C3a Receptor Inhibition Protects Brain Endothelial Cells Against Oxygen-glucose Deprivation/Reperfusion

Abstract

The complement cascade is a central component of innate immunity which plays a critical role in brain inflammation. Complement C3a receptor (C3aR) is a key mediator of post-ischemic cerebral injury, and pharmacological antagonism of the C3a receptor is neuroprotective in stroke. Cerebral ischemia injures brain endothelial cells, causing blood brain barrier (BBB) disruption which further exacerbates ischemic neuronal injury. In this study, we used an in vitro model of ischemia (oxygen glucose deprivation; OGD) to investigate the protective effect of a C3aR antagonist (C3aRA, SB290157) on brain endothelial cells (bEnd.3). Following 24 hours of reperfusion, OGD-induced cell death was assessed by TUNEL and Caspase-3 staining. Western blot and immunocytochemistry were utilized to demonstrate that OGD upregulates inflammatory, oxidative stress and antioxidant markers (ICAM-1, Cox-2, Nox-2 and MnSOD) in endothelial cells and that C3aRA treatment significantly attenuate these markers. We also found that C3aRA administration restored the expression level of the tight junction protein occludin in endothelial cells following OGD. Interestingly, OGD/reperfusion injury increased the phosphorylation of ERK1/2 and C3aR inhibition significantly reduced the activation of ERK suggesting that endothelial C3aR may act via ERK signaling. Furthermore, exogenous C3a administration stimulates these same inflammatory mechanisms both with and without OGD, and C3aRA suppresses these C3a-mediated responses, supporting an antagonist role for C3aRA. Based on these results, we conclude that C3aRA administration attenuates inflammation, oxidative stress, ERK activation, and protects brain endothelial cells following experimental brain ischemia.

Keywords: Inflammation; Ischemia; OGD; Oxidative stress; bEnd.3 cells.

Fig. 1. C3aRA administration attenuates C3aR expression level after OGD. A-D) C3aR expression was observed by immunohistochemistry and Western blot. Data shown are the mean±SEM (n=5). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 2. C3aRA treatment significantly reduces apoptotic cell death following OGD. A~D) TUNEL and Caspase-3 staining was evaluated by immunohistochemistry. Data shown are the mean±SEM (n=5). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 3. C3aRA treatment attenuates ICAM-1 expression following OGD. A) Effect of C3aR inhibition on ICAM-1 expression in the OGD induced b.End.3 cells determined by Immunohistochemistry, Scale bar: 50 µm. B) ICAM-1 color intensity was measured by Image J. The results represent the means±SEM of fold changes (n=5). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 4. C3aRA treatment reduces expression of Cox-2 in bEnd.3 cells following OGD. A) C3aRA significant reduces Cox-2 expression in OGD samples by immunocytochemistry. B) Cox-2 color intensity was measured by Image J. The results represent the means±SEM of fold changes (n=5). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 5. Effect of C3aR inhibition on gp91-phox and MnSOD expression level in endothelial cells following OGD. A) gp91-phox expression was determined by western blotting. B) gp91-phox densitometry analysis was done by Image J. C) Representative western blot showing endothelial expression of MnSOD. D) MnSOD densitometry analysis was done by Image J. The results represent the means±SEM of fold changes (n=4). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 6. C3aRA treatment attenuates tight junction protein occludin and pERK1/2 expression in endothelial cells against OGD. A) Endothelial expression of occludin was observed by Western blot. B) Occludin densitometry analysis was done by Image J. C). pERK and ERK expression assessed by Western blot. D) ERK densitometry analysis was done by Image J software (NIH). The results represent the means±SEM of fold changes (n=4). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, control group; OGD, ischemic group; Cont+C3aRA, drug control; OGD+C3aRA, drug treatment.

Fig. 7. C3a administration alone increases the expression of Caspase3, ICAM-1 and pERK ½ in bEnd.3 cells but not C3aR following OGD. A~H) C3a treatment significant enhanced the Caspase3, ICAM-1 and pERK level with OGD but not C3aR expression compared with OGD group. C3aRA treatment significantly reduced the level of these markers including C3aR with and without C3a. Color intensity was measured by Image J. The results represent the means±SEM of fold changes (n=5). ^*p<0.01, ^**p<0.001, ^***p<0.0001. Cont, Control group; Cont+C3a, Cont+C3a+C3aRA; OGD, OGD, OGD+C3a, and, OGD+C3a+C3aRA.

Fig. 8. Schematic diagram showing OGD induced vascular dysfunction and the protective role of C3aRA. Solid arrow showing increase and broken arrow showing decrease. OGD, oxygen glucose deprivation.