cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca concentration

Proteinase inhibitors, isolated from different types of , have an effect on apoptosis, angiogenesis and inflammation. The cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from . Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10–100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10–100 μM) on the membrane potential was measured with the fluorescence dye DiBAC(3) and the effect on [Ca] with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10–100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1 ± 1.8% as compared to control ( ≤ 0.05;  = 45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca of 28.4 ± 5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5 ± 0.9% as compared to control ( ≤ 0.05;  = 80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca concentration and the membrane potential.