Caspase-3 Activation Triggers Extracellular Cathepsin L Release and Endorepellin Proteolysis

Abstract

Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components. Apoptosis of endothelial cells (EC)4 is increasingly recognized as an important component of the "response to injury" process, as most clinical risk factors of atherosclerosis (such as hypertension (1, 2), hyperglycemia (3, 4), oxidized low density lipoproteins (LDLs) (5, 6) and oxidative stress (7)) induce EC apoptosis. Interventions aimed at preventing EC apoptosis in animal models of transplant vasculopathy, an immune-mediated form of atherosclerosis, prevent neointima formation, indicating that EC apoptosis is an important pro-atherosclerotic trigger (8 -13). During vascular remodeling, EC injury and apoptosis are followed by migration of ␣-smooth muscle actinpositive cells (smooth muscle cells (SMC) and myofibroblasts) that accumulate within the intima through a state of resistance to apoptosis largely dependent on Bcl-xl overexpression (14 -16).Recent evidence from our group and others suggests that apoptotic EC favor neointima formation through the release of paracrine mediators, which in turn, increase Bcl-xl expression and inhibit the apoptosis of vascular SMC and fibroblasts (17)(18)(19)(20). The production of biologically active mediators by apoptotic EC is at least partially dependent on pericellular proteolysis, leading to basement membrane and extracellular matrix (ECM) degradation with the release of cryptic anti-apoptotic factors (18 -20). A C-terminal fragment of endorepellin (perlecan domain V) released in association with EC apoptosis was found to heighten Bcl-xl expression in SMC and fibroblasts (18 -20). Perlecan is a basement membrane modular proteoglycan composed of five structural domains (21). The C-terminal domain, also called endorepellin, comprises three laminin-like globular (LG1-LG3) modules interspaced by four epiderm...

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

“…We showed previously that caspase-3 activation in ECs triggers the release of the C-terminal laminin G motif of perlecan (LG3). 8,16 Yet, fibroblasts exposed to LG3 in vitro failed to differentiate into myofibroblasts (Supplementary Figure 3). This suggested that caspase-3 activation supports the release of various mediators active on target cells.…”

“…8 Yet, in this system, LG3 failed to favor myofibroblast differentiation, indicating that caspase-3 activation in ECs promotes the release of various factors with specialized functions; some mediators, such as LG3, elicit resistance to apoptosis in fibroblasts, whereas others regulate myofibroblast differentiation.…”

“…SSC, SSC-ZVAD and SSC-DMSO were generated from C3H ECs, as described for HUVECs. 8,15,16 In brief, an equal number of ECs were exposed to the vehicle (DMSO) or ZVAD-FMK (100 mM) for 2 h, washed, kept in SS for 4 h and harvested. For all experiments, equal volumes of media conditioned by an equal number of cells (apoptotic or not) were compared.…”

“…1,3 Endothelial cell (EC) apoptosis is increasingly recognized as an early pathogenic event in fibrosis. A wide array of fibrogenic conditions, such as systemic sclerosis, 4,5 graftversus-host disease 6,7 and chronic rejection of solid allografts, 8,9 has been associated with increased EC apoptosis. Pathophysiological pathways linking endothelial apoptosis to fibrogenesis are still poorly defined.…”

“…Some models indicate that EC apoptosis favors the recruitment of professional phagocytes such as macrophages. 8,10 Upon engulfment of apoptotic cells, the macrophages produce increased amounts of transforming growth factor-b1 (TGF-b1). 11 In turn, TGF-b1 would favor myofibroblast differentiation and resistance to apoptosis in fibroblasts/myofibroblasts.…”