Bosentan and macitentan prevent the endothelial-to-mesenchymal transition (EndoMT) in systemic sclerosis: in vitro study - PubMed (original) (raw)
Bosentan and macitentan prevent the endothelial-to-mesenchymal transition (EndoMT) in systemic sclerosis: in vitro study
Claudio Corallo et al. Arthritis Res Ther. 2016.
Abstract
Background: Systemic 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.
Methods: Ten 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.
Results: After 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.
Conclusions: With 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.
Keywords: Bosentan; EndoMT; Macitentan; Microvascular endothelial cells; Systemic sclerosis.
Figures
Fig. 1
a Tubular structure formation of microvascular endothelial cells (MVECs) in Matrigel after 48 h of culture alone (before endothelial-to-mesenchymal transition [EndoMT]) or cocultures with fibroblasts. The fluorescent red images show that MVECs (post-EndoMT) have a decreased tube formation ability with respect to those treated with bosentan (BOS) and macitentan (MAC), which show a well-organized tubelike network. b The tube formation ability was measured as cells per millimeter and is expressed as the ratio of total tube length in each culture condition to the length in the culture of untreated (CTR) MVECs. Data are expressed as median (range) of six biological replicates (*p < 0.05, **p < 0.01). c Number of branching points expressed as mean ± SD of six biological replicates (*p < 0.05)
Fig. 2
a Western blot analyses (left) and the relative densitometric values (right) of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF)-A, and VEGF-A165b. Densitometric data are representative of three technical triplicates and are expressed as mean ± SD. The values of protein synthesis obtained for each treatment (bosentan [BOS] and macitentan [MAC]) were normalized to that of the untreated cells (CTR) taken as unit value by definition. Analysis of variance and Tukey’s multiple-comparisons test were performed for each group (*p < 0.05, **p < 0.01). b Quantitative real-time polymerase chain reaction analyses for CD31 (top), VEGF-A (middle), and VEGF-A165b (bottom) confirm the results observed in Western blot analysis. The results are expressed as median (range) of six biological replicates (*p < 0.05, **p < 0.01). mRNA Messenger RNA; EndoMT Endothelial-to-mesenchymal transition
Fig. 3
a Western blot analyses (left) and the relative densitometric values (right) of alpha smooth muscle actin (α-SMA), collagen type I (Col I) and transforming growth factor beta (TGF-β). Densitometric data are representative of three technical replicates and are expressed as mean ± SD. The values of protein synthesis obtained for each treatment (bosentan [BOS] and macitentan [MAC]) were normalized to that of the untreated cells (CTR), taken as unit value by definition. Analysis of variance and Tukey’s multiple-comparisons test were performed in each group (*p < 0.05, **p < 0.01). b Quantitative real-time polymerase chain reaction analyses for α-SMA (top), Col I (middle), and TGF-β (bottom) confirm results obtained by Western blot analysis. The results are expressed as median (range) of six biological replicates (*p < 0.05, **p < 0.01). mRNA Messenger RNA; EndoMT Endothelial-to-mesenchymal transition
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