing; Aortic Aneurysm, Thoracic; Aortic Valve; Bicuspid Aortic Valve Disease; Gene Expression Regulation; Heart Defects, Congenital; Heart Valve Diseases; Humans; Loeys-Dietz Syndrome; Marfan Syndrome; Mutation; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Cardiac cushion formation is crucial for both valvular and septal development. Disruption in this process can lead to valvular and septal malformations, which constitute the largest part of congenital heart defects. One of the signaling pathways that is important for cushion formation is the TGFβ superfamily. The involvement of TGFβ and BMP signaling pathways in cardiac cushion formation has been intensively studied using chicken in vitro explant assays and in genetically modified mice. In this review, we will summarize and discuss the role of TGFβ and BMP signaling components in cardiac cushion formation.

Topics: Animals; Bone Morphogenetic Proteins; Chick Embryo; Endocardial Cushions; Heart Defects, Congenital; Heart Valves; Humans; Ligands; Mice; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Ventricular Septum

Transforming growth factor β (TGFβ) regulates one of the major signaling pathways that control tissue morphogenesis. In vitro experiments using heart explants indicated the importance of this signaling pathway for the generation of cushion mesenchymal cells, which ultimately contribute to the valves and septa of the mature heart. Recent advances in mouse genetics have enabled in vivo investigation into the roles of individual ligands, receptors, and coreceptors of this pathway, including investigation of the tissue specificity of these roles in heart development. This work has revealed that (1) cushion mesenchyme can form in the absence of TGFβ signaling, although mesenchymal cell numbers may be misregulated; (2) TGFβ signaling is essential for correct remodeling of the cushions, particularly those of the outflow tract; (3) TGFβ signaling also has a role in ensuring accurate remodeling of the pharyngeal arch arteries to form the mature aortic arch; and (4) mesenchymal cells derived from the epicardium require TGFβ signaling to promote their differentiation to vascular smooth muscle cells to support the coronary arteries. In addition, a mouse genetics approach has also been used to investigate the disease pathogenesis of Loeys-Dietz syndrome, a familial autosomal dominant human disorder characterized by a dilated aortic root, and associated with mutations in the two TGFβ signaling receptor genes, TGFBR1 and TGFBR2. Further important insights are likely as this exciting work progresses.

Topics: Animals; Heart Defects, Congenital; Humans; Mesoderm; Mice; Muscle, Smooth, Vascular; Signal Transduction; Transforming Growth Factor beta; Ventricular Remodeling

Many patients with congenital cardiac disease are at risk for progressive aortic dilation. The mechanisms underlying aortic dilation in this patient cohort are described, and the similarities to the pathophysiologic alterations seen in Marfan syndrome are highlighted. Indications for treatment are discussed.

Topics: Aortic Diseases; Dilatation, Pathologic; Heart Defects, Congenital; Humans; Marfan Syndrome; Transforming Growth Factor beta

Heterotaxy is a complex set of birth defects in which the normal concordance of asymmetric thoracic and abdominal organs is disturbed. In this review the authors summarize recent research on the etiology of heterotaxy syndromes. Improved understanding of the genetic control of left-right patterning in the early embryo is leading to the identification of candidate genes that may be mutated in heterotaxy patients, and epidemiologic studies are helping to define nongenetic mechanisms of embryopathy.. Several genes have now been implicated in heterotaxy and related isolated congenital heart malformations. These studies indicate that heterotaxy can be caused by single gene mutations. They also demonstrate that there is probably extensive locus heterogeneity. Heterotaxy may be caused by teratogenic exposures, especially maternal diabetes. Isolated congenital heart defects resulting from isomerisms and disturbed looping may be caused by mutations in genes that control early left-right patterning and the earliest steps in cardiogenesis. Genes currently implicated in human heterotaxy include ZIC3, LEFTYA, CRYPTIC, and ACVR2B. Roles for NKX2.5 and CRELDA are suggested by recent case reports.. Active research on the etiology of heterotaxy is leading to a reformulation of the likely etiologies. Its complex inheritance likely results from a mix of teratogenic and single gene disorders with variable expression and incomplete penetrance.

Topics: Abnormalities, Multiple; Activin Receptors, Type II; Animals; Cell Adhesion Molecules; Extracellular Matrix Proteins; Genetic Predisposition to Disease; Heart Defects, Congenital; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Left-Right Determination Factors; Spleen; Syndrome; Transcription Factors; 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

Clubbing was first described by Hippocrates more than 2.500 years ago. It may be seen alone or as part of an entity called hypertrophic osteoarthropathy which include periostitis, arthritis and sometimes thickening and edema of the skin around the affected joints. Pulmonary diseases such as cancer, abscess, empyema, bronchiectasis and cystic fibrosis are the major diseases known to be associate with hypertrophic osteoarthropathy. Digestive tract cancer, cyanogenic congenital heart disease are well known association. Many theories have attempted to explain the appearance of this sign but few have persisted. In this article, we review characteristics, relation with etiology and the basis of the pathophysiology of hypertrophic osteoarthropathy and particularly of clubbing.

Topics: Bronchiectasis; Causality; Cystic Fibrosis; Digestive System Neoplasms; Empyema; Ferritins; Heart Defects, Congenital; Humans; Lung Abscess; Lung Neoplasms; Osteoarthropathy, Secondary Hypertrophic; Platelet-Derived Growth Factor; Prostaglandins; Transforming Growth Factor beta

Xenopus and zebrafish serve as outstanding models in which to study vertebrate heart development. The embryos are transparent, allowing observation during organogenesis; they can be obtained in large numbers; and they are readily accessible to embryologic manipulation and microinjection of RNA, DNA, or protein. These embryos can live by diffusion for several days, allowing analysis of mutants or experimental treatments that perturb normal heart development. Xenopus embryos have been used to understand the induction of the cardiac field, the role of Nkx genes in cardiac development, and the role transforming growth factor beta molecules in the establishment and signaling of left-right axis information. Large-scale mutant screens in zebrafish and the development of transgenics in both Xenopus and zebrafish have accelerated the molecular identification of genes that regulate conserved steps in cardiovascular development.

Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Embryo, Nonmammalian; Female; Gene Targeting; Genetic Techniques; Genome; Glycoproteins; Heart; Heart Defects, Congenital; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Humans; Intracellular Signaling Peptides and Proteins; Male; Models, Animal; Models, Biological; Morphogenesis; Repressor Proteins; Species Specificity; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Vertebrates; Xenopus laevis; Xenopus Proteins; Zebrafish; Zebrafish Proteins

BMP2 (bone morphogenic protein-2) is a member of the TGF-β superfamily and has essential roles in the development of multiple organs, including osteogenesis. Because of its crucial role in organ and skeletal development, Bmp2 null mice is fetal lethal. The recent report has characterized multiple patients with BMP2 haploinsufficiency, describing individuals with BMP2 sequence variants and deletions associated with short stature without endocrinological abnormalities, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. However, due to a small number of reported patients with BMP2 haploinsufficiency, the genotype and phenotype correlations are not fully understood. We experienced a family of BMP2 haploinsufficiency with a novel frameshift variant NM_001200.4: c.231dup (p.Tyr78Leufs*38) which was predicted to be "pathogenic" by the American College of Genetics and Genomics (ACGM) criteria. In addition to short stature, impaired hearing ability and minor skeletal deformities, the proband exhibited isolated dextrocardia situs solitus without cardiac anomalies and abnormal locations of other visceral organs. Our study would shed light on the crucial role of BMP2 in determining the cardiac axis, and further studies are needed to assemble more cases to elucidate BMP2 role in human heart development.

