Microparticles and MicroRNA: Novel Mediators of HIV-1 Endothelial Dysfunction

Microparticles and miRNA represent novel mediators and effectors of endothelial cell function and, in-turn, vascular health. MicroRNAs (miRNAs or miRs) are short (20-22 nucleotide) non-coding RNAs involved in the homeostatic regulation of ~60% of all gene expression. miRNAs are transferred and regulated by microparticles, small anucleoid extracellular vesicles. Alteration of cellular miRNA expression by microparticles has been mechanistically linked with the pathogenesis and progression of atherosclerotic cardiovascular disease (ASCVD). Human immunodeficiency virus (HIV)-1 infection is associated with an increased risk and prevalence of ASCVD due, in-part, to endothelial cell damage and dysfunction. Interestingly, endothelial cells are neither targets nor reservoirs of the virus. As such, the detrimental effects of HIV-1 on endothelial cell function are complex, multifactorial and poorly understood. There are currently no data regarding the direct or indirect effects of HIV-1 on microparticle and miRNA biology. Accordingly, the aims of this dissertation were to determine: 1) the effect of HIV-1 related proteins gp120 and Tat on endothelial cell senescence and expression of senescence-associated miRNA; 2) if HIV-1 gp120 and Tat stimulate the release of endothelial microparticles in vitro and if viral protein induced microparticles adversely affect endothelial cell function; and 3) whether circulating microparticles are elevated in HIV-1-seropositive adults. In addition, we determined the effects of microparticles from HIV-1-seropositive adults on endothelial cell activation, inflammation, oxidative stress, senescence and apoptosis, in vitro. To address these aims, we conducted three independent, sequential studies integrating basic and clinical approaches to optimize the translational potential of this work. The seminal and novel findings of these studies are that: 1) HIV-1 gp120 and Tat accelerate cellular senescence due, in part, to dysregulation of senescence-specific miRNA; 2) HIV-1 gp120 and Tat induce microparticle release from endothelial cells and these microparticles confer pathologic effects on endothelial cells enhancing inflammation, oxidative stress, senescence and apoptosis as well as altering cellular miRNA expression; and 3) circulating levels of microparticles are higher in HIV-1-seropositive adults and these microparticles induce a proatherogenic endothelial phenotype.</p

Effects of HIV-1 gp120 and TAT-derived microvesicles on endothelial cell function

The aims of this study were twofold. The first was to determine if human immunodeficiency virus (HIV)-1 glycoprotein (gp) 120 and transactivator of transcription (Tat) stimulate the release of endothelial microvesicles (EMVs). The second was to determine whether viral protein-induced EMVs are deleterious to endothelial cell function (inducing endothelial cell inflammation, oxidative stress, senescence and increasing apoptotic susceptibility). Human aortic endothelial cells (HAECs) were treated with recombinant HIV-1 proteins Bal gp120 (R5), Lav gp120 (X4), or Tat. EMVs released in response to each viral protein were isolated and quantified. Fresh HAECs were treated with EMVs generated under control conditions and from each of the viral protein conditions for 24 h. EMV release was higher (P < 0.05) in HAECs treated with R5 (141 ± 21 MV/µl), X4 (132 ± 20 MV/µl), and Tat (130 ± 20 MV/µl) compared with control (61 ± 13 MV/µl). Viral protein EMVs induced significantly higher endothelial cell release of proinflammatory cytokines and expression of cell adhesion molecules than control. Reactive oxygen species production was more pronounced (P < 0.05) in the R5-, X4- and Tat-EMV-treated cells. In addition, viral protein-stimulated EMVs significantly augmented endothelial cell senescence and apoptotic susceptibility. Concomitant with these functional changes, viral protein-stimulated EMVs disrupted cell expression of micro-RNAs 34a, 126, 146a, 181b, 221, and miR-Let-7a (P < 0.05). These results demonstrate that HIV-1 gp120 and Tat stimulate microvesicle release from endothelial cells, and these microvesicles confer pathological effects on endothelial cells by inducing inflammation, oxidative stress, and senescence as well as enhancing susceptibility to apoptosis. Viral protein-generated EMVs may contribute to the increased risk of vascular disease in patients with HIV-1. NEW & NOTEWORTHY Human immunodeficiency virus (HIV)-1-related proteins glycoprotein (gp) 120 and transactivator of transcription (Tat)-mediated endothelial damage and dysfunction are poorly understood. Endothelial microvesicles (EMVs) serve as indicators and potent mediators of endothelial dysfunction. In the present study we determined if HIV-1 R5- and X4-tropic gp120 and Tat stimulate EMV release in vitro and if viral protein-induced EMVs are deleterious to endothelial cell function. gp120 and Tat induced a marked increase in EMV release. Viral protein-induced EMVs significantly increased endothelial cell inflammation, oxidative stress, senescence, and apoptotic susceptibility in vitro. gp120- and Tat-derived EMVs promote a proinflammatory, pro-oxidative, prosenescent, and proapoptotic endothelial phenotype and may contribute to the endothelial damage and dysfunction associated with gp120 and Tat.National Heart, Lung, and Blood Institute [HL-131458]12 month embargo; published online: 9 May 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Effects of HIV‐1 gp120 and tat on endothelial cell sensescence and senescence‐associated microRNAs

The aim of this study was to determine, in vitro, the effects of X4 and R5 HIV-1 gp120 and Tat on: (1) endothelial cell senescence and (2) endothelial cell microRNA (miR) expression. Endothelial cells were treated with media without and with: R5 gp120 (100 ng/mL), X4 gp120 (100 ng/mL), or Tat (500 ng/mL) for 24 h and stained for senescence-associated beta-galactosidase (SA-beta-gal). Cell expression of miR-34a, miR-217, and miR-146a was determined by RT-PCR. X4 and R5 gp120 and Tat significantly increased (similar to 100%) cellular senescence versus control. X4 gp120 significantly increased cell expression of miR-34a (1.60 +/- 0.04 fold) and miR-217 (1.52 +/- 0.18), but not miR-146a (1.25 +/- 0.32). R5 gp120 significantly increased miR-34a (1.23 +/- 0.07) and decreased miR-146a (0.56 +/- 0.07). Tat significantly increased miR-34a (1.49 +/- 0.16) and decreased miR-146a (0.55 +/- 0.23). R5 and Tat had no effect on miR- 217 (1.05 +/- 0.13 and 1.06 +/- 0.24; respectively). HIV-1 gp120 (X4 and R5) and Tat promote endothelial cell senescence and dysregulation of senescence-associated miRs.National Institutes of Health (NIH) [HL131458]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Circulating Microparticles Are Elevated in Treated HIV -1 Infection and Are Deleterious to Endothelial Cell Function

Background-Circulating microparticles have emerged as biomarkers and effectors of vascular disease. Elevated rates of cardiovascular disease are seen in HIV-1-seropositive individuals. The aims of this study were to determine: (1) if circulating microparticles are elevated in antiretroviral therapy-treated HIV-1-seropositive adults; and (2) the effects of microparticles isolated from antiretroviral therapy-treated HIV-1-seropositive adults on endothelial cell function, in vitro. Methods and Results-Circulating levels of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were determined by flow cytometry in plasma from 15 healthy and 15 antiretroviral therapy-treated, virologically suppressed HIV-1-seropositive men. Human umbilical vein endothelial cells were treated with microparticles from individual subjects for 24 hours; thereafter, endothelial cell inflammation, oxidative stress, senescence, and apoptosis were assessed. Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were significantly higher (approximate to 35%-225%) in the HIV-1-seropositive compared with healthy men. Microparticles from HIV-1-seropositive men induced significantly greater endothelial cell release of interleukin-6 and interleukin-8 (approximate to 20% and approximate to 35%, respectively) and nuclear factor-jB expression while suppressing anti-inflammatory microRNAs (miR-146a and miR-181b). Intracellular reactive oxygen species production and expression of reactive oxygen species-related heat shock protein 70 were both higher in cells treated with microparticles from the HIV-1-seropositive men. In addition, the percentage of senescent cells was significantly higher and sirtuin 1 expression lower in cells treated with HIV-1-related microparticles. Finally, caspase-3 was significantly elevated by microparticles from HIV-1-seropositive men. Conclusions-Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were higher in antiretroviral therapy-treated HIV-1-seropositive men and adversely affect endothelial cells promoting cellular inflammation, oxidative stress, senescence, and apoptosis. Circulating microparticles may contribute to the vascular risk associated with HIV-1 infection.National Institutes of Health (NIH) [HL131458, HL135598]; NIH/National Center for Advancing Translational Sciences [UL1 TR001082]Open Access JournalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]