Novel microRNA pathways of impaired angiogenesis in human peripheral artery disease adipose tissue

Abstract

Background: Impaired angiogenic response in peripheral artery disease (PAD) contributes to the progression of tissue ischemia, but methods to evaluate angiogenesis at the tissue level are limited. We describe a novel approach to measure angiogenesis and identify microRNA (miR)-gene pathways utilizing adipose tissue from patients with PAD. Methods: Patients with PAD undergoing infrainguinal bypass surgery or non-PAD control patients undergoing knee replacement surgery were recruited. Subcutaneous adipose tissue was obtained at the time of surgery. In patients with PAD, adipose tissue was taken from both the proximal and the distal ends of the surgical incision. Angiogenic capacity was measured and miR sequencing was performed. Differentially expressed miRs were defined by p < 0.01 and log₂ (fold change) > 1 or < –1. The miRs that correlated with angiogenic capacity and their gene targets were identified. Results: Participants with PAD (n = 10) and control participants (n = 5) were recruited. Capillary sprouting was impaired in distal (p = 0.0014) but not proximal (p = 0.12) PAD adipose tissue. In a subset of samples (controls: n = 4, PAD: n = 6), miR sequencing revealed 56 differentially expressed miRs in distal PAD. Six miRs with a correlation to impaired capillary sprouting and related to angiogenesis using Qiagen Ingenuity Pathway Analysis (IPA) software were identified (miRs 144-3p, 15b-5p, 18b-5p, 20b-5p, 454-3p, and 363-3p). These miRs are predicted to target regulators of angiogenesis including vascular endothelial growth factor A (VEGFA), phosphatase and tensin homologue (PTEN), and cyclin-dependent kinase inhibitor 1A (CDKN1A). Conclusion: Evaluation of ischemic adipose tissue represents a novel approach to gain insight into impaired angiogenesis in PAD. Integration with miR sequencing and target analysis has the potential to identify novel pathways of impaired angiogenesis in PAD.