The Molecular Basis of Erectile Dysfunction: From Bench to Bedside

Treatment Update

TREATMENT UPDATE The Molecular Basis of Erectile Dysfunction: From Bench to Bedside Jesse N. Mills, MD, Albaha Barqawi, MD, Sweaty Koul, PhD, Hari Koul, PhD, Randall B. Meacham, MD Division of Urology, University of Colorado School of Medicine, Denver, CO Erectile dysfunction is a common problem affecting many men across all age groups. Its etiology is multifactorial. Hormonal, vascular, neurogenic, lifestyle, and psychological entities have all been implicated as causative agents. The molecular basis underlying its etiology and progression is complex and still challenges researchers in the field. Nonetheless, newly discovered common pathways and targets of its pathogenesis have opened a new era for both prevention and active treatment of the disease. This review describes some of the known molecular mechanisms contributing to erectile dysfunction and discusses the future of gene therapy for the disease. [Rev Urol. 2005;7(3):128-134] © 2005 MedReviews, LLC Key words: Erectile dysfunction • Molecular • Gene therapy • Apoptosis • Nitric Oxide rectile dysfunction (ED), defined as the inability to initiate or maintain an erection sufficient for sexual satisfaction, is a common problem. ED affects upwards of 30 million American men and is a significant public health issue.1 Although the disorder has been described for more than 1000 years, the molecular basis for ED has yet to be fully elucidated. Multiple pathophysiologic risk factors including neurogenic, vascular, endocrine, traumatic, and psychogenic events have been incriminated in the etiology of ED. Risk factors that predispose a man to ED include hypertension, E 128 VOL. 7 NO. 3 2005 REVIEWS IN UROLOGY Molecular Basis of Erectile Dysfunction diabetes, smoking, excessive alcohol consumption, pelvic surgery, hypogonadism, and the use of many medications including anti-hypertensives and psychotropics.2 A great deal of attention has been focused on ED as a harbinger of life-threatening diseases such as coronary artery disease.3 A better understanding of the molecular basis of ED, therefore, reaches beyond the field of sexual medicine. guanine, arranged into codons, sequences of 3 nucleotides. Each gene codes for a protein, the functional unit of cellular biology. Genetic diseases manifest themselves as dysfunction of one or more proteins. Therefore, molecular therapy concentrates on correction of protein dysfunction. Replacing a specific protein seems to be the most direct way of treating a genetic mutation. Insulin is an excel- A great deal of attention has been focused on ED as a harbinger of lifethreatening diseases such as coronary artery disease. A better understanding of the molecular basis of ED, therefore, reaches beyond the field of sexual medicine. Therapies for ED have been discussed for almost as long as the disease itself. The modern era of therapy arguably began with the invention of the vacuum erection device by Geddings Osbon in the 1960s. By the 1980s, intracavernosal and intraurethral deposition of prostaglandin E and other vasoactive agents became available. The serendipitous discovery in the late 1990s of the erectile-enhancing property of the class of drugs known as phosphodiesterase-subtype 5 (PDE-5) inhibitors enhanced our understanding of the molecular mechanisms of ED. Before the discovery of the PDE5 inhibitors, ED was poorly understood from a mechanistic standpoint. A Brief Review of Molecular Medicine Sequencing of the human genome, the first step in making sense of the 6 billion nucleotides that comprise the genetic information of the human through characterization of the genetic code, is one of the most important advances in modern medicine. A human gene is made up of DNA, which consists of the nucleotides adenine, thymine, cytosine, and lent example. However, since insulin is a protein that functions in the bloodstream, it is easy to replace it and get it to its target. How one might go about replacing an enzyme necessary for cartilage synthesis is more problematic. The challenge of genetic therapy is to correct the gene defect so that the body can produce more of the protein itself. For example, with diabetes, patients would no longer need to inject themselves with insulin, but would instead be able to produce the deficient protein themselves. Recent advances in gene therapy for ED have paved the way for gene therapy protocols. For instance, the technique of oligonucleotide microarray analysis has been applied to study post-radical prostatectomy rat models of ED. Microarray analysis allows one to take the messenger RNA from a tissue of interest to determine which genes are expressed, or “turned on.” The power of this technique is that it is comprehensive yet unbiased. Investigators use powerful computational tools to process expression levels of tens of thousands of genes simultaneously. Performance of microarray analysis involves extraction of RNA from a tissue of interest, converting it to a labeled form that allows it to be viewed by a laser scanner, and allowing it to bind to a specially designed silicon chip that is pre-coated with millions of