Adenosine A2A and A3 Receptors Are Involved in the Human Endothelial Progenitor Cells Migration

Abstract

Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A₂A, A₂B, and A₃ in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A₂A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A₂A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A₂A and A₃ but not A₂B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

L-arginine transport and nitric oxide synthesis in human endothelial progenitor cells

“…The fractions of CD34 + KDR -and CD34 + KDR + cells at 3 days in culture were higher (8.5 ± 0.4 and 7.0 ± 0.5 fold, respectively, p<0.05) than cells cultured for 14 days; however, a larger fraction of CD34 -KDR + cells (5.5±0.2 fold, p<0.05) was identified after 14 days compared with 3 days of culture confirming our previous reports15,16 Characterization of L-arginine transport in hEPC L-arginine transport was assessed in hEPC-3d and hEPC-14d by using L-[ 3 H]-arginine, as described in Methods. L-arginine uptake was linear determined until 60 seconds for both hEPC-3d (slope=0.00039) and hEPC-14d (slope=0.00128) and adjusted to a double Michaelis-Menten equation(Fig.…”

“…15,16,21 Briefly, fresh human peripheral venous blood was collected from eight healthy female volunteers (25-30 y), non-smokers, not receiving any medication and without any clinical diagnosis. This protocol was conducted according to the protocols defined by the Declaration of Helsinki Ethic Committee and informed consent was signed by every volunteer participating in this study.…”

“…PCM was changed every 3 days and hEPC were selected as cells attached to fibronectin-coated plates. In addition, experiments were performed as described previously in hEPC cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) 15,16 after cell starving (12 h) using PCM containing 2% of serum without growth factors. CarboxyFluorescein conjugated KDR (R&D System Inc., Minneapolis, MN, USA) as previously described 15,16 Characterization of L-arginine transport L-arginine transport was measured as described.…”

“…In addition, experiments were performed as described previously in hEPC cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) 15,16 after cell starving (12 h) using PCM containing 2% of serum without growth factors. CarboxyFluorescein conjugated KDR (R&D System Inc., Minneapolis, MN, USA) as previously described 15,16 Characterization of L-arginine transport L-arginine transport was measured as described. 22 L-arginine transport (1 min, 37°C) was initiated by the addition of HEPES-buffered Krebs solution containing unlabelled L-arginine (0.1-500 µM) and L-[ 3 H]-arginine (4 µCi/ml) and was stopped by the addition of cold phosphate buffer.…”

“…For example, on the 3 th day (hEPC-3d), hEPC are positive for CD34, CD133 and Oct4 markers, which represents an immature phenotype, while at day 14 th (hEPC-14d), hEPC lose the expression of those surface molecules and express membrane markers of mature endothelium, such as VEGFR-2. 15,16 Accordingly, hEPC exhibited a differential expression and activity of the equilibrative and concentrative nucleoside transporters, 15 showing that the cell differentiation affects the expression and function of membrane-associated transporters.…”