The pH and ionic composition of the sub-embryonic fluid of the Japanese quail (Coturnix c. japonica)

The current theory of acid-base chemistry regards [H+] as a dependent variable: changes in pH of a fluid can only occur by alteration of strong ion concentrations ([Na+], [K+], [Cl-]). The objective of this study was to determine whether manipulation of sub-embryonic fluid strong ion composition would alter [H+] in the direction predicted by theory. Changes to fluid strong ion concentrations decreased pH in the way predicted and the changes in Cl- suggest a passive distribution. Also, changes in organic anions suggest an essential role for these in acid-base chemistry of this fluid. It was notable that both Na and HCO3- were unaffected by the treatments emphasising the importance of these two ions in fluid production by the quail blastoderm

The role of the co-receptor neuropilin-1 in human vascular smooth muscle cells.

Neuropilin-1 (NRP1) is a co-receptor required for neuronal and vascular development, which binds to class 3 semaphorins and VEGFs. NRP1 has been strongly implicated in VEGF-induced endothelial cell migration. VEGF has been shown to regulate vascular smooth muscle cell (VSMC) function in vitro. Evidence from mutant mice also suggests that NRP1 disruption in vivo can affect VSMC as well as endothelial function. I therefore investigated the role of NRPs in VSMC biological functions and more particularly in their migration. Western blotting showed that NRP1 and the related molecule, NRP2, were strongly expressed in human coronary artery SMC (HCASMC), whereas the major VEGF signalling receptor VEGFR2/KDR was not detectable. A high molecular weight NRP1-immunoreactive band (>250 kDa) was also strongly expressed in HCASMC, but was not detected either in cognate Human coronary artery endothelial cells (HCAEC) or in Human umbilical vein EC. The high molecular weight species was decreased significantly by treating the SMCs with chondroitinase, an enzyme that specifically chondroitin sulphate (CS) residues found in CS proteoglycan. Treatment with heparitinase, an enzymethat specifically heparan sulphate (HS) residues also resulted in a decrease of the highmolecular weight band but to a lesser extent than chondroitinase. Finally, treatment of SMC with both enzymes caused the complete disappearance of the high molecular weight species. Hence, in SMCs, in addition to the known NRP1 species at 130 kDa, NRP1 exists as a glycosaminoglycan containing either chondroitin sulphate or heparan sulphate polysaccharide chains. Mutational analysis of candidate O-linked glycosylation sites in the NRP1 extracellular domain showed that glycosylation occurred at serine 612. The importance of this gly-cosaminoglycan (GAG) modification was assessed by generating a construct of NRP1 lacking this GAG modification, called S612A. This was done by generation of an adenovirus NRP1 mutant with an alanine residue instead of the serine found in the wild-type species, however, the over-expression of the S612A NRP1 mutant in VSMC caused no signicant difference in PDGF-induced HCASMC migration. VEGF was able to bind significantly to ECs and SMCs but did not induce a significant migratory response of SMCs in contrast to PDGF-AA and PDGF-BB. PDGF-BB-induced HCASMC migration in transwell assays was inhibited by EG3287, a NRP1-specific antagonist, which blocks the ability of VEGF-A^1^6^5 to bind to NRP1. Furthermore, the migratory response to PDGF-BB was significantly decreased by siRNA-mediated knockdown of NRP1, NRP2 or a neuropilin interacting protein (NIP1 or synectin), and by pre-treatment with soluble NRP1 or NRP1 b1 domain (NRP1 VEGF binding domain). NRP1 knockdown also inhibited the migratory response to PDGF-AA. NRP1 was found to physically interact with PDGFR\alpha, but not with PDGFR\beta, as determined by co-immunoprecipitation. PDGFR\alpha, but not PDGFR\beta, phosphorylation was decreased in response to PDGF-AA and PDGF-BB when NRP1 was knocked down in HCASMC. Intracellular signalling in response to PDGF-BB stimulation was investigated in HCASMCs with NRP1 knockdown. PDGF-BB stimulated tyrosine phosphorylation of the adapter protein p130Cas, which has been stronlgy implicated in cellular and molecular processes involved in cell migration. NRP1 knockdown reduced p130Cas phosphorylation, but had little effect on signalling pathways, such as ERK1/2, Akt, cofilin, Hsp27 and FAK. To investigate the contribution of the NRP1 intracellular domain in PDGF-induced migration and signalling, I generated a NRP1 construct lacking the intracellular domain by introducing a stop codon after the transmembrane domain. Overexpression of NRP1 lacking its C-terminus in HCASMC resulted in a decrease of PDGF-induced migration and activation of phospho-p130Cas. Furthermore, p130Cas knockdown also inhibited PDGF- induced HCASMC migration, thus reinforcing the importance of p130Cas phosphorylation in NRP1-dependent cell migration. The findings that NRP1 is strongly expressed in HCASMC in a CS-GAG and a HS-GAG modified form and plays a role in the chemotactic response to PDGF-BB, highlight the possible involvement of NRPs in neotintima formation in vasculoproliferative diseases

Neuropilins 1 and 2 mediate neointimal hyperplasia and re-endothelialization following arterial injury

