Coronary vessel development is dependent on the type III transforming growth factor beta receptor

Abstract-Transforming growth factor (TGF)␤ receptor III (TGF␤R3), or ␤-glycan, binds all 3 TGF␤ ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGF␤R1, TGF␤R2). TGF␤R3 facilitates signaling via TGF␤R1/TGF␤R2 but also has been suggested to play a unique and nonredundant role in TGF␤ signaling. Targeted deletion of Tgfbr3 revealed a requirement for Tgfbr3 during development of the coronary vessels. Coronary vasculogenesis is significantly impaired in null mice, with few vessels evident and numerous, persistent blood islands found throughout the epicardium. Tgfbr3-null mice die at embryonic day 14.5, the time when functional coronary vasculature is required for embryo viability. However, in null mice nascent coronary vessels attach to the aorta, form 2 coronary ostia, and initiate smooth muscle recruitment by embryonic day 14. Analysis of earlier developmental stages revealed defects in the epicardium. At embryonic day 13.5, these defects include an irregular and hypercellular epicardium with abundant subepicardial mesenchyme and a thin compact zone myocardium. Tgfbr3-null mice also displayed other defects in coronary development, including dysmorphic and distended vessels along the atrioventricular groove and subepicardial hemorrhage. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild-type littermates. These data demonstrate a requirement for Tgfbr3 during coronary vessel development that is essential for embryonic viability. (Circ Res. 2007;101:000-000.) Key Words: coronary vessels Ⅲ transforming growth factor ␤ receptor Ⅲ mice, null C oronary artery disease is responsible for 54% of all cardiovascular disease in the United States. 1 Coronary vessels have a unique derivation from mesothelial cells that form a transitory structure termed the proepicardium. Proepicardial cells are transferred to the heart, form the epicardium, and give rise to endothelial cells, smooth muscle cells, and cardiac fibroblasts (reviewed elsewhere 2,3 ). Endothelial cells derived from the epicardium form a vascular plexus by the process of vasculogenesis. This vascular network attaches to the aorta and recruits epicardially derived mesenchyme to become vascular smooth muscle. The identification of the molecular and cellular processes that regulate coronary vessel development may provide insight into coronary vessel disease and reveal novel therapeutic opportunities. The transforming growth factor (TGF)␤ family of growth factors regulates cell growth and differentiation in the cardiovascular system during both development and disease. 4 -6 Three ligands, TGF␤1, TGF␤2, and TGF␤3, 7-9 bind 4 cell surface proteins. These include two transmembrane serine/ threonine kinase receptors, the type I TGF␤ receptor (TGF␤R1) and the type II TGF␤ receptor (TGF␤R2). 10 -12 Several type I receptors, termed activin receptor-like kinases (ALKs), have been described. TGF␤R2 has a constitutively active cytoplasmic kinase domain and an extracellular domain that binds TGF␤1 and TGF␤3 with high affinity. Materials and Methods Generation of Null Mice A targeting vector was made to delete exon 3 that encodes the N terminus, including a portion of the extracellular ligand binding domain. Construction and validation of the targeting vector is described in the online data supplement at http://circres.ahajournals.org (see supplemental Histology, Whole-Mount Immunohistochemistry, and ␤-Galactosidase Staining Detailed methodology is described in the online data supplement. Results Deletion of Tgfbr3 Results in Embryonic Lethality Exon 3 was targeted as depicted in the online data supplement (supplemental Tgfbr3-Null Mice Display Defects in Coronary Vasculogenesis Because embryonic death coincides temporally with the known dependency of embryo viability on the formation of the coronary circulation, we examined null embryos for the presence of coronary vessels. Whole-heart immunostaining for the vascular endothelial cell marker platelet endothelial cell adhesion molecule (PECAM) (also known as CD31) at E14.0 revealed dramatically decreased immunoreactivity in nulls Percentage of Observed Defects in Embryos Tgfbr3-Null Mice Have Abnormal Epicardium and Dysmorphic Coronary Vessels The epicardium is derived from the proepicardium and contains coronary vessel precursor cells. At E13.5, the epicardium forms a tightly apposed monolayer on the surface on the atria and ventricles. In the region of the atrioventricular groove, the epicardium is separated from the myocardium by a layer of epicardial-derived mesenchyme ( Tgfbr3-Null Mice Initiate Recruitment of Smooth Muscle to Extracardiac and Coronary Vessels TGF␤ has multiple roles during blood vessel formation, including vascular smooth muscle cell recruitment. After right and left coronary arteries attach to the systemic circulation at the aorta, smooth muscle recruitment commences at the coronary ostia and progresses distally. As noted in Discussion Disruption of Tgfbr3 revealed a requirement for coronary vessel development and embryonic viability. Null embryos die at E14.5 with defects in coronary vessel development, whereas vasculature outside of the coronary circulation appears normal. Although greatly reduced in size, nascent coronary vessels attach to the aorta and initiate smooth muscle recruitment. Coronary vessel defects are coincident with epicardial abnormalities that include increased space between the epicardium and myocardium, abundant subepi- cardial mesenchyme, and persistent blood islands. In addition to coronary vessel anomalies, null embryos have OFT abnormalities and myocardial thinning. Presumably, the greatly reduced coronary vasculature in null embryos is not sufficient to adequately perfuse the heart resulting in embryonic death. Despite the abundance of data implicating TGF␤ signaling in vasculogenesis and angiogenesis, these processes appear to occur normally outside of the coronary vessels in nulls. The localization of defects to the coronary vessels may be explained by the unique derivation of these vessels (reviewed elsewhere 2,3 ). The proepicardium, adjacent to the liver rudiment, is transferred to the heart and gives rise to the epicardium as well as coronary endothelial cells, smooth muscle cells, and cardiac fibroblasts. Although targeted gene deletion in mice has uncovered roles for several molecules in coronary vessel development, none has a phenotype similar to Tgfbr3 nulls. Deletion of vascular cell adhesion molecule-1 40 or the counter receptor ␣4 integrin Defects in coronary vasculogenesis in Tgfbr3 nulls do not appear to result from defects in angioblast or endothelial differentiation because PECAM-positive cells do appear. A failure in coronary vasculogenesis could result from an insufficient population of angioblasts or endothelial cells, a possibility we cannot exclude because the numbers of these cells was not quantitated. At E14.5, we saw fewer vessels on the heart and a complete absence of large vessels formed by remodeling of the primary vascular plexus. This observation suggests a defect in angioblast or endothelial cell assembly or remodeling. TGF␤R3 contains a cytoplasmic PDZ domain that has been shown to bind glycoinositolphosphorylceramide or synectin