complimentary strands of DNA in a precisely mapped microscopic grid. Computer software that interprets a laser-scanned microscopic image of the surface of the chip allows detection and identification of minute quantities of the original RNA and thus tells the user what RNA was originally present (ie, which genes are being expressed or transcribed into RNA) in the sample. The user is provided with a genome-wide table of exactly which genes are being expressed in the tissue from which the RNA was extracted. Recent research has focused microarray technology on the rat post-prostatectomy model and identified 126 genes that demonstrated alteration of expression when compared with control animals.4 Research such as this, where microarray technology is used to elucidate the molecular basis of ED, is in its infancy, but will eventually lead to a greater understanding of all the genes that contribute to ED. More specifically, this process should improve our understanding of the genetic alterations that mediate the cavernosal cell apoptosis that accompanies perturbations to the nerve supply of the penis. So how do we transfer a gene from the test tube, where we can clone it and make it fully functional, to the target tissue? This is where the use of vectors comes into play. A vector is any system that can deliver or transfect a gene into an organism such that the organism reproduces the gene and its protein product. Several types of vectors have been used for this purpose. The vector that has been studied most extensively and that seems most promising in the area of ED is the viral vector. The goal of VOL. 7 NO. 3 2005 REVIEWS IN UROLOGY 129 Molecular Basis of Erectile Dysfunction continued viral vector technology is to engineer a virus to carry the gene of interest into the target tissue. Most viruses are naturally occurring DNA transfer devices, custom-made to enter the cell nucleus and commandeer the cell to make viral proteins and DNA to propagate the viral infection. Some RNA viruses invade the cytoplasm and take over the translational mechanisms of the cell. If a gene of interest is in- Such a vector might be injected directly into the corpus cavernosum. However, the virus might then enter the general circulation and travel to other organs where production of the protein could potentially produce unwanted side effects. How do the many recent advances in molecular genetics come to bear on the pathophysiology and management of ED? The molecular mecha- A vector is any system that can deliver or transfect a gene into an organism such that the organism reproduces the gene and its protein product. serted into a viral host and the virus is then transfected into an organism, the virus will deliver the gene to the nucleus of the cell, initiating production of the protein coded for by that gene. Useful vectors must be genetically modified from their normal state, however, so that they can no longer replicate themselves and cause infection. There are a number of caveats and limitations to this approach. Adenovirus, a strain that can cause symptoms of the common cold, is a wellstudied gene vector. Because it is a commonly encountered virus, however, many people have antibodies to various subtypes of the virus. In such a circumstance, the immune system would kill the vector before the new protein could be produced by the host cells. This immune response might not take place with the first exposure. However, by the time of the second therapeutic attempt, the body likely would have generated antibodies to the modified virus, and subsequent transfections would be vanquished by the immune system before achieving a therapeutic effect. Also, the adenovirus is not tissue specific. Take, for example, an adenoviral vector constructed to introduce a gene coding for a protein useful for improving ED. 130 VOL. 7 NO. 3 2005 nisms of ED are now beginning to be elucidated. A variety of proteins have been identified in the corpora of men suffering from ED. By enhancing the production of these proteins, we may achieve a more permanent treatment for ED than current as-needed therapies. Alternatively, by modifying the environment through such genetic manipulation, we may be able to increase patients’ responsiveness to currently available therapies. The following sections focus on the current literature on the molecular pathology associated with various common forms of ED. demonstrated.5 Although nitric oxide synthase (NOS) activity in the corpus cavernosum was not significantly altered by surgical or medical castration in one animal model, the presence of adequate androgen levels has been shown to be essential in other animal models for expression of the NOS gene inside the penis.6 Furthermore, a neuronal NOS-mediated decrease of intracavernosal pressure that was noted in hypogonadal rats was reversed by androgen replacement therapy.7 Baba and associates8 also reported a possible downregulation of both the production and activity of nitric oxide in the absence of testosterone in the rat, thus diminishing the response to peripheral stimulation via the nitric oxide pathway. A number of nitric oxide–independent mechanisms by which hypogonadism may contribute to ED have been proposed. These include smooth muscle cell degeneration and increase in apoptotic activity due to diminished androgen stimulation with associated fibrosis of the corpus cavernosum.9 An enhanced response to mediators of vasoconstriction such as
