Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis.

Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-x(L) overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-x(L) protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of alpha-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.

Apoptosis of endothelial cells triggers a caspase-dependent anti-apoptotic paracrine loop active on VSMC.

Increased endothelial apoptosis and decreased apoptosis of vascular smooth muscle cells (VSMC) are central to initiation of myo-intimal thickening. We hypothesized that apoptosis of endothelial cells (EC) induces the release of anti-apoptotic mediator(s) active on VSMC. We found that serum-free medium conditioned by apoptotic EC decreases apoptosis of VSMC compared with fresh serum-free medium. Inhibition of endothelial apoptosis during conditioning with a pan-caspase inhibitor ZVAD-FMK blocked the release of the anti-apoptotic factor(s) active on VSMC. VSMC exposed to serum-free medium conditioned by apoptotic EC showed increased ERK 1/2 phosphorylation, enhanced Bcl-xl expression, and inhibition of p53 expression. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as a C-terminal fragment of the domain V of perlecan. Serum-free medium supplemented with either a synthetic peptide containing the EGF motif of the domain V of perlecan or chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, increased ERK 1/2 phosphorylation and Bcl-xl protein levels while inhibiting apoptosis of VSMC. These results suggest that a proteolytic activity developing downstream of activated caspases in apoptotic EC initiates degradation of pericellular proteoglycans and liberation of bioactive fragments with a robust impact on inhibition of VSMC apoptosis.

Soluble Fas: a novel predictor of atherosclerosis in dialysis patients.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Disregulation of apoptosis within the vessel wall and upregulation of the Fas/Fas-ligand (Fas-L) system contribute to the development of atherosclerosis. Cross-sectional studies have suggested that elevated plasma levels of the soluble form of Fas (sFas) are associated with CVD. However, the role of sFas and sFas-L in predicting future cardiovascular events has yet to be defined. METHODS: We evaluated the role of plasma sFas and sFas-L levels as predictors of CVD in a prospective cohort of 107 chronic hemodialysis patients. RESULTS: During the study period (27 months), 53 patients (49.5%) presented with at least one cardiovascular end point. On univariate analysis, baseline sFas levels were significantly associated with the occurrence of cardiovascular end points, whereas sFas-L levels were not. Using Cox proportional hazards, increased sFas levels were associated with a significantly greater risk for cardiovascular end points (P = 0.03). This effect was independent of baseline CVD history, classic risk factors for atherosclerosis (diabetes, hypercholesterolemia, hypertension, and smoking), and markers of inflammation (C-reactive protein [CRP], soluble intercellular adhesion molecule-1). Increased CRP levels also were associated with cardiovascular end points (P = 0.04). In addition, increased cardiovascular mortality was found in patients in the highest sFas tertile compared with those in the lowest tertile (27.8% versus 8.6%; P = 0.04). CONCLUSION: Increased plasma sFas levels are predictive of future CVD. These results suggest that sFas is a novel and independent predictor of active atherosclerotic disease in patients with ESRD.

Blockade of the apoptotic machinery by cyclosporin A redirects cell death toward necrosis in arterial endothelial cells: regulation by reactive oxygen species and cathepsin D.

Blockade of the mitochondrial permeability transition pore (mPTP) by cyclosporin A (CsA) inhibits apoptosis in various cell types. However, use of CsA in humans is associated with damage to the arterial endothelium. We evaluated whether inhibition of the apoptotic machinery by CsA promotes other forms of cell death in arterial endothelial cells (EC). Exposure of human umbilical artery EC (HUAEC) to clinically relevant concentrations of CsA for up to 24 h was associated with a significant increase in necrotic features. We detected inhibition of apoptosis and a significant increase in necrosis in HUAEC exposed concomitantly to CsA and mitomycin C, a proapoptotic DNA damaging agent. We found that CsA-induced cell death is independent of caspase activation, p53 induction, and calcineurin inhibition. However, bongkrekic acid, another mPTP blocker, also increased necrosis in HUAEC. Dihydroethidium and acridine orange staining revealed increased intracellular production of reactive oxygen species (ROS) followed by lysosomal damage in HUAEC exposed to CsA. Hydroxyl radical and superoxide scavengers and inhibition of cathepsin D activity significantly attenuated CsA-induced EC death. These results suggest that inhibition of the apoptotic machinery by CsA in arterial EC favors development of a necrotic form of cell death regulated by ROS and secondary lysosomal damage.

