Inhibition of microRNA-181c-5p rescues diabetes-impaired angiogenesis through activation of key mediators of angiogenesis, cell survival and novel genes, elmo3 and trib1
Authors: Solly E L , Bouman Chen Z , Luo Y , Mulangala J , Di Bartolo B A , Nicholls S J , Psaltis P J , Bursill C A , Tan J T M 
Published: October 2024

Abstract

Introduction
Patients with diabetes have impaired angiogenesis and poor coronary collateral vessel formation post-myocardial infarction (MI), which associates with higher mortality. There is a significant unmet clinical need for new agents that stimulate angiogenesis in response to ischaemia in diabetes. We have identified that miR-181c-5p has anti-angiogenic properties, but it’s role in diabetes remains unknown.

Aim
To elucidate the role of miR-181c-5p in diabetes-impaired angiogenesis.

Methods
Human coronary artery endothelial cells were transfected with a miR-181c-5p inhibitor (antimiR-181c-5p) or negative control (antimiR-Neg), exposed to glucose (5–25mM, 48h) then underwent Matrigel tubulogenesis assay or Boyden Chamber migration assay. Levels of proteins important for angiogenesis (e.g., VEGFA, p-ERK2, p-eNOS) were determined by Western Blot. Whole transcriptome sequencing was performed in vitro to identify novel gene targets of miR-181c-5p. In vivo, diabetic mice underwent hindlimb ischaemia or wound healing surgery and were injected with antimiR-181c-5p or antimiR-Neg. Tissues were extracted early (day 3) and late (days 10-14) post-surgery. Hindlimb blood-flow reperfusion was measured by Laser Doppler imaging. Hindlimb apoptosis was assessed by TUNEL and necrotic area was assessed by H&E. Wound area was calculated daily. Neovascularisation was assessed by CD31 (capillaries) and α-actin (arterioles) immunostaining.

Results
Inhibition of miR-181c-5p increased endothelial tubule number (+28%, P&lt;0.01), tubule length (+12%, P&lt;0.01) and cell migration (+67%, P&lt;0.05). This associated with increased VEGFA (+21%, P&lt;0.05) and p-ERK2 (+32%, P&lt;0.05). Whole transcriptome and pathway analysis revealed changes to angiogenesis pathways and identified a first-time involvement of genes Elmo3 and Trib1 in the pro-angiogenic action of antimiR-181c-5p in diabetes. In vivo, inhibition of miR-181c-5p increased blood flow reperfusion to ischaemic hindlimbs (+30%, P&lt;0.001) and arteriolar density (+45%, P&lt;0.05) in diabetic mice. Mechanistically, this was associated with early changes to mediators of angiogenesis, Erk2 mRNA (+35%, P&lt;0.05), p-ERK2 (+35%, P&lt;0.05) and Trib1 mRNA (+80%, P&lt;0.05); cell survival, Bcl-2 mRNA (+44%, P&lt;0.05); and late apoptotic clearance, Elmo3 mRNA (+57%, P&lt;0.001). Furthermore, this was also associated with an increase in late stage hindlimb apoptosis (+94%, P&lt;0.05) and reduced necrotic area (-90%, P&lt;0.05) in diabetic mice. Inhibition of miR-181c-5p increased diabetic wound closure (+22%, P&lt;0.01), wound capillaries (+61%, P&lt;0.05), Bcl-2 mRNA (+52%, P&lt;0.05) and Elmo3 mRNA (+50%, P&lt;0.05) in diabetic wounds.

Conclusions
Inhibition of miR-181c-5p rescues diabetes-impaired angiogenesis by activation of angiogenesis and cell survival mediators, and through novel genes, Trib1 and Elmo3. Our findings have implications for a novel miRNA-based strategy that improves myocardial neovascularisation and the prognosis of diabetic patients post-MI.