Racial Differences In ROS Production And SOD Activity Following Induced Inflammation

Abstract

Greater hypertension incidence in Black Americans (BA) may be due to subclinical vascular dysfunction caused by inflammation and heightened levels of reactive oxygen species (ROS). At rest, BA and White Americans (WA) exhibit divergent inflammation, ROS production, and ROS clearance both in vivo and in vitro. However, racial differences in ROS production and clearance following induced inflammation are not fully elucidated. PURPOSE: To evaluate racial differences in ROS production and superoxide dismutase (SOD) activity, a major contributor to ROS clearance, in BA and WA human umbilical vein endothelial cells (HUVECs) following induced inflammation. METHODS: Following triplicate, parallel experiments with inflammation induced in eight HUVEC cell lines (n=4/group) using tumor necrosis factor-alpha (TNF-α, 50 ng/ml), cell lysate samples from Control and TNF-α treatment were collected at 4 (4H) and 24 hours (24H) post-stimulus. Fluorescence detection was used to quantify ROS production (CellROX) and viable cells alive (Hoechst) at 4H and 24H post-stimulus. Control and TNF-α-stimulated cell lysates were subsequently assayed for SOD activity and protein concentration. RESULTS: TNF-α treatment significantly increased ROS production in all HUVECs at 24H compared with 4H (CellROX/Hoechst ratio- 4H TNF-α: 1.37 ± 0.21, 24H TNF-α: 1.44 ± 0.20, p=0.0015). Notably, BA HUVECs did not exhibit a significant higher ROS production at 24H compared with 4H after TNF-α treatment (4H: 1.23 ± 0.20, 24H: 1.40 ± 0.25, p>0.05). However, WA HUVECs exhibited significantly greater ROS production at 24H compared with 4H after TNF-α treatment (4H: 1.29 ± 0.13, 24H: 1.49 ± 0.15, p=0.003). Further, BA HUVECs SOD activity was similar between conditions and time points, yet WA HUVECs exhibited significantly greater SOD activity at 24H as compared to 4H in the Control condition that was abolished with TNF-α treatment (SOD Activity (U/mg)- Control 4H: 2.86 ± 1.31, Control 24H: 5.57 ± 1.97, p=0.0034; TNF-α 4H: 2.92 ± 1.32, TNF-α 24H: 4.18 ± 1.45, p>0.05). CONCLUSION: BA and WA exhibit divergent ROS production and SOD activity following induced inflammation in vitro, suggesting higher ROS production in WA and a better ability to buffer ROS production in BA. Funded by UMD Tier 1 Grant (UMD seed grant for Sushant Ranadive)