Effect of PDE5 inhibition combined with free oxygen radical scavenger therapy on erectile function in a diabetic animal model

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1 (2003) 15, & 2003 Nature Publishing Group All rights reserved /03 $ Effect of PDE5 inhibition combined with free oxygen radical scavenger therapy on erectile function in a diabetic animal model L De Young 1,DYu 1, D Freeman 1 and GB Brock 1 1 Department of Urology, St Joseph s Health Care, Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada Phosphodiesterase (PDE) inhibitors represent an important advance in the treatment of erectile dysfunction (ED). In spite of widespread use and generally good efficacy, as a class they remain ineffective in 15 57% of men. Specific cohorts of patients with severe vascular or neurogenic basis to their ED, such as diabetic men or those who have undergone radical pelvic surgery, demonstrate lower response rates with PDE inhibition treatment. We believe that circulating levels of nitric oxide (NO) may be enhanced through delivery of adequate concentrations of free oxygen radical scavenger molecules such as vitamin E. Higher levels of NO, theoretically, should produce increased penile blood flow with the potential for a synergistic effect when combined with a PDE5 inhibitor. With this hypothesis in mind, 20 adult male Sprague Dawley streptozotocin-induced (60 mg/kg i.p.) diabetic rats were divided into four therapeutic groups (n ¼ 5). Group IFcontrol animals received peanut oil, group IIFvitamin E 20 IU/day, group IIIFsildenafil 5 mg/kg/day and group IVFvitamin E 20 IU/day plus sildenafil 5 mg/kg/day, by oral gavage daily for 3 weeks. Erectile function was assessed as a rise in intracavernous pressure following cavernous nerve electrostimulation. Penile tissue was harvested to determine the changes in tissue morphology including neuronal nitric oxide synthase, smooth muscle a-actin and endothelial cell integrity. PDE5 protein content and activity were measured. Significant increases in intracavernous pressure were measured in the animals receiving combined vitamin E plus sildenafil treatment. Immunohistochemical staining showed increases of neuronal nitric oxide synthase, endothelial cell and smooth muscle cell staining. Western blot analysis did not show significant differences of PDE5 protein between the groups. However, higher PDE5 activity was measured in the sildenafil group and lower activity of PDE5 was recorded in the cohort receiving vitamin E with sildenafil. Vitamin E enhanced the therapeutic effect of the PDE5 inhibitor in a meaningful way in this animal model of diabetes. This study indicates a potential means of salvaging erectile function among patients who are refractory to sildenafil. (2003) 15, doi: /sj.ijir Keywords: impotence; diabetes; sildenafil; vitamin E; nitric oxide; PDE5 Introduction Erectile dysfunction (ED) is experienced in men with diabetes mellitus (DM) at a greater frequency than the general population across all ages. Alteration of neural and impaired penile vascular systems is believed to be primarily responsible for ED rates approaching 75% in some reports. 1 5 In contrast to efficacy in general population of 70 89%, just slightly over 50% of diabetic men respond favorably Correspondence: G Brock, Department of Urology, St Joseph s Health Care, Lawson Health Research Institute, The University of Western Ontario, London, ON, Canada N6A 4V2. gebrock@sympatico.ca Accepted 12 December 2002 to this treatment. 6 The underlying cause of this relative treatment resistance remains poorly characterized. Over the past decade, the essential role played by nitric oxide (NO) in inducing relaxation of the penile vasculature and cavernous smooth muscle has been elucidated. 7,8 NO-dependent vascular relaxation is believed to act via activation of a cytosolic form of guanylyl cyclase producing increased amounts of cyclic guanosine monophate (cgmp). The accumulation of cgmp induces phosphorylation and calcium efflux resulting in smooth muscle relaxation. Additionally, NO can directly activate calcium-dependent potassium channels inducing hyperpolarization of the cell membrane. 9,10 These cellular changes induce a cascade of physiologic events culminating in smooth muscle relaxation, increased vascular flow and ultimately penile erection.