Topics: Animals; Bone Morphogenetic Protein 2; Dextrocardia; Dwarfism; Family; Genotype; Heart Defects, Congenital; Humans; Mice; Transforming Growth Factor beta

Endothelial-to-mesenchymal transition (EndMT) plays a critical role in the flow-induced vascular remodeling process, such as pulmonary arterial hypertension (PAH) related to congenital heart disease (CHD). NBL1 (neuroblastoma suppressor of tumorigenicity 1) is a secreted glycoprotein that has been implicated in CHD-PAH by aggravating the phenotypic transformation of smooth muscle cells. However, the underlying mechanisms regarding the interplay between NBL1 and endothelial cells in CHD-PAH remain to be fully elucidated. Thus, we aimed to identify the potential effect of NBL1 on EndMT using a novel flow-associated PAH model with

Topics: Animals; Endothelial Cells; Epithelial-Mesenchymal Transition; Familial Primary Pulmonary Hypertension; Heart Defects, Congenital; Humans; Nerve Tissue Proteins; Neuroblastoma; Pulmonary Arterial Hypertension; Rats; Transforming Growth Factor beta

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β-related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.

Topics: Animals; Cathepsin K; Disease Models, Animal; Enzyme Activation; Genetic Predisposition to Disease; Heart; Heart Defects, Congenital; Heart Valves; Humans; Lysosomal Storage Diseases; Mucolipidoses; Mutation; Transferases (Other Substituted Phosphate Groups); Transforming Growth Factor beta; Zebrafish

Recessive variants in LTBP2 are associated with eye-restricted phenotypes including (a) primary congenital glaucoma and (b) microspherophakia/megalocornea and ectopia lentis with/without secondary glaucoma. Nosology of LTBP2 pathology in humans is apparently in contrast with the consolidated evidence of a wide expression of this gene in the developing embryo. Accordingly, in previously published patients with LTBP2-related eye disease, additional extraocular findings have been occasionally reported and include, among others, high-arched palate, tall stature, and variable cardiac involvement. Anyway, no emphasis was put on such systemic manifestations. Here, we report two unrelated Roma/Gypsy patients first ascertained for a multisystem disorder mainly characterized by primary congenital glaucoma, complex congenital heart defect, tall stature, long fingers, skin striae and dystrophic scarring, and resembling Marfan syndrome. Heart involvement was severe with polyvalvular heart dysplasia in one, and transposition of great arteries, thoracic arterial tortuosity, polyvalvular heart dysplasia, and neo-aortic root dilatation in the other. Both patients were homozygous for the recurrent c.895C>T[p.(R299X)] variant, typically found in individuals of Roma/Gypsy descent with an eye-restricted phenotype. Our findings point out LTBP2 as responsible of a systemic phenotype coherent with the community of syndromes related to anomalies in genes involved in the TGFβ-pathway. Among these disorders, LTBP2-related systemic disease emerges as a distinct condition with expanding prognostic implications and autosomal recessive inheritance.

Topics: Adolescent; Child; Corneal Diseases; Ectopia Lentis; Eye Diseases, Hereditary; Female; Genetic Diseases, X-Linked; Glaucoma; Heart; Heart Defects, Congenital; Homozygote; Humans; Iris; Latent TGF-beta Binding Proteins; Male; Marfan Syndrome; Phenotype; Roma; Transforming Growth Factor beta

In a previous study, we screened thousands of long non-coding RNAs (lncRNAs) to assess their potential relationship with congenital heart disease (CHD). In this study, uc.4 attracted our attention because of its high level of evolutionary conservation and its antisense orientation to the CASZ1 gene, which is vital for heart development. We explored the function of uc.4 in cells and in zebrafish, and describe a potential mechanism of action. P19 cells were used to investigate the function of uc.4. We studied the effect of uc.4 overexpression on heart development in zebrafish. The overexpression of uc.4 influenced cell differentiation by inhibiting the TGF-beta signaling pathway and suppressed heart development in zebrafish, resulting in cardiac malformation. Taken together, our findings show that uc.4 is involved in heart development, thus providing a potential therapeutic target for CHD.

Topics: Animals; Apoptosis; Biomarkers; Cell Cycle; Cell Differentiation; Cell Line; Computational Biology; Databases, Genetic; DNA-Binding Proteins; Gene Expression; Gene Expression Profiling; Genes, Lethal; Heart Defects, Congenital; Humans; Mice; RNA, Long Noncoding; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Zebrafish

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-β-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.

Topics: Animals; Bone and Bones; Bone Morphogenetic Protein 2; Child; Child, Preschool; Chromosomes, Human, Pair 20; Cleft Palate; Craniofacial Abnormalities; Developmental Disabilities; Disease Models, Animal; Dwarfism; Female; Haploinsufficiency; Heart; Heart Defects, Congenital; Humans; Infant; Male; Mice; Mice, Knockout; Transforming Growth Factor beta

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

Topics: Cardiomyopathies; Heart Defects, Congenital; Heart Ventricles; Humans; Induced Pluripotent Stem Cells; Mutation; Myocytes, Cardiac; Phenotype; Signal Transduction; T-Box Domain Proteins; Transforming Growth Factor beta

N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis.. N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-β treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-β treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-β treatment also rescued the dofetilide-induced phenotype toward normal.. Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.

Topics: Abnormalities, Drug-Induced; Animals; Cells, Cultured; Embryo Culture Techniques; Embryonic Stem Cells; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Fetal Death; Gene Expression Regulation, Developmental; Genotype; Heart Defects, Congenital; Humans; Mice, 129 Strain; Mice, Transgenic; Morphogenesis; Mutation, Missense; Neovascularization, Physiologic; Phenethylamines; Phenotype; Potassium Channel Blockers; Signal Transduction; Sulfonamides; Transforming Growth Factor beta; Vascular Malformations

Latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-β (TGF-β) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo.

