Background: Diabetic foot ulcer (DFU) is a serious microvascular complication in diabetic patients with significant morbidity and mortality. The prevalence of chronic wounds continues to be a key focus of wound care research. Prolonged hyperglycemia-induced slow lymphangiogenesis associated with chronic oxidative stress is a major hallmark for poor wound closure in diabetic patients. Nrf2, a stress-activated transcription factor, combat oxidative stress by increasing antioxidant defense in many diseases, including diabetes. Recently we have reported the association of Nrf2 on the regulation of angiogenesis in DFU patients.
Aim: This study aims to decipher the role of miR27b on the regulation of angiogenesis in DFU through Nrf2 signaling and further validation using in vitro system.
Method: This study recruited a total of 20 patients with acute DFU and chronic DFU with no other diabetic complications. The expression of miR27b, Nrf2, HO1, SDF-1a and VEGF were studied in the tissues biopsies of study subjects using qPCR. The in vitro studies were carried out using human endothelial cells, EA.hy926 (ECs), exposed to a hyperglycemic microenvironment (High glucose with cytokine cocktail) for 24 hrs. The role of miR27b in regulating angiogenesis was demonstrated using antisense oligonucleotide targeting miR27b. Further, the association of Nrf2 with miR27b was studied using small molecule Nrf2 activators under the hyperglycemic microenvironment.
Results: The wound size, levels of CRP, and WBC counts were significantly higher in chronic than in acute DFU. We found a significant reduction in Nrf2, HO1, SDF-1α, and VEGF gene expressions and miR27b in chronic DFU subjects compared with acute DFU subjects. Further, the ECs under hyperglycemic microenvironment also showed reduced expressions of miR27b, Nrf2, HO1 and other angiogenic markers. The knockdown of miR27b using antisense revealed the positive regulation of miR27b in angiogenesis. Further, we observed a significant up-regulation of miR27b in ECs exposed to small molecule Nrf2 activators such as pterostilbene and resveratrol under a hyperglycemic microenvironment.
Discussion: Angiogenesis is the crucial step in the wound healing process which is impaired in DFU. Our results were consistent with a few earlier reports suggesting a reduced level of angiogenic markers such as SDF-1α and VEGF with the progression of DFU. We also found a decreased expression of the master regulator Nrf2 in the progression of DFU. Further, we have studied miR27b mediated Nrf2 regulation in tissue biopsies of chronic DFU patients and confirmed by in vitro studies. Inhibiting miR27b by antisense in ECs decreased angiogenic signaling, suggesting its positive role in wound healing. Of note, we confirmed the association of miR27b with the Nrf2 loop by exposing ECs with Nrf2 activators and ameliorated the dysregulated angiogenic signaling. In conclusion, our findings from this study have shown miR27b as an upstream target of Nrf2 that regulates angiogenesis in DFU through Nrf2.
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