-adrenergic stimuli is also suggested to occur in the hypogonadal environment.10 Approximately 1 in 5 men diagnosed with ED is reported to have hormonal abnormalities, mostly associated with low serum testosterone levels. Nonetheless, a consistent clinical correlation between testosterone level and the severity of ED has not been demonstrated. Molecular Basis of the Effect of Testosterone Level on Erectile Dysfunction Approximately 1 in 5 men diagnosed with ED is reported to have hormonal abnormalities, mostly associated with low serum testosterone levels. Nonetheless, a consistent clinical correlation between testosterone level and the severity of ED has not been REVIEWS IN UROLOGY Molecular Basis for Neurogenic Erectile Dysfunction Radical retropubic prostatectomy is a common therapy for early stage prostate cancer. Even in the best surgeon’s hands, ED after prostatectomy is common. With development of the nerve-sparing technique of radical prostatectomy, the incidence of post-prostatectomy ED has declined. Molecular Basis of Erectile Dysfunction Studies performed in rats in which the cavernosal nerves were transected have shown that many changes occur in the corpus cavernosum at both the tissue and cellular levels. One such study demonstrated that penile weight is significantly reduced after cavernosal nerve transection in the rat.4 Notably, however, unilateral nerve sparing seems to protect against such penile atrophy. On a molecular level, cavernosal nerve transection profoundly decreases DNA content in the cavernosal tissue. This is accompanied by up-regulation of apoptotic genes. The study of apoptosis, also referred to as programmed cell death, has exploded in the past decade, particularly as it relates to cancer. It is thought that the growth and spread of cancer is due in part to the “turning off ” of genes that induce apoptosis. Such programmed cell death seems to be one of the primary mechanisms by which abnormal cells are eliminated from the system. Apoptosis also occurs when a cell perceives decreased signaling. After a spinal cord injury, for example, the neurolysed muscles undergo severe atrophy via an apoptotic pathway involving a well-described molecular signaling system termed Fas.11 A similar phenomenon likely occurs after the cavernosal nerves are severed in a radical prostatectomy. As mentioned earlier, a number of studies have looked at molecular changes in the rat model of radical prostatectomy. However, our knowledge of neurogenic penile atrophy is not limited to the rat. Men suffering from ED after radical retropubic prostatectomy exhibit decreased penile length and circumference.12 This outcome may be due, in part, to nerve disruption–induced apoptosis and hypoxia-induced damage to the corpora, leading to smooth muscle atrophy. Molecular Basis for Vascular Erectile Dysfunction Arterial insufficiency secondary to atherosclerosis, smoking, and trauma has been shown to be an important cause of ED.13 The rat model for studying ED arising from penile ischemia involves ligation of both common iliac arteries.14 Examination of the rat penis after arterial ligation reveals many changes in penile morphology and gene expression. The nerve supply to the penis also changes profoundly following disruption of penile arterial supply in the rat. In such animals, a decrease in diameter of both myelinated and nonmyelinated fibers can be observed. Among rats subjected to iliac artery ligation, corporal smooth muscle cells have been found to be depleted of myofilaments. Additionally, there are fewer endothelial cells in the vasculo- Since testosterone serves as a stimulator of vascular endothelial growth factor, androgen deficiency may also play a contributory role in penile vascular insufficiency. Molecular Basis for Diabetes-Associated Erectile Dysfunction Diabetes is a complex disease that affects many organ systems through the effects of chronically elevated plasma glucose, which causes damage at the microvascular level. Its deleterious effects on erectile function are well documented. The common pathway that leads to diabetes-induced microvascular damage is the generation and impaired clearance of oxygenderived free radicals. The resulting oxidative stress, coupled with the hyperglycemic milieu of the microvasculature, leads to production of a Nitric oxide is known to be a potent mediator of smooth muscle relaxation, a crucial component of the erectile response. genic rat model of ED. Fewer endothelial cells would be expected to result in less nitric oxide production. Nitric oxide is known to be a potent mediator of smooth muscle relaxation, a crucial component of the erectile response. Another cause of ED is venous leak. As the name implies, this condition is characterized by an excess flow of blood from the penis during sexual stimulation, thus preventing the formation of a rigid erection.15 Although venous leak likely has multiple etiologies, it can