Effect of combination endothelial nitric oxide synthase gene ... - Nature

International Journal of Impotence Research (2004) 16, 21–29 & 2004 Nature Publishing Group All rights reserved 0955-9930/04 $25.00 www.nature.com/ijir

Effect of combination endothelial nitric oxide synthase gene therapy and sildenafil on erectile function in diabetic rats TJ Bivalacqua1,2, MF Usta1, HC Champion3, S Leungwattanakij1, PA Dabisch2, DB McNamara2, PJ Kadowitz2 and WJG Hellstrom1* 1

Department of Urology, Tulane University School of Medicine, New Orleans, Louisana, USA; 2Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisana, USA; and 3Departtment of Internal Medicine, Johns Hopkins Hospital, Baltimore, Maryland, USA

Erectile dysfunction associated with diabetes mellitus is caused in part by disordered endothelial smooth muscle relaxation, neuropathy, and a decrease in cavernosal nitric oxide synthase (NOS) activity. The purpose of this study was to determine whether a combination of sildenafil and adenoviral gene transfer of endothelial NOS (eNOS) could enhance the erectile response in diabetic rats. Five groups of animals were utilized: (1) age-matched control rats, (2) streptozotocin (STZ)induced diabetic rats (60 mg/kg i.p.), (3) STZ-rats þ sildenafil (2 mg/kg i.v.), (4) STZ-rats transfected with AdCMVbgal or AdCMVeNOS, and (5) STZ-rats transfected with AdCMVeNOS þ sildenafil (2 mg/kg i.v.). At 2 months after i.p. injection of STZ, groups 4 and 5 were transfected with the adenoviruses and 1–2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Cyclic 30 ,50 -guanosine monophosphate (cGMP) levels were assessed in the cavernosal tissue. STZ-diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve; AUC) after CNS when compared to control rats. STZ-diabetic rats þ AdCMVeNOS had a peak ICP and AUC, which were similar to control animals. STZ-diabetic rats administered sildenafil demonstrated a significant increase in peak ICP at the 5 and 7.5 V settings, while the AUC was significantly increased at all voltage (V) settings. The increase in both ICP and AUC of STZ-diabetic rats transfected with AdCMVeNOS at all V settings was greater than STZ-diabetic rats transfected with AdCMVbgal. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had a significant increase in total ICP that was greater than eNOS gene therapy alone. Cavernosal cGMP levels were significantly decreased in STZ-diabetic rats, but were increased after transfection with AdCMVeNOS to values greater than control animals. In conclusion, overexpression of eNOS and cGMP in combination with sildenafil significantly increased both the peak ICP and total ICP to CNS in the STZ-diabetic rat, which was similar to the response observed in control rats. Moreover, the total erectile response was greater in STZ-diabetic rats receiving eNOS gene therapy plus sildenafil than STZ-rats receiving sildenafil or eNOS gene therapy alone. International Journal of Impotence Research (2004) 16, 21–29. doi:10.1038/sj.ijir.3901054 Keywords: eNOS; gene therapy; sildenafil; diabetes; erectile dysfunction

Introduction According to the American Diabetes Association, diabetes mellitus affects approximately 16 million individuals in the United States. A frequent complication of diabetes is erectile dysfunction (ED),

*Correspondence: WJG Hellstrom, MD, Professor of Urology, Tulane University School of Medicine, Department of Urology SL–42, 1430 Tulane Avenue, New Orleans, LA 70112, USA. E-mail: [email protected] Received 03 September 2002; revised 03 April 2003; accepted 08 April 2003

with an estimated prevalence in diabetic men to be as high as 50–75%.1,2 The exact mechanism of ED in diabetic patients is complex and can be caused by several mechanisms including autonomic neuropathy, endothelial dysfunction, and hormonal imbalance. Penile erection is a complex neurovascular phenomenon that requires an increase in penile arterial inflow, relaxation of cavernosal smooth muscle, and restriction of venous outflow from the penis. Erectile capacity is dependent on the vascular tone of the penis. Relaxation of corporal smooth muscle is essential for normal erectile function and substantial evidence exists to implicate neuronaland endothelial-derived nitric oxide (NO) as the principal mediator of cavernosal smooth muscle