Topics: Alternative Splicing; Animals; Animals, Newborn; Cells, Cultured; Exons; Fibroblasts; Genes, Lethal; Heart Defects, Congenital; Latent TGF-beta Binding Proteins; Mice; Mice, Knockout; Protein Isoforms; Signal Transduction; Transforming Growth Factor beta

Topics: Abnormalities, Drug-Induced; Animals; Embryonic Stem Cells; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Fetal Death; Heart Defects, Congenital; Humans; Mutation, Missense; Neovascularization, Physiologic; Phenethylamines; Potassium Channel Blockers; Sulfonamides; Transforming Growth Factor beta; Vascular Malformations

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Topics: Adaptor Proteins, Signal Transducing; Animals; Female; Fetal Death; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Heart Septal Defects, Ventricular; Intercellular Signaling Peptides and Proteins; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Neuro-Oncological Ventral Antigen; RNA-Binding Proteins; Transcription Factors; Transforming Growth Factor beta

β2-Spectrin is an actin-binding protein that plays an important role in membrane integrity and the transforming growth factor (TGF)-β signalling pathway as an adaptor for Smads. Loss of β2-spectrin in mice (Spnb2(-/-)) results in embryonic lethality with gastrointestinal, liver, neural, and heart abnormalities that are similar to those in Smad2(+/-)Smad3(+/-) mice. However, to date, the role of β2-spectrin in embryogenesis, particularly in heart development, has been poorly delineated. Here, we demonstrated that β2-spectrin is required for the survival and differentiation of cardiomyocytes, and its loss resulted in defects in heart development with failure of ventricular wall thickening.. Disruption of β2-spectrin in primary muscle cells not only inhibited TGF-β/Smad signalling, but also reduced the expression of the cardiomyocyte differentiation markers Nkx2.5, dystrophin, and α-smooth muscle actin (α-SMA). Furthermore, cytoskeletal networks of dystrophin, F-actin, and α-SMA in cardiomyocytes were disorganized upon loss of β2-spectrin. In addition, deletion of β2-spectrin in mice (Spnb2(tm1a/tm1a)) prevented proper development of the heart in association with disintegration of dystrophin structure and markedly reduced survival.. These data suggest that β2-spectrin deficiency leads to inactivation of TGF-β/Smad signalling and contributes to dysregulation of the cell cycle, proliferation, differentiation, and the cytoskeletal network, and it leads to defective heart development. Our data demonstrate that β2-spectrin is required for proper development of the heart and that disruption of β2-spectrin is a potential underlying cause of congenital heart defects.

Topics: Animals; Apoptosis; Carrier Proteins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cytoskeletal Proteins; Dystrophin; Female; Heart; Heart Defects, Congenital; Male; Mice, Inbred C57BL; Microfilament Proteins; Myocytes, Cardiac; Myofibrils; Smad Proteins; Transforming Growth Factor beta

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1-/-) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1-/- aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1-/- aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.

Topics: Animals; Aortic Valve; Bicuspid Aortic Valve Disease; Calcinosis; Cell Proliferation; Cutis Laxa; Disease Models, Animal; Disease Progression; Elastic Tissue; Fibrosis; Heart Defects, Congenital; Heart Valve Diseases; Inflammation; Membrane Glycoproteins; Mice; Models, Biological; Myofibroblasts; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta; Ultrasonography

Diabetes mellitus in pregnancy causes defects in infant heart, including the outflow tracts (OFTs). Development of the aorta and pulmonary artery, which are derived from the common OFT in the embryo, is regulated by the transforming growth factor β (TGFβ) and Wnt families, and can be perturbed by hyperglycemia-generated intracellular stress conditions. However, the underlying cellular and molecular mechanisms remain to be delineated.. Female mice were induced diabetic with streptozotocin. Embryonic and fetal OFTs were examined morphologically and histologically. Cell proliferation was assessed using 5'-bromo-2'-deoxyuridine incorporation assay. Oxidative and endoplasmic reticulum (ER) stress markers and TGFβ factors were detected using immunohistochemistry. The expression of genes in the Wnt-signaling system was assessed using real-time reverse transcription polymerase chain reaction array. The role of activin-A in cell proliferation was addressed by treating embryos cultured in high glucose with activin-A.. Maternal diabetes caused complex abnormalities in the OFTs, including aortic and pulmonary stenosis and persistent truncus arteriosus. The development of the endocardial cushions was suppressed, manifested with insufficient cellularization of the tissues. Cell proliferation was significantly decreased under oxidative and ER stress conditions. The expression of genes in the Wnt signaling was significantly altered. Activin-A and Smad3 were found to be expressed in the OFT. Treatment with activin-A rescued cell proliferation in the endocardial cushions.. Maternal diabetes generates oxidative and ER stress conditions, suppresses TGFβ and Wnt signaling, inhibits cell proliferation and cellularization of the endocardial cushions, leading to OFT septal defects. Activin-A plays a role in hyperglycemia-suppressed proliferation of the endocardial cells.

Topics: Activins; Animals; Aorta; Aortic Valve Stenosis; Cardiac Output; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetes, Gestational; Embryo Culture Techniques; Embryo, Mammalian; Endocardial Cushions; Endoplasmic Reticulum Stress; Female; Gene Expression Regulation, Developmental; Glucose; Heart Defects, Congenital; Hyperglycemia; Mice; Mice, Inbred C57BL; Neural Crest; Oxidative Stress; Pregnancy; Pulmonary Artery; Pulmonary Valve Stenosis; Smad3 Protein; Streptozocin; Transforming Growth Factor beta; Truncus Arteriosus, Persistent; Wnt Proteins; Wnt Signaling Pathway

Little is known about the pathophysiology of myxomatous degeneration of the mitral valve, the pathological hallmark of mitral valve prolapse, associated with symptomatic mitral regurgitation, heart failure, and death. Excess transforming growth factor (TGF)-β signaling is known to cause mitral valve degeneration and regurgitation in a mouse model of Marfan syndrome. We examined if TGF-β signaling is dysregulated in clinical specimens of sporadic mitral valve prolapse compared with explanted nondiseased mitral valves and we tested the effects of angiotensin II receptor blockers on TGF-β signaling in cultured human mitral valve cells.. Operative specimens, cultured valve tissues, and cultured valvular interstitial cells were obtained from patients with mitral valve prolapse undergoing mitral valve repair or from organ donors without mitral valve disease. Increased extracellular matrix in diseased valve tissue correlated with an upregulation of TGF-β expression and signaling as evidenced by SMAD2/3 phosphorylation. Both TGF-β ligand and signaling mediators colocalized primarily to valvular interstitial cells suggesting autocrine/paracrine activation. In cultured valve tissue, exogenous TGF-β increased basal extracellular matrix production, whereas serological neutralization of TGF-β inhibited disease-driven extracellular matrix overproduction. TGF-β-induced extracellular matrix production in cultured valvular interstitial cells was dependent on SMAD2/3 and p38 signaling and was inhibited by angiotensin II receptor blockers.. TGF-β has a profibrotic role in the pathogenesis of sporadic mitral valve prolapse. Attenuation of TGF-β signaling by angiotensin II receptor blockers may represent a mechanistically based strategy to modulate the pathological progression of mitral valve prolapse in patients.

Topics: Angiotensin Receptor Antagonists; Benzimidazoles; Benzoates; Biphenyl Compounds; Cells, Cultured; Collagen; Elastic Tissue; Extracellular Matrix Proteins; Fibrosis; Gene Expression Regulation; Genetic Diseases, X-Linked; Heart Defects, Congenital; Humans; Losartan; Mitral Valve Insufficiency; Mitral Valve Prolapse; Myxoma; Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad3 Protein; Telmisartan; Tetrazoles; Transforming Growth Factor beta; Vimentin

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.

