In this study, we have demonstrated that gingipains induce detachment and apoptosis in endothelial cells. This is consistent with reports of gingipain-induced apoptosis in several other cell types (5
). However, the kinetics of cell adhesion molecule cleavage appear to differ between epithelial and endothelial cells (12
). While heat-stable molecules (such as butyric acid and lipopolysaccharide) produced by P. gingivalis
can induce cell death (27
), it is unlikely that the apoptosis induced in our experimental conditions was not gingipain dependent. Furthermore, there was no evidence indicating that the gingipains can induce primary necrosis in BCAEC. Interestingly, however, in BCAEC treated with W83 extract, there was the early, transient appearance of a 45-kDa cleavage product of Topo I, usually associated with necrosis (9
). Since gingipains can traverse the plasma membrane and localize within the cell (60
), it is tempting to speculate that the gingipains could directly cleave Topo I, which may have involvement in the cell death process. Surprisingly, TLCK pretreatment of W83 extracts was not able to completely inhibit VE-cadherin cleavage, most likely from incomplete gingipain inhibition. TLCK has been shown to more completely inhibit Kgp activity than Rgp activity (54
). Alternatively, other bacterial or host factors could be responsible for this cleavage, or it could also be a by-product of apoptosis. This seems unlikely, since cells treated with FLL32 extracts show no VE-cadherin cleavage, gingipains can directly cleave immunoprecipitated cadherins, and there was no VE-cadherin cleavage when apoptosis was induced with staurosporine (data not shown). To our knowledge, this is the first demonstration of gingipain-induced cleavage of VE-cadherin, N-cadherin, and integrin β1 in endothelial cells.
Since the gingipain adhesin domains can modulate P. gingivalis
adherence to epithelial cells (10
), it is not surprising that Kgp and HRgpA were able to induce morphological changes before RgpB. This would also be consistent with the report that HRgpA was more effective than RgpA(cat)
in causing the loss of integrin β1 expression on human gingival fibroblasts (61
). It is noteworthy that BCAEC treated with Kgp quickly detached and then over time reattached, with no significant loss in cell viability. It is possible that the purified Kgp was less stable than purified HRgpA or RgpB or the Kgp present in extracellular extracts from W83. Thus, in the continued presence of Kgp, with diminishing activity, the endothelial cells are able to recover, possibly through upregulation of adhesion molecules or cell survival proteins. There is evidence that Kgp can modulate gene expression in endothelial cells (52
). We cannot rule out the possibility that in the periodontal pocket, where all gingipains would be present, they synergistically cause endothelial cell damage. Kgp and HRgpA could quickly adhere to endothelial cells and cause their detachment, while HRgpA and RgpB would be responsible for inducing cell death.
Because anoikis can be induced by loss of integrin signaling following cell detachment (23
), integrin-extracellular matrix interactions are essential for cell survival and also protect endothelial cells from anoikis (2
). The rapid degradation of integrin β1 suggests that the trigger for endothelial cell death induced by gingipains might be the loss of integrin signaling, which occurs prior to cell detachment and apoptosis. This would be consistent with a recent report demonstrating that disruption of focal adhesions and the actin cytoskeleton by overexpression of integrin β1 cytoplasmic domains preceded cell detachment and was, therefore, the cause rather than a consequence of endothelial cell detachment (53
). Furthermore, it has been demonstrated that ligation of integrin β1 by antibodies protected fibroblasts from apoptosis through upregulation of phosphatidylinositol 3-kinase and Akt/protein kinase B activity (66
) and that Akt is deactivated in both caspase-dependent and caspase-independent cell death in several cell types (46
). It has even been suggested that integrins can mediate an apoptotic cell death distinct from anoikis, called “integrin-mediated death,” in which caspase-8 is recruited to unligated integrins (64
) and triggers the caspase cascade of apoptosis.
Integrin β1 can directly interact with focal adhesion kinase (45
) to transduce signals via Ras, phosphatidylinositol 3-kinase, and Akt, leading to upregulation of expression of bcl-2
), a prosurvival gene. If integrin signaling is prevented by gingipain-induced cleavage, it is likely that the levels of bcl-2
would decrease, sensitizing cells to die. Integrin β1 can also associate with Shc and signal survival through the mitogen-activated protein kinase pathway (70
). Furthermore, since integrin β1 can regulate the proapoptotic protein Bim, it is also conceivable that loss of integrin function could release Bim and signal apoptosis through the Erk pathway (57
). Integrins are associated with numerous survival pathways (reviewed in reference 63
), and we are exploring the involvement of integrin β1 in gingipain-induced endothelial apoptosis-anoikis. In the periodontal pocket, integrin β1 is expressed within the gingival epithelium and junctional epithelium and in the endothelial cells of the connective tissue (35
). Integrin β1 is consistently expressed in endothelial cells of all vessel types and sizes (49
), and several different integrin β1 molecules are expressed on the luminal side of vessels (14
). The tissue distribution of integrin β1 and the recent report that P. gingivalis
invades aortic tissue in the ApoE−/−
mouse model and accelerates atheroma development (29
) raise the possibility that gingipains can cause endothelial cell dysfunction and apoptosis in both the periodontal pocket and the cardiovascular system.
It has been suggested that VE-cadherin, which is specific for endothelial cells of almost all types of vessels (17
), promotes homotypic interactions with endothelial cells and that N-cadherin may be responsible for the anchorage of endothelial cells to other cell types. VE-cadherin acts as a seal at intercellular junctions, associating with β-catenin, plakoglobin, p120, and the actin cytoskeleton (17
), and is a target of agents that increase vascular permeability. Inhibition of its function produced more damage to the endothelial monolayer in vivo than in vitro (6
), suggesting that gingipain-induced disruption of VE-cadherin integrity may have a considerable role in the tissue damage that occurs in the periodontal pocket. It appears that the gingipains may be able to trigger endothelial cell detachment and cell death through degradation of several different molecules.
In conclusion, we have shown that gingipain-active extracts mediate the detachment and apoptosis of endothelial cells. BCAEC treated with gingipains become apoptotic, as determined by cell morphology, caspase-3 activation, annexin V positivity, and cleavage of PARP and Topo I. Gingipains cleave N-cadherin and VE-cadherin and degrade integrin β1, whereas extracts of FLL32 that have significantly reduced gingipain activity do not have cleaved CAMs. Gingipains directly cleave immunoprecipitated N-cadherin and VE-cadherin. Moreover, purified gingipains induce endothelial cell detachment and apoptosis, implicating the gingipains in vascular tissue destruction.