NF-κB Mediates FGF Signal Regulation of msx-1 Expression

AbstractThe nuclear factor-κB (NF-κB) family of transcription factors is involved in proliferation, differentiation, and apoptosis in a stage- and cell-dependent manner. Recent evidence has shown that NF-κB activity is necessary for both chicken and mouse limb development. We report here that the NF-κB family member c-rel and the homeodomain gene msx-1 have partially overlapping expression patterns in the developing chick limb. In addition, inhibition of NF-κB activity resulted in a decrease in msx-1 mRNA expression. Sequence analysis of the msx-1 promoter revealed three potential κB-binding sites similar to the interferon-γ (IFN-γ) κB-binding site. These sites bound to c-Rel, as shown by electrophoretic mobility shift assay (EMSA). Furthermore, inhibition of NF-κB activity significantly reduced transactivation of the msx-1 promoter in response to FGF-2/-4, known stimulators of msx-1 expression. These results suggest that NF-κB mediates the FGF-2/-4 signal regulation of msx-1 gene expression

Training the next generation of physician researchers – Vanderbilt Medical Scholars Program

Abstract Background As highlighted in recent reports published by the Physician-Scientist Workforce Working Group at the National Institutes of Health, the percentage of physicians conducting research has declined over the past decade. Various programs have been put in place to support and develop current medical student interest in research to alleviate this shortage, including The Vanderbilt University School of Medicine Medical Scholars Program (MSP). This report outlines the long-term program goals and short-term outcomes on career development of MSP alumni, to shed light on the effectiveness of research training programs during undergraduate medical training to inform similar programs in the United States. Methods MSP alumni were asked to complete an extensive survey assessing demographics, accomplishments, career progress, future career plans, and MSP program evaluation. Results Fifty-five (81%) MSP alumni responded, among whom 12 had completed all clinical training. The demographics of MSP alumni survey respondents are similar to those of all Vanderbilt medical students and medical students at all other Association of American Medical College (AAMC) medical schools. MSP alumni published a mean of 1.9 peer-reviewed manuscripts (95% CI:1.2, 2.5), and 51% presented at national meetings. Fifty-eight percent of respondents reported that MSP participation either changed their career goals or helped to confirm or refine their career goals. Conclusions Results suggest that the MSP program both prepares students for careers in academic medicine and influences their career choices at an early juncture in their training. A longer follow-up period is needed to fully evaluate the long-term outcomes of some participants

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function

Hypoxia Supports Epicardial Cell Differentiation in Vascular Smooth Muscle Cells through the Activation of the TGFβ Pathway

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). Results: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. Conclusion: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals

Training the next generation of physician researchers – Vanderbilt Medical Scholars Program

Abstract Background As highlighted in recent reports published by the Physician-Scientist Workforce Working Group at the National Institutes of Health, the percentage of physicians conducting research has declined over the past decade. Various programs have been put in place to support and develop current medical student interest in research to alleviate this shortage, including The Vanderbilt University School of Medicine Medical Scholars Program (MSP). This report outlines the long-term program goals and short-term outcomes on career development of MSP alumni, to shed light on the effectiveness of research training programs during undergraduate medical training to inform similar programs in the United States. Methods MSP alumni were asked to complete an extensive survey assessing demographics, accomplishments, career progress, future career plans, and MSP program evaluation. Results Fifty-five (81%) MSP alumni responded, among whom 12 had completed all clinical training. The demographics of MSP alumni survey respondents are similar to those of all Vanderbilt medical students and medical students at all other Association of American Medical College (AAMC) medical schools. MSP alumni published a mean of 1.9 peer-reviewed manuscripts (95% CI:1.2, 2.5), and 51% presented at national meetings. Fifty-eight percent of respondents reported that MSP participation either changed their career goals or helped to confirm or refine their career goals. Conclusions Results suggest that the MSP program both prepares students for careers in academic medicine and influences their career choices at an early juncture in their training. A longer follow-up period is needed to fully evaluate the long-term outcomes of some participants