Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells

Expression of non-differentiated or differentiated endothelial cell lineage makers in human endothelial progenitor cells. (A) RT-PCR for mRNA extracted from cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d). The mRNA was reversed-transcribed into cDNA, and PCRs were performed by using sequence-specific oligonucleotide primers for CD133, CD34, Oct-4, Tie-2, KDR and Lox-1, with β-actin as internal reference. Data are representative of 5 different cell cultures. (B) Gene/β-actin ratio densitometries from data in A for hEPC-3d (□) and hEPC-14d (▪). *P < 0.04 and versus corresponding values in hEPC-3d cells. Values are mean ± S.E.M. (n = 5).

Antigen expression by peripheral human endothelial progenitor cells. (A) Flow cytometry was used to identify human phycoerytrin conjugated CD34 (CD34 PE) and CarboxyFluorescein conjugated kinase insert domain receptor (KDR-FITC) antigens in human mononuclear-free cell fraction isolated from peripheral venous blood from healthy volunteers and cultured for 3 days (□) or 14 days (▪). CD34⁺/KDR⁻, CD34⁻/KDR⁺ or CD34⁺/KDR⁺ cell populations were identified. (B) CD34⁺/KDR⁻, CD34⁻/KDR⁺ or CD34⁺/KDR⁺ cell populations presented as a percentage of total number of cells. *P < 0.05 versus corresponding values in cells 3 days in culture. ^†P < 0.05 versus corresponding values in CD34⁻/KDR⁺ and CD34⁺/KDR⁺ cells. ^‡P < 0.05 versus corresponding values in CD34⁺/KDR⁺ cells. Values are mean ± S.E.M. (n = 4).

Adenosine transport in human endothelial progenitor cell. Overall adenosine transport (10 μM adenosine, 4 μCi/mL [³H]adenosine, 20 s, 22 °C) was measured in human endothelial progenitor cells (hEPCs) exposed to Krebs solution with sodium (NaCl) or where Na⁺ was equimolarly replaced by N-methylglucamine-HCl (NMG). Transport assay was performed in cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) preincubated in Krebs (30 min) without (□) or with (▪) 1 μM nitrobenzylthioinosine (NBTI, ). *P < 0.05 versus corresponding NBTI values in hEPC-3d cells, and versus values in NMG in hEPC-14d cells. Values are mean ± S.E.M. (n = 6–8).

Kinetics of adenosine transport in human endothelial progenitor cells 3 days in culture. (A) Overall adenosine transport (0.4–32 μM adenosine, 4 μCi/mL [³H]adenosine, 20 s, 22 °C) in Krebs without (○) or with (●) S-(4-nitrobenzyl)-6-thioinosine (0.1 μM, 30 min). (B) hENT1-mediated adenosine transport derived from data in A (see Methods). (C) Eadie–Hofstee transformation of transport data from B. V is adenosine transport rates. Values are mean ± S.E.M. (n = 7).

Kinetics of adenosine transport in human endothelial progenitor cells 14 days in culture. (A) Overall adenosine transport (0.4–32 μM adenosine, 4 μCi/mL [³H]adenosine, 20 s, 22 °C) in Krebs with (○, ●) or without (□, ▪) sodium in absence (○, □) or presence (●, ▪) of S-(4-nitrobenzyl)-6-thioinosine (NBTI, 1 μM, 30 min). (B) Eadie-Hofstee transformation for overall adenosine transport in Krebs containing sodium in absence of NBTI as in A. Component 1 (C1) and component 2 (C2) derived from overall adenosine transport are represented (see Methods). V is adenosine transport rates. (C) Adenosine transport mediated by a Na⁺-dependent (○) and Na⁺-independent (□) component derived from data in A. (D) Eadie-Hofstee transformation of data in C. (E) Adenosine (10 μM) transport as in A in Krebs containing increasing concentrations of extracellular sodium. (F) Hill plot of transport data in C where v is initial rate and V_max is maximal velocity of adenosine transport at varying concentrations of sodium. Values are mean ± S.E.M. (n = 7).

hENT1 and hENT2 expression in human endothelial progenitor cells. (A) hENT1 and hENT2 protein abundance in human endothelial progenitor cells 3 days (hEPC-3d) or 14 days (hEPC-14d) in culture. Upper panel: western blot (representative of other 3 experiments) for hENT1, hENT2, and β-actin (internal reference). Protein extracts from primary cultures of human umbilical vein endothelial cells (HUVEC) was used as positive control. Lower panel: densitometric ratios for hENT1/β-actin (□) or hENT2/β-actin (▪) protein abundance. (B) Expression of hENT1 (□) or hENT2 (▪) mRNA in number of copies versus 18S rRNA (internal reference) in hEPCs as in A. (C) Relative levels of hCNT1, hCNT2 and hCNT3 mRNA in hEPC-3d and hEPC-14d cells. Upper panel: real time PCR (representative of other 3 experiments) for hCNT1, hCNT2 and hCNT3. β-Actin was internal reference. mRNA extracts from MDA148 (human breast cancer cell line), CACO-2 (human epithelial colorectal adenocarcinoma cell line) and DU-145 (human prostate cancer-derived cell line) were used as positive controls for hCNT1, hCNT2 and hCNT3, respectively. Lower panel: relative mRNA levels for hCNT1, hCNT2 and hCNT3 in hEPCs versus corresponding controls. *P < 0.05 versus all other values in hEPCs. Values are mean ± S.E.M. (n = 3–7).