(604.6) Sex Differences in ROS Production and SOD Activity Following Induced Inflammation

Poster Board Number: E473

Sara Mascone (University of Maryland- College Park, University of Maryland- College Park), Katherine Kim (University of Maryland- College Park), William Evans (University of Maryland- College Park), Steven Prior (University of Maryland- College Park), Sushant Ranadive (University of Maryland- College Park)

Vascular dysfunction is closely related to and caused by oxidative stress and chronic inflammation. Oxidative stress may be augmented by inflammation due to overproduction of reactive oxygen species (ROS) and concomitant impairments in ROS clearance. Premenopausal women experience a lower incidence of vascular dysfunction compared with age-matched men. This sex difference is, in part, attributed to differences in estrogen and androgen concentrations and actions on endothelial cells. However, limited knowledge exists regarding sex differences in ROS production and ROS clearance in endothelial cells following induced inflammation. Because male human umbilical vein endothelial cells (HUVECs) are exposed to higher androgen concentrations in umbilical cord blood than female HUVECs, we sought to evaluate sex differences in ROS production and superoxide dismutase (SOD) activity (a major contributor to ROS clearance) in male and female HUVECs. We hypothesized that female HUVECs would exhibit lower ROS production and higher SOD activity than male HUVECs. Inflammation was induced in HUVEC cell lines (n=4/group) using tumor necrosis factor-alpha (TNF-α, 50ng/ml).  Cell lysate samples from time-matched control and TNF-α treatments were collected at 4 (4H) and 24 hours (24H) post-stimulus. Fluorescence detection was used to quantify ROS production and viable cells alive at 4H and 24H post-stimulus in cells stained for ROS (CellROX) and living cells (Hoechst). Data are presented as a ratio of ROS to live cells (CellROX/Hoechst ratio). Cell lysate samples were subsequently assayed for SOD activity and normalized to protein concentration. 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, female HUVECs exhibited significantly lower ROS production than male HUVECs in time-matched Control samples and following TNF-α- induced inflammation (CellROX/Hoechst ratio- Female HUVECs: 4H TNF-α: 1.20 ± 0.17, 24H TNF-α: 1.33 ± 0.14, Male HUVECs: 4H TNF-α: 1.32 ± 0.14, 24H TNF-α: 1.56 ± 0.20; p=0.0040). Further, female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF-α- induced inflammation while male HUVECs did not (female HUVECs: 4H TNF-α: 2.93 ± 1.10, 24H TNF-α: 4.82 ± 1.60, p=0.0098; male HUVECs: 4H TNF-α: 3.30 ± 1.87, 24H TNF-α: 3.72 ± 1.06 U/mg; plt;0.05). Compared with female HUVECs, male HUVECs exhibit a pro-inflammatory state following induced inflammation, potentially due to differences in exposure to androgen concentrations in fetal umbilical cord blood.

Funded by UMD Tier 1 Grant (UMD seed grant for Sushant Ranadive)