MicroRNA-181c-5p - A Novel Therapeutic Target for the Rescue of Diabetes-Impaired Angiogenesis

Abstract

Diabetes-related foot disease (DFD) accounts for up to 80% of all non-traumatic lower limb amputations and people with diabetes have a >25% lifetime risk of developing a diabetic foot ulcer (DFU). Half the patients with DFU have concurrent peripheral artery disease (PAD), which significantly increases the risk of non-healing ulcers with an 8-fold higher rate of major amputation. Despite current best practice treatment, an unacceptably high number of patients still experience devastating diabetic foot complications, highlighting an urgent need for new therapies to alleviate these health burdens. The complications of PAD and poor DFU healing are associated with impaired responses to ischaemia-driven angiogenesis. Therapeutic stimulation of angiogenesis holds promise in restoring blood supply and delivering cells and nutrients to the diabetic foot. However, despite significant effort and research, current single gene targeting therapies to stimulate angiogenesis have exhibited limited clinical efficacy. This is likely owing to the multifaceted and complex nature associated with regulating angiogenesis. MicroRNAs (miRNAs), small non-coding RNAs that post-transcriptionally regulate mRNA translation, can target multiple genes simultaneously and therefore may offer an advantage over current therapies due to their pleiotropic action. Our group previously identified an anti-angiogenic role for miR-181c-5p. Furthermore, we found that miR-181c-5p levels are strikingly elevated in Indigenous Australian males with diabetes-associated PAD, a population disproportionately impacted by diabetic vascular complications. Given the anti-angiogenic properties of miR-181c-5p, we suggested that elevated miR-181c-5p levels may contribute to diabetes-impaired angiogenic responses. However, the role of miR-181c-5p in diabetes-impaired angiogenesis remained unknown. This thesis hypothesised that miR-181c-5p is a critical driver of diabetes-impaired angiogenesis and that inhibition of miR-181c-5p will provide a novel treatment strategy for diabetic vascular complications. Using functional and mechanistic in vitro studies as well as pathophysiologically relevant pre-clinical murine surgical models in vivo, this thesis has identified, for the first time, an important role for miR-181c-5p in diabetes-impaired angiogenesis. We found that elevated miR-181c-5p levels early post-ischaemia/wounding associates with delayed angiogenic responses in diabetes. Excitingly, inhibition of miR-181c-5p increased the angiogenic propensity of human coronary artery endothelial cells (HCAECs) in vitro and rescued diabetes-impaired ischaemia-driven angiogenesis in vivo. This is shown to be mediated through restoration of key angiogenic pathways that are often impaired in diabetes. Specifically, miR-181c-5p inhibition increased the preferential phosphorylation of extracellular signal-regulated kinase 2 (ERK2), increased expression of the cell survival gene B-cell lymphoma 2 (Bcl-2) and increased endothelial nitric oxide synthase (eNOS) phosphorylation in a diabetic environment. Additionally, these results suggest that inhibition of miR-181c-5p may mediate multiple conduits, including VEGFA and Trib1, to facilitate ERK2 and eNOS activation, enhancing the induction of downstream angiogenic functional effects. In diabetic wounds, miR-181c-5p inhibition rescued diabetes-impaired wound closure and increased wound neovascularisation. Mechanistically, this was mediated through the regulation of genes that contribute to cell survival (Bcl-2) and aid in wound closure by clearing apoptotic cells from the wound sight (Elmo3) in the mid-late stages of wound healing. Additionally, we also found that miR-181c-5p inhibition regulated Elmo3 gene expression in the late stages post-ischaemic induction in diabetic hindlimbs. This highlights a potential novel role for Elmo3 in facilitating recovery across the two disease pathologies in diabetes and that this is mediated through miR-181c-5p inhibition. Collectively, our findings have identified an anti-angiogenic role for miR-181c-5p in diabetes and have demonstrated that the pleiotropic action of miR-181c-5p affects not only on angiogenesis pathways but also the time-dependent regulation of other molecules that may facilitate recovery and healing in diabetes. This presents miR-181c-5p as a novel therapeutic target for the treatment of diabetic vascular complications.