Topics: Actins; Animals; Aorta; Blood Vessels; Cattle; cdc42 GTP-Binding Protein; Cortactin; Dwarfism; Endothelial Cells; Extracellular Matrix; Face; Genetic Diseases, X-Linked; Genitalia, Male; Guanine Nucleotide Exchange Factors; Hand Deformities, Congenital; Heart Defects, Congenital; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Cardiovascular defects are the most common anomalies in diabetic embryopathy. The mechanisms underlying the manifestation of the defects remain to be addressed.. Female mice were administered streptozotocin to induce diabetes. Embryos from euglycemic (control) and hyperglycemic groups were examined for morphological and histological evaluation of malformations. Cell proliferation and programmed cell death (apoptosis) were assessed using mitotic markers (BrdU and Ki67) and TUNEL assay, respectively. Expression of eight four genes in the TGFbeta signaling system was analyzed using real-time RT-PCR.. Structural abnormalities were observed in the heart and neural tube in diabetic groups, with significantly higher malformation rates than in control groups. Moreover, malformation rates in the heart were higher than those in the neural tube. Cardiac abnormalities including dilated heart tube, smaller ventricles, conotruncal stenosis, and abnormal heart looping were seen during early morphogenesis prior to cardiac septation [embryonic day (E) 9.5-11.5]. Histological examinations showed hypoplastic myocardium and endocardial cushions. After cardiac septation (E15.5), ventricular septal defects were observed, which were manifested in the non-muscular portion of the septum. Significant decreases in cell proliferation with no differences in apoptosis were observed in the myocardium and endocardial cushions in diabetic compared to control groups. Factors in the TGFbeta signaling that regulate heart development were downregulated by maternal diabetes.. Maternal diabetes causes malformations in the heart of the embryo. The heart is more susceptible to maternal diabetic insults than the neural tube. Malformations in the heart prior to septation are associated with decreased cell proliferation, but not increased apoptosis. The TGFbeta signaling is involved in cardiac malformations in diabetic embryopathy.

Topics: Animals; Apoptosis; Cell Proliferation; Diabetes Mellitus, Experimental; Down-Regulation; Embryo, Mammalian; Embryonic Development; Female; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Mice; Myocardium; Pregnancy; Pregnancy in Diabetics; Signal Transduction; Transforming Growth Factor beta

To understand the role of TGF-β signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-β type II receptor (TβRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre(+/-)/Tgfbr2 (flox/flox) embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-β plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.

Topics: Animals; Aorta, Thoracic; Cell Differentiation; Elastic Tissue; Elastin; Extracellular Matrix Proteins; Female; Gene Knockdown Techniques; Heart Defects, Congenital; Male; Mice; Mice, Transgenic; Microfilament Proteins; Muscle Proteins; Myocytes, Smooth Muscle; Pericardium; Pregnancy; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) family members, including TGF-betas, activins, and bone morphogenetic proteins, exert diverse biological activities in cell proliferation, differentiation, apoptosis, embryonic development, and many other processes. These effects are largely mediated by Smad proteins. Smad7 is a negative regulator for the signaling of TGF-beta family members. Dysregulation of Smad7 is associated with pathogenesis of a variety of human diseases. However, the in vivo physiological roles of Smad7 have not been elucidated due to the lack of a mouse model with significant loss of Smad7 function. Here we report generation and initial characterization of Smad7 mutant mice with targeted deletion of the indispensable MH2 domain. The majority of Smad7 mutant mice died in utero due to multiple defects in cardiovascular development, including ventricular septal defect and non-compaction, as well as outflow tract malformation. The surviving adult Smad7 mutant mice had impaired cardiac functions and severe arrhythmia. Further analyses suggest that Smad2/3 phosphorylation was elevated in atrioventricular cushion in the heart of Smad7 mutant mice, accompanied by increased apoptosis in this region. Taken together, these observations pinpoint an important role of Smad7 in the development and function of the mouse heart in vivo.

Topics: Amino Acid Sequence; Animals; Arrhythmias, Cardiac; Heart; Heart Defects, Congenital; Humans; Mice; Mice, Mutant Strains; Phosphorylation; Protein Structure, Tertiary; Sequence Deletion; Smad2 Protein; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta regulates vascular development through two type I receptors: activin receptor-like kinase (ALK) 1 and ALK5, each of which activates a different downstream Smad pathway. The endothelial cell (EC)-specific ALK1 increases EC proliferation and migration, whereas the ubiquitously expressed ALK5 inhibits both of these processes. As ALK1 requires the kinase activity of ALK5 for optimal activation, the lack of ALK5 in ECs results in defective phosphorylation of both Smad pathways on TGF-beta stimulation. To understand why TGF-beta signaling through ALK1 and ALK5 has opposing effects on ECs and whether this takes place in vivo, we carefully compared the phenotype of ALK5 knock-in (ALK5(KI/KI)) mice, in which the aspartic acid residue 266 in the L45 loop of ALK5 was replaced by an alanine residue, with the phenotypes of ALK5 knock-out (ALK5(-/-)) and wild-type mice. The ALK5(KI/KI) mice showed angiogenic defects with embryonic lethality at E10.5-11.5. Although the phenotype of the ALK5(KI/KI) mice was quite similar to that of the ALK5(-/-) mice, the hierarchical structure of blood vessels formed in the ALK5(KI/KI) embryos was more developed than that in the ALK5(-/-) mutants. Thus, the L45 loop mutation in ALK5 partially rescued the earliest vascular defects in the ALK5(-/-) embryos. This study supports our earlier observation that vascular maturation in vivo requires both TGF-beta/ALK1/BMP-Smad and TGF-beta/ALK5/activin-Smad pathways for normal vascular development.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Amino Acid Substitution; Animals; Base Sequence; Blood Vessels; Colony-Forming Units Assay; DNA Primers; Female; Heart Defects, Congenital; Hematopoietic Stem Cells; Mice; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Mutagenesis, Site-Directed; Neovascularization, Physiologic; Phenotype; Pregnancy; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Yolk Sac

This study sought to investigate the influence of recipient renin-angiotensin-aldosterone system (RAAS) genotype on cardiac function, rejection, and outcomes after heart transplantation.. The RAAS influences cardiac function and up-regulates inflammatory/immune pathways. Little is known about the effect of recipient RAAS polymorphisms in pediatric cardiac transplantation.. Patients <25 years of age, after cardiac transplantation, were enrolled (2003 to 2008) and genotyped for polymorphisms in genes associated with RAAS upregulation: AGT-G, ACE-D, AGTR1-C, CYP11B2-G, and CMA-A. Presence of at least 1 high-risk allele was defined as a high-risk genotype. Univariable and multivariable associations between genotypes and outcomes were assessed in time-dependent models using survival, logistic, or linear regression models. Biopsy samples were immunostained for interleukin (IL)-6, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha during rejection and quiescence.. A total of 145 patients were studied, 103 primary cohort and 42 replication cohort; 81% had rejection, 51% had graft dysfunction, and 13% had vasculopathy, 7% died and 8% underwent re-transplantation. A higher number of homozygous high-risk RAAS genotypes was associated with a higher risk of graft dysfunction (hazard ratio [HR]: 1.5, p = 0.02) and a higher probability of death (HR: 2.5, p = 0.04). The number of heterozygous high-risk RAAS genotypes was associated with frequency of rejection (+0.096 events/year, p < 0.001) and rejection-associated graft dysfunction (+0.37 events/year, p = 0.002). IL-6 and TGF-beta were markedly upregulated during rejection in patients with >/=2 high-risk RAAS genotypes.. Recipient RAAS polymorphisms are associated with a higher risk of rejection, graft cytokine expression, graft dysfunction, and a higher mortality after cardiac transplantation. This may have implications for use of RAAS inhibitors in high-risk patients after transplantation.

