Asymmetric dimethylarginine (adma) inhibits the effect of erythropoietin on endothelial cell migration in a proinflammatory environment

Abstract

Hyperhomocysteinemia induces vascular endothelial dysfunction, an early hallmark of atherogenesis. Previously, we found a sensitizing effect of the inflammatory cytokine TNF-alpha on a promigratory action of erythropoietin (Epo), which was not observed when the molecule was modified by N-homocysteinylation with homocysteine thiolactone, the reactive derivative of homocysteine. The strong correlation found between hyperhomocysteinemia and levels of asymmetric dimethylarginine (ADMA), the amino acid formed during methylation of proteins, has been considered of interest in endothelial dysfunction. Therefore, the aim of this work was to study whether ADMA accumulation could affect the ability of different factors to induce endothelial cell migration. Wound healing assays of EA.hy926 endothelial cells stimulated by 10% fetal bovine serum (FBS), 20 ng/mL VEGF or Epo+TNF-alpha (80 ng/mL and 30 ng/mL, respectively) were performed in the presence or absence of ADMA (30 or 100 µM). Results are expressed as a percentage of the cell migration obtained in the presence of FBS. FBS: 100; Control: 22.0±2.4; ADMA: 20.1±3.4; *FBS+ADMA30: 66.6±3.5; **FBS+ADMA100: 43.3±3.9; Epo+TNF-alpha: 56.4±1.2; *Epo+TNF-alpha+ADMA30: 37.4±2.9; **Epo+TNF-alpha+ADMA100: 31.1±1.6; VEGF: 44.5±2.9; *VEGF+ADMA30: 30.3±2.1; **VEGF+ADMA100: 19.6±2.8; *P<0.05 and **P<0.01 vs. respective controls, n=4. It can be seen that the inhibition of the promigratory effects of all the factors analyzed was dependent on ADMA concentration. The results of cell viability and MTT assays confirmed that the effects of ADMA on the promigratory action of Epo+TNF-alpha and VEGF cannot be attributed to cytotoxicity or cell proliferation. These results underline the important role of homocysteine in cardiovascular pathophysiology, since increased homocysteine is involved in a direct inhibition of the enzymes responsible for ADMA degradation.