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Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.

ABSTRACT: Premature senescence of vascular endothelial cells is a leading cause of various cardiovascular diseases. Therapies targeting endothelial senescence would have important clinical implications. The present study was aimed to evaluate the potential of heme oxygenase-1 (HO-1) as a therapeutic target for endothelial senescence.Upregulation of HO-1 by Hemin or adenovirus infection reversed H2O2-induced senescence in human umbilical vein endothelial cells (HUVECs); whereas depletion of HO-1 by siRNA or HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) triggered HUVEC senescence. Mechanistically, overexpression of HO-1 enhanced the interaction between HO-1 and endothelial nitric oxide synthase (eNOS), and promoted the interaction between eNOS and its upstream kinase Akt, thus resulting in an enhancement of eNOS phosphorylation at Ser1177 and a subsequent increase of nitric oxide (NO) production. Moreover, HO-1 induction prevented the decrease of eNOS dimer/monomer ratio stimulated by H2O2 via its antioxidant properties. Contrarily, HO-1 silencing impaired eNOS phosphorylation and accelerated eNOS uncoupling. In vivo, Hemin treatment alleviated senescence of endothelial cells of the aorta from spontaneously hypertensive rats, through upregulating eNOS phosphorylation at Ser1177.HO-1 ameliorated endothelial senescence through enhancing eNOS activation and defending eNOS uncoupling, suggesting that HO-1 is a potential target for treating endothelial senescence.

SUBMITTER: Luo W

PROVIDER: S-EPMC6075439 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.

Luo Wenwei W Wang Yu Y Yang Hanwei H Dai Chunmei C Hong Huiling H Li Jingyan J Liu Zhiping Z Guo Zhen Z Chen Xinyi X He Ping P Li Ziqing Z Li Fang F Jiang Jianmin J Liu Peiqing P Li Zhuoming Z

Aging 20180701 7

<h4>Aim</h4>Premature senescence of vascular endothelial cells is a leading cause of various cardiovascular diseases. Therapies targeting endothelial senescence would have important clinical implications. The present study was aimed to evaluate the potential of heme oxygenase-1 (HO-1) as a therapeutic target for endothelial senescence.<h4>Methods and results</h4>Upregulation of HO-1 by Hemin or adenovirus infection reversed H<sub>2</sub>O<sub>2</sub>-induced senescence in human umbilical vein en ...[more]

PMID: 30048241

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Project description:An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Project description:Endothelial cells (ECs) form the inner lining of blood vessels and are central to sensing chemical perturbations that can lead to oxidative stress. The degree of stress is correlated with divergent phenotypes such as quiescence, cell death, or senescence. Each possible cell fate is relevant for a different aspect of endothelial function, and hence, the regulation of cell fate decisions is critically important in maintaining vascular health. This study examined the oxidative stress response (OSR) in human ECs at the boundary of cell survival and death through longitudinal measurements, including cellular, gene expression, and perturbation measurements. 0.5 mM hydrogen peroxide (HP) produced significant oxidative stress, placed the cell at this junction, and provided a model to study the effectors of cell fate. The use of systematic perturbations and high-throughput measurements provide insights into multiple regimes of the stress response. Using a systems approach, we decipher molecular mechanisms across these regimes. Significantly, our study shows that heme oxygenase-1 (HMOX1) acts as a gatekeeper of cell fate decisions. Specifically, HP treatment of HMOX1 knockdown cells reversed the gene expression of about 51% of 2,892 differentially expressed genes when treated with HP alone, affecting a variety of cellular processes, including anti-oxidant response, inflammation, DNA injury and repair, cell cycle and growth, mitochondrial stress, metabolic stress, and autophagy. Further analysis revealed that these switched genes were highly enriched in three spatial locations viz., cell surface, mitochondria, and nucleus. In particular, it revealed the novel roles of HMOX1 on cell surface receptors EGFR and IGFR, mitochondrial ETCs (MTND3, MTATP6), and epigenetic regulation through chromatin modifiers (KDM6A, RBBP5, and PPM1D) and long non-coding RNA (lncRNAs) in orchestrating the cell fate at the boundary of cell survival and death. These novel aspects suggest that HMOX1 can influence transcriptional and epigenetic modulations to orchestrate OSR affecting cell fate decisions.

Project description:Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

Project description:We recently reported that chronic nicotine impairs reflex chronotropic activity in female rats. Here, we sought evidence to implicate nitric oxide synthase (NOS) and/or heme oxygenase (HO) in the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate to increases (phenylephrine) or decreases (sodium nitroprusside) in blood pressure were generated in conscious female rats treated with nicotine or saline in absence and presence of pharmacological modulators of NOS or HO activity. Compared with saline-treated rats, nicotine (2 mg/kg/day i.p., for 14 days) significantly reduced the slopes of baroreflex curves, a measure of baroreflex sensitivity (BRS). Findings that favor the involvement of NOS inhibition in the nicotine effect were (i) NOS inhibition (N?-Nitro-L-arginine methyl ester, L-NAME) reduced BRS in control rats but failed to do so in nicotine-treated rats, (ii) L-arginine, NO donor, reversed the BRS inhibitory effect of nicotine. Alternatively, HO inhibition (zinc protoporphyrin IX, ZnPP) had no effect on BRS in nicotine- or control rats and failed to reverse the beneficial effect of L-arginine on nicotine-BRS interaction. Similar to female rats, BRS was reduced by L-NAME, but not ZnPP, in male rats and the L-NAME effect was not accentuated after concomitant administration of nicotine. Baroreflex dysfunction caused by nicotine in female rats was blunted after supplementation with hemin (HO inducer) but not tricarbonyldichlororuthenium(II) dimer (CORM-2), a carbon monoxide (CO) releasing molecule, or bilirubin, the breakdown product of heme catabolism. The facilitatory effect of hemin was abolished upon simultaneous treatment with L-NAME or 1H-[1], [2], [4] oxadiazolo[4,3-a] quinoxalin-1-one (inhibitor of soluble guanylate cyclase, sGC). The activities of HO and NOS in brainstem tissues were also significantly increased by hemin. Thus, the inhibition of NOS, but not HO, accounts for the baroreflex depressant of chronic nicotine. Further, hemin alleviates the nicotine effect through a mechanism that is NOS/sGC but not CO or bilirubin-dependent.

Dataset Information

Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.

Publications

Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.

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