The dual functional anti-angiogenic and anti-inflammatory peptide DA4 as a therapeutic against diabetic retinopathy

Abstract

Diabetic retinopathy (DR) has been suggested to be the most common microvascular complication of diabetes, and is the leading cause of vision loss and blindness among adults, worldwide. In 2020 there were an estimated 103.1 million adults with DR, with cases projected to rise to 160.5 million by 2045. Due to a globally aging population, increased life expectancy of those with diabetes, and increased risk for diabetes due to lifestyle and dietary trends, the burden of DR is only expected to increase in the coming years. Thus, there is a pressing need for the discovery and development of novel and effective therapies against DR. Snake venoms are regarded as a rich natural source for the discovery of biologically active molecules with diverse effects. In our work, we first set out to mine the anti-angiogenic potential of a novel peptide, DA4, isolated from the venom of the snake Deinagkistrodon acutus. We demonstrated that DA4 exhibited potent in vitro and in vivo anti-angiogenic effects. Mechanistically, the anti-angiogenic activity of DA4 was proposed to involve induction of TIMP3, which inhibited VEGF signaling, and downstream cell migratory and cell proliferative mediators such as Erk1/2, eNOS, p38, LIMK/cofilin, and Hsp27. PPP3R2 and SH2D2A, angiogenic components of the VEGF-induced signaling cascade, were also downregulated by DA4. Due to the inter-related nature of angiogenesis and inflammation, in the second part of our study, we investigated the potential anti-inflammatory effects of DA4. In vitro, we demonstrated that DA4 could inhibit major pro-inflammatory signaling pathways, including the NF-kB, MAPK, and STAT3 signaling pathways. Further, we showed that DA4 could inhibit the NLRP3 inflammasome signaling pathway, via suppression of NLRP3 gene expression. Through experiments in two different inflammation mouse models, we demonstrated the in vivo anti-inflammatory efficacy of DA4, which was comparable to other commonly used chemical inhibitors of inflammation. In the third part of the study, we investigated the therapeutic efficacy of DA4 against diabetic retinopathy, a disease where both angiogenesis and inflammation are of key importance. DA4 showed therapeutic efficacy in three different diabetic mouse models, with amelioration of key DR-related characteristic features. The anti-angiogenic and anti-inflammatory activity of DA4 was also confirmed in the retinas of DR model mice. Although retinal cell and visual function were not shown to be improved by treatment with DA4, no deleterious effects were seen. In the last part of the study, we developed and optimized a bacteria-based platform for production and purification of recombinant DA4. Functional assessments indicated that the anti-angiogenic and anti-inflammatory activity of purified recombinant DA4 was comparable to naturally isolated DA4, indicating the viability for its use in downstream in vitro and in vivo studies. Altogether, the work in this thesis presents for the first time, the anti-angiogenic and anti-inflammatory effects of the novel snake venom peptide, and its therapeutic potential against diabetic retinopathy.