eNOS gene therapy and sildenafil in diabetes TJ Bivalacqua et al 22

relaxation and penile erection.3,4 NO released by the endothelium that lines the corpus cavernosum and penile arteries that supply the penis and NO from nonadrenergic, noncholinergic (NANC) nerves bind to the soluble form of guanylate cyclase to increase cavernosal intracellular levels of cyclic 30 ,50 -guanosine monophosphate (cGMP). The enzyme that catalyzes this reaction in the endothelium and NANC neurons is termed nitric oxide synthase (NOS). The constitutive forms of the enzyme, neuronal NOS (nNOS) and endothelial NOS (eNOS), are the principal NOS isoforms involved in the induction of penile erection.5–7 Impairments in both the neurogenic and endothelium-dependent cavernosal smooth muscle relaxation exist in diabetes mellitus (DM).8–11 It is well established that the release of endothelium-derived NO and constitutive NOS activity is reduced in diabetes.1,10,12–14 Sildenafil (Viagra) is an orally active selective inhibitor of the type 5 phosphodiesterase (PDE) and inhibits the breakdown of intracellular cGMP.15 Therefore, sildenafil facilitates NO-mediated corpus cavernosum smooth muscle relaxation and thus enhances erectile responses. However, the clinical efficacy of sildenafil in diabetic patients is less than in patients with vasculogenic or psychogenic ED.16 Gene transfer approaches to the penis using adenoviral vectors have successfully accomplished sufficient transduction to enable gene expression and functional activity. Recent reports have shown that in vivo gene transfer with adenoviral vectors encoding eNOS and nNOS can reverse age-related ED in experimental animal models.17–20 Additionally, overexpression of eNOS in the aged penis results in an increase in both cavernosal NOS activity and cGMP formation.17,18,21 In the present study, we used the streptozotocin (STZ)-diabetic rat model that represents a model of type I DM. In this diabetic rat model, the penis exhibits both angiopathic vascular changes as well as neuropathic changes. These biochemical alterations are similar to molecular changes that occur in the human diabetic penis.8–11 Therefore, the aims of our study were to determine whether sildenafil or eNOS gene therapy could affect erectile dysfunction in STZinduced diabetic rats. Secondly, we sought to determine whether a combination of eNOS gene therapy and sildenafil could have a synergistic effect on erectile function in the STZ-diabetic rat.

(i.p.) injection of citrate buffer (100 mM citric acid, 200 mM disodium phosphate, pH 7.0), (2) rats receiving i.p. injection of STZ (Sigma Chemical Company, St Louis, MO, USA) in a dose of 60 mg/kg, (3) rats receiving i.p. injection of STZ (60 mg/kg) and subsequently transfected with AdCMVbgal, and (4) rats receiving i.p. injection of STZ (60 mg/kg) and subsequently transfected with AdCMVeNOS. STZdiabetic rats (Groups 2 and 4) received one intravenous (i.v.) administration of sildenafil (2 mg/kg i.v.) 10 min after the voltage-dependent erectile response to cavernosal nerve stimulation (CNS) was determined 2 months after the induction of diabetes with STZ. STZ-diabetic rats (Groups 3 and 4 received one intracavernosal injection of the adenoviruses 2 months after i.p. injection of STZ. The total body weight and blood glucose levels were determined before and after i.p. injection of STZ with blood glucose levels determined with an Accu-check blood glucose meter (Roche Diagnostics, Indianapolis, IN). Animals were considered diabetic if their blood glucose levels were greater than 200 mg/dl. The blood glucose levels and total body weight of the control and STZ-diabetic rats are summarized in Table 1. These procedures have been described previously.22,23 All procedures were performed in accordance with the NIH regulations and rats were maintained under controlled temperature and lighting.

Adenovirus vectors Two replication-deficient recombinant adenoviruses encoding nuclear-targeted b-galactosidase (AdCMVbgal) and eNOS (AdCMVeNOS), both driven by a cytomegalovirus (CMV) promoter, were generated by standard methods of the University of Iowa Gene Transfer Vector Core Laboratory, as previously described.24 Briefly, bovine eNOS was cloned by blunt-end ligation into pAdCMV4. The resultant plasmid and adenovirus backbone sequences restricted of E1 were transfected into HEK 293 cells, and plaques were isolated and amplified for analysis of eNOS expression. Recombinant adenoviruses were triple plaque-purified to assure that viral

