L-Arginine Transport and Nitric Oxide Synthesis in Human Endothelial Progenitor Cells

Abstract

Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases. In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y(+)/CAT) or system y(+)L is required for the NO synthesis via endothelial nitric oxide synthase, but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide or L-nitroarginine methyl ester, as inhibitors of y(+)/CAT system and nitric oxide synthases, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC: a high affinity transporter system (K(m)= 4.8 ± 1.1 μM for hEPC-3d and 6.1 ± 2.4 μM for hEPC-14d) and a medium affinity transporter system (K(m) = 85.1 ± 4.0 μM for hEPC-3d and 95.1 ± 8 μM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y(+)) and mRNA for 2 subunits of y(+)L system, yLAT1, and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), and endothelial nitric oxide synthase expression (both mRNA and protein) were observed in hEPC-14d compared with hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.

Overexpression of LOXIN Protects Endothelial Progenitor Cells From Apoptosis Induced by Oxidized Low Density Lipoprotein

“…Cells were characterized as described before. 15,30,31 Media was changed every 3 days, and 16 hours before experiment cells were exposed to media without FBS. This study was approved by the Ethical Committee belonging to the Universidad de Concepcion.…”

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

“…Indeed, it is well known that hWMSCs may secrete angiopoietin-1, angiogenin, interleukin 8 (IL-8), monocyte chemotactic protein-1 (MCP-1), serpin E1 (plasminogen activator inhibitor 1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and trombospondrina-1 [27] . A combination of those factors (and perhaps many others) may participate in the healing effect of conditioned medium derived from hWMSC-End [36] . In our study we also found that CM recovered from hWMSCs or hWMSC-End30d cultures increased the formation of capillary-like structures in HUVEC cultured on Matrigel, supporting that pro-angiogenic factor(s) might taken part in the promotion of wound healing.…”

“…Cells were lysed and treated in a cation ion-exchange resin Dowex-50W (50X8-200, Na + form), to separate L-[ 3 H]-citrulline present in H 2 O eluates from L-[ 3 H]-arginine retained in the column. When required, N G -nitro-L-arginine methyl ester (L-NAME, 100 µM), a NOS inhibitor, was added to cell cultures 30 min before experiments [36] .…”

Vascular Regeneration by Endothelial Progenitor Cells in Health and Diseases

“…The original description of hEPCs by Asahara et al was based on (1) the ability of hEPC to adhere to fibronectin-coated surfaces and (2) the surface expression of both immature stem cells (CD34, CD45, VEGFR2, or Flk-1) and mature endothelial cell (EC) markers (CD31, Eselectin, and angiopoietin receptor Tie-2) [20]. In addition, the expression of endothelial nitric oxide synthase (eNOS), the synthesis of nitric oxide (NO), and the ability to incorporate lowdensity lipoproteins (LDL) have been also associated with differentiation of hEPC toward endothelial cells [22].…”