Effects Of Candesartan On Endothelial Progenitor Cell And Oxidative Stress In Salt-loaded Hypertensive Rats

Objective The study investigated effects of Angiotensin II (Ang II) receptor blocker (ARB), candesartan on peripheral blood endothelial progenitorcell (EPC) number and function and oxidative stress in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP) in vivo and aimed to investigate potential antioxidative mechanism that underlies ARB improving EPC dysfunction and to provide new theoretical and experimental evidences for ARB in the treatment of cardiovascular diseases.Methods Twelve-week-old WKY rats (WKY, n=6) and SHRSP were fed high-salt diet (8% NaCl) for 2 weeks, while SHRSP were randomly divided into 4 groups. At the same time of high salt diet, SHRSP were treated with different treatments. Untreated SHRSP (SHRSP, n=6) received only dissolvent. SHRSP treated with candesartan (Can, n=6) received candesartan (1 mg/kg/day) orally for 2 weeks. SHRSP treated with Tempol (Tem, n=6) received Tem (1mmol/L) in drinking water for 2 weeks. SHRSP treated with trichlormethiazide (TCM, n=6) received TCM (1.6 mg/kg/day) orally for 2 weeks. Peripheral blood mononuclear cells (MNC) were isolated by a density-gradient centrifuge method. MNC were stained with a fluorescein isothiocyanate (FITC)–conjugated anti-CD34 monoclonal antibody for flow cytometric analysis. MNC with CD34+ were assayed as tentative EPC. MNC were also cultured to assay EPC colony formation and migration function. EPC colonies were observed and numerated under low power microscope. EPC coloies were detected expression of endothelial marker CD31 by immunofluorescence technique. Oxidative stress in non-adherent MNC (containing EPC) was evaluated by thiobarbituric acid reactive substance (TBARS) assay. Rat renal total RNA of renal cortex, aorta and left ventricular tissue were extracted and reversed to cDNA. NAD(P)H oxidase subunits were evaluated by quantitative Real time PCR. Aorta tissues were stained with periodic acid-schiff stain (PAS) and gp91phox in aorta was detected by immunohistochemical labeling. Periferal blood from fourteen-week-old WKY rats and SHRSP were used to culture EPC colony. EPC colonies were picked out under microscope, and cellular total RNA was extracted. Renin-Angiotesin system(RAS)components mRNA expression in EPC colonies were detected by RT-PCR.Results1. Systolic blood pressure was significantly higher in SHRSP group than in WKY group. Treatment with candesartan, Tem and TCM significantly decreased systolic blood pressure in salt-loaded SHRSP (P<0.01). But there were no significant difference between Candesartan and TCM treatment.2. The percentage of CD34+ cells (% MNC) was significantly (P<0.01) lower in SHRSP than in WKY group (0.40±0.02% vs 1.08±0.10%). Candesartan and Tem administration markedly (P<0.01) increased the percentage of CD34+ cells in salt-loaded SHRSP compared to that in untreated salt-loaded SHRSP (0.84±0.09%,0.61±0.08% vs 0.40±0.02%), while TCM treatment did not increase the percentage of CD34+ cells (0.38±0.13% vs 0.40±0.02%) in salt-loaded SHRSP.3. MNC were cultured to EPC colony formation cells and assayed positive for CD31 by immunofluorescence. The number of EPC colonies was significantly (P<0.01) lower in SHRSP group than in WKY group (5.3±3.0 vs 32.2±8.6). Cndesartan and Tem administration markedly (P<0.01) increased the number of EPC colonies in salt-loaded SHRSP compared to that in untreated salt-loaded SHRSP (198.5±46.9, 45.3±6.3 vs 5.3±3.0). However, TCM treatment did not increase the number of EPC colonies (3.8±0.4 vs 5.3±3.0) in salt-loaded SHRSP.4. Chemotaxis assay showed non-adherent MNC (containing EPC) chemotaxis to SDF-1and VEGF were significantly impaired in SHRSP group compared to those in WKY group (P<0.01). Candesartan treatment increased their chemotaxis to SDF-1 in salt-loaded SHRSP,while Tem and TCM had no such effects. Expression of VEGF mRNA was lower in aorta from SHRSP group than in aorta from WKY group. Treatment with both candesartan and Tem significantly increased expression of VEGF mRNA in salt-loaded SHRSP (P<0.01).5. The TBARS score was significantly (P<0.05) higher in non-adherent MNC (containing EPC) from SHRSP group than in MNC from WKY group. Candesartan and Tem administration significantly decreased the TBARS score in MNC from salt-loaded SHRSP (P<0.01). However, TCM treatment did not affect the increased TBARS score.6. Expressions of gp91phox, p22phox and p47phox mRNA in renal cortex, heart and aorta of SHRSP group were significantly (P<0.01) higher than those in WKY group. Candesartan amd Tem administration significantly decreased mRNA expression of NADPH oxidase subunits in cardiovascular organs from salt-loaded SHRSP (P<0.01), whereas TCM treatment did not affect mRNA levels of NADPH oxidase subunit.7. Histopathology and immunohistochemistry showed vascular injury in SHRSP group was heavier than in WKY group. Candesartan and Tem, but not TCM, reduced vascular injury in salt-loaded SHRSP and downregulated gp91 phox expression in aorta.8. Expression of renin, cathepsin D, chymase, AT1R and AT2R mRNA were detected in EPC colonies from SHRSP and WKY rats, but angiotensinogen and ACE mRNAs were not detected in EPC colonies.Conclusion1. Rat peripheral blood MNC are cultured to EPC colony formation in vitro and immunofluorescence assay shows EPC colony cells positive for endothelial marker CD31.2. Candesartan treatment enhances EPC number and EPC colony formation, improves non-adhehent MNC (containing EPC) migration function, decreases thiobarbituric acid reactive substance (TBARS) in non- adhehent MNC , and downregulates NADPH oxidase subunit mRNAs in cardiovascular organs from salt-loaded SHRSP. Increased VEGF expression in aorta in candesartan treated group suggests candesartan may increase mobilization of EPC to injured aorta via VEGF, which may contribute to repair of cardiovascular vascular damage.3. The antioxidant, Tempol also has similar effects of partly improving EPC dysfunction and antioxidation in salt-loaded SHRSP,while the diuretic, TCM has no such effects.4. Candesartan treatment enhances EPC number and improves EPC dysfunction in salt-loaded SHRSP at least partially through its potential antioxidative mechanism, independently of its blood pressure reduction.5. Candesartan treatment reduces EPC impairment by oxidative stress and ameliorates EPC colony formation by blocking RAS via AT1R on EPC in salt-loaded SHRSP.