be induced in the animal model by castration.16 Androgen deficiency is thought to induce venous leak by generating smooth muscle atrophy in the corpus cavernosum. It seems, therefore, that in addition to decreasing libido, androgen deprivation induces ED at the cellular level. class of compounds termed advanced glycation end products (AGEs). AGEs are a heterogeneous group of products formed by non-enzymatic Maillard reactions between a protein’s primary amino group and carbohydrate-derived aldehyde groups. AGEs bind with a receptor, RAGE, which mediates binding of AGEs to endothelial cells, thereby inhibiting endothelialmediated smooth muscle relaxation via a nitric oxide pathway.17 The end result of this molecular cascade is impaired vasodilatation in any microvascular area, ranging from coronary vasculature to the retina to the penis.18 Emerging research offers an interesting hypothesis regarding diabetesinduced ED. Rather than its resulting from the “turning off ” of genes necessary for smooth muscle relaxation, VOL. 7 NO. 3 2005 REVIEWS IN UROLOGY 131 Molecular Basis of Erectile Dysfunction continued a growing body of literature suggests that the opposite occurs. A signaling pathway involved in calcium channeling, called RhoA/Rho kinase, is turned on in diabetic rats.19 This leads to vasoconstriction of the microvasculature of the penis and chronic detumescence. Therefore, instead of thinking of diabetes as the inability to achieve erection, one might view it as the persistence of the flaccid state. This principle suggests that inhibiting this detumescence factor would lead to enhanced erections, an approach that has been shown to work in the diabetic rat.19 The chronic detumescence theory and the more traditional tumescence inhibition theory of diabetes-related ED are not mutually exclusive. Indeed, both mechanisms likely contribute to diabetesinduced ED. Apoptosis plays a critical role in diabetes-associated ED20 just as it does in neurogenic ED. Assessment of the rat diabetic penis reveals decreased expression of the anti-apoptosis gene Bcl-2 in cavernosal tissue.21 Tissue deficient in Bcl-2 expression is likely to have the cellular homeostasis shifted toward cell death and organ degeneration. Therefore, it appears that enhanced programmed cell death in the penis further contributes to diabetes-related ED. Therapeutic Applications of Molecular Genetics The most exciting outcome of our evolving understanding of the molecular basis of ED is the development of new therapies for sexual dysfunction. Human trials using gene therapy in the management of a variety of diseases have been under way since 1990. A major setback occurred in 1999 with the death of a young man undergoing intrahepatic administration of a recombinant adenovirus vector encoded with the ornithine transcarbamylase (OTC) gene. The 132 VOL. 7 NO. 3 2005 patient was born without the OTC gene, which is necessary for urea metabolism. He died from multi-organ failure 4 days after administration of the vector. In spite of this event, however, hundreds of human gene therapy trials are currently being conducted.22 Given the knowledge that has been gleaned from studying the mechanism of the phosphodiesterase inhibitors, it should not be surprising that much of the effort in gene therapy for the treatment of ED has focused on replenishing supplies of nitric oxide in the corpus cavernosum. Nitric oxide, as noted earlier, is the cellular currency that leads to smooth muscle relaxation, vasodilatation, and subsequent erection. A number of strategies have been devised to increase circulating levels of penile nitric oxide. Some laboratories (PnNOS) to increase erectile response in the aging rat. They demonstrated that expression of the gene was detectable 56 days after transfection. This group also addressed a lingering concern related to gene therapy, which is where, other than the target organ, the gene construct may travel and be expressed. In this experiment, the investigators transfected a plasmid cDNA construct of PnNOS into rat corpora cavernosa. They demonstrated that the penis retained the highest level of expression and that there was no sign of expression in the liver, heart, or kidney. There was, however, weak expression in the rat lung.24 Another strategy for applying gene therapy to the management of ED involves increasing nitric oxide levels through enhancement of penile vasculature. Human studies in cardiac revascularization using recombinant Much of the effort in gene therapy for the treatment of ED has focused on replenishing supplies of nitric oxide in the corpus cavernosum. are focusing on transfecting isoforms of NOS directly into the corpora cavernosa of rats. Bivalacqua and colleagues19 have shown that recombinant endothelial nitric oxide synthase (eNOS) carried in an adenovirus vector can reverse diabetes-induced ED in the rat. These authors demonstrated that the intracavernosal pressure achieved during stimulation of the cavernosal nerves was much lower in diabetic rats than controls. They then showed that injecting an eNOS vector into the corpora cavernosa of diabetic rats reversed this decline in intracavernosal pressure. They also showed