Abstract
BackgroundOxidative stress plays a pivotal role in the development of endothelial dysfunction and the pathophysiology of atherosclerosis. P66shc is an adaptor protein that acts as a redox‐sensitive enzyme involved in mitochondrial reactive oxygen species (ROS) production. Upon endothelial cell activation, protein kinase C‐β2 (PKC‐β2) mediates Ser36 phosphorylation of p66shc, thereby allowing p66shc translocation from the cytosol to the mitochondria. Furthermore, we have shown that African American (AA) endothelial cells display higher levels of inflammation and oxidative stress markers compared to Caucasian American (CA) endothelial cells. Therefore, the purpose of this study was to investigate racial differences in p66shc activity in response to tumor necrosis factor‐α (TNF‐α)‐induced endothelial cell activation.MethodsHuman umbilical vein endothelial cells (HUVECs) from three AA donors and three CA donors were cultured and incubated with TNF‐α (10ng/ml, 4hr) to assess racial differences in p66shc, PKC‐β2 and endothelial nitric oxide synthase (eNOS) protein levels and bioactivity.ResultsEnzymatically active eNOS (p‐eNOSS1177) was significantly lower in AA HUVECs under both basal and TNF‐α incubation conditions (p= 0.01, Fig. 1A). Additionally, p‐Ser36 p66shc/total p66shc was significantly lower in AA HUVECs compared to CA HUVECs (p= 0.04, Fig. 3C). However, there was no group or treatment effect on any on the other variables measured.Conclusionp66shc represents an important molecular pathway that mediates ROS production and plays an integral role in the development of cardiovascular disease. According to our results, racial differences in endothelial function cannot be explained by p66shc. However, future research should focus on assessing cytosolic vs. mitochondrial p66shc levels in AA endothelial cells compared to CA.eNOS protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOS. B) Densiometric quantification and representative Western blot of eNOS. C) p‐eNOS bioactivity. (* p= .01 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1p66shc protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐p66shc. B) Densiometric quantification and representative Western blot of p66shc. C) p‐p66shc bioactivity. (* p= .04 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2
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