Novel Fibrogenic Pathways Are Activated in Response to Endothelial Apoptosis: Implications in the Pathophysiology of Systemic Sclerosis

Abstract

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.

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

“…Co-incubation with PP2 and LY2940002, inhibitors of SFK and PI3K, respectively, blocked CTGFdependent aSMA upregulation in human and mouse fibroblasts ( Figure 7b and Supplementary Figure 5). In agreement with our previous work, 15,16 heightened Akt phosphorylation was found in fibroblasts exposed to SSC (Figure 7c). CTGF also increased Akt phosphorylation in fibroblasts, which was inhibited in the presence of PP2 (Figure 7c).…”

“…Surprisingly, blockade of TGF-b signaling with a pan-TGF-b-neutralizing antibody did not prevent myofibroblast differentiation induced by either SSC or recombinant CTGF. These results are in keeping with our previous work 15 showing low TGF-b1 levels in SSC, and indicate a predominant role of TGF-b1-independent pathways in our system.…”

“…The fibrogenic signature of SSCs would then serve to validate that the mediator identified by comparative proteomics reproduces the fibrogenic signaling pattern induced by SSC. SSC induces myofibroblast differentiation through PI3K-dependent pathways, 15 but upstream PI3K activators have not yet been characterized. Src-family kinases (SFKs) and focal adhesion kinase (FAK) are prime candidates as they are known PI3K activators and have been associated with fibrosis.…”

“…Previously we showed that serum-free medium conditioned by apoptotic ECs (SSC) induces myofibroblast differentiation in vitro. 15 To verify the ability of SSC to promote fibrogenesis in vivo, mouse ECs (mECs) were isolated and cultured to generate mouse SSCs (mSSCs) (Supplementary Figure 2a). Serum starvation for 4 h also induced a significant apoptotic response in mEC (Supplementary Figure 2b).…”

“…15,16 In the present work, we aimed to establish the fibrogenic role of this paracrine loop in vivo and to identify the paracrine mediators mediating this fibrogenic activity. We demonstrate that caspase-3 activation in apoptotic ECs triggers CTGF secretion, which in turn induces myofibroblast differentiation and fibrogenesis through TGF-b1-independent pathways.…”