Poorly controlled complement activation is now associated with many pathologies, and recent evidence implicates complement dysregulation in the pathogenesis of several neovascular ocular diseases, including diabetic retinopathy. Although evidence that complement activation may contribute to vascular pathology is promising, crosstalk between the complement system and neovascularisation remains largely unclear. Taking a step back to understand the role of complemen in diabetic retinopathy would not only provide a more in-depth knowledge of the cellular and molecular mechanisms involved in disease pathogenesis, but it could also highlight novel therapeutic targets for DR and other vasoproliferative diseases in the eye.
The objectives of the studies presented in this thesis were to test to test the hypothesis that complement has a novel function in angiogenesis which is separate from its characterised role as an immune surveillance system within the ocular environment. This work is specifically focused on exploring the role of the central component of the alternative complement pathway, complement factor B in regulating retinal angiogenesis. Initially, CFB expression was characterised using rodent models and human patient samples. In the retina of STZ-induced diabetic mice there was no difference in CFB expression between buffer-injected controls and STZ-induced diabetic mice. Similarly, characterisation of systemic CFB levels in human patient serum samples revealed no differences between control and diabetic patients at different stages of retinopathy. However, analysis of ocular CFB levels in human aqueous samples, revealed a higher expression in diabetic patients with retinopathy compared to diabetic patients without. The well-established mouse model of oxygen induced retinopathy was carried out to study CFB expression in pathologic vessel formation. RT-qPCR analysis demonstrated that CFB was significantly upregulated in mice retina subject to oxygen-induced retinopathy. To establish the impact of CFB on retinal vascular cell function and angiogenesis, in vitro and ex vivo angiogenesis assays were carried out. Results demonstrate that CFB was able to promote human retinal endothelial cell viability, proliferation, tube formation, Transwell migration, and aortic ring sprouting, thus indicating a pro-angiogenic role of CFB. Finally, to elucidate the mechanism through which CFB exerts its pro-angiogenic function the relationship between CFB and the VEGF signalling system was investigated using in vitro cultures of human retinal endothelial cells. Observations from these preliminary mechanistic studies revealed that CFB mediates VEGF gene expression, and VEGFR2 gene and protein expression. And that the pro-angiogenic function of CFB is upstream of, and acts through the VEGF signalling pathway. Several reports have implicated complement and complement related proteins in the development of DR. However, few studies have investigated the more precise role played by CFB and the mechanism(s) by which complement mediates its tissue damaging effects. Collectively, results from this study clearly indicate a pro-angiogenic role of CFB in DR associated neovascularisation.