Table 1 Weight and blood glucose levels in control and STZdiabetic rats

Materials and methods Development of diabetes Adult male CD rats (Harlan Sprague-Dawley, San Diego, CA, USA) were divided into four groups: (1) age-matched control rats receiving intraperitoneal International Journal of Impotence Research

Initial weight (g) Final weight (g) Initial blood glucose (mg/dl) Final blood glucose (mg/dl) n

Control rats

Diabetic rats

32976 454713* 10575 9676

32775 252713* 10274 404712*

Po0.05 value is significantly different from that obtained at the start of the study.

eNOS gene therapy and sildenafil in diabetes TJ Bivalacqua et al

suspensions were free of wild-type virus, and virus titers were determined by plaque assay on HEK 293 cells. After purification, the virus was suspended in PBS with 3% sucrose and kept at 701C until use. AdCMVbgal was used as a control virus in the present study.

In vivo gene delivery to the corpora cavernosa At 2 months after the rats developed diabetes, the STZ-diabetic rats were anesthetized with sodium pentobarbital (30 mg/kg i.p.) and placed in a supine position on a thermoregulated surgical table. Using sterile technique, the penis was exposed. A volume of 20 ml of AdCMVbgal (1 108 parts/ml) or AdCMVeNOS (1 108 parts/ml) was injected into the corpus cavernosum with a 30-gauge needle attached to a microliter syringe, as previously described.17,18,25 Rats did not show any overt signs of systemic (fever, dyspnea, tachycardia) or local (purulent discharge, erythema, edema) infection when observed any day after transfection.

Measurement of cavernosal tissue cGMP levels Cavernosal cGMP levels were measured in control and STZ-diabetic rats 1–2 days after instillation of AdCMVbgal and AdCMVeNOS into the corpus cavernosum. Corpus cavernosal tissue was rinsed with PBS, quick frozen in liquid nitrogen, and stored at 701C until determination of cGMP levels. The samples were assayed for cGMP using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA), as previously described.18 Cavernosal cGMP levels are expressed as picomoles per milligram of protein.

left jugular vein was cannulated (PE-50 tubing) for the administration of fluids. The shaft of the penis was freed of skin and fascia, and by removing part of the overlying ischiocavernous muscle exposure of the right crus was performed. A 25-gauge needle filled with 250 U/ml of heparin and connected to PE-50 tubing was inserted into the right crura and connected to a pressure transducer to permit continuous measurement of intracavernosal pressure (ICP). The bladder and prostate were exposed through a midline abdominal incision. The right major pelvic ganglion and cavernosal nerve were identified posterolateral to the prostate on one side, and an electrical stimulator with a stainless-steel bipolar hook was placed around the cavernosal nerve. MAP and ICP were measured with a pressure transducer connected to a data acquisition system (Biopac Systems, Santa Barbara, CA, USA) for continuous measurement of MAP and ICP pressures. The cavernosal nerve was stimulated with a square pulse stimulator (Grass Instruments, Quincy, MA, USA). Each rat underwent CNS at a frequency of 15 Hz and pulse width of 30 s. The application of 2.5, 5, and 7.5 V was used in the current protocol to achieve a significant and consistent erectile response. The duration of stimulation was 1 min with rest periods of 2–3 min between subsequent stimulations. The total erectile response or total ICP was determined by the area under the erectile curve (AUC; mmHg s) from the beginning of CNS until the ICP returned to baseline or prestimulation pressures. The ratio between the maximal ICP and MAP obtained at the peak of erectile response was calculated to normalize for variations in systemic blood pressure. These procedures have been described previously.18,23 The Tulane University School of Medicine Animal Care and Use Committee has approved all procedures used in the present study.

23

Drug preparation Measurement of erectile responses Control, STZ-diabetic, and STZ-diabetic rats 1–2 days after adenovirus administration were anesthetized with thiopentobarbital (100 mg/kg i.p.) and placed on a thermoregulated surgical table. The trachea was cannulated (PE-240 polyethylene tubing) to maintain a patent airway, and the animals breathed room air enriched with 95% O2/5% CO2. A carotid artery was cannulated (PE-50 tubing) for the measurement of mean systemic arterial pressure (MAP). Systemic arterial pressure was measured continuously with a Viggo-Spectramed transducer (Viggo Spectramed, Oxnard, CA, USA), which was attached to a data acquisition system (Biopac Systems, Santa Barbara, CA, USA), and connected to a computer simultaneously recording MAP. The

Sildenafil (Stanford Research Institute, Menlo Park, CA, USA) was dissolved in 0.9% saline with sonication and stored at 201C in brown amber bottles.

