An essential role for connexin43 gap junctions in mouse coronary artery development

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.     

Avian coronary endothelium is a mosaic of sinus venosus‐ and ventricle‐derived endothelial cells in a region‐specific manner

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein‐Tol2 system and fluorescent dye labeling, four types of quail‐chick chimeras were made and quail‐specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium‐derived inner cells and the other was surface mesothelium‐derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region‐specific manner, and the sources of ECs were basically the same as those observed in mice.     

bves: A novel gene expressed during coronary blood vessel development

We have used a subtractive method to clone novel messages enriched in the heart. Here we show that one such message, bves (blood vessel/epicardial substance) is a novel protein that is highly conserved between chicken and mouse. The bves message is detected at high levels in early chick hearts. Using anti-Bves antibodies, we show expression in cells of the proepicardial organ, migrating epicardium, epicardial-derived mesenchyme, and smooth muscle of the developing intracardiac arterial system, including the coronary arteries. Our data suggest that Bves is an early marker of developing vascular smooth muscle cells. In addition, the expression pattern of Bves protein reveals the patterning of intracardiac vascular smooth muscle and possible insights into the cellular regulation of smooth muscle differentiation during vasculogenesis.     

Induction of proepicardial marker gene expression by the liver bud

Cells of the coronary vessels arise from a unique extracardiac mesothelial cell population, the proepicardium, which develops posterior to the sinoatrial region of the looping-stage heart. Although contribution of the proepicardial cells to cardiac development has been studied extensively, it remains unresolved how the proepicardium is induced and specified in the mesoderm during embryogenesis. It is known, however, that the proepicardium develops from the mesothelium that overlays the liver bud. Here, we show that the expression of proepicardial marker genes - Wt1, capsulin (epicardin, pod1, Tcf21) and Tbx18, can be induced in na?ve mesothelial cells by the liver bud, both in vitro and in vivo. Lateral embryonic explants, when co-cultured with the liver bud, were induced to express these proepicardial marker genes. The same induction of the marker genes was detected in vivo when a quail liver bud was implanted in the posterior-lateral regions of a chick embryo. This ectopic induction of marker gene expression was not evident when other endodermal tissues, such as the lung bud or stomach, were implanted. This inductive response to the liver bud was not detectable in host embryos before stage 12 (16-somite stage). These results suggest that, after a specific developmental stage, a large area of the mesothelium becomes competent to express proepicardial marker genes in response to localized liver-derived signal(s). The developmentally regulated competency of mesothelium and a localized inductive signal might play a role in restricting the induction of the proepicardial marker gene expression to a specific region of the mesothelium. The data might also provide a foundation for future engineering of a coronary vascular progenitor population.     

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.     

Semaphorin3D regulates invasion of cardiac neural crest cells into the primary heart field

The primary heart field in all vertebrates is thought to be derived exclusively from lateral plate mesoderm (LPM), which gives rise to a cardiac tube shortly after gastrulation. The heart tube then begins looping and additional cells are added from other embryonic regions, including the secondary heart field, cardiac neural crest and the proepicardial organ. Here we show in zebrafish that neural crest cells invade and contribute cardiac myosin light chain2 (cmlc2)-positive cardiomyocytes to the primary heart field. Knockdown of semaphorin3D, which is expressed in the neural crest but apparently not in LPM, reduces the size of the primary heart field and the number of cardiomyocytes in the primary heart field by 20% before formation of the primary heart tube. Sema3D morphants have subsequent complex congenital heart defects, including hypertrophic cardiomyocytes, decreased ventricular size and defects in trabeculation and in atrioventricular (AV) valve development. Neuropilin1A, a semaphorin receptor, is expressed in LPM but apparently not in the neural crest, and nrp1A morphants have cardiac development defects. We propose that a population of sema3D-dependent neural crest cells follow a novel migratory pathway, perhaps toward nrp1A-expressing LPM, and serve as an important early source of cardiomyocytes in the primary heart field.     

The neuroimmune semaphorin-3A reduces inflammation and progression of experimental autoimmune arthritis

