Letters

Quercetin Mitochondriotropic Derivatives Antagonize Nitrate Tolerance and Endothelial Dysfunction of Isolated Rat Aorta Rings
Miriam Durante 1, Giampietro Sgaragli 1, Lucia Biasutto 2, 3, Andrea Mattarei 2, 4, Fabio Fusi 1 1 Dipartimento di Scienze della Vita, Universit degli Studi di Siena, Siena, Italy 2 CNR Istituto di Neuroscienze, Padova, Italy 3 Dipartimento di Scienze Biomediche, Universit degli Studi di Padova, Padova, Italy 4 Dipartimento di Scienze Chimiche, Universit degli Studi di Padova, Padova, Italy

Abstract
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Key words rat aorta rings glyceryl trinitrate tolerance endothelial dysfunction quercetin

Abbreviations
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GTN: glyceryl trinitrate ACh: acetylcholine QTA-3BTPI: 3,4,5,7-tetra-acetyl-3-(4-O-triphenylphosphoniumbutyl) quercetin iodide Q-3BTPI: 3-(4-O-triphenylphosphoniumbutyl) quercetin iodide TPP+: positively charged triphenylphosphonium Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica

Oxidative stress, tolerance, and endothelial dysfunction of coronary and resistance arteries are responsible for the loss of effectiveness of organic nitrates in the course of therapy of cardiovascular diseases. Oxidative stress originates in mitochondria where reactive oxygen species are produced in excess thus damaging complex I of the respiratory chain [1] and aldehyde dehydrogen-

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Chronic use of glyceryl trinitrate is limited by serious side effects, inter alia tolerance and endothelial dysfunction of coronary and resistance arteries. The natural flavonoid quercetin has been shown to counteract the development of glyceryl trinitrate tolerance in vitro. Two mitochondriotropic, 4-O-triphenylphosphoniumbutyl derivatives of quercetin (QTA-3BTPI and Q-3BTPI) were compared to quercetin for protection against glyceryl trinitrate-induced tolerance and endothelial dysfunction of isolated rat aorta rings. Both QTA-3BTPI and Q-3BTPI significantly counteracted the reduced vascular responsiveness to both glyceryl trinitrate and acetylcholine caused by prolonged exposure of the vessel to glyceryl trinitrate itself, their potency being much greater than that of quercetin. QTA-3BTPI, however, turned out to cause endothelial dysfunction per se. Since Q-3BTPI antagonized in vitro nitrate tolerance and endothelial dysfunction of vessels, this encourages assessing whether this effect is displayed also in vivo during long-term glyceryl trinitrate treatment.

ase-2, which bioactivates GTN [2]. Interventions with antioxidants to safeguard vascular redox balance might delay or even prevent the development of GTN-induced side effects by scavenging radicals. In order to be effective, however, antioxidants, once absorbed and distributed to tissues, should accumulate in mitochondria where oxidative damage originates. Mitochondriotropic agents can be obtained by linking a membrane-permeable TPP+ moiety to antioxidants [3]. Owing to the electrical potential difference (negative inside) across the plasma and the inner mitochondrial membrane, these lipophilic cations accumulate in cytoplasm and several-hundredfold into mitochondria [4]. The antioxidant, dietary flavonoid quercetin, found in fruits, vegetables, and beverages, at high M, nonphysiological concentrations, prevents in vitro tolerance to nitrates [5]. It was hypothesized that once conjugated to TPP+, it would accumulate in mitochondria thus preventing GTN unwanted effects. To this end, two derivatives of quercetin, namely Q-3BTPI and its acetylated analogue QTA-3BTPI, were synthesized [6]. The effects of physiological concentrations of quercetin and equivalent concentrations of its TPP+ derivatives on GTN tolerance as well as on GTN-induced endothelial dysfunction were investigated on isolated rat aorta rings. Both GTN and ACh relaxed concentration-dependently phenyl" Fig. 1 and Table 1), whereas QTAephrine-contracted vessels (l 3BTPI, Q-3BTPI, quercetin, and TPP+ (10 nM-1 M) were devoid of effects (data not shown). Exposure of the rings to 10 M GTN for 60 min caused a significant ~ 10-fold reduction of both GTN " Table 1) and efficacy (l " Fig. 1), effects conand ACh potency (l sidered as an index of tolerance and endothelial dysfunction, respectively (see [7]). Although the vasorelaxing effect of GTN was not altered by the presence of QTA-3BTPI, Q-3BTPI, quercetin, or TPP+ (data not shown), incubation of the rings with either 100 nM and 1 M QTA-3BTPI or 1 M Q-3BTPI significantly reduced the ability of " Fig. 1 a and Table 1). On the contrary, GTN to induce tolerance (l " Table 1) had no efincubation with quercetin or TPP+ alone (l fects. Taken together, these data indicate that: 1) the significant reversal of tolerance was not due to an altered response of the rings to GTN caused by quercetin derivatives; 2) acetylation of the OH groups of quercetin improved potency, probably by facilitating its access to mitochondria; 3) reversal of tolerance was not due to the release of TPP+, per se ineffective, but the TPP+ moiety was key to the activity of quercetin derivatives, since quercetin alone did not reverse tolerance. Recently Suri et al. [8] showed that quercetin and its metabolite quercetin-3-sulfate significantly reduce the development of tolerance in pig coronary arteries, although at concentrations 10- to 100-fold higher than those employed here, which might have favored mitochondrial accumulation of quercetin [9]. Conversely, QTA-3BTPI and Q-3BTPI reverted GTN tolerance at much lower concentrations, comparable to the levels of quercetin attained in human plasma after the intake of flavonoid rich foods [10, 11], due to their michondriotropic features [6]. It is hypothesized that mitochondrial accumulation of these drugs reduces the concentration of radicals, particularly O2, responsible for the development of tolerance to GTN. It cannot be ruled out, however, that quercetin and its derivatives might activate heme oxygenase-1 [12], responsible for antagonizing the development of tolerance following treatment with pentaerythrityl tetranitrate but not with GTN [13]. Evidence that chronic nitrate treatment causes endothelial dysfunction, resulting in a decreased efficacy of vasodilators acting on endothelium (e.g., ACh), is now substantial. Dysregulation of

Letters

Table 1 Effects of quercetin, QTA-3BTPI, and Q-3BTPI on GTN and ACh vasorelaxing activity. Drug GTN relaxation pIC50 (M)
Control 10 M GTN GTN + 100 nM QTA-3BTPI GTN + 1 M QTA-3BTPI GTN + 100 nM Q-3BTPI GTN + 1 M Q-3BTPI GTN + 1 M quercetin GTN + 1 M TPP+ 7.44 0.09 6.39 0.12*** 7.07 0.18## ** 7.10 0.15# ** 6.14 0.25** 7.01 0.10## 6.55 0.20* 6.32 0.29**

ACh relaxation pIC50 (M)

7.11 0.11 6.26 0.12** 6.87 0.16# 6.02 0.13** 6.93 0.13## 6.81 0.21# 7.20 0.14## 6.18 0.08**

" Fig. 1. GTN relaxation represents Experimental conditions are the same as those in l

GTN tolerance, whereas ACh relaxation represents endothelial dysfunction. Data represent the mean SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, # p < 0.05,
##

p < 0.01 vs. GTN, one way ordinary ANOVA and Dunnetts post-test

Fig. 1 Effects of quercetin, QTA-3BTPI, and Q-3BTPI on GTN-induced tolerance and endothelial dysfunction. Rat aorta rings with intact endothelium, preincubated for 15 min with antioxidants, were incubated with vehicle (control) or 10 M GTN for 60 min and washed for 30 min in the absence or presence throughout of QTA-3BTPI, Q-3BTPI, or quercetin and then challenged with 0.3 M phenylephrine. As the contraction had reached a stable value, GTN (a) or ACh (b) was added at cumulative concentrations, and the ensuing relaxation was recorded. On the ordinate axis, the response is reported as percentage of the contraction obtained with phenylephrine (Phe; 100 %). Data represent the mean SEM. a n = 1023, b n = 724. Efficacy: ** p < 0.01, *** p < 0.001 vs. control, # p < 0.05 vs. GTN, one way ordinary ANOVA and Dunnetts post-test.

Material and Methods

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key enzymes and the resulting production of O2 [14], which rapidly reacts with and inactivates NO, appear to be crucial for endothelial dysfunction [1517]. Antioxidants preserve the sensitivity of the vasculature to organic nitrates in different animal experimental models but proved ineffective in clinical settings [14]. Promising results, however, emerged from the study of the mitochondriotropic antioxidant MitoQ [18]. Here, the mitochondriotropic quercetin derivatives [6], as well as quercetin, were able to restore the pharmacological response to ACh in preparations " Fig. 1 b and Table where endothelial dysfunction was achieved (l 1), thus displaying endothelial protection. This might be due to the capacity of quercetin to increase NO bioavailability either by scavenging intracellular O2 or by impairing tissue O2 generation [1921]. If this hypothesis is correct, the ambivalent redox behaviour of polyphenols was also shared by the two mitochon-

Isometric tension recording was performed in endothelium-intact rat aorta rings (34 mm wide; [22]). In vitro tolerance to GTN or endothelial dysfunction was developed in rings exposed to 10 M GTN for 60 min, followed by 30 min washout. Thereafter, the effect of cumulatively increasing concentrations of either GTN or ACh was assessed against a submaximal contraction to phenylephrine (0.3 M) in the presence and absence of QTA3BTPI, Q-3BTPI, quercetin, or TPP+ (preincubated for 105 min before phenylephrine addition). Acquisition and analysis of data were accomplished using LabChart 4 software (AD Instruments Ltd.) and GraphPad Prism version 5.04 (GraphPad Software, Inc.), respectively. QTA-3BTPI and Q-3BTPI were synthesized as previously described in Mattarei et al. [6].

Supporting information
Detailed protocols for the contractile studies are available as Supporting Information.

Durante M et al. Quercetin Mitochondriotropic Derivatives

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driotropic quercetin derivatives, which would act either as antioxidants if used at 0.11 M concentrations on rat aorta rings or, conversely, as cytotoxic pro-oxidants if used at concentrations 1 M on cultured cancer cells [6]. Furthermore, this ambivalent feature might account for the QTA-3BTPI-induced endothelial dysfunction. In fact, QTA-3BTPI per se reduced the vasorelaxing effect of ACh in a concentration-dependent manner (pIC50 values of 6.41 0.13 at 100 nM, n = 9, and of 5.94 0.12 at 1 M, n = 8; " Table 1), whereas Q-3BTPI, quercetin, p < 0.01 vs. control, see l or TPP+ (100 nM-1 M) had no effects (data not shown). Therefore, further studies are needed to unravel the mode of action of these compounds. In conclusion, the hypothesis that GTN tolerance and endothelial dysfunction are two distinct phenomena occurring at mitochondrial and cytosolic level, respectively [14], helps to explain the different behavior displayed by quercetin and its derivatives on GTN-induced side effects. Q-3BTPI is superior to quercetin in counteracting in vitro tolerance to GTN and the associated endothelial dysfunction: when proved successful in vivo, it may help reduce the phenomena that limit the clinical use of nitrates in terms of doses and frequency of administration.

Letters
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Acknowledgements
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We wish to thank Dr. Maddalena Simone for her assistance in some preliminary experiments, and the Fondazione Cassa di Risparmio di Padova e Rovigo for support (Excellence grant Developing a pharmacology of polyphenols).

Conflict of Interest
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The authors declare no conflict of interest.

References
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Bibliography DOI http://dx.doi.org/10.1055/s-0032-1328293 Published online Planta Med Georg Thieme Verlag KG Stuttgart New York ISSN 00320943 Correspondence Dr. Fabio Fusi Dipartimento di Scienze della Vita Universit degli Studi di Siena via A. Moro 2 53100 Siena Italy Phone: + 39 05 77 23 44 38 Fax: + 39 05 77 23 44 46 fabio.fusi@unisi.it

Durante M et al. Quercetin Mitochondriotropic Derivatives

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