AIMS: Neuropilins 1 and 2 (NRP1 and NRP2) play crucial roles in endothelial cell migration contributing to angiogenesis and vascular development. Both NRPs are also expressed by cultured vascular smooth muscle cells (VSMCs) and are implicated in VSMC migration stimulated by PDGF-BB, but it is unknown whether NRPs are relevant for VSMC function in vivo. We investigated the role of NRPs in the rat carotid balloon injury model, in which endothelial denudation and arterial stretch induce neointimal hyperplasia involving VSMC migration and proliferation. METHODS AND RESULTS: NRP1 and NRP2 mRNAs and proteins increased significantly following arterial injury, and immunofluorescent staining revealed neointimal NRP expression. Down-regulation of NRP1 and NRP2 using shRNA significantly reduced neointimal hyperplasia following injury. Furthermore, inhibition of NRP1 by adenovirally overexpressing a loss-of-function NRP1 mutant lacking the cytoplasmic domain (ΔC) reduced neointimal hyperplasia, whereas wild-type (WT) NRP1 had no effect. NRP-targeted shRNAs impaired, while overexpression of NRP1 WT and NRP1 ΔC enhanced, arterial re-endothelialization 14 days after injury. Knockdown of either NRP1 or NRP2 inhibited PDGF-BB-induced rat VSMC migration, whereas knockdown of NRP2, but not NRP1, reduced proliferation of cultured rat VSMC and neointimal VSMC in vivo. NRP knockdown also reduced the phosphorylation of PDGFα and PDGFβ receptors in rat VSMC, which mediate VSMC migration and proliferation. CONCLUSION: NRP1 and NRP2 play important roles in the regulation of neointimal hyperplasia in vivo by modulating VSMC migration (via NRP1 and NRP2) and proliferation (via NRP2), independently of the role of NRPs in re-endothelialization

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

VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy- Terminal NRP1 Fragments That Regulate Angiogenic Signaling

OBJECTIVE: NRP1(neuropilin-1) acts as a coreceptor for VEGF (vascular endothelial growth factor) with an essential role in angiogenesis. Recent findings suggest that posttranslational proteolytic cleavage of VEGF receptors may be an important mechanism for regulating angiogenesis, but the role of NRP1 proteolysis and the NRP1 species generated by cleavage in endothelial cells is not known. To characterize NRP1 proteolytic cleavage in endothelial cells, determine the mechanism, and investigate the role of NRP1 cleavage in regulation of endothelial cell function. APPROACH AND RESULTS: NRP1 species comprising the carboxy (C)-terminal and transmembrane NRP1 domains but lacking the ligand-binding A and B regions are constitutively expressed in endothelial cells. Generation of these C-terminal domain NRP1 proteins is upregulated by phorbol ester and Ca2+ ionophore, and reduced by pharmacological inhibition of metalloproteinases, by small interfering RNA-mediated knockdown of 2 members of ADAM (a disintegrin and metalloproteinase) family, ADAMs 9 and 10, and by a specific ADAM10 inhibitor. Furthermore, VEGF upregulates expression of these NRP1 species in an ADAM9/10-dependent manner. Transduction of endothelial cells with adenoviral constructs expressing NRP1 C-terminal domain fragments inhibited VEGF-induced phosphorylation of VEGFR2 (VEGF receptor tyrosine kinase)/KDR and decreased VEGF-stimulated endothelial cell motility and angiogenesis in coculture and aortic ring sprouting assays. CONCLUSIONS: These findings identify novel NRP1 species in endothelial cells and demonstrate that regulation of NRP1 proteolysis via ADAMs 9 and 10 is a new regulatory pathway able to modulate VEGF angiogenic signaling

Microporous Biodegradable Films Promote Therapeutic Angiogenesis

Peripheral arterial disease and critical limb ischemia are common symptoms of cardiovascular disease. Vascular surgery is used to create a bypass around occluded blood vessels to improve blood flow to ischemic muscle, thus avoiding the need for amputation. Attempts to vascularize tissues by therapeutic angiogenesis using delivery of exogenous angiogenic agents are underwhelming. A material-based approach that provides an endogenous stimulus capable of promoting angiogenesis and increased tissue perfusion would provide a paradigm shift in treatment options available. It is reported here that microporous biodegradable films produced using thermally induced phase separation provide a localized biophysical stimulus of proangiogenic genes in vivo that is associated with increased blood vessel density and restoration of blood flow to ischemic tissue. These findings show, for the first time, that acellular, nonfunctionalized biodegradable biomaterials can provide an innovative, material-based approach for therapeutic angiogenesis to enhance tissue reperfusion in vivo

Neuropilin 1 mediates epicardial activation and revascularization in the regenerating zebrafish heart

Unlike adult mammals, zebrafish can regenerate their heart. A key mechanism for regeneration is the activation of the epicardium, leading to the establishment of a supporting scaffold for new cardiomyocytes, angiogenesis and cytokine secretion. Neuropilins are co-receptors mediating signaling of kinase receptors for cytokines known to play critical roles in zebrafish heart regeneration. We investigated the role of neuropilins in response to cardiac injury and heart regeneration. All four neuropilin isoforms nrp1a, nrp1b, nrp2a and nrp2b were upregulated by the activated epicardium and a nrp1a knockout mutant showed a significant delay in heart regeneration and displayed persistent collagen deposition. The regenerating hearts of nrp1a mutants were less vascularized and epicardial-derived cell migration and re-expression of the developmental gene wt1b was impaired. Moreover, cryoinjury-induced activation and migration of epicardial cells in heart explants was reduced in nrp1a mutant. These results identify a key role for Nrp1 in zebrafish heart regeneration, mediated through epicardial activation, migration and revascularization

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

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

Endosome-to-plasma membrane recycling of VEGFR2 receptor tyrosine kinase regulates endothelial function and blood vessel formation.

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis