Effects Of Metalloproteinases Structural Domains On The Integrity And Viability Of Endothelial Cells (EC) In Vitro. 

¹Zamuner, SF; ¹Olivo,RA; ²Assakura, MT; ³Silva, CA; ³Serrano, SMT; ¹Teixeira, CFP

1 Laboratório de Farmacologia, 2 Laboratório de Bioquímica e Biofísica, 3 Laboratório Especial de Toxinologia Instituto Butantan, SP – Brasil.

Metalloproteases are involved in the local and systemic toxic effects of Viperidae snake venoms. Snake venom metalloproteinases (SMVP) are classified in four groups basead on their domain constitution. In this study we evaluated the effects of native MPA, isolated from B. moojeni venom, recombinant D-Bothrostatin (D-Bt) (disintegrin domain), DC-HF3 (disintegrin and cysteine-rich domain), and F12  medium (control) on the integrity and viability of EC monolayers. Human umbilical cord EC (ECV-304) were cultured in F12 medium-10% SFB and seeded in 96 well plates. After reaching confluence (48h) EC monolayers were incubated with 5-25 ug/mL MPA, D-Bt, DC-HF3, and F12 for 30, 60 and 120 min before evaluation of EC injury and viability. Integrity of monolayers was quantified by staining with crystal violet, and determination of O.D. at 620 nm. EC viability was evaluated by lactate dehidrogenase (LDH) activity. MPA, D-Bt or DC-HF3 did not affect EC viability. However, MPA (25ug/mL), but not D-Bt and DC-HF3, caused detachment of EC monolayers (22,80%), from 30-120 min of incubation. Concentration of 10 ug/mL of MPA reduced EC monolayers integrity from 60 to 120 min. This data indicate the ability of MPA, but not D-Bt or DC-HF3 to affect the integrity of EC monolayers. This effect is not related to death of cells. The lack of effects of the disintegrin domain and disintegrin cysteine-rich domains on EC monolayers suggests that the catalytic domain is relevant for SVMP to display effects on EC.

Support: FAPESP, Capes, CNPq

Correspondence to: szamuner@butantan.gov.br