Background/Purpose:

The accumulation of myofibroblasts in fibrotic tissue plays an important role in the pathogenesis of scleroderma (SSc) fibrosis. Recent studies have shown that myofibroblasts can originate from endothelial cells through Endo-MT. TGFβ is involved in the generation of tissue fibrosis through multiple pathways including Endo-MT. MicroRNA-126 (miR-126) is expressed mainly in microvascular endothelial cells (MVECs), and has been shown to inhibit TGFβ induced FoxO3/Smad 4 signaling by direct repression of phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2), a negative regulator of the PIK3/Akt signaling pathway. In this study we investigated the expression levels of miR-126 and TGFβ Endo-MT responses in SSc and control MVECs.

Methods:

MVECs were isolated from involved SSc skin and matched healthy subjects. The expression of miR-126 was detected by qPCR and in situ hybridization followed by quantitative densitometry analysis. Endo-MT was induced by TGFβ1 (10ng/ml) for 48 hours and assessed by expression levels of Mesenchymal Mark genes which include CNN1(Sm-Calp), Collagen type1 (COL1), fibronectin (FN), NOTCH3 and vimentin (VIM) using qPCR. The expression levels of PIK3R2 were examined by qPCR and western analysis. The expression of miR-126 was inhibited in control-MVECs by transfecting cells with hsa-miR-126 inhibitor and enhanced in SSc-MVECs by transfecting cells with hsa-miR-126 Mimic.

Results:

miR-126 expression levels in SSc-MVECs and skin biopsies were significantly down regulated by over 10 fold in SSc-MVECs and 2.32 fold in SSc skin when compared to control. Co-localization of miR-126 and endothelial specific marker CD31 were also observed in skin biopsies. The base line expression of mesenchymal marker genes was similar in SSc and control-MVECs. Addition of TGFβ to control MVECs resulted in increased mRNA expression levels of FN (1.75 folds ± 0.12), COL1 (2.11 folds±0.16), SM-calponin (1.65 folds ±0.10). Whereas an enhanced responses to TGFβ were seen in SSc-MVECs with 3.52 fold ± 0.32 for FN, 4.63 fold ± 0.50 for COL1 and 8.66 fold ± 0.68 for Sm-Calp. Control-MVECs transfected with miR-126 inhibitor repressed miR-126 expression levels by 78% for up to 96 hours as measured by qPCR. This was associated with significant upregulation of mRNA and protein expression levels of PIK3R2 and enhanced TGFβ-induced COL1, FN and Sm-Calp mRNA expression levels. Whereas overexpression of miR-126 by hsa-miR-126 Mimic transfection to SSc-MVECs resulted in upregulation of miR-126 expression levels and reduced expression of PIK3R2, it also resulted in significantly decreased TGFβ1-induced COL1, FN and Sm-Calp mRNA expression.

Conclusion:

The data demonstrate that miR-126 is a crucial regulator of TGFβ- induced Endo-MT. Down-regulation of microRNA-126 in SSc-MVECs enhances Endo-MT induced by TGFβ by directly targeting PIK3R2. Inhibition of miR-126 in control-MVECs resulted in increased Endo-MT responses to TGFβ, whereas forced expression of miR-126 in SSc-MVECs reduced TGFβ-Endo-MT responses. Upregulation of MiR-126 expression may be an effective therapeutic strategy in SSc and other fibrosis diseases in which Endo-MT plays a pathogenetic role.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/down-regulation-of-microrna-126-in-scleroderma-microvascular-endothelial-cells-enhances-the-transition-of-endothelial-to-mesenchymal-cells-endo-mt-induced-by-tgf%ce%b2-through-down-regulating-pi3ak/