Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro

Abstract

Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N 5,N 10-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 μM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 μM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 μM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.

Zinc Oxide Nanoparticles Impair the Integrity of Human Umbilical Vein Endothelial Cell Monolayer <I>In</I> <I>Vitro</I>

“…20 Annexin V binds specifically to phosphatidylserine; some of supramagnetic materials used as contrast agents for magnetic resonance imaging (MRI) can be decorated with annexin V and used for non-invasive atheromatous lesions imaging. 32 However, the ZnO-NP induced changes in the cell membrane asymmetry and should be considered cautiously in the imaging approach.…”

“…20 Cultures at 3-5 passages with a confluence of minimum 70% were treated with commercially available ZnO-NPs (544906, Sigma-Aldrich, Steinheim, Germany). NPs were dispersed in ultrapure water (stock solution 10 mg/ml) and sonicated for 5 min at 40 kHz (Ultrasonic Cleaner, Branson 200; Taiwan).…”

Microparticles, Vascular Function, and Atherothrombosis

“…12,13 Caspase activities have been identified in different MPs, and thus could represent an attempt for cells to escape cellular apoptosis. 14 -16 Non-standard Abbreviations and Acronyms HUVEC 177 Nomura et al 178 Stampfuss et al 179 … Thrombin Sapet et al, 18 Simoncini et al 20 Barry et al, 60 Dale et al, 180 Chang et al, 181 … … Collagen … Barry et al, 60 Boilard et al, 182 Chang et al, 181 … … Homocysteine Sekula et al 183 Olas 187 Curtis et al, 19 Abid Hussein et al, 15 Wang et al, 22 Nomura et al, 178 Piguet et al 188 Schecter et al 189 Jungel et al 190 Oxidative stress Vince et al, 191 Endothelial MP formation and release have received significant attention over the past recent years, and different signaling pathways have been identified depending on the stimuli (Table). 17 Clearly, endothelial MP shedding can occur independently of endothelial apoptosis.…”

Acceleration of New Biomarkers Development and Discovery in Synergistic Diagnostics of Coronary Artery Disease

“…PS is specifically bound to annexin V and is recommended as a distinguish marker for MP enumeration (Bulut et al, 2009;Shah et al 2009). However, a number of evidence suggests that some vesicles derived from endothelial cells are PS-negative by annexin-V labelling (Jimenez et al, 2003;Sekuła et al, 2011). In platelet-poor plasma obtained from healthy donors, 80% of platelet-derived MPs failed to bind annexin V (Connor et al, 2010).…”