Semaphorin-3A (Sema3A), a member of a large family of conserved proteins originally implicated in axon guidance, is expressed by activated T cells and downmodulates T cell activation in vitro. This study examined the effect and mechanism of action of Sema3A overexpression in a mouse model of collagen-induced arthritis. Prophylactic i.p. administration of plasmid DNA encoding Sema3A markedly reduced the incidence, disease severity, and articular inflammation compared with control plasmid without insert. Treatment of Sema3A reduced anticollagen IgG levels and suppressed collagen-specific proinflammatory cytokine (IFN-γ and IL-17) release, but increased IL-10 concentration in the serum. In line with results in arthritic mice, Sema3A expression is defective in CD4(+) T cells derived from patients with rheumatoid arthritis. In contrast, increased expression of the Sema3A receptor neuropilin-1 (NP-1) is detected in the same cells. The CD4(+)NP-1(+) T cells are a T cell subset involved in the control of the immune responses. They express greater amounts of IL-10 and show suppressive activities on autologous CD4(+) T cells. Sema3A acted directly on CD4(+)NP-1(+) T cells, because it could increase IL-10 production and influence the regulatory function on CD4(+) T cell growth. Therefore, I propose that Sema3A increases the CD4(+)NP-1(+) T cell ability to suppress alloresponses, that its transient expression is altered in rheumatoid inflammation, and that reintroduction of Sema3A is sufficient to attenuate collagen-induced arthritis, supporting its therapeutic potential in the treatment of autoimmune disorders.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages

The axon guidance cues semaphorins (Semas) and their receptors plexins have been shown to regulate both physiological and pathological angiogenesis. Sema4A plays an important role in the immune system by inducing T cell activation, but to date, the role of Sema4A in regulating the function of macrophages during the angiogenic and inflammatory processes remains unclear. In this study, we show that macrophage activation by TLR ligands LPS and polyinosinic-polycytidylic acid induced a time-dependent increase of Sema4A and its receptors PlexinB2 and PlexinD1. Moreover, in a thioglycollate-induced peritonitis mouse model, Sema4A was detected in circulating Ly6C(high) inflammatory monocytes and peritoneal macrophages. Acting via PlexinD1, exogenous Sema4A strongly increased macrophage migration. Of note, Sema4A-activated PlexinD1 enhanced the expression of vascular endothelial growth factor-A, but not of inflammatory chemokines. Sema4A-stimulated macrophages were able to activate vascular endothelial growth factor receptor-2 and the PI3K/serine/threonine kinase Akt pathway in endothelial cells and to sustain their migration and in vivo angiogenesis. Remarkably, in an in vivo cardiac ischemia/reperfusion mouse model, Sema4A was highly expressed in macrophages recruited at the injured area. We conclude that Sema4A activates a specialized and restricted genetic program in macrophages able to sustain angiogenesis and participates in their recruitment and activation in inflammatory injuries.     

BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoderm.     

Thyroid hormone receptor-α and vascular function

Thyroid hormone (TH) treatment exerts beneficial effects on the cardiovascular system: it lowers cholesterol and LDL levels and enhances cardiac contractile function. However, little is known about the effect of TH on vascular function or the functional role of TH receptors (TRs) in the regulation of vascular tone. We have investigated the contribution of TRs to vascular contractility in the heart. Among different TR subtype-specific knockout (KO) mice, vascular contraction was significantly enhanced in coronary arteries isolated from TRα KO compared with wild-type mice, while chronic TH treatment significantly attenuated coronary vascular contraction. We found that TRα is the predominant TR in mouse coronary smooth muscle cells (SMCs). Coronary SMCs isolated from TRα KO mice exhibited a significant decrease in K(+) channel activity, whereas TH treatment increased K(+) channel activity in a dose-dependent manner. These data suggest that TRα in SMCs has prominent effects on regulation of vascular tone and TH treatment helps decrease coronary vascular tone by increasing K(+) channel activity through TRα in SMCs.     

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.     

Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.     

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.     

Plexin-D1/Semaphorin 3E pathway may contribute to dysregulation of vascular tone control and defective angiogenesis in systemic sclerosis

IntroductionThe vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis.MethodsSema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynaud''s phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides.ResultsSerum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs.ConclusionsOur findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.     

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.     

A 3-D model of coronary vessel development

Coronary vascular disease is one of the leading causes of mortality and morbidity in the United States. Therefore, a mechanistic understanding of coronary vessel morphogenesis would aid in the innovation of new therapies targeting vascular disorders. Moreover, a functionally equivalent in vitro model system allows for the delineation of the molecular mechanisms that regulate coronary vessel development. In this study, we present a novel in vitro model system. This three-dimensional (3-D) model system consists of a tubular scaffold, which is engineered from type-I collagen and has been optimized to support the growth of embryonic cardiac tissues. In this report, proepicardial (PE) cells, the developmental precursors of coronary vessels, have been isolated from several model species and cultured on this scaffold. In this model system, the PE cells were able to recapitulate several aspects of coronary vessel morphogenesis including epicardial formation, the epicardial to mesenchymal transformation, and de novo coronary vessel development or vasculogenesis. The differentiation of PE cells was characterized using a variety of specific protein markers. The potential uses of this novel coronary developmental model are discussed.     

Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.