Saturday, September 7, 2024
9/7/2024
Peripheral artery disease (PAD), an arterial occlusive disease that impedes blood flow to the lower extremities, can develop into critical limb ischemia (CLI), characterized by chronic ischemic rest pain with high risk for amputation and cardiovascular complications. Patients with diabetes are disproportionally afflicted by both PAD and CLI with limited medical interventions to improve limb perfusion. Angiogenesis is impaired in diabetic patients and the mechanisms controlling this process are not fully understood. MicroRNAs (miRNAs) are small, non-coding RNAs capable of repressing gene expression and are involved in a variety of pathophysiological processes with important therapeutic potential, though their role in angiogenic signaling pathways in diabetic CLI remains poorly defined. Because miRNAs exhibit high conservation across species, sequencing of miRNAs from the plasma of human subjects with diabetes and increasing severity of PAD and diabetic mice with limb ischemia was used to identify overlapping, new miRNA targets including a top candidate miR-130b. Preliminary gain and loss-of-function studies revealed that miR- 130b overexpression rapidly promoted proliferation, migration, and sprouting in endothelial cells (ECs), whereas miR-130b inhibition exerted anti-angiogenic effects. ECs exposed to high glucose downregulated miR-130b and co-transfection of miR-130b under high glucose conditions accelerated EC wound closure. Local delivery of miR-130b mimics into ischemic muscles of diabetic db/db mice following femoral artery ligation (FAL) promoted revascularization by increasing angiogenesis and markedly improved limb necrosis and amputation. Mechanistically, overlapping downregulated transcripts from RNA-seq and miRNA prediction algorithms identified that miR-130b directly targeted and repressed inhibin-b-A (INHBA), a subunit involved in the formation of activin A, and downstream Smad2 signaling. Indeed, ectopic delivery of siRNA targeting Inhba in db/db ischemic muscles following FAL improved revascularization and limb necrosis, recapitulating the phenotype of miR-130b delivery. These observations provide the foundation for the central hypothesis that the miR-130b-INHBA signaling axis may serve as a critical regulator of EC angiogenic responses for patients with PAD and diabetes at risk of developing CLI. To better understand the precise role of miR-130b in INHBA signaling and angiogenesis, we will in Aim1 delineate the molecular basis for miR-130b’s ability to regulate INHBA signaling and EC angiogenic functions. In Aim2, we will determine the effect of altered miR-130b expression in experimental critical limb ischemia in diabetic mice. Finally, in Aim3 we will assess the expression of the miR-130b-INHBA signaling axis in a unique cohort of human subjects with CLI with or without diabetes. Our studies will address a major gap in our understanding of diabetic CLI and inform how miR-130b- INHBA mediated control of EC angiogenic functions may provide new targets for therapy.
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