High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells

Abstract

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.

The Effects of Type 2 Diabetes Mellitus on Organ Metabolism and the Immune System

“…Chettab et al have shown that the expression of ICAM-1 as well as the production of IL-8, are significantly increased in HUVECs cultured in HG medium compared to cells cultured in normal glucose (NG, 5.5 mM) conditions (52). Bammert et al found out that incubation of HUVECs with HG media promotes the generation of endothelial microparticles (EMPs) that, when added to normally cultured HUVECs, downregulate the expression of anti-apoptotic microRNA miR-Let7a, but enhance the synthesis of active caspase-3 and cause cell apoptosis (53). Several microRNAs, including miR-21, miR-26a, miR-30, miR-92a, miR-126, miR-139, miR-199a, miR-222, and miR-let7d, regulate vascular homeostasis.…”

<p>Interfering Human Papillomavirus E6/E7 Oncogenes in Cervical Cancer Cells Inhibits the Angiogenesis of Vascular Endothelial Cells via Increasing miR-377 in Cervical Cancer Cell-Derived Microvesicles</p>

“…The expressions of MV markers (CD63 and CD9) were detected, and the results showed that the isolation was successful ( Figure 1B). Meanwhile, miRNAs including let-7a-5p, 28 miR-103a-3p, 29 miR-191-5p, 30 and miR-26a-5p 31 have been reported to be related to cell apoptosis, proliferation, migration, and angiogenesis of HUVECs. Therefore, the expressions of let-7a-5p, miR-103a-3p, miR-191-5p, miR-377, and miR-26a-5p were compared in CC-MVs with or without interfering E6/E7.…”

Effect of Endothelial Microparticles Induced by Hypoxia on Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells by Delivering MicroRNA-19b

“…The pelleted EMPs were re-suspended in endothelial cell medium at a concentration of 1.0 × 10 7 MP/ml. HUVECs seeded on 6-well plates were treated with an equal number of EMPs (50 μl)[ 20 ] for 24 h under normoxic condition. Finally, HUVECs were used for migration and tube formation assays or were collected for real-time polymerase chain reaction (RT-PCR) and quantitative protein assay.…”