Abstract
Purpose :
Complement activation and deposition has been reported in the eyes of diabetic retinopathy (DR) patients leading to pro-inflammatory changes that may play a role in retinal vascular damage. The initial factors contributing to complement activation are not well understood. Exosomes, membrane vesicles (40-200nm) that are secreted into extracellular environment were recently shown to contribute to pro-inflammatory changes in other tissues. This study was designed to elucidate the role of exosomes in activation of complement pathway contributing to pro-inflammatory changes in the pathogenesis of DR.
Methods :
Exosomes from control or STZ induced diabetic C57/B6 mice and Wistar rats were isolated via ultracentrifugation method, and further purified via density gradient separation. To quantify the exosomes, Static Light Scattering (SLS) was used. Classical complement activation was performed in vitro using C1 activation assay and C1S activity was measured by Western blot. Similar analyses were used to investigate the exosomes from plasma of control and type II diabetic patients.
Results :
After removal of 80% of the exosome from the rat plasma we found a 60% reduction of immunoglobulin compared with whole plasma. This indicates that most of the immunoglobulins in circulation bind to the exosomes. In the mouse model, we found a two folds (paired t-test, p<0.05) increase in the amount of immunoglobulin in diabetic compared to control exosomes (n=3) under equal loading conditions. Moreover, purified immunoglobulin-containing human plasma exosomes induced C1S activation and inhibition of C1 led to a reduction of complement activation by 9±2 fold (n=3)
Conclusions :
Our results demonstrate that there is higher amount of circulating immunoglobulins bound to exosomes in diabetes compared to control. Additionally, immunoglobulin-containing exosomes in circulation activate classical complement pathway. We propose that increased level of immunoglobulin on diabetic plasma exosomes may trigger activation of downstream complement pathway and production of pro-inflammatory anaphylatoxin contributing to the development of DR.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.