Background/Purpose: SSc vasculopathy is characterized by endothelial injury and deficient endothelial-dependent vasodilation leading to occlusive and proliferative vascular outcomes. Platelet activation and elevated circulating platelet markers have long been observed in SSc. Moreover, dysregulated endothelial Nitric Oxide (NO) pathway is believed to be a major step in the pathogenesis of SSc vasculopathy. In this study, we examined platelet adhesion to microvascular endothelial cells (MVECs) and the epigenetic regulation involved in enhanced platelet adhesion, deficient endothelial nitric oxide synthase (eNOS) expression, and the crucial role played by microRNA-126 (miR-126) in this process. 

Methods: MVECs were isolated from involved SSc skin (n=7) and matched healthy subjects. Platelet adhesion to MVECs was determined by the Calcein AM method. The expression levels of eNOS, miR-126, and DNA methyltransferase-1 (Dnmt1) were measured by qPCR or Western blotting (WB). L-NAME was used as NO synthase antagonist. The effect of Dnmt1 on eNOS mRNA expression was examined by transfecting SSc-MVECs with Dnmt1 specific siRNA and irrelevant control siRNA. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. NOS3 promoter methylation was detected by bisulfite DNA sequencing. 

Results: MiR-126 expression levels were significantly downregulated by 6.48 ±1.22 folds in SSc-MVECs compared to control (P< 0.01). SSc-MVECs supported platelet adhesion at a higher level than control cells (10.16+/-2.8 platelet/ EC vs. 3.3 +/-0.94 in control cells, mean +/-SD, P< 0.001). Adding L-NAME to control MVECs resulted in enhanced platelet adhesion in a dose-dependent fashion. eNOS expression levels were significantly reduced in SSc-MVECs (mean 32% +/- 2.4 of normal values, P< 0.001), Dnmt1 expression levels were significantly higher in SSc-MVECs (2.3 folds +/- 0.2, < 0.001). eNOS underexpression in SSc cells was related to heavy DNA methylation of the promoter CpG islands as shown by promoter sequence analysis of DNA after bisulfite modification. Transfection of SSc-MVECs with siRNA specific for Dnmt1 resulted in 80% decreases in the expression levels in association with increased eNOS expression levels. Since Dnmt1 3’UTR contains a miR-126 binding site suggesting that miR-126 regulates DNA methylation by directly targeting Dnmt1. Thus, the upregulation of miR-126 in SSc-MVECs resulted in a significant reduction of Dnmt1 and upregulation of eNOS expression levels. While the inhibition of miR-126 expression levels in control MVECs resulted in enhanced Dmnt1 expression and decreased eNOS expression levels.

Conclusion: The data demonstrate that defective miR-126 expression in SSc-MVECs leads to upregulation of Dmnt1 expression and downregulation of eNOS expression that is associated with defective NO release and enhanced platelet/endothelial interaction.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigenetic-regulation-of-microrna-126-in-scleroderma-is-associated-with-upregulation-of-dna-methyltransferase-1-repression-of-endothelial-nitric-oxide-synthase-expression-and-enhanced-platelet-adhes/