Statistics All hemodynamic data are expressed as mean7 s.e.m. and were analyzed using a one-way analysis of variance (ANOVA) with repeated measures and Neumann-Keuls post hoc test for multiple group comparisons (Statview, Abacus Concepts, Inc., Berkeley, CA, USA). A P-value of less than 0.05 was used as the criterion for statistical significance. International Journal of Impotence Research


Results

baseline levels, was significantly lower (Po0.05) in STZ-diabetic rats at all voltage settings (Figure 1).

Effect of diabetes on the erectile response At 2 months after the induction of diabetes, there was a significant decrease (Po0.05) in erectile function in STZ-diabetic rats (n¼10; Figure 1). We elicited penile erection in control and STZ-diabetic rats by stimulating the cavernosal nerve for 1 min. The magnitude of the increase in ICP after cavernosal nerve stimulation in the STZ-diabetic rats was significantly lower (Po0.05) than the age-matched control animals at all levels of stimulation (Figure 1). The total ICP (AUC) of penile erection, as measured from the start of CNS until the ICP returned to

Figure 1 Bar graph depicting the voltage-dependent erectile response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for 1 min in age-matched control and STZ (60 mg/kg i.p.) diabetic rats. n indicates the number of experiments; *Po0.05, response significantly different from control rats.

International Journal of Impotence Research

Influence of sildenafil on erectile responses in diabetes The effects of sildenafil on erectile responses to CNS were investigated in STZ-diabetic rats and these data are summarized in Figures 2 and 3. At 2 months after the induction of diabetes with streptozotocin (60 mg/kg i.p.), there was a significantly lower (Po0.05) voltage-dependent cavernosal nerve-in-

Figure 2 Bar graph depicting the voltage-dependent erectile response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for 1 min in control rats and before and after administration of the type 5 cGMP-specific PDE inhibitor sildenafil in a dose of 2 mg/kg i.v. in STZ-diabetic rats. n indicates the number of experiments; *Po0.05, response significantly different from control rats; **Po0.05, response significantly different from STZ-diabetic rats.


within 5–10 s, while the STZ-diabetic rats stimulation curve had a significantly lower erectile response with regard to the duration of the curve that was shorter (Figure 3). After the administration of sildenafil (2 mg/kg i.v.), STZ-diabetic rats’ stimulation curve approached that of the control animals and the duration of the erectile response was longer than the STZ-diabetic rats (Figure 3).

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Influence of eNOS gene therapy and sildenafil on erectile responses in diabetes

Figure 3 Representative ICP tracing after CNS at the 5 V setting for 1 min in (a) control rats, (b) STZ-diabetic rats, and (c) STZdiabetic rats treated with sildenafil (2 mg/kg i.v.).

duced erectile response (ICP/MAP and total ICP) in STZ-diabetic animals when compared to the agematched control rats (n¼9; Figure 2). After voltage-dependent (2.5, 5 and 7.5 V) erectile responses were determined, sildenafil was administered intravenously at a dose of 2 mg/kg. Intravenous administration of sildenafil (2 mg/kg) resulted in a significant decrease (Po0.05) in MAP to 7276 mmHg from a baseline level of 8877 mmHg in the STZ-diabetic rats. The decrease in systemic arterial pressure in response to sildenafil was rapid in onset, with the MAP returning to preinjection baseline within 15–20 min. At 10 min after the administration of sildenafil, the magnitude of the increased ICP/MAP in response to cavernosal nerve stimulation was increased significantly (Po0.05) at the 5 and 7.5 V settings (Figure 2). The total ICP (AUC) was significantly increased at all voltage settings (Figure 2). However, the erectile responses after sildenafil treatment were still significantly lower than the erectile responses in the control rats (Figure 2). A representative intracavernosal tracing after stimulation of the cavernosal nerve at 5 V for 1 min in control (a), STZ-diabetic (b), and STZdiabetic rats after the administration of sildenafil (c; 2 mg/kg i.v.) is depicted in Figure 3. Age-matched control animals had a rapid increase in ICP after cavernosal nerve stimulation that reached a plateau

The effect of overexpression of eNOS via adenoviral gene transfer of eNOS to the corpus cavernosum of STZ-diabetic rats in addition to the i.v. administration of the type 5 cGMP-specific PDE inhibitor sildenafil was investigated 2 months after induction of diabetes with streptozotocin (60 mg/kg i.p.). At 1–2 days after transfection with AdCMVbgal or AdCMVeNOS, STZ-diabetic rats’ erectile responses to CNS were investigated and these data are summarized in Figures 4 and 5. There was a significantly lower (Po0.05) voltage-dependent cavernosal nerve-induced erectile response (ICP/MAP and total ICP) in STZ-diabetic rats transfected with AdCMVbgal when compared to the control rats (n¼9; Figure 4). The magnitude of the increase in ICP (ICP/MAP) in response to CNS in the STZdiabetic rats transfected with AdCMVbgal was significantly lower (Po0.05) than the control rats, whereas rats transfected with AdCMVeNOS had a greater response to CNS that was similar to the response obtained in the control rats (Figure 4). However, the total ICP in the AdCMVeNOS-transfected STZ-diabetic rats was still significantly lower than the control rats. There was no statistical difference between STZ-diabetic rats and STZdiabetic rats transfected with AdCMVbgal erectile responses (data not shown). After voltage-dependent (2.5, 5, and 7.5 V) erectile responses were determined in the STZ-diabetic rats transfected with AdCMVeNOS, sildenafil was administered intravenously at a dose of 2 mg/kg. Intravenous administration of sildenafil (2 mg/kg) resulted in a significant decrease (Po0.05) in MAP to 6774 mmHg from a baseline level of 8375 mmHg in the AdCMVeNOS-transfected STZ-diabetic rats. After the administration of sildenafil, there was no change in the magnitude of the increase ICP/MAP in response to CNS at all voltage settings (Figure 4). However, the total ICP (AUC) was significantly increased at the 5 and 7.5 V settings (Figure 4). A representative intracavernosal tracing after stimulation of the cavernosal nerve at 5 V for 1 min in control (a), STZ-diabetic rats transfected with AdCMVbgal (b), and STZ-diabetic rats transfected with AdCMVeNOS before (c) and after (d) International Journal of Impotence Research


Figure 4 Bar graph depicting the voltage-dependent erectile response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for 1 min in control rats, STZ-diabetic rats transfected with AdCMVbgal, and AdCMVeNOS before and after administration of the type 5 cGMP-specific PDE inhibitor sildenafil at a dose of 2 mg/kg i.v. In vivo erection experiments were conducted 1–2 days after transfection with adenoviruses. n indicates the number of experiments; *Po0.05 response significantly different compared to control rats; **Po0.05 response significantly different compared to STZ-diabetic rats transfected with AdCMVbgal; r indicates Po0.05, response significantly different compared to STZ-diabetic rats transfected with AdCMVeNOS.

administration of sildenafil (2 mg/kg i.v.) is depicted in Figure 5. STZ-diabetic rats transfected with AdCMVeNOS had an increase in ICP after CNS that was similar to the responses exhibited in control rats. However, after the administration of sildenafil (2 mg/kg i.v.), AdCMVeNOS-transfected STZ-diabetic rats’ AUC or total ICP was significantly longer (Po0.05) than the AdCMVeNOS-transfected STZdiabetic rats before treatment with sildenafil (Figure 5). International Journal of Impotence Research

Figure 5 Representative ICP tracing after CNS at the 5 V setting for 1 min in (a) control rats, (b) STZ-diabetic rats transfected with AdCMVbgal, (c) STZ-diabetic rats transfected with AdCMVeNOS, and (d) STZ-diabetic rats transfected with AdCMVeNOS and treated with sildenafil (2 mg/kg i.v.).

Cavernosal cGMP levels Cavernosal tissue concentrations of cGMP were measured in control and in STZ-diabetic rats 1–2 days after transfection with AdCMVbgal and AdCMVeNOS, and these data are summarized in Figure 6. Cavernosal cGMP levels were significantly lower (Po0.05) in the STZ-diabetic rats when compared to control rats (n¼4; Figure 6). Gene transfer of eNOS to the diabetic penis resulted in cavernosal cGMP concentrations that were significantly higher (Po0.05) when compared to STZdiabetic rats transfected with AdCMVbgal (n¼4; Figure 6). Cavernosal cGMP levels were similar in STZ-diabetic rat cavernosal tissue and STZ-diabetic rat corpus cavernosum transfected with AdCMVbgal (data not shown).


