Snake venom metalloproteinases: action in endothelial cells.

Fritzen, M.¹; Modesto, J.C.A.¹; Ventura, J.S.¹; Moura da Silva, A.M.²; Selistre de Araújo, H.S.³; Chudzinski-Tavassi, A.M.¹

1 Laboratório de Bioquímica e Biofísica; 2 Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP; 3 Dep. Ciências Fisiológicas da Universidade Federal de São Carlos, SP, Brazil.

In this study we have compared the activity on human umbilical vein endothelial cells (HUVECs) of six snake venom metalloproteinases (SVMPs): four PI (P141 from Bothrops insularis, B151 and RQ2 from Bothrops brazili and ACLF from Agkistrodon contortrix laticinctus) and two PIII (Berythractivase from Bothrops erythromelas and Jarharagin from Bothrops jararaca). Inhibition and induction of apoptosis and viability were determined by immunofluorescence cell staining with acridine orange and ethidium bromide in cells incubated for 48h with medium RPMI containing 1% or 10% fetal bovine serum (FBS) with or without SVMPs. For proliferation activity cells were plated in RPMI containing 10% FBS with or without SVMPs and 72h later cells were detached and counted by phase-contrast microscopy. Expression of ICAM-1 and DAF were analysed by flow cytometric after incubation of HUVECs with SVMPs (50mg/ml) during 1h. The concentrations of nitric oxide in the supernatants were determined by chemiluminescence in gaseous phase, elicited by the reaction of NO with ozone, after nitrate and nitrite reduction with VCl3 saturated solution in 1M HCl. In HUVECs, B151 and Jarharagin induced monolayer cell detachment and cell clustering. The percentage of apoptotic cells induced by B151 (26.0±1.9) and Jarharagin (30.2±3.5) was greater than that seen in control samples (17.5±2.0). A decrease in cells number was observed in the proliferation assays induced by Jarharagin (64.2±0.9x10³ vs 28.6±1.7x10³ n=3). Berythractivase, P141 and RQ2 did not disrupt cells, neither induced apoptosis nor cell proliferation. ACLF showed a tendency to inhibit apoptosis but it was not statistically significant. Flow cytometric analysis showed that the incubation of HUVECs with SVMPs produced an increased expression of ICAM-1 (17.8±1 vs. 51.3±4 arbitrary units of fluorescence (UAF), p<0.05, n=4) induced by ACLF and DAF (46±3 vs. 73±14 and 68±9 UAF, p<0.05, n=3) induced by B151 and ACLF respectively. SVMPs showed a tendency to increased NO in the supernantants of HUVECs, however P141 (11±1), Berythractivase (10.4±1) and Jarharagin (10.5±1) were statistically significant than that seen in control samples (6.1±0.5mM). These results showed that although these SVMPs share structural similarities they exert different functions related to cell survival, proliferation and expression of diferent molecules involved in biological responses.

Supported by: FAPESP.

Correspondence to: amchudzinski@butantan.gov.br