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To study the genomic changes in senescent porcine coronary arterial endothelial cells by multiple passaging in vitro

ABSTRACT: 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

ORGANISM(S): Sus scrofa

PROVIDER: GSE8974 | GEO | 2007/09/08

SECONDARY ACCESSION(S): PRJNA102429

REPOSITORIES: GEO

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Project description:The nitric oxide synthase (NOS) co-factor, tetrahydrobiopterin (BH4), is a redox-active molecule that regulates nitric oxide (NO) and reactive oxygen species (ROS) production by NOS, and is an example of redox-dependent signalling in the endothelium that underlies both the maintenance of vascular homeostasis and the development of cardiovascular disease (CVD). Loss of endothelial BH4 is observed in CVD states and results in decreased NO production and increased ROS generation by endothelial NO synthase (uncoupling). Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial uptake of BH4, highlighting the need to explore the wider roles of BH4 in order to find novel therapeutic targets. In this study we aim to elucidate the effects of BH4 depletion on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme GTPCH in vitro. Knockdown of GTPCH (and, therefore, intracellular BH4 levels) by >90% lead to a striking induction of ROS generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4 depleted GCH fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Furthermore, this BH4-dependent, mitochondrial-derived ROS oxidized mitochondrial BH4 and is seen alongside distinct changes in the thioredoxin and glutathione antioxidant pathways, revealed by mass spectrometry using an unbiased proteomic approach. These changes are accompanied by a modest increase in mitochondrial size, attenuated respiratory function, and marked changes in the cellular metabolic profile; including succinate accumulation. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and metabolism.

Project description:Chen2006 - Nitric Oxide Release from Endothelial Cells This model is described in the article: Theoretical analysis of biochemical pathways of nitric oxide release from vascular endothelial cells. Chen K, Popel AS. Free Radic. Biol. Med. 2006 Aug; 41(4): 668-680 Abstract: Vascular endothelium expressing endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which has a number of important physiological functions in the microvasculature. The rate of NO production by the endothelium is a critical determinant of NO distribution in the vascular wall. We have analyzed the biochemical pathways of NO synthesis and formulated a model to estimate NO production by the microvascular endothelium under physiological conditions. The model quantifies the NO produced by eNOS based on the kinetics of NO synthesis and the availability of eNOS and its intracellular substrates. The predicted NO production from microvessels was in the range of 0.005-0.1 microM/s. This range of predicted values is in agreement with some experimental values but is much lower than other rates previously measured or estimated from experimental data with the help of mathematical modeling. Paradoxical discrepancies between the model predictions and previously reported results based on experimental measurements of NO concentration in the vicinity of the arteriolar wall suggest that NO can also be released through eNOS-independent mechanisms, such as catalysis by neuronal NOS (nNOS). We also used our model to test the sensitivity of NO production to substrate availability, eNOS concentration, and potential rate-limiting factors. The results indicated that the predicted low level of NO production can be attributed primarily to a low expression of eNOS in the microvascular endothelial cells. This model is hosted on BioModels Database and identified by: BIOMD0000000676. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.