Figure 6 Bar graph demonstrating cGMP levels in cavernosal tissue of control and STZ-diabetic rats transfected with AdCMVbgal or AdCMVeNOS. n indicates the number of experiments; *Po0.05 indicates that cGMP levels are significantly different when compared to control rats. **Po0.05 indicates that cGMP levels are significantly different when compared to STZdiabetic rats transfected with AdCMVbgal.

Discussion The results of the present study demonstrate, for the first time, that sildenafil increases both the peak ICP and total ICP erectile responses in STZ-diabetic rats in vivo. Additionally, this study demonstrates that adenoviral-mediated gene transfer of eNOS to the STZ-diabetic rat penis increases erectile responses in vivo as a result of an increase in cGMP formation. Moreover, the combination of eNOS gene therapy and sildenafil in STZ-diabetic rats resulted in a synergistic erectile response that was greater than either of these therapies alone. Cavernosal cellular responses are regulated by cyclic 30 ,50 -adenosine monophosphate (cAMP) and cGMP. The cellular levels of cAMP and cGMP are determined by the relative synthetic activities of adenylate and guanylate cyclase and the degradative activities of the cyclic nucleotide PDEs in cavernosal tissue.26,27 PDE enzymes are responsible for the hydrolysis of cAMP and cGMP, which in their active state induce smooth muscle relaxation. PDE inhibitors increase intracellular levels of cAMP and cGMP by hindering the hydrolytic activity of PDE, thus maintaining the vasodilator activity of cAMP and cGMP. Sildenafil, a selective type 5 PDE inhibitor, inhibits the hydrolysis of cGMP in the corpus cavernosum, thereby increasing cavernosal smooth muscle relaxation and prolonging penile erection.28 Sildenafil has been shown to enhance NO-mediated corpus cavernosum smooth muscle relaxation and penile erection both in vitro and in vivo.29–32 The effect of sildenafil on normal erectile responses to CNS has been previously studied in the penile

vasculature of the rat and, in that study, sildenafil was found to prolong the decay period of erections but did not increase the amplitude.33 In the present study, we found in the STZ-diabetic rats both an increase in the total ICP or decay period of erections as well as an increase in the amplitude (ICP/MAP) of the erectile response to CNS, suggesting that sildenafil therapy improves both erectile parameters in STZ-diabetic rats. The process of penile erection is contingent on an increase in arterial inflow and restricted venous outflow from the penis, coordinated by relaxation of the penile corpus cavernosum. The principal mediator responsible for the relaxation of the corpus cavernosum is NO, which is produced in the endothelium and NANC nerves by eNOS and nNOS, respectively. Manifestation of ED may result from a number of vascular, neurological, and hormonal complications. The prevalence of ED in diabetic patients has been reported to be as high as 50–75%.1 Since NO plays a dominant role in erectile physiology, most studies propose that diabetic-related ED is a result of disordered endothelial smooth muscle relaxation and NANC-related neuronal defects in the corpus cavernosum of the penis.8,23 Recently, cGMP formation in the corpus cavernosum of diabetic rabbits was found to be significantly reduced.34 This finding is consistent with the reduction in cGMP levels found in the STZ-diabetic rats 2 months after induction of diabetes in the present study. Sildenafil is an effective and well-tolerated treatment for ED in men with diabetes.15,35,36 However, sildenafil does not have the same efficacy in improving erectile function as measured by the International Index of Erectile Function in diabetic patients when compared to other ED etiologies.16 Sildenafil requires at least partial function of NANC penile nerves to be effective, and, therefore, men who have DM or have undergone non-nerve-sparing radical prostatectomy are less likely to respond.37 Recently, sildenafil significantly enhanced sodium nitroprusside- and electrical field stimulationmediated corpus cavernosum smooth muscle relaxation in rabbit diabetic corpus cavernosum in vitro, suggesting that sildenafil can have beneficial effects on impaired diabetic cavernosal smooth muscle relaxation.34 In the present study, we examined the effect of sildenafil on erectile function in STZ-diabetic rats in vivo. STZ-diabetic rats had a significant reduction in erectile function 2 months after i.p. injection of STZ. After a voltage-dependent (2.5, 5, and 7.5 V) erectile response was determined, sildenafil was administered at a dose of 2 mg/kg intravenously and the voltage series was repeated. Sildenafil increased the peak ICP at the 5 and 7.5 V settings and the total ICP at all voltage settings studied in the STZ-diabetic rat. However, this increase was still statistically different from the age-matched control rat’s erectile

