Project description:Nitric oxide helps to prevent endothelial dysfunction and senescence. This study aimed to define genomic and proteomic changes in cultured porcine senescent endothelial cells and their resemblance with those observed in regenerated endothelial cells. Senescent endothelial cells were produced by passaging primary porcine coronary arterial endothelial cells until passage four. The protein presence of endothelial nitric oxide synthase, cyclic GMP levels [basal and during stimulation (bradykinin and A23187)], were reduced. The mRNA expression level was measured by microarray assays. Genes related to oxidative stress [superoxide dismutase (MnSOD), glutathione peroxidase 3, glutathione S-transferase M1] were downregulated, extracellular matrix components (type III collagen, thrombospondin 1 and 3, transforming growth factor ?) upregulated and nuclear factor kappa B (NF?B)-signaling pathway [IkappaB, TNF receptor-associated factor 1 and 5 (TRAF1 and 5)] activated in senescent cells. The differential gene expression of MnSOD and TRAF5 was confirmed at the protein level by Western blotting and biochemical assay (MnSOD). The basal and stimulated (by tumor necrosis factor-?)?levels of NF?B were augmented as demonstrated by electrophoretic mobility shift assay. In summary, cultured senescent endothelial cells exhibit reduced nitric oxide production, and decreased antioxidative, proliferative capacities, augmented expression of extracellular matrix components and activation of NF?B. These molecular changes do not exactly mimick those occurring during endothelial regeneration in vivo. Experiment Overall Design: Totally 8 samples were analyzed with 4 replicates each for control (cells at passage one) and test (cells at passage four) samples.
Project description:Nitric oxide helps to prevent endothelial dysfunction and senescence. This study aimed to define genomic and proteomic changes in cultured porcine senescent endothelial cells and their resemblance with those observed in regenerated endothelial cells. Senescent endothelial cells were produced by passaging primary porcine coronary arterial endothelial cells until passage four. The protein presence of endothelial nitric oxide synthase, cyclic GMP levels [basal and during stimulation (bradykinin and A23187)], were reduced. The mRNA expression level was measured by microarray assays. Genes related to oxidative stress [superoxide dismutase (MnSOD), glutathione peroxidase 3, glutathione S-transferase M1] were downregulated, extracellular matrix components (type III collagen, thrombospondin 1 and 3, transforming growth factor beta) upregulated and nuclear factor kappa B (NFKB)-signaling pathway [IkappaB, TNF receptor-associated factor 1 and 5 (TRAF1 and 5)] activated in senescent cells. The differential gene expression of MnSOD and TRAF5 was confirmed at the protein level by Western blotting and biochemical assay (MnSOD). The basal and stimulated (by tumor necrosis factor-alpha) levels of NFKB were augmented as demonstrated by electrophoretic mobility shift assay. In summary, cultured senescent endothelial cells exhibit reduced nitric oxide production, and decreased antioxidative, proliferative capacities, augmented expression of extracellular matrix components and activation of NFKB. These molecular changes do not exactly mimick those occurring during endothelial regeneration in vivo. Keywords: disease state comparison
Project description:Phenotypic heterogeneity among arterial ECs is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in their atherosusceptibility. To explore EC heterogeneity, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary and iliac artery ECs. We demonstrate that in vivo the endothelial transcriptional profile of a coronary artery (the right coronary artery) is intrinsically different from that of a major conduit vessel (the external iliac artery), and that this difference is consistent with former vessel being more prone to atherosclerosis. Keywords: coronary atherosclerosis, endothelial heterogeneity, microarray, gene expression Endothelial cells were freshly harvest from right coronary, left and right iliac arteries from four pigs. RNA were isolated and expression profiles were obtained using olig microarrays.
Project description:Atherosclerotic plaques tend to form in the major arteries at certain predictable locations. As these arteries vary in atherosusceptibility, inter-arterial differences in endothelial cell (EC) biology are of considerable interest. To explore the origin of differences observed between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of harvested porcine coronary (CECs) and iliac (IECs) artery ECs grown in static culture out to passage four. Fewer differences were observed between the transcriptional profiles of CECs and IECs in culture compared to in vivo, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. One-class significance of microarrays revealed that most in vivo interarterial differences disappeared in culture, as fold differences after passaging were not significant for 85% of genes identified as differentially expressed in vivo at a 5% false discovery rate. However, the three homeobox genes HOXA9, HOXA10, and HOXD3 remained under-expressed in coronary endothelium for all passages by at least 9, 8, and 2-fold, respectively. Continued differential expression despite removal from the in vivo environment suggests that primarily heritable or epigenetic mechanism(s) influence transcription of these three genes. Quantitative real-time polymerase chain reaction confirmed expression ratios for seven genes associated with atherogenesis and over- or under-expressed by 3-fold in CECs relative to IECs. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial endothelium. The transcriptional differences observed here may provide new insights into pathways responsible for coronary artery susceptibility. Endothelial cells were freshly harvested from right coronary and iliac arteries from four pigs. Cells were cultured out to passage four. RNA was isolated after each passage and expression profiles were obtained using oligo microarrays.
Project description:Phenotypic heterogeneity among arterial ECs is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in their atherosusceptibility. To explore EC heterogeneity, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary and iliac artery ECs. We demonstrate that in vivo the endothelial transcriptional profile of a coronary artery (the right coronary artery) is intrinsically different from that of a major conduit vessel (the external iliac artery), and that this difference is consistent with former vessel being more prone to atherosclerosis. Keywords: coronary atherosclerosis, endothelial heterogeneity, microarray, gene expression