2 348 The central role of NO in this sequence of events has led investigators to examine the potential impact of increased and decreased levels of NO. The presence of oxygen free radicals inactivates NO and reduces its physiologic impact. 11,12 NO has a biological half-life of less than 30 s in which it undergoes oxidation to nitrite and nitrate. Within the penis, the source of NO appears to be derived from both the vascular endothelium and from neural sources. 13 Impaired NO activity and endothelial dysfunction have been reported in diabetic animal models and human volunteers. 14,15 Additionally, increased autooxidation of glucose and oxidation of lowdensity lipoproteins has been described in diabetes, which may result in overproduction of free-radical species leading to smooth muscle dysfunction. 13,16,17 Direct inactivation of NO, largely by a superoxide anion believed to be present in higher than normal concentrations in diabetes, may also play a role in producing impaired cavernosal relaxation. 18 Sildenafil, a PDE5 inhibitor, has proven to be an effective agent for the treatment of male ED across a broad range of etiologies. 19 Sildenafil acts by selectively inhibiting the cgmp-specific Type 5 phosphodiesterase. This is the major isozyme metabolizing cgmp in the corpus cavernosum. 20 This inhibition of cgmp breakdown acts synergistically with the release of NO from endothelial and neural sources to elevate cgmp, thus enhancing and maintaining penile smooth muscle relaxation. 21 The efficacy of sildenafil in the general ED population is 74 97%, but only 50 56% among diabetic patients. 6,22,23 The potential impact of higher concentrations of NO coupled to prolonged daily treatment with sildenafil is currently unknown. Vitamin E (a-tocopherol) is a lipid-soluble antioxidant and free oxygen radical scavenger. 24 It has been shown to enhance endothelial cell function, trap free oxygen radicals, 25 inhibit monocyte endothelial adhesion and cytokine release. 26 Additionally, recent reports describe inhibition of platelet adhesion and aggregation by a protein kinase C-dependent mechanism. 27,28 In vivo as well as in vitro evidence demonstrates that vitamin E treatment can reverse protein kinase C activation which is responsible for an important component of glucose-induced vascular dysfunction in diabetes. 29,30 Vitamin E has also been reported to improve NO-mediated arterial relaxation, maintain cell membrane integrity and protein stability. 31,32 The potential impact of combining vitamin E and sildenafil to treat diabetes induced ED (ED-DM) remains to be determined. This animal study was designed to gain insight into the potential clinical utility of this approach. In this report, we describe our experience within diabetic animals and plan to compare these results to a nondiabetic cohort in future work. Materials and methods Diabetic model: 20 adult male Sprague Dawley rats aged weeks received streptozotocin (60 mg/kg i.p.) in ph 4.5 citric acid buffer to induce diabetes. Rats that tested positive for glycosuria at 48 h were divided into the following four therapeutic groups: (I) control (peanut oil), (II) vitamin E 20 IU/day, (III) sildenafil (Pfizer Canada) 5 mg/kg/day and (IV) vitamin E plus sildenafil. The sildenafil dose was based on results from our previous dose response studies showing 5 mg/kg/day to be the optimal erectogenic dose of sildenafil in this animal model. Peanut oil was chosen as the vehicle for its low natural content of a-tocopherol. The dose of 20 IU per animal per day of vitamin E was chosen, based on a literature review which demonstrated that a therapeutic range exists between 2 and 120 IU. Treatments were by daily oral gavage for 3 weeks. All treatments were stopped at least 20 h before evaluation of erectile function. The final dose of sildenafil was administered 20 h preceding the electrostimulation experiments. Given that at this time point greater than 5 half-lives of sildenafil had expired in this animal model, changes in blood pressure (BP) attributable to the vasoactive effects of sildenafil are not expected and therefore BP was not measured. Evaluation of erectile function The blood glucose level was measured by tail vein sampling at the time of the erectile function assessment. The lateral-prostatic