Abstract
BackgroundSystemic sclerosis (SSc) is characterized by early vascular abnormalities and subsequent fibroblast activation to myofibroblasts, leading to fibrosis. Recently, endothelial-to-mesenchymal transition (EndoMT), a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, has been reported in SSc. In the present study, we evaluated the ability of endothelin-1 (ET-1) dual receptor antagonists bosentan (BOS) and macitentan (MAC) to antagonize EndoMT in vitro.MethodsTen women with limited SSc were enrolled. They underwent double skin biopsy (affected and nonaffected skin). Fibroblasts and microvascular endothelial cells (MVECs) were isolated from biopsies. We performed mono- or coculture of MVECs (isolated from nonaffected skin) with fibroblasts (isolated from affected skin and stimulated with ET-1 and transforming growth factor beta [TGF-β]). In cocultures, the MVEC layer was left undisturbed or was preincubated with BOS or MAC. After 48 h of coculture, MVECs were analyzed for their tube formation ability and for messenger RNA and protein expression of different vascular (CD31, vascular endothelial growth factor-A [VEGF-A], VEGF-A165b) and profibrotic (alpha-smooth muscle actin [α-SMA], collagen type I [Col I], TGF-β) molecules.ResultsAfter 48 h, MVECs showed a reduced tube formation ability when cocultured with SSc fibroblasts. CD31 and VEGF-A resulted in downregulation, while VEGF-A165b, the antiangiogenic isoform, resulted in upregulation. At the same time, mesenchymal markers α-SMA, Col I, and TGF-β resulted in overexpression in MVECs. Tube formation ability was restored when MVECs were preincubated with BOS or MAC, also reducing the expression of mesenchymal markers and restoring CD31 expression and the imbalance between VEGF-A and VEGF-A165b.ConclusionsWith this innovative EndoMT in vitro model realized by coculturing nonaffected MVECs with affected SSc fibroblasts, we show that the presence of a myofibroblast phenotype in the fibroblast layer, coupled with an ET-1-TGF-β synergic effect, is responsible for EndoMT. BOS and MAC seem able to antagonize this phenomenon in vitro, confirming previous evidence of endothelium-derived fibrosis in SSc and possible pharmacological interference.
Highlights
Systemic sclerosis (SSc) is characterized by early vascular abnormalities and subsequent fibroblast activation to myofibroblasts, leading to fibrosis
Tube formation ability of microvascular endothelial cell (MVEC) In our in vitro endothelial-to-mesenchymal transition (EndoMT) model, MVECs cocultured with affected fibroblasts showed a significant impairment in tube formation, probably due to the persistent stimulation of the myofibroblast phenotype (Fig. 1a)
The analysis of tube formation ratio evidenced significant improvement in tube formation when MVECs were preincubated with BOS (p < 0.01) and MAC (p < 0.01). These data were confirmed by analysis of the number of branching points (Fig. 1c) that were statistically increased when MVECs were incubated with BOS (p < 0.05) and MAC (p < 0.05)
Summary
Systemic sclerosis (SSc) is characterized by early vascular abnormalities and subsequent fibroblast activation to myofibroblasts, leading to fibrosis. Endothelial-to-mesenchymal transition (EndoMT), a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, has been reported in SSc. In the present study, we evaluated the ability of endothelin-1 (ET-1) dual receptor antagonists bosentan (BOS) and macitentan (MAC) to antagonize EndoMT in vitro. EndoMT is a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype and express mesenchymal cell products such as alpha smooth muscle actin (α-SMA) and collagen type I (Col I) [8, 9]. Considering the synergistic action of ET-1 and TGF-β to induce EndoMT, we investigated the potential of ET-1 dual receptor antagonists bosentan (BOS) and macitentan (MAC) to antagonize EndoMT in our in vitro coculture model
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