Enhanced development of caspase-independent cortical cell death during cold storage in kidneys of non-heart-beating donors.

BACKGROUND: Understanding the mechanisms of injury associated with cardiac arrest is essential for defining strategies aimed at improving preservation and function of kidneys harvested in non-heart-beating (NHB) donors. METHODS: We standardized a model of NHB donors in rats and studied the kinetics and types (apoptosis vs. necrosis) of renal cell death developing during cold storage. Using quantitative polymerase chain reaction, immunoblotting, and caspase inhibition, we also studied the molecular pathways regulating renal cell death in this model. RESULTS: The kinetics and extent of cell death developing in cortical tubules during cold storage were found to be increased in non-heart-beating (NHB) kidneys. Apoptosis of cortical tubules predominated in NHB kidneys exposed to 10 hr of cold storage, whereas necrosis increased after longer periods of cold ischemia. Shortly after cardiac arrest, a rapid up-regulation of Bax and Hsp 70 was found at the protein level in NHB kidneys. After 24 hr of cold storage, induction of Bax was maintained, whereas protein levels of Hsp70 returned to levels comparable to heart-beating (HB) controls. Also, mRNA levels of Bax were found to increase during cold storage in NHB kidneys. Cortical cell death was found to be largely caspase-independent but responsive to hydroxyl-radical scavenging with dimethyl sulfoxide (DMSO). CONCLUSIONS: Cardiac arrest promotes activation of death-inducing molecules such as Bax and is associated with increased development of caspase-independent renal cell death during cold storage. Developing strategies, such as free radical scavenging, aimed at inhibiting cell death during cold storage, could prove useful for improving preservation of NHB kidneys.

Soluble Fas is a marker of peripheral arterial occlusive disease in haemodialysis patients.

BACKGROUND: Peripheral arterial occlusive disease (PAOD) including lower-extremity and cerebrovascular atherosclerosis is a leading cause of morbidity in haemodialysis patients. Recent evidence suggests that the expression of Fas, a molecule implicated in the initiation of apoptosis in various cell types, is increased at sites of atherosclerotic plaques. However, the significance of plasma levels of the soluble form of Fas (sFas) as a marker of peripheral arterial disease has yet to be defined. METHODS: The present report is based on a cross-sectional analysis of baseline data from an ongoing prospective study designed to evaluate the role of sFas as marker of PAOD in end-stage renal disease (ESRD). We evaluated the association between sFas levels and evidence of PAOD in a cohort of 107 chronic haemodialysis patients. RESULTS: Compared with subjects without evidence of disease (n=56), subjects with PAOD (n=51) had significantly higher plasma levels of sFas (30.0+/-8.9 vs 26.4+/-9.5 ng/ml; P=0.04). Using multiple regression, sFas was found to be associated with PAOD independently of classical risk factors for atherosclerosis (hypercholesterolaemia, diabetes, hypertension, and smoking), markers of inflammation (e.g. C-reactive protein, intercellular cell adhesion molecule type 1), and other risk factors (e.g. age, gender). An increase of one quintile in the plasma concentration of sFas was associated with an odds ratio of PAOD of 1.69 (95% CI: 1.09--2.63, P=0.01). In addition, models that incorporated sFas were significantly better at predicting PAOD than models limited to classical risk factors for atherosclerosis, alone or in combination with CRP levels (P=0.01). CONCLUSIONS: Increased plasma levels of sFas are associated with established PAOD. These results suggest that sFas may represent a novel and independent marker of atherosclerosis.

Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells.

The mechanisms of cytoprotection conferred by stress preconditioning remain largely uncharacterized in endothelial cells (EC). We report that stress preconditioning of EC with serum starvation induces the release of soluble mediator(s) that confer resistance to apoptosis, increase proliferation, and enhance angiogenesis in a second set of "non-preconditioned" EC. Preconditioning was found to target specifically the mitochondrial control of apoptosis in EC with increased protein levels of Bcl-2, decreased protein levels of Bax, and decreased cytosolic release of cytochrome c. Regulators of apoptosis acting upstream and downstream of the mitochondria such as p53, cIAP-1, cIAP-2, and XIAP were not altered. Mediators classically associated with preconditioning in other cell types such as adenosine, opioids, and nitric oxide are not implicated in this cytoprotective loop. Blockade of protein kinase C-dependent signaling inhibited cytoprotection of EC. Further characterization of this paracrine pathway should provide insights into the molecular regulation of preconditioning in endothelial cells.