gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Reversal of eNOS uncoupling and upregulation of eNOS expression as a new therapeutic approach to cardiovascular diseases

Meeting Contribution

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  • corresponding author H. Li - Department of Pharmacology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
  • U. Förstermann - Department of Pharmacology, University Medical Center, Johannes Gutenberg University, Mainz, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf05

DOI: 10.3205/10dgaf05, URN: urn:nbn:de:0183-10dgaf053

Published: March 23, 2011

© 2011 Li et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) represents a vasoprotective principle. Cardiovascular diseases are associated with decreased NO bioavailability and eNOS uncoupling (i.e., uncoupling of oxygen reduction from NO synthesis in eNOS). An uncoupled eNOS produces superoxide instead of NO. Therefore, compounds that increase eNOS protein levels are only beneficial when guaranteeing eNOS functionality. Recently, we have found compounds that maintain eNOS functionality in disease, and, at the same time, upregulate expression of the enzyme. Midostaurin upregulates eNOS expression through PKC-independent mechanisms and reduces NADPH oxidase expression via PKC inhibition. Oxidative stress-mediated oxidation of tetrahydrobiopterin (BH4), the essential eNOS cofactor, is likely to be a major cause for eNOS uncoupling. By reducing NADPH oxidase-mediated oxidative stress, midostaurin reverses eNOS uncoupling in spontaneously hypertensive rats and in atherosclerosis-prone apolipoprotein E knockout (apoE-KO) mice, which is associated with NO-mediated vasodilation and blood pressure reduction. AVE9488 and AVE3085 are two small-molecular weight eNOS transcription enhancers. Both compounds stimulate eNOS transcription in endothelial cells in vitro and in vascular tissues in vivo. Importantly, treatment of apoE-KO mice with AVE9488 enhances vascular BH4 levels and reverses eNOS uncoupling. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduces cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduces atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Also resveratrol reverses eNOS uncoupling and enhances eNOS expression. In conclusion, compounds combining eNOS upregulation with eNOS recoupling may have therapeutic potential for cardiovascular diseases.


Introduction

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is an anti-hypertensive, antithrombotic and anti-atherosclerotic principle [1]. Cardiovascular diseases such as hypertension, atherosclerosis and diabetes mellitus are associated with decreased NO bioactivity due to reduced NO production by eNOS and/or the increased inactivation of NO following reaction with superoxide [2].

The reduction in the production of NO by eNOS is a result of the inhibition of eNOS enzymatic activity and/or dysfunction of the enzyme (i.e., eNOS uncoupling). Under a number of pathological conditions, the enzymatic reduction of molecular oxygen by eNOS is no longer coupled to L-arginine oxidation, resulting in the production of superoxide rather than NO. This phenomenon is referred to as eNOS uncoupling [3]. A number of potential mechanisms have been reported to contribute to eNOS uncoupling. Among these, the NOS cofactor tetrahydrobiopterin (BH4) is likely to act as the major “coupling switch”, and BH4 deficiency seems to be the primary cause for eNOS uncoupling in pathophysiology. Thus, reversal of eNOS uncoupling and upregulation of eNOS expression may represent a new therapeutic approach to cardiovascular diseases.


Methods

Expression of eNOS was analyzed in cultured human endothelial cells and in cardiovascular tissues of experiment animals by quantitative real-time RT-PCR and Western blot. eNOS uncoupling was studied by measuring reactive oxygen species production in the presence of NOS inhibitor L-NAME.


Results

Midostaurin (4'-N-benzoyl staurosporine, CGP41251, PKC-412) is a glycosidic indolocarbazole analog of staurosporine. Midostaurin upregulates eNOS expression through PKC-independent mechanisms [4], [5], [6] and reduces NADPH oxidase expression via PKC inhibition [6]. By reducing NADPH oxidase-mediated oxidative stress, midostaurin increases BH4 levels and reverses eNOS uncoupling in spontaneously hypertensive rats and in atherosclerosis-prone apoE-KO mice. This is associated with an increased NO-mediated vasodilation and blood pressure reduction [5], [6]. However, the therapeutic use of the PKC inhibitor is limited by significant systemic toxicity in vivo.

