Abstract
Purpose :
Complement activation and deposition reported in the eyes of diabetic retinopathy (DR) patients is thought to play a role in vascular damage. The initial factors contributing to complement activation in DR are, however, not well understood. Exosomes, membrane vesicles (40-200nm) that are secreted into extracellular environment, were shown to cargo complement proteins in plasma. This suggests that exosomes may participate in complement induced vascular damage. The present study aims to elucidate the role of plasma exosomes in activation of the complement pathway in DR pathogenesis.
Methods :
Exosomes were isolated from control or STZ-induced diabetic C57/B6 mice, Wistar rats, and patients’ plasma via ultracentrifugation or ExoQucik method, and further purified via density gradient separation. Static Light Scattering (SLS) was used to quantify the exosomes. Classical complement activation was measured in vitro by C1 activation assay and C1s activity was determined by Western blot. In vitro, rat control or diabetic tail artery plasma with or without exosomes were used to treat the human retinal endothelial cells (HREC). Cytotoxicity was measured by LDH assay and Trypan blue assay. MAC deposition on HREC was observed via immunofluorescence staining.
Results :
Most of plasma immunoglobulins were found to be bound to exosomes in mouse, rat and human blood. SLS and western blot for exosome makers quantification showed significantly higher number of plasma exosomes in diabetic mice (1.14 x10^11/mL) when compared to control (0.735x10^11/mL) (n=9, p<0.05). Importantly, diabetic mouse plasma had higher exosome-bound IgG levels when compared to control (n=9, p<0.05). Immunoglobulin bound to exosomes caused activation of the classical [JMB1] complement complex, C1. Treatment of HREC with diabetic rat plasma lead to cytotoxicity via MAC deposition. Moreover, removal of exosomes from diabetic plasma reduced HREC cytotoxicity and MAC deposition(n=3, p<0.05), and adding back the exosomes exacerbated cytotoxicity.
Conclusions :
Diabetes-induced increase in immunoglobulin-laden exosomes in circulation can activate classical complement pathway and induce retinal endothelial cells damage via MAC deposition, suggesting that exosomes play an important role in vascular damage in DR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.