space was dissected utilizing a lower abdominal midline incision. The major pelvic ganglion and cavernous nerve were identified, isolated and hooked with a stainless-steel bipolar electrode. Through a sagittal perineal incision, the penile crus was exposed. A 23-G needle filled with heparin (250 IU/ml) and connected to Tygon tubing was inserted into the penile crus. The microsurgery procedure was facilitated by the use of a Zeiss SR operating stereomicroscope. Intracavernosal pressure was evoked with 0.2 ms pulses of 2 ma at 20 Hz for a 40 s duration and recorded using LabVIEW 2 software (National Instruments, Austin, TX, USA). Three electrostimulations were replicated at intervals of 10 min. The animals were killed using pentobarbital (200 mg/kg i.p.), following which penile tissue was harvested for analysis. The animal experimental protocol was approved by the University Council on Animal Care

3 Animal Use Subcommittee, University of Western Ontario, London, Canada. Histological fixation Penile tissue was fixed in cold fresh 2% formaldehyde 0.1 M phosphate buffer (ph 8) for 4 h and cryoprotected in 15% sucrose for 20 h at 41C, then embedded in OCT compound (Tissue-Tek, Sakura, USA) and stored at 701C. The OCT embedded tissues were cut into 5 mm section and adhered to superfrost plus slides (Fisher Scientific, Nepean, ON, Canada). Immunostaining for nerves positive for neuronal nitric oxide synthase (nnos), for endothelial cells and for smooth muscle a-actin was performed. The sections were air-dried for 10 min, hydrated in PBS buffer for 10 min, and then treated with 0.3% hydrogen peroxide in methanol for 5 min. After two rinsing with water and PBS buffer, the sections were blocked with 3% goat serum in PBS buffer for 3 h at room temperature (RT). The sections were then incubated in blocking buffer at 41C overnight with primary antibody mouse anti-nnos, anti-cd31 (Transduction Lab BD PharMingen, Mississauga, ON, Canada), mouse antia-actin (Roche Diagnostics, Quebec, Canada) 1 : 100, 1 : 500 dilution and 1 : 300 dilution, respectively. The sections were washed three times with PBS buffer and incubated with the secondary antibody Biotin Conjugated Goat anti-mouse IgG (Sigma, St Louis, MO, USA) 1 : 250 dilution in PBS with 1% BSA for 2 h at RT. Following three washings in PBS buffer, the sections were incubated with anti-biotin clone BN-34 Peroxidase Conjugate IgG Fraction (Sigma, St Louis, MO, USA) for 2 h. The antigen localization was visualized by using DAB Peroxidase substrate (Sigma, St Louis, MO, USA). The sections were counterstained with hematoxylin and dehydrated through graded alcohols to xylene and coverslipped. Western blot for protein analysis Frozen penile tissues stored at 701C were washed once in cooled PBS buffer and homogenized in a modified ph 7.2 tissue protein extracting buffer (Pierce, Rockford, IL, USA) containing 0.5 mm EDTA, 10 mm HEPES, M sucrose, a protease inhibitor mix and 20 mg/ml PMSF. All procedures were carried out on ice using an Ultra Turrax homogeniser at high speed. Soluble supernatant was separated by centrifugation at rpm for 10 min. The protein concentration was determined by using BCA Protein Assay reagents (Pierce Rockford, IL, USA) according to the manufacturer s protocol. A sample containing 20 mg of protein was mixed in 6 gel sample buffer and boiled for 5 min. The proteins were separated on 10% polyacrylamide gel electrophoresis and transferred to Hyboud-ECL nitrocellulose membrane (Amersham, Piscataway, NJ, USA) in ph 8.3 buffer containing 25 mm Tris, 250 mm glycine and 20% methanol. Membranes were blocked overnight at 41C with 5% nonfat dry milk and 4% goat serum in Tris-buffered saline (ph 7.4) containing 0.05% Tween 20 (TBS-T). After three washes in TBS-T (10 min), the membranes were incubated with primary mouse anti- PDE5 antibody 1 : 500 in 5% nonfat milk and 4% goat serum in TBS-T at RT for 2 h. After five washes in TBS-T (10 min 5), the membranes were incubated (2 h at 221C) with anti-mouse horseradish peroxidase-conjugated IgG antibody (final dilution 1 : 3000). Antibody binding was detected using ECL (Amersham-Pharmacia Biotech, England) by exposing blots to radiographic film for s. The radiographic film was scanned by using Hewlett- Packard ScanJet 4200C and the density was