Effect Of Angiotensin II On The Biological Activities Of Endothelial Progenitor Cells Derived From Rat Bone Marrow

Background and ObjectivePrevious studies have identified a cell population termed endothelial progenitor cells (EPCs), isolated from bone marrow (BM), peripheral blood (PB), and umbilical cord blood (UCB). EPCs, also called angioblasts, have the capacity to differentiate into mature endothelial cells (ECs), and are considered as precursors of endothelial cells. Laboratory evidence indicated that EPCs participate not only in the process of vasculogenesis, the term applied to the formation of embryonic blood vessels, but also in the process of postnatal neovascularization and repair of injured endothelium. EPCs could be mobilized and home to foci of vascular injury or ischemia, affecting the vascular repair capacity and local perfusion. Nevertheless, it remains unclear about the effects of several factors on biological activities of EPCs, which plays a crucial role in clinical application of EPCs.Recently, the number and function of EPCs have been shown to inversely correlate with risk factors for cardiovascular disease, such as hypercholesterolemia, diabetes and C-reactive protein (CRP). Numerous clinical and experimental studies have demonstrated that angiotensin II (AngII) is critically involved in the development of cardiovascular diseases, but it remains to be determined whether AngII may regulate the biological activities of EPCs. On the other hand, increasing evidence has demonstrated the role of AngII in angiogenesis, suggesting that AngII may have a close relationship with EPCs. AngII promotes ECs proliferation, inhibits apoptosis, upregulates adhesion molecules, and potently augments endothelial nitric oxide synthase (eNOS) mRNA expression and thus increasing nitric oxide (NO) release. Therefore, we hypothesize that AngII may have similar effects on EPCs as well. In the present study, we examined the effects of AngII at different concentrations on proliferation, apoptosis, NO release, and adhesion potential in isolated EPCs derived from rat bone marrow. We also evaluated the possible mechanisms of several effects, so as to contribute to the better therapy of ischemia diseases.MethodsPart one: Isolation, differentiation and identification of rat BM-derived EPCs in vitro. Hollow femurs and tibias of rat were prepared by standard surgical procedures and whole bone marrow was harvested by flushing marrow. Total mononuclear cells (MNCs) were isolated from cell suspension of bone marrow by Ficoll density gradient centrifugation. Isolated cells subsequently were plated onto dishes in medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). EPCs were characterized as adherent cells double positive for CD133 and vascular endothelial growth factor type 2 receptor (VEGFR-2/Flk-1) by fluorescent staining under a laser scanning confocal microscope.Part two: Effect of AngII on the proliferation of BM-EPCs and possible mechanism. First passage cells were collected and divided into different groups. A series of concentrations of AngII were added into each group respectively. The change of EPCs proliferative activity after respective intervention was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Reverse transcription-polymerase chain raction (RT-PCR) was used to determine the change of Flk-1 mRNA expression in each group.Part three: Effects of AngII on apoptosis, NO release, and adhesion potential of isolated EPCs. All of the above parameters were determined after incubation with different concentrations of AngII. First, the proportion of apoptotic EPCs after serum starvation was determined by manually counting pyknotic nuclei after Hoechst staining. Second, the amount of NO released by EPCs was measured using a NO assay kit according to the manufacturer's protocol. Third, EPCs adhesion activity assay was performed by staining with crystal violet.Results1. Rat BM-derived EPCs were cultured and identified in vitro successfully.2. In the presence of VEGF, AngII at diverse concentration enhanced the proliferative proficiency of EPCs (P<0.05) in a dose-dependent manner and increased Flk-1 mRNA expression, compared with control. The effect was blocked by protein kinase C (PKC) inhibitor or valsartan, an AngII type 1 receptor (AT1R) agonist (P<0.05).3. Compared with control, AngII at diverse concentration significantly reduced the rate of EPCs apoptosis induced by serum starvation (P<0.05), elevated the amount of NO secretion (P<0.05), and increased the number of EPCs attaching to culture dishes in a short period (P<0.05). Moreover, all of these AngII-mediated effects on EPCs were attenuated by the pretreatment with valsartan (P<0.05).ConclusionsAngII at certain concentration improved EPCs biological activities through enhancing EPCs proliferative activity, inhibiting apoptosis, promoting NO release, and augmenting adhesion potential. Furthermore, AngII mediated these favorable effects through AT1R. The ability of AngII to regulate EPCs activities may represent a possible mechanism that links AngII with angiogenesis.