Effects Of Homocysteine On Number And Activity Of Endothelial Progenitor Cells From Peripheral Blood And Analysis Of Its Mechanism

Homocysteine (Hcy), a sulfur-containing amino acid, is an intermediate metabolite of methionine. Recently, hyperhomocysteinemia has been recognized as a major risk factor of cardiovascular diseases. Although the mechanism by which Hcy damages the vessels wall and induces atherosclerosis is ill understood, a growing body of evidence suggested that endothelial dysfunction played a major role. Endothelial dysfunction ultimately loses a balance between the magnitude of injury and the capacity for repair. A variety of evidence suggested that circulating endothelial progenitor cells (EPCs) constituted one aspect of this repair process.EPCs are a cell population that have the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but have neither acquired characteristic mature endothelial markers nor formed a lumen. Laboratory evidence suggested that these precursors participated in postnatal neovascularization and reendothelialization. Recently, studies have demonstrated that risk factors for coronary artery disease (CAD) correlate with a reduced number and functional activity of circulating EPCs. In addition, there is evidence to suggest that smoking, hypertension, hypercholesterolemia and diabetes affect EPCs numbers and functions.On the basis of these considerations, We hypothesized that Hcy not only directly injured endothelial cell but also affected EPC numbers and functions at the same time, thus influenced endothelial repair process and disturbed the balance between the magnitude of injury and the capacity for repair, which led to endothelial dysfunction andpromoted the progression of CAD. To test this hypothesis, we measured the numbers and activity of EPCs exposed to homocysteine at first. Then, the numbers and activity of EPCs from peripheral blood of patients with hyperhomocysteinemia were determined. Finally, we studied the mechanisms by which Hcy reduced EPCs numbers and activity.Part 1: Effects of homocysteine on number and activity of endothelialprogenitor cells from peripheral bloodThe aim of this study is to investigate whether Hcy has influences on EPCs number and activity. Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days cultured, attached cells were stimulated with Hcy (to make a series of final concentrations: 10μmol/L, 50μmol/L, 100μmol/L, 200μmol/L) or vehicle control for the respective time points (6h, 12h, 24h and 48h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then adherent cells were counted. Incubation of isolated human MNCs with Hcy dose and time dependently decreased the number of EPCs, maximum at 200μmol/L, 24h (35.7±6.7 vs 62.5±10.6, P<0.01). In addition, Hcy dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. In conclusion, hyperhomocysteine may induce the reduction of EPCs with decreased functional activity.Part 2 Changes in the number and activity of circulating endothelial progenitor cells in patients with hyperhomocysteinemiaHyperhomocysteinemia (HHcy) contributes to atherosclerosis and coronary artery diseases by inducing endothelial cell injury and dysfunction. Recent studies provided increasing evidence that EPCs participated in ongoing endothelial repair. The changes of EPCs in patients with HHcy have not been elucidated so far. Therefore, we investigated the number and functional activity of EPCs in patients with HHcy. Human EPCs were isolated and cultured from patients with HHcy (n=30) and matched volunteers (n=30). Circulating EPCs were enumerated as AC133~+KDR~+ cells via fluorescence-activated cell sorter analysis. Additionally, EPCs were expanded from human blood in vitro and identified by Dil-acLDL uptake and lectin staining by direct fluorescent staining under a laser scanning confocal microscope. EPCs migration activities were determined by modified Boyden chamber assay. EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes, then adherent cells were counted. A significant decrease was observed in circulating EPCs (AC133~+KDR~+ cells) numbers in patients with HHcy compared with control subjects (63.9±11.7 cells/ml vs 91.5±14.2 cells/ml blood, P<0.01). In addition, the numbers of EPCs also decreased in patients with HHcy after ex vivo cultivation (36.1±6.5 vs 51.5±8.3 EPCs/x200 field, P<0.01). Both circulating EPCs and differentiated EPCs were inversely correlated with total Hcy levels. In addition, EPCs from patients with HHcy were significantly impaired in their migratory capacity and ability to adhere to fibronectin compared with controls. The present study demonstrated that EPCs numbers and functional capacity were impaired in patients with HHcy.Part 3 Homocysteine accelerates senescence and reduces proliferation of endothelial progenitor cellsOur previous studies have shown that Hcy reduced EPCs numbers and impaired functional activity. However, the mechanisms by which Hcy reduces EPCs numbers and activity remain to be determined. Recent studies have demonstrated that reduced EPCs numbers and activity was associated with EPCs senescence which involved telomerase activity. Therefore, we investigated whether Hcy accelerated the onset of EPCs senescence through telomerase inactivation, leading to cellular dysfunction. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic β-galactosidase staining. Hcy dose-dependently accelerated the onset of EPCs senescence in culture. Moreover, Hcy decreased proliferation of EPCs as assessed by BrdU incorporation assay and colony-forming capacity. To get further insights into the underlying mechanisms of these effects induced by Hcy, we measured telomerase activity and determined the phosphorylation of Akt by using western blot. Hcy significantly diminished telomerase activity and Akt phosphorylation. Taken together, the results of the present study demonstrated that Hcy accelerated the onset of EPCs senescence, leading to cellular dysfunction. The effect of Hcy might be dependent on telomerase inactivation, and Akt dephosphorylation also appeared to play a major role. In addition, atorvastatin had a preventative effect against Hcy-induced EPCs senescence.