The University of Toledo Translation Journal of Medical Sciences 
Rheumatology Abstract, Department of Medicine Research Symposium UTJMS 2023 May 05; 11(1):e1-e1 

Downregulation of Microrna-126 in Scleroderma is 
Associated with Epigenetically Mediated Nitric Oxide 
Synthase Repression and Enhanced Platelet/Endothelial 
Interaction 
Yongqing Wang, PhD1* and Bashar Kahaleh, MD2

1Division of Pulmonology, Department of Medicine, The University of Toledo, 
Toledo, OH 43614 
2Division of Rheumatology, Department of Medicine, The University of Toledo, 
Toledo, OH 43614 

*Corresponding author: yongquing.wang2@utoledo.edu

Published: 05 May 2023 

Introduction: SSc vasculopathy is characterized by deficient endothelial nitric oxide (eNOS) and 
enhanced platelet adhesion to endothelial cells. In this study, we examined the epigenetic regulation 
involved in enhanced platelet adhesion, deficient eNOS expression, and the role of miRNA-126 in this 
process.  

Methods: Platelet adhesion to MVECs was determined by the Calcein AM method. eNOS, NOS3, miR-
126, DNA methyltransferase-1 (Dnmt1) expression were measured by qPCR and WB. L-NAME was 
used as NO antagonist. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by 
hsa-miR-126 Mimic.  

Results: MiR-126 expression levels were significantly downregulated in SSc-MVECs. 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). Addition of L-NAME to control MVECs resulted in enhanced 
platelet adhesion in a dose-dependent fashion. NOS3 expression levels were significantly reduced in SSc 
cells, Dnmt1 expression levels were significantly higher in SSc cells. NOS3 under expression 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. Upregulation of MiR126 in SSc MVECs resulted 
in the reduction of Dnmt1 and upregulation of NOS3 expression levels, while the inhibition of MiR 126 
expression levels in control MVECs resulted in decreased NOS3 levels and enhanced Dmnt1 levels.  

Conclusion: The data demonstrate that defective miR-126 expression in SSc MVES leads to 
upregulation of Dmnt1 expression and downregulation of NOS3 expression that is associated with 
defective NO release and enhanced platelet/endothelial interaction. 

https://dx.doi.org/10.46570/utjms.vol11-2023-621 

https://dx.doi.org/10.46570/utjms.vol11-2023-621
mailto:yongquing.wang2@utoledo.edu