Effects Of Angiotensin Ⅱ On Endothelial Progenitor Cells From Peripheral Blood And Its Mechanisms

Objective: Endothelial progenitor cells (EPC) are a cell population of endothelial precursors that have the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but have not yet acquired characteristic mature endothelial markers . Recent studies demonstrated that EPCs participated not only during embryonic neovascularization, but also in postnatal neovascularization and reendothelialization. It was reported that some growth factors and drugs, such as Stem cell factor (SCF), vascular endothelial growth factor (VEGF), HMG-CoA reductase inhibitors (statins), granulocyte-macrophage colony-stimulating factor (GM-CSF) could mobilize EPCs from the bone marrow into peripheral circulation and promote EPCs proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Angiotensin II (Ang II), the main effector of the rennin-angiotensin system (RAS), is well known to cause potent increases in systemic and local blood pressure, to influence renal tubules to retainsodium and water. However, studies have shown that Ang II can modify cellular functions unrelated to these physiological events. Over the past decade, numerous studies have shown that Ang II can potentiate the proliferation and differentiation of a variety of cell types. Ang II has been shown to regulate cell growth of hematopoietic progenitor cells(HPC) and vascular smooth muscle cells (SMC). Ang II may also contribute to vessel growth regulation. In the present study, we observed whether Ang II influences the number of EPCs isolated from peripheral blood. We examined the effect of Ang II on the proliferation, migration , adhesion and in vitro vasculogenesis capacity of EPCs. Moreover, the effect of Ang II on the expression of VEGF receptor kinase domain-containing receptor (KDR) mRNA was evaluated. We also examined whether the angiotensin type 1 (AT1) receptor antagonist (valsartan) can inhibit the effect of Ang II on EPC biological activity. Therefore, We investigated whether Ang II stimulates the number and function of EPCs through the induction of KDR mRNA expression by ATI receptor antagonist.Methods:1. Isolation and cultivation of EPCs: Total mononuclear cells (MNCs)were isolated from 20ml of peripheral blood by Ficoll density gradientcentrifugation. After 7 days in culture, adherent cells were incubated with various concentrations of Ang II (10"5mol/L, 10"7mol/L, 10"9mol/L) orvehicle control in the medium as above for 24h or with 10" mol/L Ang II for the respective time points (6 h, 12 h, 24 h and 48 h). In other group, cells were pretreated with valsartan (10"5mol/L) for l/2h, then AngII(10"5mol/L) was added to the culture medium for 24h.2. Characterization of EPCs: To detect the uptake of cells were incubated with 1,1' -dioctadecyl-3,3,3' ,3' -tetramethylindocarbocyanine -labeledacetylated low density lipoprotein (DiLDL) at 37 °C for lh. Samples were viewed with an inverted fluorescent microscope (Leica) and further demonstrated by laser scanning confocal microscope (LSCM, Leica). Cells demonstrating double-positive for both UEA-1 and DiLDL were identified as differentiating EPCs.3. Phenotypical analysis of EPCs: 2 * 105 adherent cells were incubated with phycoerythrin-labeled monoclonal antibodies against human VEGF R2 (KDR), AC 133 and CD34 at 4°C for 30min.Then, the cells were resuspended in 300 uL PBS. Quantitative analyses were performed using a FACS SCAN flow cytometer and CellQuest software.4. Adhesion assay of EPCs: After centrifugation and resuspension in 500 uL Ml 99, identical cell numbers were replated onto fibronectin-coatedculture dishes and incubated at 37°C for 30 minutes. Adherent cells were counted by independent blinded investigators.5. Migration assay of EPCs: 2x 104 EPCs resuspended in 50uL Ml99 were placed in the upper chamber of a modified Boy den chamber. 25uL Ml 99 and human recombinant VEGF (50 ng/mL) were placed in the lower compartment of the chamber. After 24h incubation at 37°C, cells migrating into the lower chamber were counted.6. Proliferation assay of EPCs: MTT assay was used to evulate the effects of Ang II on the proliferative capacity of EPCs. EPCs were supplemented with lOuL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,5g/ L)per well and incubated for 4h.Then the supernatant was discarded by aspiration and the EPCs preparation was shaked with 150uL DMSO for 10 minutes, before the OD value was measured at 490nm.7. In vitro vasculogenesis assay: In vitro vasculogenesis assay was performed with In Vitro Angiogenesis Assay Kit. ECMatrix? solution was mixed with 10 * ECMatrix? Diluent to solidify. EPCs were harvested and 5><104 EPCs were replated on top of the solidified matrix solution. Cells were incubated at 37°C for 24h. Tubule formation was inspected under an inverted light microscope at 200* magnification. Five independent fieldswere assessed for each well, and the average number of tubules/200 x fielddetermined.8. KDR mRNA expression assay of EPCs: Total RNA was extracted byusing Trizol RNA extraction kit. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed.Results:1. Effect of Ang II on EPCs number: The number of EPCs significantly increased after treatment with Ang II for 6h (p < 0.05), with a peak at 24h (P <0.01) and decreased a little at 48h, but it increased significantly compared with control group (P <0.01). Ang II had a peak effect at 10"5mmol/l concentration^ <0.01).2. Effect of Ang II on the adhesive capacity of EPCs: Ang II dose- and time-dependently improved the adhesive capacity of isolated EPCs (P <0.05, PO.01). The number of adhered cells increased after 6h (P <0.05) ,with a peak at 24h (P<0.01) and at 10~5mmol/l concentration (P <0.01).3. Effects of Ang II on the Migratory Capacity of EPCs: Ang II dose-and time-dependently enhanced EPCs migratory activity. The migratory activity was enhanced by Ang II treatment at 12h(P <0.01), with a peak at 24h(P< 0.01) and at 10"5mmol/l concentration.4. Effects of Ang II on the proliferative capacity of EPCs: Ang II dose-and time-dependently improved EPCs proliferative activity. The proliferative capacity of EPCs improved at l2h(P <0.05), with a peak at 24h (P <0.01) and at 10"5mmol/l concentration (P <0.01) and decreased at 48h, but it increased significantly compared with control group (P <0.01) .5. Effects of Ang II on EPCs In vitro Vasculogenesis: In vitro vasculogenesis assay was used to investigate the ability of EPCs to participate in neovascularization, which is the most important activity of EPCs. Ang II significantly increases number of tube-like structures. Number of tube-like structures increased in a dose dependent response to Ang II at 24h of incubation, with peak production at 10"5mol/L Ang II. Moreover, Ang II made tube-like structures qualitatively different and more complex than control group.6. Effects of Ang II on KDR mRNA expression of EPCs: Ang II stimulated KDR mRNA expression in a dose- and time-dependent manner, with a maximal increase at 24h (P <0.01) and at 10" mmol/1 concentration (PO.01).7. Effects of valsartan on the changes of number and activity of EPCs induced by Ang II: Addition of valsartan(10"5mol/l) in combination withAng II significantly inhibited the effects of Ang II alone on EPCs. There was no statistic difference compared with control group(p>0.05).Conclusions:1. Ang II increases the number of EPCs in a dose- and time-dependent manner.2. Ang II dose- and time-dependently improves the adhesive capacity,migratory activity ,proliferative activity of EPCs.3. Ang II potentiates in vitro vasculogenesis capacity of EPCs.4. Ang II stimulates KDR mRNA expression in a dose- and time-dependent manner.5. ATI-specific receptor antagonist (valsartan) significantly inhibits the effects of Ang II on EPCs,suggesting that these effects occurred through up-regulation of KDR mRNA expression via the ATI receptor.6. Ang II may be an essential component for an in vitro expansion culture system.