The zebrafish cardiac endothelial cell – roles in development and regeneration

In zebrafish, the spatiotemporal development of the vascular system is well described due to its stereotypical nature. However, the cellular and molecular mechanisms orchestrating post embryonic vascular development, the maintenance of vascular homeostasis or how coronary vessels integrate into the growing heart are less well studied. In the context of cardiac regeneration, if the central cellular mechanism by which the heart regenerates a fully functional myocardium relies on the proliferation of pre-existing cardiomyocytes, the epicardium and the endocardium are also known to play key roles in the regenerative process. Remarkably, revascularisation of the injured tissue occurs within a few hours after cardiac damage, thus generating a vascular network acting as a scaffold for the regenerating myocardium. The activation of the endocardium leads to the secretion of cytokines, further supporting the proliferation of the cardiomyocytes.Although epicardium, endocardium and myocardium interact with each other to orchestrate heart development and regeneration, in this review, we will be focussing on recent advancements in the understanding of the development of the endocardium and the coronary vasculature in zebrafish as well as their pivotal roles in the heart regeneration process

Smooth muscle cell-specific knockout of neuropilin-1 impairs postnatal lung development and pathological vascular smooth muscle cell accumulation

Neuropilin 1 (NRP1) is important for neuronal and cardiovascular development due to its role in conveying class 3 semaphorin and vascular endothelial growth factor signaling, respectively. NRP1 is expressed in smooth muscle cells (SMCs) and mediates their migration and proliferation in cell culture and is implicated in pathological SMC remodeling in vivo. To address the importance of Nrp1 for SMC function during development, we generated conditional inducible Nrp1 SMC-specific knockout mice. Induction of early postnatal SMC-specific Nrp1 knockout led to pulmonary hemorrhage associated with defects in alveogenesis and revealed a specific requirement for Nrp1 in myofibroblast recruitment to the alveolar septae and PDGF-AA-induced migration in vitro. Furthermore, SMC-specific Nrp1 knockout inhibited PDGF-BB-stimulated SMC outgrowth ex vivo in aortic ring assays and reduced pathological arterial neointima formation in vivo. In contrast, we observed little significant effect of SMC-specific Nrp1 knockout on neonatal retinal vascularization. Our results point to a requirement of Nrp1 in vascular smooth muscle and myofibroblast function in vivo, which may have relevance for postnatal lung development and for pathologies characterized by excessive SMC and/or myofibroblast proliferation

VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine, the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser(612). HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1 Delta C). NRP1 co-immunoprecipitated with PDGFR alpha (PDGF receptor alpha), and immunofluorescent staining indicated that NRP1 and PDGFR alpha co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFR alpha activation. NRP1-specific siRNA, Ad.NRP1 Delta C and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130(Cas) (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130(Cas) knockdown inhibited HCASMC migration. Chemotaxis and p130(Cas) phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130(Cas) phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFR alpha and via selective mobilization of a novel p130(Cas) tyrosine phosphorylation pathway

The Zebrafish Cardiac Endothelial Cell—Roles in Development and Regeneration

In zebrafish, the spatiotemporal development of the vascular system is well described due to its stereotypical nature. However, the cellular and molecular mechanisms orchestrating post-embryonic vascular development, the maintenance of vascular homeostasis, or how coronary vessels integrate into the growing heart are less well studied. In the context of cardiac regeneration, the central cellular mechanism by which the heart regenerates a fully functional myocardium relies on the proliferation of pre-existing cardiomyocytes; the epicardium and the endocardium are also known to play key roles in the regenerative process. Remarkably, revascularisation of the injured tissue occurs within a few hours after cardiac damage, thus generating a vascular network acting as a scaffold for the regenerating myocardium. The activation of the endocardium leads to the secretion of cytokines, further supporting the proliferation of the cardiomyocytes. Although epicardium, endocardium, and myocardium interact with each other to orchestrate heart development and regeneration, in this review, we focus on recent advances in the understanding of the development of the endocardium and the coronary vasculature in zebrafish as well as their pivotal roles in the heart regeneration process

The Zebrafish Cardiac Endothelial Cell—Roles in Development and Regeneration

In zebrafish, the spatiotemporal development of the vascular system is well described due to its stereotypical nature. However, the cellular and molecular mechanisms orchestrating post-embryonic vascular development, the maintenance of vascular homeostasis, or how coronary vessels integrate into the growing heart are less well studied. In the context of cardiac regeneration, the central cellular mechanism by which the heart regenerates a fully functional myocardium relies on the proliferation of pre-existing cardiomyocytes; the epicardium and the endocardium are also known to play key roles in the regenerative process. Remarkably, revascularisation of the injured tissue occurs within a few hours after cardiac damage, thus generating a vascular network acting as a scaffold for the regenerating myocardium. The activation of the endocardium leads to the secretion of cytokines, further supporting the proliferation of the cardiomyocytes. Although epicardium, endocardium, and myocardium interact with each other to orchestrate heart development and regeneration, in this review, we focus on recent advances in the understanding of the development of the endocardium and the coronary vasculature in zebrafish as well as their pivotal roles in the heart regeneration process.</jats:p

Emerging Roles for Neuropilin-2 in Cardiovascular Disease

Cardiovascular disease, the leading cause of death worldwide, is predominantly associated with atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterised by the narrowing of large to medium-sized arteries due to a build-up of plaque. Atherosclerotic plaque is comprised of lipids, extracellular matrix, and several cell types, including endothelial, immune, and vascular smooth muscle cells. Such narrowing of the blood vessels can itself restrict blood flow to vital organs but most severe clinical complications, including heart attacks and strokes, occur when lesions rupture, triggering the blood to clot and obstructing blood flow further down the vascular tree. To circumvent such obstructions, percutaneous coronary intervention or bypass grafts are often required; however, re-occlusion of the treated artery frequently occurs. Neuropilins (NRPs), a multifunctional family of cell surface co-receptors, are expressed by endothelial, immune, and vascular smooth muscle cells and are regulators of numerous signalling pathways within the vasculature. Here, we review recent studies implicating NRP2 in the development of occlusive vascular diseases and discuss how NRP2 could be targeted for therapeutic intervention

Emerging Roles for Neuropilin-2 in Cardiovascular Disease

Cardiovascular disease, the leading cause of death worldwide, is predominantly associated with atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterised by the narrowing of large to medium-sized arteries due to a build-up of plaque. Atherosclerotic plaque is comprised of lipids, extracellular matrix, and several cell types, including endothelial, immune, and vascular smooth muscle cells. Such narrowing of the blood vessels can itself restrict blood flow to vital organs but most severe clinical complications, including heart attacks and strokes, occur when lesions rupture, triggering the blood to clot and obstructing blood flow further down the vascular tree. To circumvent such obstructions, percutaneous coronary intervention or bypass grafts are often required; however, re-occlusion of the treated artery frequently occurs. Neuropilins (NRPs), a multifunctional family of cell surface co-receptors, are expressed by endothelial, immune, and vascular smooth muscle cells and are regulators of numerous signalling pathways within the vasculature. Here, we review recent studies implicating NRP2 in the development of occlusive vascular diseases and discuss how NRP2 could be targeted for therapeutic intervention.</jats:p