Effects Of Remnant Lipoproteins On Angiogenesis Of Circulating Endothelial Progenitor Cells

Background and Objective:Remnant lipoproteins (RLPs) are closely associated with coronary heart disease and can induce endothelial dysfunction through oxidative mechanisms. Many risk factors accelerate the onset of endothelial progenitor cells (EPCs) senescence via increased oxidative stress. In this study, we investigated the effect of RLPs on EPCs senescence and function.Methods:RLPs were isolated from postprandial plasma of hypertriglyceridemic patients by use of the immunoaffinity gel mixture of anti-apoA-1 and anti-apoB-100 monoclonal antibodies. The mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolated cells were suspended in Medium 199 supplement with 20% fetal blood serum and bovine pituitary extract for culturing, differentiating and proliferating. The expressions of specific antigens on cell surface, such as CD133,KDR,CD34,CD31 and vWF were analyzed by immunocytochemistry on day 10. To assess the onset of senescence, SA-β-gal activity was measured. The numbers of SA-β-gal positive cells were counted manually in each sample under bright field illumination. Modified Boyden chamber assay and MTT assay was used to measure the migration and proliferation function of EPCs.The adhesion assay was performed by replating on fibronectin-coated dishes, then adherent cells were counted. Matrigel Network Formation Assay was performed by replating on Matrigel-coated dishes, then the number of closed loops formed by capillary tubes network was counted.Results:peripheral blood mononuclear cells cultivated with M199 supplement with growth factors generated EPCs as expected, which were characterized by typical morphology and phenotype. EPCs became senescent as determined by senescence-associated acidicβ-galactosidase (SA-β-Gal) staining after ex vivo cultivation without any stimulation. Co-incubation with RLPs accelerated the increase in SA-β-Gal-positive EPCs. The acceleration of RLPs-induced EPCs senescence occurred dose-dependently with a maximal effect when EPCs were treated with RLPs at 100μg/mL (P<0.01). RLPs decreased adhesion, migration and proliferation capacities of EPCs as assessed by adherence to fibronectin, modified Boyden chamber technique and MTT assay (P<0.01), respectively. Moreover RLPs suppressed capillary-like tube formation on Matrigel for all cells tested. However, RLPs-induced EPCs senescence and dysfunction were significantly inhibited by treatment of atorvastatin (ATR,1.0μmol/L)(P<0.01).Conclusions:RLPs accelerate the onset of EPCs senescence via increased oxidative stress, accompanying with the impaired adhesion, migration, proliferation and suppressed capillary-like tube formation capacity. ATR prevents RLPs-induced senescence and EPCs dysfunction. Background and Objective:Hindlimb ischemia model is the widely used animal model, for it involved in the study of angiosclerosis and embolism in lower limb. Recentely, it becames an original tendency that encouraging the neovascularization by EPCs transplantation in animal hindlimb ischemia model. In this study, we investigated the effects of RLPs on therapeutic angiogenesis of circulating endothelial progenitor cells transplanted in hindlimb-ischemic nude mouse.Methods:8-10 weeks old BALB/c male nude mice were operated according to Heeschen's methods(Circulation,2004; 109:1615-22) to obtain hind limb ischemia model.One day after surgery, 1×106 of DAPI labeled EPCs in 200μL of M199 medium were injected into the caudal vein of mice. A laser Doppler perfusion imager (Moor Instruments), which maps tissue blood flow by the shift in the laser light frequency, was used for serial noninvasive physiological evaluation of neovascularization. Each mouse was followed by serial recording of surface blood flow of hind limb on days 0,3,7,14, and 21. Tissue capillary density was determined as a histological evaluation of neovascularization. To assess the onset of senescence, SA-β-gal activity was measured. The numbers of SA-β-gal positive cells and capillaries were counted manually in each sample under bright field illumination.Results::We establish nude mouse hindlimb ischemia mode with obvious ischemia in mouse's hindlimb. EPCs transplantation significantly reduce foot necrosis and auto-amputation. Administration of EPCs treated with ATR resulted in a significantly lower rate of limb loss (necrosis of tissue above knee or autoamputation) compared with EPCs treated with RLPs and with control (P<0.01). Serial analysis of laser Doppler perfusion imaging revealed a greater increase of limb perfusion in the ischemic limb of mice injected with EPCs that were treated with ATR than with RLPs(P<0.01). Administration of EPCs treated with ATR increased capillary density of ischemic skeletal muscle compared with EPCs that were treated with RLPs(P<0.01).Conclusions:RLPs accelerate the onset of EPCs senescence and reduce therapeutic angiogenesis of circulating EPCs transplanted in hindlimb-ischemic nude mouse. ATR improves the angiogenesis ability of these EPCs. Background and Objective:MicroRNAs (miRNAs) are small (18-24nt) non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis, proliferation and neovascularization. We investigated the possible mechanism of effects of remnant lipoproteins on angiogenesis of circulating endothelial progenitor cells using an oligonucleotide microchip for microRNA profiling.Methods:After 10 days of culture, EPCs were treated with/ without RLPs,the later were served as control. An oligonucleotide microchip was used to find miRNAs that were differential expressed. Total RNA was extracted using mirVana RNA Isolation Kit for miRNA microarray. Assess the purity of the RNA adopting Agilent RNA 6000 Nano Assay. Between two groups of the fingerprints, Agilent Microarray Scanner and Agilent Feature Extraction Softwarewas performed to screen the markedly differentially expressed genes.Results:Four miRNAs differentially expressed between these two groups were detected. Three are upregulated:hsa-miR-148b*,hsa-miR-155,and hsa-miR-513c, One is downregulated, that is hsa-miR-574-3p (RLPs treated vs. control). Conclusions:MiRNAs may regulate the effect of remnant lipop-roteins on angiogenesis of circulating endothelial progenitor cells.