Soluble guanylate cyclase stimulation fosters angiogenesis and blunts myofibroblast-like features of scleroderma endothelial cells

In scleroderma (systemic sclerosis, SSc), peripheral microvasculopathy and angiogenesis impairment advance in parallel with the development of tissue fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-myofibroblast transition (EndMT). Soluble guanylate cyclase (sGC) stimulation was found to counteract transforming growth factor (TGF)β-induced fibroblast-to-myofibroblast differentiation with significant antifibrotic effects in various experimental models of tissue fibrosis. Here, we investigated the effects of pharmacological stimulation of sGC on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-dMVECs). To determine whether sGC stimulation affected cell viability and proliferation, five lines of SSc-dMVECs and five lines of healthy dermal MVECs (H-dMVECs) were challenged with the sGC stimulator MK-2947 and assayed by annexin V/propidium iodide flow cytometry and WST-1, respectively. To study angiogenesis and EndMT, MK-2947-treated SSc-dMVECs were subjected to wound healing and capillary-like tube formation assays, and analyzed for the expression of endothelial and myofibroblast markers by quantitative real-time PCR, immunoblotting and immunofluorescence. Cell contractile ability was investigated by collagen gel contraction assay. In other experiments, H-dMVECs were preincubated with MK-2947 before induction of EndMT through administration of recombinant human TGFβ or serum from SSc patients. MK-2947 treatment did not affect H-dMVEC viability/proliferation, while it significantly increased SSc-dMVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-dMVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in gene and protein expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 myofibroblast markers was also found in MK-2947-treated SSc-dMVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-dMVECs to contract collagen gels and reduced phosphorylated-ERK1/2 protein levels. Finally, treatment with MK-2947 efficiently protected H-dMVECs against TGFβ- and SSc serum-induced EndMT. These findings demonstrate for the first time that pharmacological stimulation of sGC effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-dMVECs, thus providing new evidence for repurposing sGC stimulators for the treatment of SSc-related skin fibrosis and peripheral vascular manifestations.