Alternagin-C, a Disintegrin-like Protein, Induces Vascular Endothelial Cell Growth Factor (VEGF) Expression and Endothelial Cell Proliferation in Vitro

Abstract

Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, interacts with the major collagen I receptor, the ␣ 2 ␤ 1 integrin, inhibiting collagen binding. Here we show that ALT-C also inhibits the adhesion of a mouse fibroblast cell line (NIH-3T3) to collagen I (IC 50 2.2 M). In addition, when immobilized on plate wells, ALT-C supports the adhesion of this cell line as well as of human vein endothelial cell (HUVEC). ALT-C (3 M) does not detach cells that were previously bound to collagen I. ALT-C (5 nM) induces HUVEC proliferation in vitro, and it inhibits the positive effect of vascular endothelial growth factor (VEGF) or FGF-2 on the proliferation of these cells, thus suggesting a common mechanism for these proteins. Gene expression analysis of human fibroblasts growing on ALT-C-or collagen-coated plates showed that ALT-C and collagen I induce a very similar pattern of gene expression. When compared with cells growing on plastic only, ALT-C up-regulates the expression of 45 genes including the VEGF gene and downregulates the expression of 30 genes. Fibroblast VEGF expression was confirmed by RT-PCR and ELISA assay. Up-regulation of the VEGF gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates Akt/PKB phosphorylation, a signaling event involved in endothelial survival and angiogenesis. In conclusion, ALT-C acts as a survival factor, promoting adhesion and endothelial cell proliferation.Cell attachment to the extracellular matrix depends mostly on the integrins, a large family of glycoproteins expressed at the cell surface (1). Integrins are heterodimers formed of noncovalently associated ␣-and ␤-subunits (2). In many cells in culture, integrin-mediated adhesion results in specialized adhesion sites, named focal contacts (3). In these sites, structural and signaling proteins such as integrins, cytoskeletal proteins, and kinases are concentrated and initiate signal transduction pathways (4). Aggregation of integrin receptors, ligand occupancy, and tyrosine kinase-mediated phosphorylation are the key events that results in diverse processes such as cell migration and differentiation, tissue remodeling, cell proliferation, angiogenesis, and tumor cell invasion and metastasis (1, 5).Antagonists of integrins have been developed in order to provide powerful therapeutic approaches for the treatment of several types of cancer, such as antibodies to the ␣ v integrin (6). Synthetic peptides with the sequence Arg-Gly-Asp (RGD) can competitively block the binding of several integrins to their ligands and efficiently reduce platelet aggregation and the number of experimental metastasis (7). RGD peptides induce the disassembly of focal contacts in melanoma cells and disrupt the actin cytoskeleton (8). Disintegrins are small peptides derived from viperidae snake venoms with an internal RGD or KGD motif (9). Disintegrins can bind to integrins and interfere with integrin function. In platelets, disintegrins...

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

“…ALT-C was reported to induce in vitro VEGF expression by fibroblasts, but not by HUVEC (18). However, in the present study VEGF expression was not detected either at mRNA or protein level, suggesting that this effect may be cell-specific.…”

“…These peptides represent a family of cysteine-rich proteins, isolated from snake venoms, and are known to inhibit cell-to-matrix and cell-to-cell interactions mediated by integrins (2,17) . Most disintegrins contain a RGD/KGD sequence within a hairpin loop maintained by disulfide bonds and are very potent inhibitors of αIIbβ3 integrindependent platelet aggregation as well as cell-to-matrix interactions involved in tumor cell metastasis and angiogenesis (2,18,19) .…”

“…ALT-C induces integrin-mediated signaling and chemotaxis in neutrophils and also strongly leads to human vein endothelial cells (HUVEC) proliferation in vitro by up-regulating the expression of some growth factors, including vascular endothelial growth factor (VEGF) (2,18,20,21) .…”

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

“…ALT-C also inhibited the adhesion of mouse fibroblasts to collagen I (20) and several tumor cell lines such as human cervix epithelioid carcinoma (HeLa), human bladder epithelioid carcinoma (ECV-304/ T24), and the estrogen-independent breast human carcinoma (MDA-MB-231) (Terruggi CHB, Bérard M, Crépin M and Selistrede-Araujo HS, unpublished data). These results suggest that the α 2 ß 1 integrin is one of the major collagen receptors in these cells.…”

Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

“…b 1 integrin can pair with different a-subunits to form two principal receptors for type I collagen, namely a 2 b 1 and a 1 b 1 integrin, and a 2 b 1 integrin is a functional cellular receptor for type I collagen (Jokinen et al 2004). Results of numerous studies have demonstrated that a 2 b 1 integrin is involved in the adhesion of mouse fibroblasts to type I collagen (Cominetti et al 2004) and a 2 b 1 integrin rather than a 1 b 1 integrin mediates cell adhesion to unhydroxylated type I collagen (Perret et al 2003), indicating that the interaction between cells and unhydroxylated type I collagen is a 2 b 1 -specific. In mesenchymal stem cells, it has been suggested that high levels of type I collagen are required to maintain the cells in an undifferentiated state .…”