Background/Purpose: Impaired angiogenesis in SSc is a crucial component of disease pathology that occurs in spite of upregulation of VEGF and other proangiogenic factors. MicroRNA-126 (miR-126) is expressed mainly in MVECs throughout the capillaries. MiR-126 regulates angiogenic signaling and responses to VEGF by direct repression of negative regulators of VEGF signalling pathway, including the Sprouty-related protein-1 (Spred1), and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2). In this study we investigated the expression levels of miR-126 and VEGF responses in SSc and control MVECs.

Methods: <span’arial’,’sans-serif’;”>MVECs were isolated from involved SSc skin and matched healthy subjects. The expression of miR-126 was measured by qPCR. The expression levels of VEGF inhibitors Spred1 and PIK3R2 were examined by qPCR and western analysis. The angiogenic potentials of MVECs were tested in a matrigel-based tube formation assay and in scratch test migration assay. <span’arial’,’sans-serif’;”>The expression of miR-126 was inhibited in control-MVECs by transfecting cells with has-miR-126 inhibitor and enhanced in SSc-MVECs by transfecting cells with has-miR-126 Mimic. 

Results:   miR-126 expression levels in SSc-MVECs and exosomes were significantly down regulated by over 10 folds in SSc-MVECs and 5 folds in SSc-exosomes (1.2 x10⁷ miR-126 molecules/1million RNU44 molecules in control-MVECs and 8×10⁵ in SSc-MVECs). Increased mRNA expression levels of Spred1 (2.5 folds ±0.2) and PIK3R2 (3.4±0.3 folds) in SSc-MVECs were noted by qPCR and confirmed by western analysis. Addition of VEGF (50ng/ml) to control-MVECs resulted in robust tube formation with and average branch length of (246.83±28.69 um) whereas diminished responses were seen in SSc-MVECs (88.58±15.46uM). Addition of VEGF enhanced control-MVECs migration and resulted in 50% wound closure in 6 hours, while this response was significantly diminished in SSc-MVECs (5%).  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 Spred1 and PIK3R2 and reduced angiogenic capacity to levels comparable to that of SSc-MVECs. has-miR-126 Mimic transfection to SSc-MVECs resulted in upregulation of miR-126 expression, reduced expression of Spred1 and PIK3R2, and enhanced angiogenic potentials to levels comparable to control-MVECs.  

Conclusion: <span’arial’,’sans-serif’;”>The data demonstrate impaired angiogenic potential of SSc-MVECs and diminished response to VEGF that was associated with significant down regulation of miR-126 in SSc-MVECs. Inhibition of miR-126 in control-MVECs resulted in impaired responses to VEGF, whereas forced expression of miR-126 in SSc-MVECs upregulated VEGV responses and normalized the angiogenic potential of SSc-MVECs. The data suggest that down regulation of miR-126 play a major role in impaired angiogenesis potential of SSc-MVECs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/down-regulation-of-microrna-126-in-scleroderma-microvascular-endothelial-cells-mvecs-is-associated-with-impaired-responses-to-vascular-endothelial-growth-factor-vegf-and-defective-angiogenesi/