Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis

J Cell Mol Med. 2021 Jul;25(14):7078-7088. doi: 10.1111/jcmm.16727. Epub 2021 Jun 17.


Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA-126 (miR-126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty-related protein-1 (SPRED1), and phosphoinositide-3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR-126 expression was measured by qPCR and in situ hybridization. Matrigel-based tube assembly was used to test angiogenesis. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. Epigenetic regulation of miR-126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR-126 promoter region. MiR-126 expression, as well as EGFL7 (miR-126 host gene), in SSc-MVECs and skin, was significantly down-regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR-126 in NL-MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR-126 in SSc-MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR-126 and EGFL7 expression levels in SSc-MVECs. Heavy methylation in miR-126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down-regulation of miR-126 results in impaired VEGF responses.

Keywords: VEGF; angiogenesis; endothelial cells; epigenetics; microRNA-126.

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Cells, Cultured
  • DNA Methylation
  • Down-Regulation
  • EGF Family of Proteins / genetics
  • EGF Family of Proteins / metabolism
  • Endothelial Cells / metabolism*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism
  • Epigenesis, Genetic*
  • Humans
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Neovascularization, Physiologic
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Scleroderma, Systemic / genetics
  • Scleroderma, Systemic / metabolism*
  • Scleroderma, Systemic / pathology
  • Vascular Endothelial Growth Factor A / genetics*
  • Vascular Endothelial Growth Factor A / metabolism


  • Adaptor Proteins, Signal Transducing
  • Calcium-Binding Proteins
  • EGF Family of Proteins
  • EGFL7 protein, human
  • MIRN126 microRNA, human
  • MicroRNAs
  • SPRED1 protein, human
  • Vascular Endothelial Growth Factor A
  • phosphoinositol-3 kinase regulatory subunit 2, human