Mechanistic insights into folic acid-dependent vascular protection: Dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: A novel HPLC-based fluorescent assay for DHFR activity

Gao, Ling; Chalupský, Karel; Stefani, Enrico; Cai, Hua

doi:10.1016/j.yjmcc.2009.07.025

Cited by 92 publications

(136 citation statements)

References 40 publications

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“…of several diseases, such as hypercholesterolemia (50), diabetes (15,51), hypertension (19,52,53), and atherosclerosis (54). Taken together, these observations again show the importance of the NOX-eNOS uncoupling pathway in vascular diseases, specifically under conditions of irregular hemodynamic flow.…”

Section: Discussionmentioning

confidence: 69%

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

Siu

Gao

Cai

2016

Journal of Biological Chemistry

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The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS.

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Section: Discussionmentioning

confidence: 69%

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

Siu

Gao

Cai

2016

Journal of Biological Chemistry

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“…For Western blotting, ϳ20 -40 g of proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with phosphorylated ERK1/2, ERK1/2, eNOS, and eNOS s1179 (1:1,000) antibodies following standard Western blotting procedure (12).…”

Section: Methodsmentioning

confidence: 99%

Induction of cardioprotection by small netrin-1-derived peptides

Cai

2015

American Journal of Physiology-Cell Physiology

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Li Q, Cai H. Induction of cardioprotection by small netrin-1-derived peptides. Am J Physiol Cell Physiol 309: C100 -C106, 2015. First published April 29, 2015; doi:10.1152/ajpcell.00332.2014.-We have shown that netrin-1 induces potent cardioprotection via extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent endothelial nitric oxide synthase (eNOS)/NO activation. The present study investigated cardioprotective effects of small netrin-1-derived peptides. We synthesized three laminin (Lam) V peptides and found those time dependently increased phosphorylation of ERK1/2 and eNOSs1179 in endothelial cells at the same molar concentration used for netrin-1. Preperfusion with Lam V peptides induced a substantial reduction in infarct size (control 39.3 Ϯ 0.2% vs. 14.6 Ϯ 2.3%, 23.0 Ϯ 2.8%, and 18.8 Ϯ 0.8% for V1, V2, and V3, respectively). Furthermore, reperfusion with all three also induced potent cardioprotection (control 37.6 Ϯ 1.3% vs. 17.6 Ϯ 3.2%, 20.6 Ϯ 1.7%, and 15.8 Ϯ 2.0% for V1, V2, and V3, respectively), implicating that these peptides are consistently beneficial whenever they are delivered to the heart. Based on the sequence alignment, we found a region of high sequence homology and synthesized smaller peptides [V1-9 amino acid (aa), V2-10aa, and V3-11aa]. These smaller peptides markedly reduced infarct size during reperfusion (V1-9aa 16.8 Ϯ 2.2%, V2-10aa 18.6 Ϯ 1.7%, and V3-11aa 16.7 Ϯ 3.0% vs. control 37.6 Ϯ 1.3%). A negative control V3-16aa with no sequence homology failed to protect the heart. Of note, the core area has the characteristic sequence of: Cx(1-2)Cx(3-4)Tx(0 -1)G. Furthermore, all three smaller peptides induced NO production in endothelial cells that could in turn diffuse to cardiomyocytes to promote survival. Combined applications of V1-9aa and V2-10aa synergistically induced more NO production. Taken together, these data strongly suggest that small netrin-1-derived peptides are highly effective in protecting the heart against myocardial ischemia-reperfusion injury and has the potential to be developed into peptide drugs directly applicable to the treatment of myocardial infarction.netrin-1-derived small peptides; cardioprotection; ischemia-reperfusion injury; I/R; ERK1/2; endothelial nitric oxide synthase; eNOS MYOCARDIAL INFARCTION (MI) occurs when blood cannot flow to part of the heart because of occlusion of coronary arteries, and the heart muscle is injured as a result, which leads to impaired cardiac function (13). The conventional therapy for MI is to restore blood perfusion as soon as possible to salvage some of the myocardium (8). However, reperfusion itself can paradoxically cause myocardial injury, at least in part mediated by increased oxidative stress (3, 9). Until now, no pharmacological agents have been proven clinically effective in reducing reperfusion injury. We have previously shown that a novel protein, netrin-1, can function as a potent cardioprotective agent (5,20,21,26). It induces cardioprotection against ischemia-reperfusion (I/R) injury both ex vivo and in...

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“…These critical roles of eNOS in endothelial cell growth and survival also implicate BH4 in angiogenesis. There is increasing evidence that BH4, as an essential eNOS cofactor, improves endothelial cell proliferation (81,87,165,241) and tube formation (81,87). In vitro supplementation with the BH4 precursor sepiapterin has been shown to enhance proliferation in coronary endothelial cell from diabetic rats (165), bovine aortic endothelial cells (241), and HUVEC (87).…”

Section: Fig 7 Thementioning

confidence: 99%

Tetrahydrobiopterin in Cardiovascular Health and Disease

Bendall

Douglas

McNeill

et al. 2014

Antioxidants & Redox Signaling

188

187

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Tetrahydrobiopterin (BH4) functions as a cofactor for several important enzyme systems, and considerable evidence implicates BH4 as a key regulator of endothelial nitric oxide synthase (eNOS) in the setting of cardiovascular health and disease. BH4 bioavailability is determined by a balance of enzymatic de novo synthesis and recycling, versus degradation in the setting of oxidative stress. Augmenting vascular BH4 levels by pharmacological supplementation has been shown in experimental studies to enhance NO bioavailability. However, it has become more apparent that the role of BH4 in other enzymatic pathways, including other NOS isoforms and the aromatic amino acid hydroxylases, may have a bearing on important aspects of vascular homeostasis, inflammation, and cardiac function. This article reviews the role of BH4 in cardiovascular development and homeostasis, as well as in pathophysiological processes such as endothelial and vascular dysfunction, atherosclerosis, inflammation, and cardiac hypertrophy. We discuss the therapeutic potential of BH4 in cardiovascular disease states and attempt to address how this modulator of intracellular NO-redox balance may ultimately provide a powerful new treatment for many cardiovascular diseases. Antioxid. Redox Signal. 20, 3040-3077.

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Mechanistic insights into folic acid-dependent vascular protection: Dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: A novel HPLC-based fluorescent assay for DHFR activity

Abstract: Background-Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious.

Cited by 92 publications

References 40 publications

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

Induction of cardioprotection by small netrin-1-derived peptides

Tetrahydrobiopterin in Cardiovascular Health and Disease

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