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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.

Abbreviations

BB:

binding buffer

CAD:

coronary artery disease

FA:

folic acid

FBS:

fetal bovine serum

FITC:

fluorescein isothiocyanate

Hcy:

homocysteine

HHcy:

hyperhomocysteinemia

HUVEC:

human umbilical vein endothelial cells

Met:

methionine

MI:

myocardial infarction

MP(s):

microparticle(s)

MTHFR:

N5,N10-methylenetetrahydrofolate reductase

PECAM-1:

platelet endothelial cell adhesion molecule-1

PBS:

phosphate buffered salt solution

PE:

phycoerythrin

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Correspondence to Ewa Stępień.

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Sekuła, M., Janawa, G., Stankiewicz, E. et al. Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro . Cell Mol Biol Lett 16, 69–78 (2011). https://doi.org/10.2478/s11658-010-0040-2

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  • DOI: https://doi.org/10.2478/s11658-010-0040-2

Key words

  • Flow cytometry
  • Homocysteine
  • Hyperhomocysteinemia
  • Microparticles
  • Human umbilical vein endothelial cells
  • Methylenetetrahydrofolate reductase