Plasma MPO concentration of DM patients was significantly higher than healthy controls. though B2R-mediated activation of PI3K and EGFR signaling pathways. < 0.001). Furthermore, DM patient plasma myeloperoxidase (MPO) concentrations were significantly higher than for controls (Figure ?(Figure1C;1C; 4.3 1.0 ng/mL vs 2.5 0.6 ng/mL, < 0.001). Pearson correlation analyses showed that plasma MPO concentration was inversely correlated with the B2R expression level on CD34+ cells (Figure ?(Figure1D;1D; = ?0.619; = 0.001). Open in a separate window Figure 1 Expression of B2R on circulating CD34 positive cells of DM patients and healthy controlsA. Graph Npy showing that the percentage of circulating CD34 positive cells also immunopositive for B2R in DM patients (= 13) was significantly lower than in healthy controls (= 13). B. Representative flow cytometry analysis of B2R positive cells within the population of circulating CD34+ cells of both DM patients and healthy controls. M1 stand for B2R positive cells. C. Plasma MPO concentration of DM patients was significantly higher than healthy AVN-944 controls. D. Pearson correlation analyses showing the correlation of plasma MPO concentrations with AVN-944 B2R expression of CD34+ cells (= ? 0.619, = 0.001) *< 0.001; B2R: Bradykinin receptor 2. DM: Diabetes Mellitus. MPO: Plasma Myeloperoxidase. Characterization of cultured hEPCs Human umbilical cord blood-derived mononuclear cells (MNCs) were separated by density-gradient centrifugation. Double staining for FITC-lectin and acLDL-Dil showed that human EPCs (hEPCs) were able to uptake acLDL-Dil, which binds to an endothelial cell-specific lectin. Immunofluorescence showed that these hEPCs expressed CD34, kinase domain receptor (KDR), and CD105, but not CD45. hEPCs were immunopositive for CD34, KDR, CD105, and B2R, but not AVN-944 CD45 by flow cytometry (Figure ?(Figure22). Open in a separate window Figure 2 Phenotypic characterization of cultured hEPCs including analysis of B2R expressionA. Photomicrographs showing that the adherent cells intensively took up acLDL-Dil and bound an endothelial-specific lectin, as assessed using fluorescence microscopy. (Original magnification: 400). B. Representative flow cytometry analyses of hEPCs for expression of cell surface markers. The hEPCs of passage 3 were positive for CD34, KDR, and CD105, but were negative for CD45. C. Representative flow cytometry analysis of hEPCs for the expression of B2R. hEPCs: Human Endothelial Progenitor Cells. acLDL-Dil; acetylated low-density lipoprotein; KDR: vascular endothelial growth factor receptor. BK inhibits oxidative stress-induced senescence -galactosidase (SA-Gal) staining revealed that 300 M H2O2 significantly induced hEPC senescence (mean SEM, 49.6 8.2 cells/field vs 6.4 1.1 cells/field, < 0.05). Furthermore both 0.1 nM and 1.0 nM BK dramatically inhibited H2O2-induced hEPC senescence compared to cells treated with H2O2 alone (20.4 1.7 cells/field vs 6.4 1.1 cells/field for 0.1 nM BK; 18.0 6.0 cells/field vs 6.4 1.1 cells/field for 1.0 nM BK; < 0.05). No statistical difference in oxidative stress-induced senescence was found between 0.1 and 1.0 nM BK treatment groups (> 0.05, Figure ?Figure33). Open in a separate window Figure 3 BK inhibits oxidative stress induced senescence of hEPCsA. SA-Gal staining showing the degree of senescence in untreated controls. B. SA-Gal staining showing senescent hEPCs following H2O2 induced oxidative stress. C. Effect of 0.1 nM and 1nM BK on SA-Gal staining of senescent hEPCs. D. Histograph showing the number of senescent cells per microscopic field. (Original magnification 200 ; = 5 for each group) demonstrating that both concentrations of BK significantly inhibit oxidative stress induced senescence in hEPCs. *< 0.05 vs control and both concentrations of BK, #> 0.05 vs 1.0 nM BK) BK: Bradykinin; hEPCs: Human Endothelial Progenitor Cells; SA-Gal: -galactosidase. BK suppresses H2O2-induced intracellular oxygen radical production Examination of intracellular oxygen radicals visualized using dichlorofluorescein diacetate (DCFH-DA) probes incubated with H2O2-induced senescent hEPCs showed that the senescent cells had significantly higher levels than normal controls (mean fluorescence intensities: 0.143 0.014/pixel vs 0.034 0.001/pixel, < 0.05). Also, we found that treatment with BK at 0.1 nM (mean fluorescence intensities: 0.063 0.002/pixel vs 0.143 0.014/pixel, < 0.05) and 1.0 nM (mean fluorescence intensities: 0.060 0.003/pixel vs 0.143 0.014/pixel, < 0.05) suppressed the generation of intra-cellular oxygen radicals compared to hEPCs treated with H2O2 alone. No statistical differences were found between cells treated with the 2 2 concentrations of BK (> 0.05, Figure ?Figure44). Open in a separate window Figure.