Marc-André Raymond

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-xL 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-xL protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of α-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.

Increased Risk of Thrombotic Microangiopathy in Patients Receiving a Cyclosporin–Sirolimus Combination

A single‐center cohort study of kidney and kidney–pancreas recipients was conducted to evaluate the association between new immunosuppressive regimens and risk of thrombotic microangiopathy (TMA). From January 1st,1996 to December 31, 2002, 368 patients received a kidney or kidney–pancreas transplant at our center. Four immunosuppressive regimens were evaluated as potential risk factors of TMA: cyclosporin + mycophenolate mofetil (CsA + MMF), cyclosporin + sirolimus (CsA + SRL), tacrolimus + myophenolate mofetil (FK + MMF), and tacrolimus + sirolimus (FK + SRL). Thirteen patients developed biopsy‐proven TMA in the absence of vascular rejection. The incidence of TMA was significantly different in the four immunosuppressive regimens studied (p < 0.001). The incidence of TMA was highest in the CsA + SRL group (20.7%). The relative risk of TMA was 16.1 [95% confidence interval (CI): 4.3–60.8] for patients in the CsA + SRL group as compared with those in the FK + MMF group. We also investigated in vitro the pathophysiological basis of this association. The CsA–SRL combination was found to be the only regimen that concomitantly displayed pro‐necrotic and anti‐angiogenic activities on arterial endothelial cells. We propose that this combination concurs to development of TMA through dual activities on endothelial cell death and repair.

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

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 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.

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).

Epidermal Growth Factor and Perlecan Fragments Produced by Apoptotic Endothelial Cells Co‐Ordinately Activate ERK1/2‐Dependent Antiapoptotic Pathways in Mesenchymal Stem Cells

Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum‐free medium conditioned by apoptotic EC increases extracellular signal‐regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl‐xL protein levels and poly (ADP‐ribose) polymerase cleavage) of human MSC. A C‐terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with β1‐integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering β1‐integrin and EGF receptor‐dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response. STEM CELLS 2010;28:810–82028:810–820

ROLES OF DNA TOPOISOMERASES IN THE REGULATION OF R-LOOP FORMATION IN VITRO

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.