The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant proteins present in Bothrops snake venoms and possess multiple biological functions including the regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Molecular size exclusion chromatography, affinity chromatography followed by ion exchange chromatography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrinogenolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of 2.4 μg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Akt and eNOS phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications.

Background: Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia . In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo .