Daily Treatment with Sildenafil Reverses Endothelial Dysfunction and Oxidative Stress in an Animal Model of Insulin Resistance

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1 european urology 53 (2008) available at journal homepage: Sexual Medicine Daily Treatment with Sildenafil Reverses Endothelial Dysfunction and Oxidative Stress in an Animal Model of Insulin Resistance Delphine Behr-Roussel a, Alexandra Oudot a, Stéphanie Caisey a, Olivier L.E. Coz a, Diane Gorny a, Jacques Bernabé a, Chris Wayman b, Laurent Alexandre a, François A. Giuliano c, * a Pelvipharm, Domaine CNRS, 1 Avenue de la terrasse, Bâtiment 5, Gif sur Yvette, France b Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, CT13 9NJ, United Kingdom c Raymond Poincaré Hospital, Neuro-Uro-Andrology Unit, Dept. of Physical Medicine and Rehabilitation, Garches, France Article info Article history: Accepted November 6, 2007 Published online ahead of print on November 20, 2007 Keywords: Sildenafil Insulin resistance Vascular function Endothelium Oxidative stress Abstract Objectives: Patients with insulin resistance exhibit endothelial dysfunction with decreased nitric oxide (NO) production and increased oxidative stress. We postulated that daily sildenafil improved endothelial function in fructosefed rats. Methods and results: Wistar rats were fed a standard or fructose-enriched diet (FFR) for 9 wk. From weeks 6 8, sildenafil was administered twice daily (sc, 20 m g/kg), followed by a 1-wk washout. Concentration-response curves (CRCs) to endothelium-dependent (acetylcholine [Ach] and A23187) and -independent (sodium nitroprusside [SNP]) relaxing agents were performed on isolated precontracted aortas and superior mesenteric arteries (SMAs). Vascular cyclic guanosine monophosphate (cgmp) content, urinary excretion of nitrates/ nitrites (NOx) and 8-isoprostanes (IPT), and plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) were evaluated. Relaxations to ACh were significantly reduced in aortas and SMAs of FFR. Sildenafil restored AChinduced relaxations in aortas and provoked a significant leftward shift of the CRC to ACh in SMAs, whereas it did not modify the enhanced relaxations to SNP in FFR. IL-6, TNF-a, vascular cgmp, and urinary NOx levels were not modified by the fructose or sildenafil treatment. Urinary IPT levels were significantly elevated in FFR and normalized by sildenafil. Conclusions: Endothelial dysfunction and oxidative stress associated with insulin resistance can be reversed by daily sildenafil, even 1 wk after treatment cessation. # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Neuro-Uro-Andrology Unit, Dept of Physical Medicine and Rehabilitation, Raymond Poincaré Hospital, 104 bd Raymond Poincaré, Garches, France. Tel ; Fax: address: giuliano@cyber-sante.org (F.A. Giuliano) /$ see back matter # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 european urology 53 (2008) Introduction Insulin resistance (IR) has been implicated in the pathogenesis of obesity and the metabolic syndrome (MetS) as defined either by the International Diabetes Federation (IDF), National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII), or European Group for the study of Insulin Resistance (EGIR) criteria [1]. In fact, MetS in both children and adults is a known independent cardiovascular risk factor [1], and patients with MetS exhibit impaired endothelium-dependent vasodilation [2]. It is now recognized that these disturbances in endothelial function are principal players in cardiovascular diseases [3]. It was reported that fructose consumption might be a contributing factor to the development of obesity and the accompanying metabolic abnormalities observed in MetS [4], and fructose has also been associated with angiopathy in patients with diabetes [5]. Interestingly, high fructose consumption by animals leads to impaired glucose tolerance, IR, hyperinsulinemia, and hypertriglyceridemia [6], with even a mild elevation of blood pressure found by some authors [7] but not confirmed by others [8]. In a previous study, we evidenced the beneficial effects of daily sildenafil on erectile function in normal rats and further characterized these effects in vitro to be via improved cavernosal endothelial function [9]. Because previous work had shown the central role of the Akt-dependent phosphorylation of endothelial nitric oxide synthase (enos) in mediating penile erection [10], we hypothesized that chronic sildenafil may actually exert a direct or indirect effect beyond phosphodiesterase (type) 5 (PDE5) inhibition and thus modulate the transduction pathway leading to the activation of enos. Indeed, the serine/threonine specific protein kinase Akt could be involved in the upregulation of enos activity by a calcium-independent mechanism, resulting in the phosphorylation of enos and increase in enos activity [11]. Thus, a plausible explanation of the beneficial effects of daily sildenafil on endothelial reactivity may be that it leads to an increased expression of phosphorylated enos over total enos and active Akt over total Akt, as recently evidenced in cavernosal tissues [12]. Furthermore, it was suggested that daily treatment with PDE5 inhibitors could have additional and prolonged beneficial effects on endothelial function in patients with increased cardiovascular risks [13]. The benefit of this therapy could even be sustained 2 wk after discontinuation of therapy [13]. In this study, we aimed to investigate whether chronic sildenafil could improve systemic endothelium-dependent relaxations in an experimental model of IR by in vitro isometric tension studies on aortic and superior mesenteric arterial (SMA) rings. Moreover, because oxidative stress has been suggested to contribute to IR [14] and associated endothelial dysfunction [15], we sought to determine the effects of chronic sildenafil on a potentially relevant biomarker of endothelial dysfunction, urinary 8-isoprostanes (IPT) content, a direct marker of nonenzymatic in vivo lipid peroxidation and the most reliable and clinically relevant marker of oxidative stress available to date [16]. 2. Methods 2.1. Chronic treatment of rats and monitoring of endothelial biomarkers All procedures were performed in accordance with the legislation on the use of laboratory animals (NIH publication No 85-23, revised 2002) and animal care regulations in force in France. Male Wistar rats (Charles River, L Arbresle, France, g) were given standard chow (Reference TD.03102; CONT) or an isocaloric fructose-enriched diet containing 18.3% protein, 60.3% fructose, and 5.2% lard expressed in % diet by weight (Reference TD.89247; fructose-fed rats, FFR) for the following 9 wk (Teklad Labs, Madison, WI, USA). Subcutaneous injections with saline (CONT, FFR) or sildenafil mesylate 20 mg/kg (FFR+SIL) (Pfizer, Sandwich, UK) were performed twice a day (40 mg kg 1 day 1 in total) between weeks 6 and 8 to achieve clinical relevant plasma exposures (circa 20 nmol/l unbound, known to give efficacy in man; a 100-mg dose of sildenafil in man yields a free plasma concentration of 30 nmol/l [Pfizer Inc, data on file]) [9,12,17]. A 1-wk washout period ensued for proper elimination of all circulating sildenafil and metabolites [12]. At the end of the treatment period (week 8) and after 1 wk of washout from the treatment (week 9), blood samples were taken after a 5-h fasting period. Glycemia was measured on whole blood (Accu-Chek active; Roche Diagnostics, France) and triglycerides, interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) concentrations were measured on plasma samples with the use of commercialized colorimetric assay kits (Sigma, St Louis, MO, USA, and Pierce, IL, USA, respectively). Oral glucose tolerance tests (OGTTs) were performed on overnight fasted rats gavaged with a solution of glucose 1 g/kg. Blood glucose was measured at 0, 10, 20, 30, 60, and 90 min after gavage and normalized by the initial overnight fasted glycemia for each rat. The area under the curve (AUC; in arbitrary units) was then calculated by using the plotted OGTT curve using normalized glycemia values. Twenty-four hour urine samples were obtained from overnight fasted rats at week 9. Samples to be used for IPT determination were stored at 80 8C in the presence of 0.005% BHT to prevent further oxidative formation of IPT and purified through an affinity column before immunoenzymatic determination (Cayman Chemical, Ann Arbor, MI, USA). The concentration of nitrates/nitrites (collectively referred to as

3 1274 european urology 53 (2008) NOx) was determined with the use of a commercially available assay kit (Cayman Chemical, Ann Arbor, MI, USA). All urinary results were normalized by the clearance of creatinine to limit variability in the assays due to changes in renal excretory function Isometric tension studies in aortic and mesenteric rings and cgmp content After the 1-wk washout period, rats were terminally anesthetized with an intraperitoneal injection of urethane (1.2 g/kg). For cgmp content determination, samples taken from the thoracic aorta and the SMA, homogenized in a glass potter at 4 8C in a 50 mmol/l phosphate buffer containing 1 mmol/l theophylline (ph 7.4) and supernatants assayed for cgmp (Cayman Chemical, Ann Arbor, MI, USA). Results were normalized by the protein content. Meanwhile, aortic and SMA rings were placed in 5-ml organ baths filled with Krebs solution (in mmol/l: NaCl 118.0; KCl 4.6; CaCl 2 2.5; KH 2 PO 4 1.2; MgSO 4 1.2; glucose 11.1; NaHCO ; HEPES, 20.85, ph ) at 37 8C, bubbled with 95% O 2-5%CO 2, and connected to a forcedisplacement transducer. Concentration-response curves (CRCs) to endothelium-dependent eliciting agents such as acetylcholine (Ach; mol/l to 10 5 mol/l) and A23187, a calcium ionophore (10 10 mol/l to 10 6 mol/l), or encotheliumindependent relaxing agents such as sodium nitroprusside (SNP; mol/l to 10 6 mol/l) were constructed in the presence of indomethacin (10 5 mol/l) on phenylephrine (PE)- induced precontracted tissues (10 6 mol/l or mol/l, depending on the individual responses of each tissue to maintain comparable levels of stimulated forces between groups) Calculations and statistical analysis Values are expressed as mean standard error of the mean of n experiments. Biochemical determinations were compared by using a one-way analysis of variance (ANOVA) statistical analysis followed by Newman-Keuls complementary analysis with p < 0.05 considered significant. Tissue contractile responses were expressed as the absolute change in maximal developed tension (in grams) normalized per gram tissue weight, and relaxations as the percentage of change in phenylephrine (PE)-induced tone. Comparison of these curves were made by using a two-way ANOVA statistical analysis test with p < 0.05 considered significant. Concentrations inducing 50% of the maximal effect were expressed as pd 2 values and calculated with the use of GraphPad Prism 1. sildenafil corrected this hyperglycemic response while treatment lasted ( p < 0.05, one-way ANOVA; Fig. 1). This effect, however, wore off 1 wk after cessation of sildenafil treatment. Moreover, chronic sildenafil treatment significantly countered the pronounced hypertriglyceridemia secondary to the fructose feeding in FFR at week 8 ( p < 0.001, one-way ANOVA; Table 1), and this effect was maintained even after the 1-wk washout period (week 9, p < 0.05, one-way ANOVA; Table 1) Isometric tension studies All experiments were performed after the 1-wk washout from sildenafil treatment. In all experiments, care was taken to obtain comparable precontraction levels with PE before inducing concentration-dependent relaxant responses to ACh, A23187, or SNP (Table 2). In aortic rings, maximal relaxation responses to ACh and A23187 were significantly reduced in FFR compared with CONT ( p < 0.001) although pd 2 values were unchanged. Interestingly, the degree of relaxation elicited by SNP was greater in FFR compared with CONT ( p < 0.001). Strikingly, endothelial relaxations induced by ACh and A23187 were restored in FFR treated chronically with sildenafil compared with saline-treated FFR ( p < 0.001) without a significant change in pd 2. Conversely, relaxations elicited by SNP were unchanged in tissues from sildenafil-treated FFR compared with saline-treated FFR (Fig. 2, Table 2). 3. Results Body weight changes, fasting glycemia, and plasma IL-6 and TNF-a levels remained statistically unchanged in the various treatment groups (Table 1). Conversely, FFR displayed an impaired OGTT as indicated by a greater hyperglycemic response and AUC compared with control rats both at weeks 8 and 9. Strikingly, chronic treatment with Fig. 1 Area under the curve of normalized glycemia (% change from initial overnight fasted glycemia) following oral glucose tolerance tests performed at the end of the chronic treatment with sildenafil (week 8) and after the 1-wk washout period (week 9) in control rats (CONT; empty bar), and fructose-fed rats (FFR) that received saline (full bars) or sildenafil (20 mg kg S1 d S1 sc 2T d S1, horizontal stripes) for 3 wk. * p < 0.05 versus CONT, one-way analysis of variance.

4 european urology 53 (2008) Table 1 Plasma levels of glucose, triglycerides, and proinflammatory markers interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) after 3 wk of chronic treatment with sildenafil or after the 1-wk week washout period from sildenafil (week 9) CONT (n = 12) FFR (n = 12) FFR+SIL (n = 14) Week 8 Body weight (g) Fasting glycemia (mg/dl) Triglycerides (equivalent triolein, mmol/l) W 0.42 * 0.71 W 0.09 z Creatinine clearance (ml/min) W 0.03 y Plasma IL-6 concentration (pg/ml) Plasma TNF-a concentration (pg/ml) Week 9 Body weight (g) Fasting glycemia (mg/dl) Triglycerides (equivalent triolein, mmol/l) W 0.40 * 1.01 W 0.13 y Creatinine clearance (ml/min) Plasma IL-6 concentration (pg/ml) Plasma TNF-a concentration (pg/ml) CONT, control rats; FFR, fructose-fed rats treated with saline; FFR+SIL, fructose-fed rats treated with sildenafil. Data are presented as mean standard error of the mean (SEM). * p < 0.01 vs. CONT. y p < z p < vs. FFR, one-way analysis of variance followed by Newman-Keuls analysis. In rings of SMA, relaxation responses to ACh were significantly diminished in FFR compared with CONT ( p < 0.01), although pd 2 and maximal developed tension (E max ) values were unchanged (Table 2). Relaxations to A23187 were, however, similar in CONT and FFR, whereas relaxations to SNP were significantly enhanced in FFR compared with CONT ( p < 0.001). Chronic treatment with sildenafil dramatically enhanced the sensitivity and responsiveness of these mesenteric arteries to Ach, with pd 2 values significantly augmented in sildenafiltreated FFR compared with saline-treated FFR ( p < 0.01). It also enhanced relaxations to A23187 ( p < 0.01), whereas responses to SNP were unchanged (Fig. 2, Table 2) Biochemical determinations Tissue cgmp content from unstimulated aortas and mesenteric arteries prepared after a 1-wk washout from sildenafil therapy demonstrated similar concentrations in each group whatever the type of vessel examined (not significant [NS], one-way ANOVA; Fig. 3). On the other hand, neither the fructose diet nor the sildenafil treatment modified significantly the urinary NOx excretion levels be it after 3 wk of sildenafil treatment (data not shown) or after the 1 wk of washout (NS, one-way ANOVA; Fig. 4, left panel). Conversely, urinary IPT levels were dramatically increased following the fructose diet (FFR, vs. CONT, , p < 0.01, one-way ANOVA). Chronic sildenafil treatment restored normal levels of urinary IPT excretion even after treatment cessation (FFR+SIL, ng ml 1 24 h 1, p < 0.01 vs. FFR, one-way ANOVA; Fig. 4, right panel). 4. Discussion A high-fructose diet in rats leads to impaired glucose tolerance, IR, hyperinsulinemia, and hypertriglyceridemia [6], with even a mild elevation of blood pressure found by some authors [7] but not confirmed by others [8], a set of symptoms that resembles MetS in humans. In humans, each of the above abnormalities is associated with endothelial dysfunction, let alone when all these risk factors are combined. Here, we confirmed the elevation of triglycerides and the lack of fasting hyperglycemia repeatedly found in FFR [18], as well as impaired glucose tolerance and endothelial dysfunction at the level of the aorta and the SMA. Different susceptibilities and kinetics to developing IR in rats have been reported, with, for example some animals exhibiting resistance to the effect of the diet [8]. Furthermore, diet duration and composition may also play a critical role in inducing metabolic changes. A wide variety of different diets have been used in the literature, which were distinct in the percentage of fructose in the diet, as well as the percentage of associated fat. Interestingly, however, our results are in accordance with previous studies

5 Table 2 Isometric tension studies on isolated rings of aortas and mesenteric arteries from control rats or FFR treated chronically with saline or sildenafil Acetylcholine A23187 Sodium nitroprusside E max pd 2 E max pd 2 Precontraction tension (g/g ww) E max (%) pd 2 Precontraction tension (g/g ww) Precontraction tension (g/g ww) Aortas CONT FFR W 4.7 y W 4.2 y W 4.2 y FFR+SIL W 3.0 z W 3.9 z * 1276 european urology 53 (2008) Mesenteric arteries CONT FFR FFR+SIL W 0.08 * Emax, maximal developed tension; pd2, concentrations inducing 50% of maximal effect; CONT, control rats; FFR, fructose-fed rats treated with saline; FFR+SIL, fructose-fed rats treated with sildenafil. p < vs. CONT, z p < vs. FFR, two-way analysis of variance, followed by Bonferroni analysis. p < 0.01 vs. FFR, one-way analysis of variance, followed by Newman-Keuls analysis. y reporting impaired glucose tolerance associated with impaired responses to endothelium-dependent vasodilators as well as decreased enos activity within the vascular wall [6,19,20]. It is noteworthy that, indeed, endothelial dysfunction has been directly related to each of the independent risk factors present in the MetS (ie, hypertriglyceridemia, IR, or hypertension). In this context, we postulated that chronic treatment with sildenafil could upregulate the NOS/cGMP pathway and improve endothelial function in fructose-fed rats. It was suggested that endothelial dysfunction present in obese and prediabetic men may be elicited by a low-grade inflammatory state characterized by higher circulating concentrations of proinflammatory cytokines [21]. We thus sought to determine levels of circulating TNF-a and IL-6, both defined as precursory inflammatory markers of inflammation. Interestingly, in FFR rats, both markers were similarly expressed in FFR compared with normal rats. Moreover, sildenafil treatment had no effect on the expression of these markers. It is thus unlikely that such a mechanism could be held responsible for the observed beneficial effects of chronic sildenafil treatment in FFR rats. Interestingly, chronic selective hypertriglyceridemia, an independent risk factor in the development and progression of atherosclerosis, has been described to lead to endothelial dysfunction associated with increased oxidative stress and subsequent decrease in bioavailable NO [22]. Our results are in accordance with this description because we have shown in these hypertriglyceridemic rats both an increase in urinary IPT levels, a direct marker of nonenzymatic in vivo lipid peroxidation and the most reliable and clinically relevant marker of oxidative stress available to date [16], as well as altered acetylcholine-induced endothelium-dependent relaxations. More strikingly, chronic treatment with sildenafil was able to restore normal levels of triglycerides and glycemia after oral glucose challenge, while reversing endothelial dysfunction and correcting increased levels of urinary IPT. Interestingly, it has been very recently described that restoration of normal endothelial function via correction of oxidative stress in fructose-fed rats could, in fact, raise adiponectin levels and thus participate in the regulation of triglyceridemia [23]. The occurrence of such a phenomenon may well have occurred in the present study as well although it remains yet to be determined. This sustained effect is all the more striking in that it seems to be exerted beyond PDE5 inhibition because we could not detect significantly modified cgmp levels in

6 european urology 53 (2008) Fig. 2 Concentration-response curve to acetylcholine (Ach; 10 S10 10 S5 mol l S1, upper panel), A23187 (10 S10 10 S6 mol l S1, middle panel), or sodium nitroprusside (SNP; 10 S10 10 S6 mol l S1, lower panel) in aortic rings (left panel) and superior mesenteric arteries (SMAs; right panel) from control rats (CONT; full circle), or fructose-fed rats (FFR) that received saline (open squares) or sildenafil (20 mg kg S1 d S1 sc 2T d S1, full squares) for 3 wk. All experiments were carried out after a 1-wk washout period from chronic sildenafil treatment. ** p < 0.01, *** p < 0.001, two-way analysis of variance. parallel in vascular tissues after 1 wk of washout from sildenafil treatment. Because NO rapidly oxidizes to nitrite and predominantly nitrate, the in vivo production of NO can be monitored indirectly by measuring NOx levels. Measurement of plasma NOx reflects the level of systemic NO production but also depends on the level of glomerular filtration and proximal tubule NO generation because there is substantial tubular reabsorption of NOx [24]. So, a more straightforward and useful measurement of the total NO production can be obtained by quantifying the 24-h urinary NOx excretion in animals on a controlled diet after a suitable period of fasting [25].

7 1278 european urology 53 (2008) Fig. 3 Vascular cyclic guanosine monophosphate (cgmp) content in segments from aortas (left panel) and SMAs (right panel) of control rats (empty bar), and fructose-fed rats (FFR) that received saline (full bars) or sildenafil (20 mg kg S1 d S1 sc 2T d S1, horizontal stripes) for 3 wk (weeks 6 8). Tissue was harvested after a 1-wk washout period from chronic sildenafil treatment (week 9). Not significant, one-way analysis of variance. We sought to determine these urinary NOx levels to characterize an expected defective vascular NO production in the FFR that would parallel the functional endothelial dysfunction observed in vitro in isometric tension studies. However, we could not detect any defect in excretion of urinary NOx. It may well be that our method of detection, although standard, was not refined enough to detect such an effect. However, another likely hypothesis for this finding could be based on the increased state of oxidative stress present in the vasculature of FFR, which is reported to contribute to IR [14] and associated endothelial dysfunction [15]. Indeed, a key alteration in endothelial cell phenotype is an increased formation of reactive oxygen species. This alteration can be partly due to the uncoupling of endothelial NO synthase such that it generates more superoxide anions than NO. Consequently, endothelial vasodilator mechanisms are impaired because superoxide anions have been reported to scavenge NO within the vascular wall to reduce its biological half-life, thus attenuating vascular NO dependent relaxations [26]. Moreover, NO and superoxide anions then combine to form peroxynitrite, which, in turn, transforms into nitrates [27]. Thus, while NO release and/or bioavailability might be impaired in FFR, leading to an impaired vasoreactivity, excessive production of superoxide anions may occur, thus yielding stable urinary NOx levels. An increased inducible nitric oxide synthase (inos) activity in these insulinresistant rats could be another explanation because recent reports have shown that inos is associated with IR [28]. Confirming these hypotheses, we have assayed urinary IPT, a direct marker of nonenzymatic in vivo lipid peroxidation and thus oxidative stress, and have clearly evidenced increased levels of urinary IPT following fructose overload in FFR. Moreover, previous work had shown that chronic delivery of PDE5 inhibitors could modulate the transduction pathway leading to the Akt-dependent phosphorylation of enos [9,12], resulting in an increased capacity of enos to produce NO [11]. Thus, a plausible explanation for the beneficial Fig. 4 Urinary excretion of nitrates/nitrites (NOx; left panel) and 8-isoprostane (IPT; right panel) in control rats (CONT; empty bar), and fructose-fed rats (FFR) that received saline (full bars) or sildenafil (20 mg kg S1 d S1 sc 2T d S1, horizontal stripes) for 3 wk (weeks 6 8). Urine samples were collected after a 1-wk washout period from chronic sildenafil treatment (week 9). ** p < 0.01 vs. CONT, p < 0.01 vs. FFR, one-way analysis of variance followed by Newman-Keuls analysis.

8 european urology 53 (2008) effects of chronic sildenafil on endothelial reactivity could be that it leads to an increased expression of phosphorylated enos. We could not perform such experiments here to confirm this hypothesis because the tissues were destined for in vitro isometric tension studies. However, chronic treatment with sildenafil was able to both restore endothelium-dependent NO-dependent relaxations at various sites of the vascular tree and, concomitantly, bring levels of urinary IPT excretion back to normal, even 1 wk after cessation of the treatment. It may well be that chronic sildenafil treatment has restored a normal balance between NO and superoxide anion production by favoring Akt-dependent phosphorylation of enos, as previously suggested [9,12]. Moreover, sildenafil has been shown to inhibit superoxide formation via gp91phox in vitro [29] and reduce oxidative stress via improved oxygenation, and hence improved endothelial function/no production in vivo [30,31]. Thus, the unifying hypothesis for sildenafil beneficial effects on endothelial function would be a regulation of free radical generation and NO bioavailability. This hypothesis may also well explain why no increase in NOx excretion levels could be detected following chronic sildenafil therapy. The improvement of endothelial function following daily administration of sildenafil could play a key role in an improved clinical outcome in patients with IR. Given that the severity of endothelial dysfunction seems to correlate with systemic cardiovascular status [32], maintaining or restoring a correctly functioning endothelium at the systemic level might be of utmost importance to the patient presenting one or more cardiovascular risk factors. In fact, in a very recent study (reported in the form of an abstract), diabetic patients with erectile dysfunction were administered sildenafil daily during 4 wk (50 mg the first week, then 100 mg the last 3 wk) [33]. Most interestingly, chronic treatment with sildenafil had no significant effect on the levels of two inflammatory cytokines (IL-6 and IL-8), whereas it significantly decreased the level of IPT in these patients. Thus, this study and ours provide further support for the predictive value of endothelial reactivity and associated biological markers of oxidative stress such as urinary IPT as surrogate markers in future clinical trials addressing cardiovascular risks. Furthermore, it provides more evidence toward a putative vasculoprotective effect of chronic treatment with sildenafil, thus supporting the proposal of cardiovascular health benefits following such a mode of administration, which would deserve future clinical trials [34 36]. Conflicts of interest This work was supported by a restricted grant from Pfizer Ltd. References [1] Nilsson PM, Engstrom G, Hedblad B. 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Antioxidant treatment associated with sildenafil reduces monocyte activation and markers of endothelial damage in patients with diabetic erectile dysfunction: a double-blind, placebo-controlled study. Eur Urol 2007;52: [32] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362: [33] Burnett AL, Strong T, Musicki B, Bivalacqua TJ, Jin L. Serum biomarkers of endothelial function and oxidative stress after daily dosing of sildenafil in type 2 diabetic men with erectile dysfunction. J Urology 2007;177:955. [34] Bella AJ, Deyoung LX, Al Numi M, Brock GB. Daily administration of phosphodiesterase type 5 inhibitors for urological and nonurological indications. Eur Urol 2007;52: [35] Hatzimouratidis K, Hatzichristou D. Phosphodiesterase type 5 inhibitors: the day after. Eur Urol 2007;51: [36] Ückert S, Hedlund P, Andersson KE, et al. Update on phosphodiesterase (PDE) isoenzymes as pharmacologic targets in urology: present and future. Eur Urol 2006;50: Editorial Comment on: Daily Treatment with Sildenafil Reverses Endothelial Dysfunction and Oxidative Stress in an Animal Model of Insulin Resistance Ian Eardley St James University Hospital, Leeds, UK ian.eardley@btinternet.com Over the past few years, urologists have become increasingly aware of the so-called metabolic syndrome, a combination of conditions including glucose intolerance, hyperlipidaemia, hypertension, and obesity. A number of different definitions of the metabolic syndrome exist, but the fundamental features are common to all definitions [1].A number of studies have correlated the presence of metabolic syndrome with cardiovascular risk and in the urologic arena with erectile dysfunction (ED) and more recently with lower urinary tract symptoms (LUTS). One of the core features of the metabolic syndrome is insulin resistance, which is characterised by a diminished ability of cells to respond to the actions of insulin in transporting glucose from the bloodstream into tissues. Insulin resistance typically heralds the onset of type 2 diabetes. In this paper, an animal model of insulin resistance is used to investigate the effect of chronic dosing of sildenafil. The main findings are that sildenafil reverses many of the pathophysiologic effects of insulin resistance, most notably the associated endothelial dysfunction and the oxidative stress associated with insulin resistance [2].

10 european urology 53 (2008) Over the past few years, several studies have demonstrated the beneficial effect of phosphodiesterase type 5 (PDE5) inhibitors on endothelial function [3] and this study suggests that the mechanism by which this occurs is via regulation of free radical generation and nitric oxide bioavailability. We know that there is an epidemiologic association between ED and subsequent cardiovascular disease [4] and it is well recognised that a relationship exists between endothelial dysfunction and cardiovascular disease. Given the recently identified association between the metabolic syndrome and LUTS [5], this paper suggests a rationale for hypothesising that if PDE5 inhibitors are used in a chronic dosing regime, either for ED or LUTS, there may be additional cardiovascular benefits. Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation 2004;109: [2] Behr-Roussel D, Oudot A, Caisey S, et al. Daily treatment with sildenafil reverses endothelial dysfunction and oxidative stress in an animal model of insulin resistance. Eur Urol 2008;53: [3] Rosano GM, Aversa A, Vitale C, et al. Chronic treatment with tadalafil improves endothelial function in men with increased cardiovascular risk. Eur Urol 2005;47: [4] Thompson IM, Tangren C, Goodman PJ, et al. Erectile dysfunction and subsequent cardiovascular disease. JAMA 2005;294: [5] Rohrmann S, Smit E, Giovannucci E, Platz EA. Association between markers of the metabolic syndrome and lower urinary tract symptoms in the Third National Health and Nutrition Examination Survey (NHANES III). Int J Obesity 2005;29: References [1] Grundy SM, Brewer HB, Cleeman JI, et al. Definition of the metabolic syndrome. Report of the National Heart, DOI: /j.eururo DOI of original article: /j.eururo