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 β‐galactosidase (SA‐β‐gal). Cell expression of miR‐34a, miR‐217, and miR‐146a was determined by RT‐PCR. X4 and R5 gp120 and Tat significantly increased (~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.
The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 105 cells/condition. Cells were incubated with media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144+ expression) and concentration was determined by flow cytometry. HUVECs (3×106 cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5±0.1 vs 1.0±0.1 ng/mL) and staurosporine-stimulated (2.2±0.2 vs 1.4±0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (~140%) by high glucose EMPs (0.43±0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression.
What is the central question of this study? Are there sex-related differences in the number of circulating endothelial microparticles (EMPs) and microparticle microRNA expression in middle-aged adult humans? What is the main finding and its importance? Although the numbers of circulating endothelial microparticles do not differ between middle-aged men and women, there are sex-related differences in the expression of miR-125a in activation-derived EMPs and miR-34a in apoptosis-derived EMPs. Differences in circulating endothelial microparticle microRNA content may provide new insight into the sex-related disparity in the risk and prevalence of vascular disease in middle-aged adults. The aims of this study were to determine: (i) whether circulating concentrations of endothelial microparticles (EMPs) differ in middle-aged men compared with women; and (ii) whether there are sex-related differences in microRNA expression in EMPs. Peripheral blood was collected from 30 sedentary adults: 15 men (56 ± 6 years old) and 15 women (56 ± 5 years old). Endothelial microparticles were defined by markers of activation (CD62e ) or apoptosis (CD31 /CD42b ) by flow cytometry. Expression of microRNA (miR-34a, 92a, 125a and 126) in activation- and apoptosis-derived EMPs was measured by RT-PCR. Circulating activation- (33 ± 31 versus 39 ± 35 microparticles μl ) and apoptosis-derived EMPs (49 ± 54 versus 42 ± 43 microparticles μl ) were not significantly different between men and women. Expression of miR-125a (2.23 ± 2.01 versus 6.95 ± 3.99 a.u.) was lower (∼215%; P < 0.05) in activation-derived EMPs, whereas expression of miR-34a (1.17 ± 1.43 versus 0.38 ± 0.35 a.u.) was higher (∼210%; P < 0.05) in apoptosis-derived EMPs from men compared with women. Expression of microRNA in circulating EMPs may provide new insight into sex-related differences in cardiovascular disease.
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