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Published on: March 24, 2017
A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like
Vijay Chaitanya Ganta1, Min Hyub Choi1, Anna Kutateladze1
1From Cardiovascular Research Center (V.C.G., M.H.C., B.H.A.), Department of Biology (A.K.), Department of Pharmacology (T.E.F.), Department of Public Health Sciences (C.R.F.), and Department of Cardiology (B.H.A.), University of Virginia, Charlottesville.
Background:
Currently, no therapies exist for treating and improving outcomes in patients with severe peripheral artery disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and to reduce tissue loss in genetic PAD models. However, the cell-specific function, downstream mechanisms, or signaling involved in miR93-mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD, and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1 activation/polarization state. In the present study, we identified a novel mechanism by which miR93 regulates macrophage polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental PAD.
Methods:
In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation conditions) and in vivo experiments in preclinical PAD models (unilateral femoral artery ligation and resection) were conducted to examine the role of miR93-interferon regulatory factor-9-immunoresponsive gene-1 (IRG1)-itaconic acid pathway in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery.
Results:
In vivo, compared with wild-type controls, miR106b-93-25 cluster-deficient mice (miR106b-93-25-/-) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like macrophages after experimental PAD. Intramuscular delivery of miR93 in miR106b-93-25-/- PAD mice increased angiogenesis, arteriogenesis, and the extent of perfusion, which correlated with more M2-like macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like polarization even under M1-like polarizing conditions (hypoxia serum starvation). Delivery of bone marrow-derived macrophages from miR106b-93-25-/- to wild-type ischemic muscle decreased angiogenesis, arteriogenesis, and perfusion, whereas transfer of wild-type macrophages to miR106b-93-25-/- had the opposite effect. Systematic analysis of top differentially upregulated genes from RNA sequencing between miR106b-93-25-/- and wild-type ischemic muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage polarization. The 3' untranslated region luciferase assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not an miR93 target but that interferon regulatory factor-9, which can regulate IRG1 expression, is an miR93 target. In vitro, increased expression of interferon regulatory factor-9 and IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential.
Conclusions:
miR93 inhibits interferon regulatory factor-9 to decrease IRG1-itaconic acid production to induce M2-like polarization in ischemic muscle to enhance angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.
Insights
MicroRNA93 (miR93) promotes M2 macrophage polarization, enhancing blood vessel growth and perfusion in severe peripheral artery disease (PAD). This study reveals miR93
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Immunology
Background:
- Severe peripheral artery disease (PAD) lacks effective therapies.
- MicroRNA93 (miR93) shows potential in preclinical PAD models by modulating angiogenesis and reducing tissue loss.
- The precise mechanisms of miR93 in ischemic muscle neovascularization, particularly its role in macrophage polarization, remain unclear.
Purpose of the Study:
- To elucidate the cell-specific function and downstream signaling of miR93 in promoting angiogenesis and arteriogenesis in experimental PAD.
- To investigate the novel mechanism by which miR93 regulates macrophage polarization (M1/M2 state) to revascularize ischemic muscle.
Main Methods:
- In vitro studies using macrophages, endothelial cells, and skeletal muscle cells under normal and hypoxic conditions.
- In vivo experiments in preclinical PAD models involving unilateral femoral artery ligation and resection.
- Examination of the miR93-interferon regulatory factor-9 (IRF9)-immunoresponsive gene-1 (IRG1)-itaconic acid pathway's role in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery.
Main Results:
- miR106b-93-25 cluster-deficient mice exhibited reduced angiogenesis and arteriogenesis with increased M1-like macrophages in experimental PAD.
- Intramuscular delivery of miR93 in deficient mice enhanced angiogenesis, arteriogenesis, and perfusion, correlating with increased M2-like macrophages.
- miR93 was found to target IRF9, which regulates IRG1 and itaconic acid production, thereby modulating macrophage polarization and endothelial angiogenic potential.
Conclusions:
- miR93 inhibits IRF9, leading to decreased IRG1-itaconic acid production.
- This pathway induces M2-like macrophage polarization in ischemic muscle.
- Enhanced M2 polarization promotes angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.
