Systemic Sclerosis Sera Impair Angiogenic Performance of Dermal Microvascular Endothelial Cells: Therapeutic Implications of Cyclophosphamide

Abstract

In systemic sclerosis (SSc), dermal capillaries are progressively lost with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. Clinical studies reported that intravenous cyclophosphamide (CYC) may improve SSc-related peripheral microvascular damage. Recently, we showed that CYC treatment may normalize SSc sera-induced abnormalities in endothelial cell-matrix interactions. Our objective was to evaluate in vitro the effects of sera from treatment-naïve or CYC-treated SSc patients on dermal blood microvascular endothelial cell (dMVEC) angiogenesis, migration, proliferation and apoptosis. dMVECs were challenged with sera from 21 SSc patients, treatment-naïve (n = 8) or under CYC treatment (n = 13), and 8 healthy controls. Capillary morphogenesis on Geltrex matrix was significantly reduced upon challenge with sera from naïve SSc patients compared with healthy controls. When dMVECs were challenged with sera from CYC-treated SSc patients, their angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity and chemotaxis in Boyden chamber were both significantly decreased in the presence either of naïve or CYC-treated SSc sera compared with healthy sera. WST-1 assay revealed that cell proliferation was significantly decreased in dMVECs challenged with sera from naïve SSc patients compared with healthy sera. Conversely, dMVEC proliferation was not impaired in the presence of sera from CYC-treated SSc patients. Accordingly, the percentage of TUNEL-positive apoptotic dMVECs was significantly higher in the presence of sera from naïve SSc patients than healthy controls, while CYC-treated SSc sera did not induce dMVEC apoptosis. Levels of the angiostatic mediators endostatin, pentraxin 3, angiostatin and matrix metalloproteinase-12 were all significantly elevated in sera from naïve SSc patients compared with sera from both healthy controls and CYC-treated SSc patients. In SSc, CYC treatment might boost angiogenesis and consequently improve peripheral microangiopathy through the normalization of the endothelial cell-matrix interactions, reduction of endothelial cell apoptosis and rebalance of dysregulated angiostatic factors.

Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis

“…Therefore, we herein investigated whether MMP-12-dependent uPAR-D1 cleavage could be implicated in the induction of EndoMT by SSc sera. Consistent with previous reports,40 43 MMP-12 levels were raised in SSc sera (see online supplementary figure S2A). Treatment of H-dMVECs with SSc sera resulted in uPAR-D1 cleavage already after 24 hours (see online supplementary figure S2B).…”

“…In addition, here we clearly demonstrate that a prolonged treatment with sera from patients with SSc is capable of sustaining the EndoMT process in H-dMVECs. Of note, shorter time treatments with SSc sera have previously been shown to impair angiogenesis and survival of H-dMVECs 29 39 40. Mechanistically, our present findings show that MMP-12-dependent cleavage of uPAR, a process that has been deeply implicated either in the impaired angiogenic performance of SSc-dMVECs or in fibroblast-to-myofibroblast differentiation,35 41 42 takes part in the pro-EndoMT effect exerted by SSc sera.…”

“…Previous studies have demonstrated that treatment with sera from patients with SSc impairs the angiogenic performance of H-dMVECs in vitro 29 39 40. Nevertheless, whether these antiangiogenic effects may be in part related to the induction of the EndoMT process has never been investigated.…”

Decreased expression of neuropilin-1 as a novel key factor contributing to peripheral microvasculopathy and defective angiogenesis in systemic sclerosis

“…In our study, we observed that the proangiogenic NRP1 receptor was constitutively downregulated in dermal SSc-MVECs and that treatment with SSc sera could significantly reduce NRP1 expression in H-MVECs, which is in line with the reported antiangiogenic properties of SSc sera 2 31 32 37 41. Strikingly, we also found that NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic process comparable to that of cells treated with SSc sera, further supporting the implication of NRP1 deficiency in the disturbed angiogenesis of SSc.…”

“…H-MVECs stimulated with healthy sera produced an abundant network of branching cords (figure 5). On the contrary, as previously reported,31 32 angiogenesis was significantly reduced on challenge with SSc sera (p<0.01 vs basal H-MVECs) (figure 5). The addition of recombinant human VEGF-A165 or anti-VEGF-A165b blocking antibodies to SSc sera significantly increased H-MVEC angiogenesis compared with cells treated with SSc sera alone (both p<0.05) (figure 5).…”