27



response. Our findings demonstrate that inhibition of the type 5 PDE enzymes can partially normalize the diminished erectile response in the STZ-diabetic rat and suggests that this pharmacological drug can partially compensate for the diabetes-induced reduction in cavernosal cGMP. To our knowledge, this is the first report demonstrating that sildenafil can improve diabetic erectile dysfunction in vivo. Alterations in the NO/cGMP system are also present in the natural aging process and contribute to age-associated alterations in erectile function. Several groups have demonstrated that adenoviral gene therapy can be used to restore NO production in the rat corpus cavernosum to improve erectile function.18–20,38 Recently, adenovirus-mediated gene transfer of eNOS to diabetic rabbit aorta augmented vascular reactivity and partially restored impaired smooth muscle relaxation.39 Therefore, a gene transfer approach can be used to restore impaired cavernosal smooth muscle relaxation seen in diabetic corpus cavernosum. In this study, we have successfully transferred the eNOS gene to STZ-diabetic rat penises, which resulted in an increase in cGMP formation in the corpus cavernosum. This increase in cGMP formation after adenoviral-mediated gene transfer of eNOS resulted in an increase in ICP/MAP and total ICP after CNS in STZ-diabetic rats to values that were similar to the control animals. However, the total ICP of erectile response in STZ-diabetic rats transfected with AdCMVeNOS was still lower than control animals. We next wanted to determine if the administration of sildenafil to eNOS-transfected STZ-diabetic rats could increase the total ICP. In theory, this combination therapy should prove beneficial because eNOS-transfected STZ-diabetic rats have an increase in cavernosal cGMP, the second messenger molecule sildenafil selectively inhibits the catabolism in cavernosal smooth muscle cells. Consequently, this combination should result in an increase in the total duration of erectile response. After the administration of sildenafil to eNOS-transfected STZ-diabetic rats, the total ICP was significantly greater than the total ICP before treatment at the 5 and 7.5 V settings. Thus, these data suggest that when cGMP is overexpressed by eNOS gene therapy in cavernosal tissue and an inhibitor of the PDE 5 enzyme is present in the tissue, penile erectile response is significantly enhanced due to a synergistic effect of this combination pharmacological and gene therapy. In the present study, we have used an adenoviral vector, which has a transduction efficiency of a particular gene in the corpus cavernosum of the rat for approximately 7–10 days.38 This adenovirus has a peak expression of the eNOS gene at 1–2 days; therefore, we used this time point to determine all biochemical and physiological changes in the STZdiabetic rat. Gene therapy trials using adenoviral vectors have not been conducted for the treatment of


ED. However, the use of similar viral vectors such as ‘gutless’ adenoviral and adeno-associated viral vectors, as well as ex vivo expanded marrow stromal cells (stem cells) that have the potential to differentiate into endothelial and smooth muscle cells, are the focus of future experiments. Development of better vectors to deliver specific genes to the penis, which cause no local immune response and longer durations of expression, will be the selected vectors used in the application of gene therapy for the treatment of ED.

Conclusion In summary, the results of the present study demonstrate for the first time that sildenafil can improve erectile responses in STZ-diabetic rats in vivo. Moreover, the combination of eNOS gene therapy and sildenafil enhanced the total ICP response to CNS in STZ-diabetic rats to a value greater than either therapy alone. Therefore, it is reasonable to conclude that using oral PDE 5 inhibitors in combination with eNOS gene therapy could represent an exciting new form of therapy for the treatment of diabetic ED.

Acknowledgements We thank Drs Donald D Heistad and Beverley Davidson and the University of Iowa Vector Core Laboratory for preparation of the virus. This work was supported in part by a Young Investigator Award from the International Society of Impotence Research and Pfizer Inc. and the American Medical Association to TJ Bivalacqua.

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