Topics: Biopsy; Cardiomyopathies; Child; DNA; Echocardiography; Female; Follow-Up Studies; Gene Expression Regulation; Genotype; Graft Rejection; Heart Defects, Congenital; Heart Transplantation; Humans; Interleukin-6; Male; Myocardium; Polymerase Chain Reaction; Retrospective Studies; Risk Factors; Time Factors; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Homologous

Abnormalities of embryonic patterning are hypothesized to underlie many common congenital malformations in humans including congenital heart defects (CHDs), left-right disturbances (L-R) or laterality, and holoprosencephaly (HPE). Studies in model organisms suggest that Nodal-like factors provide instructions for key aspects of body axis and germ layer patterning; however, the complex genetics of pathogenic gene variant(s) in humans are poorly understood. Here we report our studies of FOXH1, CFC1, and SMAD2 and summarize our mutational analysis of three additional components in the human NODAL-signaling pathway: NODAL, GDF1, and TDGF1. We identify functionally abnormal gene products throughout the pathway that are clearly associated with CHD, laterality, and HPE. Abnormal gene products are most commonly detected in patients within a narrow spectrum of isolated conotruncal heart defects (minimum 5%-10% of subjects), and far less commonly in isolated laterality or HPE patients (approximately 1% for each). The difference in the mutation incidence between these groups is highly significant. We show that apparent gene dosage discrepancies between humans and model organisms can be reconciled by considering a broader combination of sequence variants. Our studies confirm that (1) the genetic vulnerabilities inferred from model organisms with defects in Nodal signaling are indeed analogous to humans; (2) the molecular analysis of an entire signaling pathway is more complete and robust than that of individual genes and presages future studies by whole-genome analysis; and (3) a functional genomics approach is essential to fully appreciate the complex genetic interactions necessary to produce these effects in humans.

Topics: Amino Acid Sequence; Animals; Body Patterning; Case-Control Studies; Codon; Cohort Studies; DNA Mutational Analysis; Embryo, Nonmammalian; Epidermal Growth Factor; Forkhead Transcription Factors; GPI-Linked Proteins; Growth Differentiation Factor 1; Heart Defects, Congenital; Holoprosencephaly; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Models, Biological; Molecular Sequence Data; Mutation; Neoplasm Proteins; Nodal Protein; Pilot Projects; Sequence Homology, Amino Acid; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Zebrafish

Geleophysic dysplasia is an autosomal recessive disorder characterized by short stature, brachydactyly, thick skin and cardiac valvular anomalies often responsible for an early death. Studying six geleophysic dysplasia families, we first mapped the underlying gene to chromosome 9q34.2 and identified five distinct nonsense and missense mutations in ADAMTSL2 (a disintegrin and metalloproteinase with thrombospondin repeats-like 2), which encodes a secreted glycoprotein of unknown function. Functional studies in HEK293 cells showed that ADAMTSL2 mutations lead to reduced secretion of the mutated proteins, possibly owing to the misfolding of ADAMTSL2. A yeast two-hybrid screen showed that ADAMTSL2 interacts with latent TGF-beta-binding protein 1. In addition, we observed a significant increase in total and active TGF-beta in the culture medium as well as nuclear localization of phosphorylated SMAD2 in fibroblasts from individuals with geleophysic dysplasia. These data suggest that ADAMTSL2 mutations may lead to a dysregulation of TGF-beta signaling and may be the underlying mechanism of geleophysic dysplasia.

Topics: Abnormalities, Multiple; Biological Availability; Cell Line; Child; Child, Preschool; Extracellular Matrix Proteins; Growth Disorders; Hand Deformities, Congenital; Heart Defects, Congenital; Heart Valves; Humans; Mutation; Transforming Growth Factor beta

Mutations in the genes encoding transforming growth factor-beta receptor types I and II (TGFBR1 and TGFBR2, respectively) are commonly identified in patients with Loeys-Dietz syndrome, as well as some patients with Marfan's syndrome or familial thoracic aortic aneurysms and dissections. This suggests that there is considerable phenotypic heterogeneity associated with mutations in these genes. Because bicuspid aortic valve (BAV) is a congenital heart defect in patients with Loeys-Dietz syndrome, this study was conducted to investigate whether variants in TGFBR1 or TGFBR2 are responsible for sporadic BAV. Analysis of these genes in 35 patients with BAVs identified only known single-nucleotide polymorphisms or novel synonymous or intronic substitutions. In conclusion, mutations in TGFBR1 and TGFBR2 rarely cause sporadic BAV.

Topics: Adolescent; Adult; Aorta, Thoracic; Aortic Valve; Child; Child, Preschool; Dilatation, Pathologic; DNA; Female; Heart Defects, Congenital; Humans; Infant; Male; Mutation; Polymerase Chain Reaction; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Proliferation; DNA, Complementary; Embryo, Mammalian; Feedback, Physiological; Heart; Heart Defects, Congenital; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Humans; LIM-Homeodomain Proteins; Mice; Multipotent Stem Cells; Myocardium; Myocytes, Cardiac; Oligonucleotide Array Sequence Analysis; Phenotype; Smad1 Protein; Transcription Factors; Transforming Growth Factor beta

Msx1 and Msx2 are highly conserved, Nk-related homeodomain transcription factors that are essential for a variety of tissue-tissue interactions during vertebrate organogenesis. Here we show that combined deficiencies of Msx1 and Msx2 cause conotruncal anomalies associated with malalignment of the cardiac outflow tract (OFT). Msx1 and Msx2 play dual roles in outflow tract morphogenesis by both protecting secondary heart field (SHF) precursors against apoptosis and inhibiting excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the conotruncal cushions. During incorporation of SHF precursors into the OFT myocardium, ectopic apoptosis in the Msx1-/-; Msx2-/- mutant SHF is associated with reduced expression of Hand1 and Hand2, which from work on Hand1 and Hand2 mutants may be functionally important in the inhibition of apoptosis in Msx1/2 mutants. Later during aorticopulmonary septation, excessive proliferation in the OFT cushion mesenchyme and myocardium of Msx1-/-; Msx2-/- mutants is associated with premature down-regulation of p27(KIP1), an inhibitor of cyclin-dependent kinases. Diminished accretion of SHF precursors to the elongating OFT myocardium and excessive accumulation of mesenchymal cells in the conotruncal cushions may work together to perturb the rotation of the truncus arteriosus, leading to OFT malalignment defects including double-outlet right ventricle, overriding aorta and pulmonary stenosis.

Topics: Animals; Apoptosis; Body Patterning; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Movement; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Female; Fetal Heart; Gene Expression Regulation, Developmental; Genes, Homeobox; Heart Defects, Congenital; Homeodomain Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; MSX1 Transcription Factor; Mutation; Neural Crest; Pregnancy; Signal Transduction; Transforming Growth Factor beta

Latent TGF-beta binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-beta and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-beta activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-beta activity in heart organogenesis.

