Figure 4:

IgG from high fat diet (HFD)-fed mice and IgG hyposialylated by neuraminidase treatment inhibit eNOS activation by VEGF in human endothelial cells via FcγRIIB. A. ¹⁴C-L-arginine conversion to ¹⁴C-L-citrulline under basal conditions or in response to VEGF (100ng/ml) was measured in intact primary human aortic endothelial cells over 15min. The effects of IgG isolated from wild-type mice fed a control diet (Con IgG) or HFD (HFD IgG), or Con IgG treated with neuraminidase (NA) (NA IgG) were evaluated. B-C. Parallel experiments were performed in cells transfected with either control siRNA or FcγRIIB-targeting siRNA. Effective loss of FcγRIIB protein with siFcγRIIB transfection was confirmed by immunoblot analysis, and findings for two samples per group are shown in B. D-E. Total eNOS protein abundance was evaluated by immunoblot analysis following 48h treatment with Con IgG, HFD IgG or NA IgG. Findings for 3 samples per group are shown (D), and summary data are provided (E). F-H. Changes in eNOS Ser1177 and Thr495 phosphorylation in response to VEGF (100ng/ml) were evaluated by immunoblot analysis in cells treated with Con IgG, HFD IgG or NA IgG. Representative findings are shown (F) and summary data are provided (G,H). In bar graphs values are mean±SEM, in G and H, n=4. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.