{'fraction_non_alphanumeric': 0.044, 'fraction_numerical': 0.03636363636363636, 'mean_word_length': 5.457746478873239, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effects of HIV‐1 gp120 and TAT on Endothelial Microparticle Release and Oxidative Stress

Human immunodeficiency virus (HIV)‐1 infection is associated with an increased risk and prevalence of atherosclerotic cardiovascular disease (CVD). A major factor underlying the increased CVD burden with HIV‐1‐infection is endothelial damage and dysfunction. Although the mechanisms underlying the HIV‐1‐related vasculopathy are not fully understood, HIV‐1 associated viral proteins are known to have deleterious effects on the endothelium. Endothelial cell release of microparticles and endothelial cell oxidative stress are key characteristics of a proatherogenic endothelial phenotype. The direct effect of HIV‐1 viral proteins on endothelial microparticle release and oxidative stress, however, is currently unknown. The aim of the present study was to determine the effects of X4 and R5 HIV‐1 gp120 and TAT on endothelial cell microparticle release and oxidative stress. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated at a density of 2 × 106 cells/condition. HUVECs were treated with media alone or media containing either lavgp120 (X4; 100 ng/mL), Balgp120 (R5; 100 ng/mL) or TAT (500 ng/mL) for 24 h to generate endothelial microparticles (EMPs). Viral protein stimulated EMPs were pelleted by centrifugation and resuspended in culture media. Characterization and concentration of EMPs was determined by flow cytometry using VE‐Cadherin (CD 144‐PE). To assess oxidative stress, intracellular reactive oxygen species (ROS) production was determined. To do so, 2.5 ×104 HUVECs were plated in 96‐well tissue culture plates and allowed to adhere overnight. Cells were then incubated with 2′,7′ –dichlorofluorescin diacetate (DCFDA; 25 uM), washed and stimulated with media in the presence or absence of viral proteins. All measurements were done in triplicate and calculated as percent (%) of control by fluorescence spectroscopy. Treatment of HUVECs with X4 (117±28 MP/μL), R5 (105 ± 24 MP/μL) and TAT (101±36 MP/μL) induced markedly higher (~105%) EMP release compared with untreated (51±13 MP/μL) cells. Additionally, X4 (135 ±11%), R5 (139±13%) and TAT (142±7%) significantly increased (~40%) ROS production compared with untreated cells. Of note, the magnitude of increase in EMP release and ROS production was not significantly different between viral proteins. These results indicate that HIV‐1 gp120 and TAT induce EMP release and increase oxidative stress in endothelial cells. Viral protein augmentation of cellular ROS may be the impetus for EMP release, and requires further study. EMP generation and increased endothelial oxidative burden likely contribute to atherogenic effects of HIV‐1 viral proteins.