Topics: Animals; Animals, Newborn; Cell Differentiation; Extracellular Matrix; Gene Expression Regulation; Gene Targeting; Heart; Heart Defects, Congenital; Latent TGF-beta Binding Proteins; Mice; Mice, Knockout; Neural Crest; Protein Isoforms; Transforming Growth Factor beta

The complex cardiac defects that occur in heterotaxy result from abnormal left-right patterning. Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy, HTX1. We previously have generated a targeted deletion of the murine Zic3 locus and demonstrated that these knockout mice correctly model HTX1. Fifty percent of Zic3 null embryos have cardiac looping anomalies at embryonic day 10.5 to 14.5, with ventral looping and sinistral looping as the predominant phenotypes. The penetrance of these phenotypes is increased in mice that are also haploinsufficient for Nodal. Zic3(+/-); Nodal (+/-) compound heterozygous mice are born in significantly reduced numbers (P=0.0001), indicating a genetic interaction between the loci. Furthermore, an upstream Nodal enhancer is responsive to Zic3 in both Xenopus and mouse. These studies provide evidence that Zic3 interacts genetically with Nodal in left-right patterning and subsequent cardiac development and delineate a critical Zic3-responsive enhancer required for mediating Nodal expression at the node.

Topics: Animals; Enhancer Elements, Genetic; Genetic Diseases, X-Linked; Heart; Heart Defects, Congenital; Homeodomain Proteins; Mice; Mice, Inbred C57BL; Nodal Protein; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20-25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46-60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis.

Topics: Animals; Base Sequence; Body Patterning; DNA, Complementary; Female; Gene Expression Regulation, Developmental; Heart Defects, Congenital; In Situ Hybridization; Male; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Mutation; Nodal Protein; Pregnancy; Signal Transduction; Transforming Growth Factor beta; Ubiquitin; Ubiquitin-Protein Ligases

Development of the aorta and pulmonary artery is a complex process involving multiple molecular genetic pathways that modulate morphogenesis of the outflow tracts and the anastomosis of branch vessels. Recent genetic studies of the cardiovascular system demonstrate that congenital and adult onset progressive disorders of the great vessels such as aneurysms are components of generalized vascular, cardiac, and extracardiovascular syndromes. Current paradigms suggest that aortic disease is founded in patterning anomalies of the conotruncus that occur in utero. These aberrations can be consequences of genetic aberrations in transcriptional regulation of signal transduction both within and outside the developing great vessels.

Topics: Blood Vessels; Electrophysiology; Genetic Diseases, Inborn; Heart Defects, Congenital; Humans; Phenotype; 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

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Cardiovascular System; Cell Differentiation; Embryo, Mammalian; Gene Expression Regulation, Developmental; Gestational Age; Heart Defects, Congenital; Heart Ventricles; Mice; Mice, Knockout; Morphogenesis; Myocardium; Neural Crest; Pericardium; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Growth Factor; Signal Transduction; Somites; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.

Topics: Amino Acid Sequence; Animals; Body Patterning; Heart; Heart Defects, Congenital; Intercellular Signaling Peptides and Proteins; Kupffer Cells; Molecular Sequence Data; Nodal Protein; Proteins; Signal Transduction; Transforming Growth Factor beta; Xenopus Proteins; Zebrafish; Zebrafish Proteins

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2(-/-) mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2(-/-) mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2(-/-) mice, and that such malformations may be the sole manifestation of a laterality defect.

Topics: Animals; Body Patterning; Crosses, Genetic; DNA-Binding Proteins; Gene Expression; Heart; Heart Defects, Congenital; Homeobox Protein PITX2; Homeodomain Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nodal Protein; Nuclear Proteins; Organogenesis; Repressor Proteins; Trans-Activators; Transcription Factor AP-2; Transcription Factors; Transforming Growth Factor beta

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.

Topics: Animals; Body Patterning; Cilia; DNA-Binding Proteins; Embryo, Mammalian; Forkhead Transcription Factors; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Homeobox Protein PITX2; Homeodomain Proteins; Left-Right Determination Factors; Mice; Mice, Knockout; Myocardium; Nodal Protein; Nuclear Proteins; Transcription Factors; Transforming Growth Factor beta

Axes formation is a fundamental process of early embryonic development. In addition to the anteroposterior and dorsoventral axes, the determination of the left-right axis is crucial for the proper morphogenesis of internal organs and is evolutionarily conserved in vertebrates. Genes known to be required for the normal establishment and/or maintenance of left-right asymmetry in vertebrates include, for example, components of the TGF-beta family of intercellular signalling molecules and genes required for node and midline function. We report that Notch signalling, which previously had not been implicated in this morphogenetic process, is required for normal left-right determination in mice. We show, that the loss-of-function of the delta 1 (Dll1) gene causes a situs ambiguous phenotype, including randomisation of the direction of heart looping and embryonic turning. The most probable cause for this left-right defect in Dll1 mutant embryos is a failure in the development of proper midline structures. These originate from the node, which is disrupted and deformed in Dll1 mutant embryos. Based on expression analysis in wild-type and mutant embryos, we suggest a model, in which Notch signalling is required for the proper differentiation of node cells and node morphology.

Topics: Abnormalities, Multiple; Animals; Body Patterning; Cell Differentiation; Embryonic and Fetal Development; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Homozygote; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; Mice, Mutant Strains; Notochord; Organizers, Embryonic; Random Allocation; Receptor, Notch1; Receptor, Notch2; Receptors, Cell Surface; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

We have identified and characterized a new zebrafish gene, southpaw, that is required for visceral and diencephalic left-right asymmetry. southpaw encodes a new member of the nodal-related class of proteins, a subfamily within the transforming growth factor beta superfamily of secreted factors. southpaw is expressed bilaterally in paraxial mesoderm precursors and then within the left lateral plate mesoderm. At late somite stages, left-sided southpaw expression transiently overlaps the left-sided expression domains of other genes that mark the developing heart, such as lefty2. We have injected morpholinos to block the translation of the southpaw mRNA or to block splicing of the southpaw pre-mRNA. These morpholinos cause a severe disruption of early (cardiac jogging) and late (cardiac looping) aspects of cardiac left-right asymmetry. As the left-right asymmetry of the pancreas is also affected, southpaw appears to regulate left-right asymmetry throughout a large part of the embryo. Consistent with the morphological changes, the left-sided expression domains of downstream genes (cyclops, pitx2, lefty1 and lefty2) are severely downregulated or abolished within the lateral plate mesoderm of Southpaw-deficient embryos. Surprisingly, despite the absence of southpaw expression in the brain, we find that early diencephalic left-right asymmetry also requires Southpaw activity. These observations lead to a model of how visceral organ and brain left-right asymmetry are coordinated during embryogenesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Body Patterning; Diencephalon; DNA, Complementary; Gene Expression Regulation, Developmental; Heart Defects, Congenital; In Situ Hybridization; Mesoderm; Models, Biological; Molecular Sequence Data; Mutation; Nodal Protein; Oligodeoxyribonucleotides, Antisense; Organizers, Embryonic; Pancreas; Sequence Homology, Amino Acid; Transforming Growth Factor beta; Zebrafish; Zebrafish Proteins

Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect.

