Synergism of gingipains in endothelial cell adhesion disruption and apoptosis

The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells (BCAEC) and human microvascular endothelial cells (HMVEC) to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis . Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin beta1 was observed in immunoblots of cell lysates. Loss of cell adhesion, as well as N- and VE-cadherin and integrin beta1 cleavage, could be inhibited or significantly delayed by preincubation of gingipain-active extracts with the cysteine protease inhibitor TLCK. Furthermore, purified gingipains also induced endothelial cell detachment; however, Kgp-induced detachment was temporary and only HRgpA and RgpB induced apoptosis. Apoptosis-associated events, including Annexin-V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and Topoisomerase I (Topo I) were observed in endothelial cells after detachment. We have demonstrated that Kgp activity can be inhibited by synthetic peptide caspase inhibitors. Using z-VAD-FMK to inhibit Kgp activity and leupeptin to inhibit Rgp activity, we further investigated the specific roles of the gingipains in producing the observed morphological changes. In the presence of z-VAD-FMK or leupeptin, gingipain-induced cell detachment was only delayed. There was partial cleavage of N-cadherin and delayed cleavage of VE-cadherin. Degradation of integrin beta1 was inhibited only in the presence of z-VAD-FMK. Interestingly, in the absence of active caspases, both gingipain-active extracts and purified HRgpA and RgpB induce apoptotic morphology, suggesting the gingipains can induce apoptosis by a caspase-independent mechanism. Collectively, our results indicate that the gingipains, at concentrations naturally occurring in the periodontal pocket, can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells with HRgpA and RgpB triggering caspase-dependent and caspase-independent apoptosis. These results have further clarified the role P. gingivalis plays in tissue destruction occurring in the periodontal pocket.