The Anti-Inflammatory and Pro-Angiogenic Capacity of High-Density Lipoproteins in Diabetic Wound Healing

Abstract

Diabetes is the one of the most common metabolic disorders and is associated with a range of complications including diabetic foot ulcers (DFU) [1]. It is estimated that 25% of patients with diabetes develop DFU and of these a quarter do not heal resulting in amputation. This places them at risk of lower limb amputation [1]. There are currently no therapies that actively promote wound healing. It is increasingly being recognised that to improve biologically complex wound healing, an effective therapy requires pleiotropic actions that targets multiple facets of impaired wound healing such as prolonged inflammation and poor revascularisation. High-density lipoproteins (HDL) possess significant wound healing properties and regulate multiple important wound healing mechanisms including anti-inflammatory and pro-angiogenic effects. HDL has traditionally been viewed as an atheroprotective protein, with a well-established inverse relationship between plasma HDL-cholesterol levels and the risk of myocardial infarction (MI). Efforts to pharmacologically raise HDL in large-scale clinical trials have failed to show benefit on MI risk. This redirected the focus from HDL-cholesterol levels to HDL functionality as a better predictor of disease. The relationship between HDL functionality and diabetic wound healing is yet to be explored. In this thesis we aimed to 1) determine the anti-inflammatory properties of topically applied reconstituted HDL (rHDL) in a murine model of wound healing and to track its fate, 2) compare changes in the functionality of HDL in patients with and without diabetes post-toe/s amputation over time, and 3) determine the relationship between HDL functionality and wound closure in patients with and without diabetes and an acute toe amputation/s. Firstly, we utilised a murine model of diabetic wound healing to assess the effect of topical rHDL on wound inflammation. Diabetic mice had delayed wound healing and higher levels of wound macrophages, when compared to non-diabetic mice. Topical application of rHDL completely rescued diabetes-impaired wound healing but had no effect on wound macrophage content. There were, however, significant reductions in the mRNA levels of inflammatory mediators Rela and Ccl2, and CCL2 protein in diabetic wounds following topical rHDL, 72hrs post-wounding. Overall, this study showed topical rHDL exhibits anti-inflammatory effects in diabetic wounds. We next conducted a clinical study that determined changes in HDL functionality of patients with and without diabetes at the time of amputation (baseline), 1- and 6-months post amputation/s and tracked wound closure. We found that HDL isolated from patients with diabetes had impaired cholesterol efflux, anti-inflammatory and pro-angiogenic functionality, when compared to non-diabetic HDL at 1- and 6-months post-toe amputation/s. We identified a significant positive correlation between HDL-cholesterol levels and the rate of wound closure. Furthermore, there were significant negative correlations between the anti-inflammatory effects of HDL for Ccl2 and Icam1, and the rate of wound healing in patients with diabetes 1-month post-amputation. In conclusion, this thesis has demonstrated that topical rHDL has anti-inflammatory effects in diabetic wounds. This adds to its already well-characterised pro-angiogenic effects, making rHDL a potential wound healing therapy with important pleiotropic properties. Our clinical study revealed that HDL from patients with diabetes and toe amputations had impaired functionality. Furthermore, we identified for the first time that the anti-inflammatory functionality of HDL inversely correlated with wound closure rate, highlighting its potential as new predictive marker of healing. In conclusion, rHDL presents as an exciting new topical agent for improving wound healing in people with diabetes, and the anti-inflammatory capacity of endogenous HDL shows potential as a predictive biomarker for diabetic wound healing outcomes.