Topics: Animals; Animals, Newborn; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Mice; Mice, Mutant Strains; Mice, Transgenic; Myocytes, Cardiac; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta2

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder because of mutations in the genes coding for endoglin (HHT1) or ALK-1 (HHT2). The disease is associated with haploinsufficiency and a murine model was obtained by engineering mice that express a single Endoglin allele. Of a total of 171 mice that were observed for 1 year, 50 developed clinical signs of HHT. Disease prevalence was high in 129/Ola strain (72%), intermediate in the intercrosses (36%), and low in C57BL/6 backcrosses (7%). Most mice first presented with an ear telangiectasia and/or recurrent external hemorrhage. One-third of mice with HHT showed severe vascular abnormalities such as dilated vessels, hemorrhages, liver and lung congestion, and/or brain and heart ischemia. Disease sequelae included stroke, hydrocephalus, fatal hemorrhage, and congestive heart failure. Thus the murine model reproduces the multiorgan manifestations of the human disease. Levels of circulating latent transforming growth factor (TGF)-beta1 were significantly lower in the 129/Ola than in the C57BL/6 strain. Intercrosses and 129/Ola mice expressing reduced endoglin also showed lower plasma TGF-beta1 levels than control. These data suggest that modifier genes involved in the regulation of TGF-beta1 expression act in combination with a single functional copy of endoglin in the development of HHT.

Topics: Abnormalities, Multiple; Animals; Antigens, CD; Blood Vessels; Brain; Cerebral Hemorrhage; Disease Models, Animal; Endoglin; Gastrointestinal Hemorrhage; Genes; Heart Defects, Congenital; Heart Failure; Hemorrhage; Heterozygote; Liver; Lung; Lung Diseases; Mice; Mice, Inbred C57BL; Receptors, Cell Surface; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Cell Adhesion Molecule-1

In vertebrates, specification of anteroposterior (A/P) and left-right (L/R) axes depends on TGFbeta-related signals, including Nodal, Lefty, and BMPs. Endoproteolytic maturation of these proteins is probably mediated by the proprotein convertase SPC1/Furin. In addition, precursor processing may be regulated by related activities such as SPC4 (also known as PACE4). Here, we show that a proportion of embryos lacking SPC4 develop situs ambiguus combined with left pulmonary isomerism or complex craniofacial malformations including cyclopia, or both. Gene expression analysis during early somite stages indicates that spc4 is genetically upstream of nodal, pitx2, lefty1, and lefty2 and perhaps maintains the balance between Nodal and BMP signaling in the lateral plate that is critical for L/R axis formation. Furthermore, genetic interactions between nodal and spc4, together with our analysis of chimeric embryos, strongly suggest that during A/P axis formation, SPC4 acts primarily in the foregut. These findings establish an important role for SPC4 in patterning the early mouse embryo.

Topics: Abnormalities, Multiple; Animals; Body Patterning; Bone Morphogenetic Proteins; Chromosome Mapping; Embryonic and Fetal Development; Gene Expression Regulation, Developmental; Heart Defects, Congenital; Mice; Mice, Knockout; Nodal Protein; Proprotein Convertases; Serine Endopeptidases; Signal Transduction; Transforming Growth Factor beta

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.

Topics: Activin Receptors, Type II; Animals; Avian Proteins; Body Patterning; DNA-Binding Proteins; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Heart; Heart Defects, Congenital; Hedgehog Proteins; Homeobox Protein PITX2; Homeodomain Proteins; Left-Right Determination Factors; Nodal Protein; Nuclear Proteins; Paired Box Transcription Factors; Proteins; Quail; Receptors, Growth Factor; Retinoids; Signal Transduction; Somites; Tissue Distribution; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Vitamin A; Vitamin A Deficiency

Placental development is profoundly influenced by oxygen (O(2)) tension. Human cytotrophoblasts proliferate in vitro under low O(2) conditions but differentiate at higher O(2) levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal-fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1alpha and ARNT subunits, activates many genes involved in the cellular and organismal response to O(2) deprivation. Analysis of Arnt(-/-) placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt(-/-) trophoblasts. Specifically, Arnt(-/-) placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O(2) levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt(-/-) animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt(-/-) embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Cell Division; Cell Hypoxia; Cells, Cultured; DNA-Binding Proteins; Endothelium, Vascular; Female; Heart; Heart Defects, Congenital; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Transgenic; Nuclear Proteins; Placenta; Polyploidy; Pregnancy; Receptors, Aryl Hydrocarbon; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta3; Trophoblasts; Yolk Sac

Determination of the left-right position (situs) of visceral organs involves lefty, nodal and Pitx2 genes that are specifically expressed on the left side of the embryo. We demonstrate that the expression of these genes is prevented by the addition of a retinoic acid receptor pan-antagonist to cultured headfold stage mouse embryos, whereas addition of excess retinoic acid leads to their symmetrical expression. Interestingly, both treatments lead to randomization of heart looping and to defects in heart anteroposterior patterning. A time course analysis indicates that only the newly formed mesoderm at the headfold-presomite stage is competent for these retinoid effects. We conclude that retinoic acid, the active derivative of vitamin A, is essential for heart situs determination and morphogenesis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Homeobox Protein Nkx-2.5; Homeobox Protein PITX2; Homeodomain Proteins; Left-Right Determination Factors; Mesoderm; Mice; Mice, Transgenic; Nodal Protein; Nuclear Proteins; Paired Box Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Xenopus Proteins

Topics: Activins; Animals; Congenital Abnormalities; Embryonic Induction; Heart; Heart Defects, Congenital; Hedgehog Proteins; Humans; Lung; Morphogenesis; Nodal Protein; Oligopeptides; Peptides; Proteins; Situs Inversus; Trans-Activators; Transforming Growth Factor beta; Twins, Conjoined

A molecular pathway leading to left-right asymmetry in the chick embryo has been described, in which FGF8 is a right determinant and Sonic Hedgehog a left determinant. Here evidence is presented that the Fgf8 and Sonic Hedgehog genes are required for left-right axis determination in the mouse embryo, but that they have different functions from those previously reported in the chick. In the mouse FGF8 is a left determinant and Sonic Hedgehog is required to prevent left determinants from being expressed on the right.

Topics: Animals; Body Patterning; Chick Embryo; Embryo, Mammalian; Embryonic and Fetal Development; Embryonic Induction; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Hedgehog Proteins; Homeobox Protein PITX2; Homeodomain Proteins; Left-Right Determination Factors; Lung; Mesoderm; Mice; Mutation; Nodal Protein; Nuclear Proteins; Paired Box Transcription Factors; Proteins; Recombinant Proteins; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

The rightward looping of the primary heart tube is dependent upon upstream patterning events that establish the vertebrate left-right axis. In Xenopus, a left-sided Vg1 signaling pathway has been implicated in instructing cells to adopt a 'left-sided identity'; however, it is not known whether 'right-sided identity' is acquired by a default pathway or by antagonism of Vg1 signaling. Here, we propose that an antagonistic, BMP/ALK2/Smad-mediated signaling pathway is active on the right side of the Xenopus embryo. Truncated ALK2 receptor expression on the right side of the blastula elicits heart reversals and altered nodal expression. Consistent with these findings, constitutively active ALK2 (CA-ALK2) receptor expression on the left side of the blastula also elicits heart reversals and altered nodal expression. Coexpression of CA-ALK2 with mature Vg1 ligand results in predominantly left-sided nodal expression patterns and normal heart looping, demonstrating that the ALK2 pathway can 'rescue' left-right reversals that otherwise occur following right-sided misexpression of mature Vg1 ligand alone. Results with chimeric precursor proteins indicate that the mature domain of BMP ligands can mimic the ability of the ALK2 signaling pathway to antagonize the Vg1 pathway. Consistent with the observed antagonism between BMP and Vg1 ligands, left-sided ectopic expression of Xolloid results in heart reversals. Moreover, ectopic expression of Smad1 or Smad7 identified two downstream modulators of the BMP/ALK2 signaling pathway that also can regulate cardiac orientation. Collectively, these results define a BMP/ALK2-mediated pathway on the right side of the Xenopus embryo and, moreover, suggest that left-right patterning preceding cardiac morphogenesis involves the activation of two distinct and antagonistic, left- and right-sided TGF(beta)-related signaling pathways.

