Pellet-Many, C.A.P.; (2010) The role of the co-receptor neuropilin-1 in human vascular smooth muscle cells. Doctoral thesis , UCL (University College London).

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Abstract

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.

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