determined with the Histogram function of Corel PhotoPaint 8 (Corel, Ottawa, Canada). PDE5 activity assay by HPLC A measure of 50 mg of protein was suspended in reaction buffer (0.1 M Tris-HCl, 10 mm MgCl 2 and 0.1 M KCl ph 8.3) as modified from Spoto et al. 33 To this mixture was added cgmp to a final concentration of 100 mm and then incubated at 371C for 30 min. The reaction was stopped by immersion of the reaction tubes in boiling water bath for 5 min. The boiled samples were then immersed in an ice bath and stored at 201C until analysis. Analysis was conducted by ion-paired HPLC on a Novapak- C18 ( mm 2 ) column. The cgmp remaining in each reaction mixture was quantified by external standardization against authentic cgmp. The PDE5 concentration in the sample was determined indirectly by comparing cgmp levels to those generated from a standard curve formed from purified PDE5 (Transduction Lab BD PharMingen, Mississauga, ON, Canada) and cgmp. Statistical analysis Values are expressed as mean7s.e. of five experiments for each group. Data were compared by twotailed t-tests, a ¼ Results The animals in all the four groups showed persistent hyperphagia, polydipsia and polyuria consistent 349

4 350 Table 1 Body weight, blood glucose and serum triglycerides Control Vitamin E Sildenafil VitE+Sil Body weight (g) Blood glucose (mm/l) Serum triglycerides (mm/l) Penile Pressure (cmh2o) Penile Response C VitE Sil VitE+Sil Treatment Figure 1 Penile pressure measured in response to electrostimulation of the cavernous nerve in diabetic rats. The mean maximum intracavernous pressure7s.e. of five experiments are: C (control) cmh 2 O, VitE (vitamin E) cmh 2 O, Sil (sildenafil) cmh 2 O and VitE+Sil (vitamin E plus sildenafil) cmh 2 O. All treatment groups showed a significant increase compared with the control group, Po0.05. VitE+Sil has significant higher penile pressure increase compared with VitE only, Po0.05. with poorly controlled diabetes. No differences in body weight loss or hyperglycemia were measured between the groups. Diabetes was confirmed by the elevated serum glucose measured prior to killing and ICP testing. Interestingly, in spite of similar elevations in glucose, the animals receiving vitamin E and vitamin E plus sildenafil demonstrated lower serum triglyceride levels than animals receiving peanut oil and/or sildenafil alone (Table 1). The maximal intracavernous pressure during electrostimulation of the cavernous nerve is depicted in Figure 1. The peak intracavernous pressure (PICP) rose significantly in all the treatment groups compared with controls. However, the group receiving vitamin E combined with sildenafil showed the greatest increase, with the smallest intragroup variation among the three active treatment arms, but failed to reach our lab s historical normal value for age-matched-nondiabetic animals. The sildenafil treated group showed a wide intragroup variation in PICP, which is consistent with clinical experience among diabetic men with ED, in whom the efficacy of sildenafil is 50 60%. The responder animals did not display improved sugar control or other evident biochemical parameters distinct from the nonresponders. The histological examination was performed using a Zeiss microscope with a computerized imaging system (Northern Eclipse: Empix, Canada). The reviewer was blinded to the groups and the counts were repeated for consistency. The sum of positive staining NO synthase nerve fibers was calculated as the total from the dorsal nerve, an area adjacent to dorsal vein, and two regions from the right and the left posterior corpus cavernosum in the field of view at 400 power. All the treatment groups showed a significant higher positive staining for nnos compared to the nontreated diabetic animals (control). The most meaningful change was measured among the animals that received a combination of vitamin E and sildenafil. Vitamin E, sildenafil and vitamin E plus sildenafil groups showed a 36, 44 and 110% higher value over controls. In the sildenafil group, the largest increases in nerve fiber staining were measured within the corpora cavernosa (Table 2). Penile cavernosum sinusoidal endothelial cell staining was altered in all the groups to varying degrees (compare with our nondiabetic age-matched animals). Among the