Recently, we have described vascular effects of two pentacyclic triterpenoid acids that occur in various plants: betulinic acid [7] and ursolic acid [8]. We found that these triterpenoid acids upregulate eNOS expression, and at the same time, reduce NADPH oxidase expression in human endothelial cells through PKC-independent mechanisms [7], [8] (Figure 1 [Fig. 1]). The triterpenoids thus have the potential to reverse eNOS uncoupling. In addition, betulinic acid also enhances eNOS enzymatic activity by phosphorylation of eNOS at serine 1177 and dephosphorylation of eNOS at threonine 495 (authors’ unpublished data). Both compounds are devoid of prominent in vivo toxicity (at least in rodents) [9]. Their therapeutic potential in cardiovascular disease needs to be further investigated in in vivo studies.

Two small-molecular-weight eNOS transcription enhancers – AVE9488 (4-fluoro-N-indan-2-yl-benzamide) and AVE3085 (2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide) – have been identified in a high throughput screening [10]. These compounds stimulate eNOS transcription in endothelial cells in vitro and in vascular tissues in vivo. Importantly, treatment of apoE knockout mice with AVE9488 enhances vascular BH4 levels and reverses eNOS uncoupling. In apoE-KO mice, but not in eNOS-KO mice, treatment with AVE9488 reduces cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduces atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice [10]. Moreover, AVE9488 reverses impaired functional activity of EPCs from patients with ischemic cardiomyopathy [11], and improves cardiac remodeling and heart failure after experimental myocardial infarction [12]. Despite the promising results in the above in vivo experiments, the long-term therapeutic benefit of AVE9488 and AVE3085 is still unknown.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic phytoalexin found in red grapes and several other plants [13]. Resveratrol has been shown to prevent or slow the progression of a wide variety of diseases including cancer and cardiovascular diseases [14]. Resveratrol also extends the lifespan of various organisms from yeast to vertebrates [15]. No significant in vivo toxicity has been reported for resveratrol [16].

We have previously demonstrated that resveratrol increases eNOS expression in human endothelial cells [17]. Recent data from our laboratory demonstrate that resveratrol can also reverse eNOS uncoupling [18]. As a polyphenolic compound, resveratrol has been shown to scavenge several types of radicals (including hydroxyl, superoxide and metal-induced radicals [14]. However, the direct antioxidant effect of resveratrol is poor; the protective effects of resveratrol against oxidative injury are likely to be attributed mostly to the upregulation of endogenous cellular antioxidant system rather than its direct ROS scavenging activity. Treatment with resveratrol leads to an upregulation of superoxide dismutases SOD1, SOD2, SOD3, glutathione peroxidase 1 (GPx1) and catalase in the hypercholesterolemic apoE-KO mice as well as in cultured human endothelial cells [18], [19] (Figure 1 [Fig. 1]). At the same time, the expression and activity of NADPH oxidases are downregulated [18], [19]. As a result, peroxynitrite levels and BH4 oxidation are reduced by resveratrol. Importantly, resveratrol also increases BH4 levels by upregulating GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme for BH4 biosynthesis [18] (Figure 1 [Fig. 1]). The resulting reversal of eNOS uncoupling, along with the enhanced expression levels and enzymatic activity (eNOS phosphorylation and eNOS deacetylation), is likely to contribute to the protective effects of trans-resveratrol.


Discussion

In addition to the abovementioned compounds, some established drugs have also been shown to reverse eNOS uncoupling and enhance eNOS expression. These include the 3-hydroxy-3-methylgulutaryl-coenzyme A reductase inhibitors (statins), the renin inhibitor aliskiren, the angiotensin-converting enzyme (ACE) inhibitors, the angiotensin AT1 receptor blockers, and the aldosterone antagonist eplerenone [2]. The resulting improvement of NO bioactivity and endothelial function is likely to be part of their pleiotropic actions. Novel pharmacological approaches to prevent or reverse eNOS uncoupling are of therapeutic interest.


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