Vascular breakdown in diabetic retinopathy: Role of connexin43 and possible interventions

Abstract

Background: Diabetic retinopathy (DR) is a chronic vascular disease of the retina that can lead to vision loss. Current DR therapies, including anti-vascular endothelial growth factors, mainly target late-stage vascular defects and long-term treatments have been shown to contribute to geographic atrophy and retinal ganglion cell death. Therefore, alternative treatments that target upstream disease mechanisms are needed. Recent studies have shown that connexin43 hemichannels play a role in the pathogenesis of chronic inflammatory diseases and blocking these channels can alleviate vascular leak and inflammation associated with these conditions. However, there is conflicting literature with regards to connexin43 expression in DR. This is believed to be because most animal models used are hyperglycaemia-only models that do not take the significance of inflammation in the disease pathogenesis into account. Therefore, the main aim of this thesis was to develop a new DR model that combines both hyperglycaemia and inflammation and use it to investigate the role of connexin43 hemichannels in the disease pathology. Methods: Using immunohistochemistry, the effect of hyperglycaemia and inflammation on connexin43 expression patterns was evaluated in primary human microvascular endothelial cells as well as in Akita (Ins-/-) and Akimba (Ins-/-,hVEGF+/-) mouse models compared to human donor retinas with confirmed DR diagnosis. Using a retinal pigment epithelial cell line, the role of connexin43 hemichannels was then evaluated in terms of pro-inflammatory cytokine and ATP release as well as expression of inflammatory proteins, in particular to resolve the role of connexin43 hemichannels in the innate immune system inflammasome pathway. Building upon this work, a new model of DR was created by intravitreally injecting pro-inflammatory cytokines, IL-1β and TNF-α, into non-obese diabetic (NOD, hyperglycaemic) mice. The effect of connexin43 hemichannel block to prevent the development of DR signs was studied by administering a well characterised connexin43 hemichannel blocker, Peptide5, at the same time as the pro-inflammatory cytokines. Retinas were evaluated using in vivo ocular assessment techniques and immunohistochemistry. Results: Connexin43 levels increased in human donor retinas with confirmed DR diagnosis correlating with the expression patterns seen in in vitro and in vivo models only when hyperglycaemic and inflammatory processes were combined. Blocking connexin43 hemichannels using Peptide5 prevented the amplification and perpetuation of inflammation (in particular the NLRP3 inflammasome pathway) by blocking the ATP autocrine feedback loop that activates the inflammasome/inflammation cycle. NOD mice intravitreally injected with the pro-inflammatory cytokines developed vascular leak and oedema accompanied by pronounced inflammation of the retina, vitreous and optic nerve, signs that have previously been reported as advanced signs of DR. Intravitreal Peptide5 treatment resulted in reduced vascular leak, oedema and inflammation supporting the role of connexin43 in the disease pathogenesis. Conclusions: Taken together, the findings presented in this thesis have proven that the pathology of DR most likely arises from a combination of hyperglycaemia and inflammation. More significantly, connexin43 hemichannels play a role in the pathogenesis of DR such that connexin43 hemichannel block should be considered as a potential upstream approach for DR treatment.