Effects Of Nitroglycerin On The Proliferation Of Endothelial Progenitor Cells From Peripheral Blood Of Patients With Coronary Artery Disease

Objective:The vascular endothelium plays a critical role in maintaining cardiovascular homeostasis. Recently, increasing evidence has revealed that endothelial dysfunction is the major contributor for the initiation and progression of atherosclerosis and coronary artery disease (CAD). Endothelial progenitor cells (EPCs) can contribute to vascular repair and inhibit the progression of atherosclerotic lesion. Circulating EPCs were impaired in patients with established CAD. Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest, and apoptosis. Nitroglycerin (NTG) has long been the principal therapeutic agent for the treatment of acute angina and congestive heart disease. As NO donor drugs, NTG has been suggested to be metabolized to NO and supplements exogenous NO attributed to its biology effect, but continuous therapy with NTG increases vascular oxidative stress and endothelial dysfunction. The aim of this research was to investigate the effects and mechnisms of NTG on proliferation and NTG tolerance of human peripheral blood-derived EPCs.Method:1.Human peripheral blood was collected from healthy volunteers for isolation of mononuclear cells.Peripheral-blood derived mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque Plus.The mononuclear cells were cultured in the endothelial differentiation medium consisting of M199 supplemented with 20% FBS,10ng/mL recombinant human VEGF (R&D Systems), 5ng/mL fibroblast growth factor, 100g/mL streptomycin, and 500g/mL penicillin on fibronectin-coated dishes. After 7 days of culture, adherent cells were used for determination of their endothelial lineage characteristics,EPCs were evaluated for the uptake of acetylated low-density lipoprotein (LDL) and the binding of lectin (FITC-UEA-I). EPCs took up Dil-ac-LDL and were positively stained with FITC-UEA-1 at the seventh day. And the growth curve of EPCs was analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.2.EPCs from patients with CAD were dealt with seven concentrations of nitroglycerin, as 0.0 mg/L(control),0.3 mg/L,1.0 mg/L,2.0 mg/L,7.5 mg/L,15.0 mg/L,20.0 mg/L, respectively. For evaluation of EPCs proliferation, MTT assay and direct counting were performed after 72h.The Nitric Oxide.ONOO- and the vascular endothelial growth factor-A(VEGF-A) in supernatant were tested by ELISA assay.3.EPCs were further pretreated with phosphatidylinositide-3-kinase(PI3K) inhibitor (LY294002,50μmol/L) for 12 hours before treating with nitroglycerin(7.5 and 20.0 mg/L),Culture supernatants were collected and assayed for NO and VEGF-A using NO/VEGF-A ELISA kit,ROS in adherent cells were determined by CM-H2DCFDA,and the expression of Akt/eNOS was tested by western blot analysis.4.EPCs were pretreated with β-mercaptoethanol (βME) (50μmol/L) for 4 hours before treating with nitroglycerin(20.0 mg/L),then EPCs proliferation,ROS and the expression of Akt/eNOS were tested.Four independent experiments were performed.All date are expressed as the mean ±standard deviation.After testing for normal distribution of variables,the one way analysis of variance test was used for repeated measures. Statistical analyses were performed using SPSS17.0(SPSS,Chicago,USA) software.A P value of <0.05 was considered statistically significant.Results:1.Cells began to adhere the bottom of the well after 24 h culture.Colonies and spindle-shaped cells were observed on the 7 day(Figure lA).At day 14 colony formation starts with a central aggregate of round cells, surrounded by spindle-shaped cells radiating away from the central core(Figure 1B).And after 1-2 day paving stone sample cells were formed(Figure 1C). Then, the attached EPCs were evaluated for the uptake of lectin (FITC-UEA-I)(green)(Figure. 1D) and binding of acetylated LDL labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine (red) (Figure. 1E).And the attached EPCs bind both(yellow)(Figure 1F).The growth curve of EPCs:At day 1-5 after resedeed at a density of 2 ×105/mL in medium 199,a significant linear increase in capacity of EPCs prolif eration was observed and at day 7 reached the maximum capacity(Figure 2)2.Compared with control (0.0 mg/L NTG), the number and proliferative activity of EPCs reached a maximum at 7.5 mg/L NTG, In parallel, maximal VEGF-A protein and eNOS and Akt phosphorylation expression was seen at a dose of 7.5 mg/L,However, no change of ONOO- and ROS level was observed when EPCs incubated with 0.3-7.5 mg/L of NTG.While there was a significant reduction of the number and proliferative activity of EPCs following treatment with high dose of NTG (20.0 mg/L),VEGF-A protein and eNOS and Akt phosphorylation expression were declined,and the production of ONOO- and ROS by cultured EPCs showed a significant increase.A significant linear increase in NO level was observed with NTG dose-dependent manner. The findings suggested that the amount of NO released from moderate concentrations of nitroglycerin(meanwhile the level of ROS is low) up-regulated the expressions of p-Akt and p-eNOS,then contributed to the secretion of VEGF-A,which enhanced EPCs proliferation significantly. However, the reduction of VEGF-A was associated with excess ROS and ONOO-levels when treated with the higher concentrations of NTQthen EPCs proliferation is decreased(Figure3,4,5,6).3.Pre-incubation with β-mercaptoethanol (βME) down-regulated ROS production and up-regulated eNOS and Akt phosphorylation expression when treated with 20mg/L NTG(Figure5,7).Conclusion:1. The effects of NTG on the in vitro proliferation of EPCs is closely related with the doses, moderate doses of NTG enhanced the proliferation of EPCs, however, high dose of NTG (20.0 mg/L) inhibitedthe proliferation of EPCs.2.Moderate doses of NTG-induced exogenous NO production contributed to enhancing proliferation of EPCs in vitro via PI3K/Akt pathway, but high dose of NTG(20.0 mg/L) induces nitroglycerin tolerance, eNOS and Akt phosphorylation expression were down-gratulated and inhibit proliferation of EPCs.3.β-mercaptoethanol ameliorates nitroglycerin tolerance via PI3K/Akt pathway.