animals receiving vitamin E and vitamin E plus sildenafil, greater endothelial marker CD31 staining of the endothelial lining was measured. A significant increase in the proportion of positive staining of penile smooth muscle a-actin was measured in the sildenafil and vitamin E plus sildenafil groups compared with control and vitamin E groups. The observed staining patterns were similar to our previous work on age-matched nondiabetic animals. Penile nnos staining of control animals is compared to the animals receiving vitamin E plus sildenafil in Figure 2. An increase in both the number and intensity of nnos positive staining was measured among those animals receiving combined vitamin E plus sildenafil treatment. The sinusoidal endothelial cell staining of the vitamin E plus sildenafil group showed an increase in the number and intensity of endothelial cell staining relative to the control. These data strongly demonstrate that the vitamin E and sildenafil cohort had improved NOS presence histochemically, supported in part by the physiologic assessment of increased response to cavernous nerve stimulation. Western blot analysis of PDE5 protein levels from penile tissue extracts showed a trend toward lighter bands of PDE5 protein in vitamin E plus sildenafil and control groups compared with sildenafil and vitamin E treatment groups. The strongest bands

5 Table 2 Summary of immunohistological results 351 Control Vitamin E Sildenafil VitE+Sil nnos % Endothelial cell %SMa-actin Immunohistological examination of mid-shaft penile sections was performed using nnos monoclonal antibody, anti-rat CD31 (PECAM- 1) monoclonal antibody and anti-smooth muscle a-actin monoclonal antibody. The sum of positive staining of nnos within the dorsal nerve and three other sites in penile corpus cavernosum (power 400) is reported. A significant increase was measured in all three treatment groups (vitamin E, sildenafil and vitamin E plus sildenafil) compared with controls. Higher increases were seen in the vitamin E plus sildenafil group compared with either vitamin E or sildenafil alone. The proportion of positive staining of endothelial cells within the major penile sinusoids (power 400) of cavernosal sections of diabetic rat penile tissue demonstrated significant increases in the vitamin E and the vitamin E plus sildenafil groups compared with controls. The greatest increase measured was in the vitamin E plus sildenafil group compared with vitamin E alone. The proportion of positive staining of smooth muscle a-actin to the cross-sectional areas of mid-shaft penile sections (power 25) was significantly increased in sildenafil and vitamin E plus sildenafil groups compared with controls. a c 50µM d b 50µM PDE5 95KDa Vitamin E Control Vit E + Sildenafil Sildenafil Densitometric Value C VitE Sil VitE+Sil Treatment Figure 3 Effect of sildenafil on PDE5 protein concentration was assessed by Western blot analysis of penile tissue extract using anti-pde monoclonal antibody. A measure of 20 mg of total protein was loaded on a 10% SDS gel and proteins transferred to Hybond- ECL membrane. Positive bound antibody was detected using ECL. Right panel: densitometric analysis of four penile extracts in each group. Control demonstrates lower PDE protein, but there is no significant difference between the groups. 50µM 50µM Figure 2 Immunostaining for nnos in dorsal nerve: (a, control; b, vitamin E+sildenafil). The antigen localization was visualized by using DAB peroxidase substrate. Increased positive staining (dark colour) is seen in section b compared to control. Immunostaining of endothelial cell CD31 of corpora cavernosa c, control; d, vitamin E+sildenafil. Note strong positive staining (arrows) in section d compared to control (magnification 400). were shown in the vitamin E group. However, there is no statistical difference in the densitometry readings of the four groups (Figure 3). Lower PDE5 activity in diabetic animal model has been reported by others using an enzyme activity assay. 34 HPLC analysis of PDE5 activity (Figure 4) from penile tissue extracts shows a significantly higher PDE5 activity in the sildenafil treatment group compared with the other groups. The lowest activity was recorded in the vitamin E with sildenafil group. Discussion PDE5 inhibitors are now considered as first-line therapy for the management of ED across a wide PDE5 Activity (pmol/min) PDE5 Activity C VitE Sil VitE+Sil Treatment Figure 4 PDE5 by activity assay: 50 mg protein from penile tissue extract was reacted with 100 mm cgmp at 371C for 30 min and the reaction was stopped by boiling the mixture for 5 min. The remaining cgmp was separated by HPLC with a 5cm 3.9 mm Novapak C18 column. cgmp was converted to PDE5 equivalents by comparison to a standard reaction curve constructed from varying concentrations of purifed PDE5 and a fixed concentration of cgmp. Note that the significant higher activity of PDE5 was seen in the sildenafil group; however, the lower activity was seen in vitamin E plus sildenafil group. Po0.05 with mean7s.e. of five tissue extracts in each group.

6 352 range of etiologies. Sildenafil, tadalafil and vardenafil have all undergone extensive testing among men with ED due to hypertension, peripheral vascular disease, nerve injury and other causes. These agents have consistently demonstrated impressive response rates in all cohorts of patients. Diabetic men with ED have displayed impaired response rates across all of the therapeutic trials. 35,36 In our study, the animals that received daily sildenafil by gavage demonstrated an improved response to cavernous nerve stimulation compared with controls. This, in part, supports the finding by others that sildenafil enhanced electrical field stimulation-mediated relaxation and restored cgmp formation in a rabbit diabetes model. 37 The underlying cause of reduced efficacy of PDE5 inhibitors in clinical practice among diabetic men remains to be fully elucidated. In this report it appears clear that at the early stage of diabetes, addition of sildenafil by daily gavage to a cohort of diabetic rats enhanced the animals ability to increase intracavernous pressure in response to cavernous nerve stimulation. The treatment started just 2 days after diabetes was induced and continued for 3 weeks. We chose this early time point to evaluate penile function without extensive end organ damage, believing that these animals would be the most likely to show a reversible effect of therapy. It is important to note that all electrostimulation studies were performed at least 20 h after the last sildenafil dose; in this way, reducing the chance for direct sildenafil induced BP changes. The greatest response was measured in animals receiving the combination of sildenafil and vitamin E, supporting our initial hypothesis that a reactive oxygen scavenger may enhance PDE5 inhibitor efficacy through a prolongation of NO half-life as well as other potential physiologic effects. There is supporting evidence from the literature concerning the role of vitamin E in the aorta, lung and other organs. 38,39 Vitamin E is an active nonenzymatic antioxidant, highly lipid soluble, associated with structures such as cell membranes, and chemically deactivates relatively hydrophobic reactive species. Vitamin E appears to be the first line of defense against peroxidation of polyunsaturated fatty acids contained in cellular and subcellular membrane phospholipids. Vitamin E binds to a variety of active oxidant species such as singlet oxygen, superoxide free radicals and peroxyl free radicals that form stable molecules. 40 In this study, we did not measure penile samples for levels of NO or the concentration of reactive oxygen species. Both of these parameters would be valuable pieces of information to help determine the exact impact that vitamin E, in combination with sildenafil, plays on the host. We plan to incorporate measures of reactive oxygen species in future work. Interestingly, we measured a rise in PDE5 activity among the animals receiving sildenafil alone and this increased activity was negatively correlated to electric-evoked penile pressure in the study. This finding may be a result of a negative feedback of the host in response to chronic exposure to a potent inhibitor such as sildenafil. Chronic elevation of cgmp in the sildenafil group may activate protein kinase G (PKG) which can phosphorylate PDE5 and increase the affinity of PDE5 allosteric site for cgmp, thereby increasing PDE5 catalytic activity causing a decrease in cgmp levels This has been reported with the camp-pka crossreaction to increase PDE5 catalytic activity. 44 The addition of vitamin E may mitigate this rise. This result may indicate that in diabetes-induced ED, chronic use of sildenafil may alter the PDE-cGMP system by increasing usually reduced levels of PDE5 activity resulting in reduced cgmp levels. This may be one of the factors contributing to some ED-DM patients being refractory to PDE5 inhibition treatment. In contrast, the vitamin E plus sildenafil treatment group showed the lowest PDE5 activity. We speculate that vitamin E may alter the metabolism of the diabetic rats through the protein kinase signaling pathway, resulting in decreased PDE5 activity, leading to enhanced penile tissue relaxation. The reduction of endothelial and smooth muscle density in the corpora cavernosa of diabetic rat and reduced nitric oxide synthase (NOS) presence in both dorsal nerve and corpora cavernosa as shown in our study is supported in the literature The addition of daily vitamin E supplements to sildenafil appeared to act in a synergistic fashion, increasing the response to electric-stimulated penile pressure and an improved nnos, endothelial cells and smooth muscle content. Although the triglyceride levels did not achieve statistical significance, the observed differences among our groups may have in part contributed to the measured changes. To our knowledge, this is the first report on the chronic use of sildenafil and vitamin E in a diabetic animal model that examines physiologic, morphologic and enzyme changes as they relate to erectile function. This report illustrates that diabetes induced ED is a multifaceted condition affecting neural, muscular, vascular and metabolic functions. The vitamin E treated group showed improved endothelial cell staining as did the vitamin E plus sildenafil group. These findings may indicate that free oxygen radicals are important factors that contribute to the physiological and pathological changes in diabetes induced ED. In brief, vitamin E was shown to protect penile tissue injury and preserved nerve function as demonstrated by the nnos staining pattern and measured rise in intracorporal pressure. There exists support in the literature for the potential neurotrophic role of vitamin E in ischemic animal models. Gonzalez-Perez et al 48 have shown a beneficial effect of vitamin E on neural remodeling in an ischemic rat model. Additionally, vitamin E has been shown to

7 reduce oxidative stress in congestive heart failure, the mechanism is not yet known, but may have in our work contributed to the improved intracorporal pressure rises. 49 Finally, recent evidence strongly supports the potential benefit of vitamin E within the diabetic population. As described by Evans et al, 50 hyperglycemia within the diabetic population may lead to activation of kinases and stressactivated-protein kinases responsible for many of the pathological changes observed in this population. 50 As such, the utility of antioxidants is proposed as a new strategy for treatment. Conclusion Vitamin E combined with sildenafil enhanced erectile function better than either vitamin E or sildenafil alone in an animal model of diabetes. This is the first study to use both sildenafil and vitamin E to treat streptozotocin-induced diabetes in rats. Our results show significant improvements in penile intracavernous pressure and improved physiologic capacity (smooth muscle markers, endothelial cell and nnos staining) in the vitamin E plus sildenafil group. Early treatment with sildenafil plus vitamin E demonstrates positive changes in intracavernous pressure, nnos, smooth muscle a-actin endothelial cell staining and an apparent decrease in PDE5 activity. Further work evaluating the levels of free oxygen radicals and the impact of other scavenger molecules seems clinically relevant for this large cohort of men who remain poorly responsive to current PDE5 inhibition therapy. References 1 Benet AE, Melman A. The epidemiology of erectile dysfunction. Urol Clin N Am 1995; 22: Maatman TJ, Montague DK, Martin LM. Erectile dysfunction in men with diabetes mellitus. Urology 1987; 29: Hakim LS, Goldstein I. Diabetic sexual dysfunction. Endocrinol Metab Clin N Am 1996; 25: Kolodny RC, Kahn CB, Goldstein HH, Barnett DM. 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