Topics: Activin Receptors, Type I; Animals; Body Patterning; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; DNA-Binding Proteins; Embryo, Nonmammalian; Female; Gene Expression Regulation, Developmental; Glycoproteins; Heart; Heart Defects, Congenital; Metalloendopeptidases; Nodal Protein; Receptors, Growth Factor; Signal Transduction; Smad Proteins; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Vertebrates; Xenopus laevis; Xenopus Proteins

During vertebrate embryogenesis, a left-right axis is established. The heart, associated vessels and inner organs adopt asymmetric spatial arrangements and morphologies. Secreted growth factors of the TGF-beta family, including nodal, lefty-1 and lefty-2, play crucial roles in establishing left-right asymmetries [1] [2] [3]. In zebrafish, nodal signalling requires the presence of one-eyed pinhead (oep), a member of the EGF-CFC family of membrane-associated proteins [4]. We have generated a mutant allele of cryptic, a mouse EGF-CFC gene [5]. Homozygous cryptic mutants developed to birth, but the majority died during the first week of life because of complex cardiac malformations such as malpositioning of the great arteries, and atrial-ventricular septal defects. Moreover, laterality defects, including right isomerism of the lungs, right or left positioning of the stomach and splenic hypoplasia were observed. Nodal gene expression in the node was initiated in cryptic mutant mice, but neither nodal, lefty-2 nor Pitx2 were expressed in the left lateral plate mesoderm. The laterality defects observed in cryptic(-/-) mice resemble those of mice lacking the type IIB activin receptor or the homeobox-containing factor Pitx2 [6] [7] [8] [9], and are reminiscent of the human asplenic syndrome [10]. Our results provide genetic evidence for a role of cryptic in the signalling cascade that determines left-right asymmetry.

Topics: Alleles; Animals; Animals, Newborn; Dextrocardia; Embryonic and Fetal Development; Fetal Heart; Gene Expression Regulation, Developmental; Genotype; Growth Substances; Heart Defects, Congenital; Homeobox Protein PITX2; Homeodomain Proteins; Intercellular Signaling Peptides and Proteins; Left-Right Determination Factors; Mesoderm; Mice; Mice, Knockout; Morphogenesis; Nodal Protein; Nuclear Proteins; Paired Box Transcription Factors; Recombinant Fusion Proteins; Signal Transduction; Spleen; Syndrome; Transcription Factors; Transforming Growth Factor beta; Transposition of Great Vessels; Viscera; Zebrafish Proteins

FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.

Topics: Abnormalities, Multiple; Activins; Amino Acid Isomerases; Animals; Brain; Cardiomyopathy, Dilated; Carrier Proteins; DNA-Binding Proteins; Female; Fetal Death; Gene Deletion; Heart Defects, Congenital; Heart Septal Defects; Heat-Shock Proteins; Inhibins; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Tacrolimus Binding Proteins; Transforming Growth Factor beta

Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.

Topics: Animals; Body Patterning; DNA-Binding Proteins; Embryonic and Fetal Development; Fetal Proteins; Gene Expression Regulation, Developmental; Genes, Lethal; Heart Defects, Congenital; Hedgehog Proteins; Hepatocyte Nuclear Factor 3-beta; Left-Right Determination Factors; Mice; Mutation; Neural Tube Defects; Nodal Protein; Notochord; Nuclear Proteins; Proteins; Somites; T-Box Domain Proteins; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

The midline has a theoretical role in the development of left-right asymmetry, and this is supported by both genetic analyses and experimental manipulation of midline structures in vertebrates. The mouse brachyury (T) gene encodes a transcription factor which is expressed in the developing notochord and is required for its development. T/T mice lack a mature notochord and have a dorsalised neural tube. We have examined the hearts of T/T mice and have found consistent morphological abnormalities, resulting in ventrally displaced ventricular loops, and a 50% incidence of inverted heart situs. Three TGF-beta related genes, lefty-1, lefty-2 and nodal, are expressed asymmetrically in mouse embryos, and are implicated in the development of situs. We find that nodal, which is normally expressed around the node and in left lateral plate mesoderm in early somite embryos, is completely absent at this stage in T/T embryos. In contrast, lefty-1 and lefty-2, which are normally expressed in the left half of prospective floorplate and left lateral plate mesoderm, respectively, are both expressed in T/T embryos only in a broad patch of ventral cells in, and just rostral to, the node region. These results implicate the node as a source of instructive signals driving expression of nodal and lefty-2 in the left lateral plate mesoderm, and being required for normal looping and situs of the heart.

Topics: Animals; DNA-Binding Proteins; Embryonic and Fetal Development; Fetal Proteins; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Left-Right Determination Factors; Mice; Mice, Mutant Strains; Myocardium; Nodal Protein; T-Box Domain Proteins; Transcription Factors; Transforming Growth Factor beta

The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.

Topics: Abnormalities, Multiple; Animals; Bone and Bones; Cleft Palate; Craniofacial Abnormalities; Cyanosis; Ear, Inner; Embryonic Induction; Epithelium; Eye Abnormalities; Genes, Homeobox; Heart Defects, Congenital; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Transforming Growth Factor beta; Tretinoin; Urogenital Abnormalities

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Chick Embryo; Drug Implants; Heart Defects, Congenital; Microscopy, Electron, Scanning; Transforming Growth Factor beta

Maternal sources of transforming growth factor-beta 1 (TGF-beta 1) are shown here to contribute to the normal appearance and perinatal survival of TGF-beta 1 null newborn mice. Labeled TGF-beta 1 crossed the placenta and was recovered intact from various tissues after oral administration to mouse pups. TGF beta-1 protein was also detected in cells recovered from breast milk. In immunohistochemical analyses, TGF-beta 1 null embryos and null newborn pups born to TGF-beta 1 heterozygotes stained positive for TGF-beta 1, whereas those born to a null female were negative and had severe cardiac abnormalities. These results suggest an important role for maternal sources of TGF-beta 1 during development and, more generally, provide evidence for maternal rescue of targeted gene disruption in the fetus.

Topics: Animals; Animals, Newborn; Embryonic and Fetal Development; Female; Fetus; Heart Defects, Congenital; Heterozygote; Homozygote; Maternal-Fetal Exchange; Mice; Milk; Pregnancy; Transforming Growth Factor beta