that administering sildenafil to the transfected rats increased intracavernosal pressure and the duration of response. Magee and associates23 used the penile form of neuronal nitric oxide synthase REVIEWS IN UROLOGY vascular endothelial growth factor (VEGF) are currently in phase II trials.25 VEGF is a multifunctional protein, stimulating angiogenesis, inhibiting apoptosis, and increasing vascular permeability. The hypothesis is that, since endothelial cells synthesize nitric oxide, increasing the quantity of endothelial cells in the target organ will increase nitric oxide production and subsequent vasodilatation. This can prevent ischemia in the heart and, similarly, might improve erections in the penis. To that end, Lin and associates26 have evaluated transfection of VEGF into the rat penis to reverse vasculogenic ED via an increase in the levels of eNOS and inducible nitric oxide synthase (iNOS). Other researchers found that, among rats with surgically induced penile venous leak, they Molecular Basis of Erectile Dysfunction could increase intracavernosal pressures after transfecting recombinant VEGF via an adenovirus vector.27 They also noted an increase in regeneration of penile smooth muscle and nerves as well as endothelial cell hypertrophy and hyperplasia. In a more recent application, VEGF was demonstrated to inhibit apoptosis after intracavernous injection, effectively restoring pressure in diabetic rat penile crura.28 Gene therapy in the treatment of ED has not been evaluated in human trials, but Christ and colleagues29 presented to the National Institutes of Health committee on human gene transfer a proposal to transfer human hslo/maxi-K gene. This gene is responsible for increasing the response of smooth muscle to minimal levels of endogenous muscle relaxants such as nitric oxide in the corpora cavernosa of patients with ED. An Update From Our Laboratory We have developed rat models to study the effects of injecting recombinant vascular endothelial growth factor into the corpora cavernosa of rats in the presence and absence of a PDE-5 inhibitor. Our models include a cavernous neurotomy model, an internal iliac arterial ligation model, and a diabetic model. We hypothesize that the combination of intracavernosal delivery of gene product with daily administration of a PDE-5 inhibitor will down-regulate pro-apoptotic factors and up-regulate anti-apoptotic factors. We plan to look at a variety of markers to detect these changes. Additionally, we have cloned VEGF from human prostate cancer cells and genetically engineered a VEGF-GFP (green fluorescent protein) fusion protein expression system suitable for eukaryotic expression, and we are in the process of generating replication-deficient adenoviral vector systems (unpublished data, 2004). The hope is that we will develop an animal model to test the efficacy of daily PDE-5 inhibitor intake combined with less frequently administered gene therapy to counteract the effects of some common etiologies for erectile dysfunction. Conclusions As clinicians, we have entered an exciting era in medicine in general, and specifically in the management of ED. The sequencing of the human genome has accelerated our knowledge of the molecular basis of human physiology and disease. As is the case with many chronic disorders, it is likely that the future treatment of ED will gradually transition from symptomatic asneeded therapies to preventive strategies. Ongoing advances in molecular biology and gene therapy will certainly play a large role in this treatment revolution. Acknowledgement The authors wish to thank Jason C. Mills, MD, PhD, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, for help in editing the section “A Brief Review of Molecular Medicine.” References 1. 2. Melman A, Gingell JC. The epidemiology and pathophysiology of erectile dysfunction. J Urol. 1999;161:5-11. Melman A, Rehman J. Pathophysiology of erectile dysfunction. Mol Urol. 1999;3:87-102. Main Points • Multiple pathophysiologic risk factors including neurogenic, vascular, endocrine, traumatic, and psychogenic events have been incriminated in the etiology of erectile dysfunction (ED). Risk factors that predispose a man to ED include hypertension, diabetes, smoking, excessive alcohol consumption, pelvic surgery, hypogonadism, and the use of many medications including antihypertensives and psychotropics. • A number of nitric oxide independent-mechanisms by which hypogonadism may contribute to ED have been proposed. These include smooth muscle-cell degeneration and increased apoptotic activity due to diminished androgen stimulation with associated fibrosis of the corpus cavernosum. An enhanced response to mediators of vasoconstriction such as
-adrenergic stimuli is also suggested to occur in the hypogonadal environment. • Men suffering from ED after radical retropubic prostatectomy exhibit decreased penile length and circumference. This outcome may be due, in part, to nerve disruption–induced apoptosis and hypoxia-induced damage to the corpora, leading to smooth muscle atrophy. • Venous leak, a cause of ED, likely has multiple etiologies; it can be induced in the animal model by castration. Androgen deficiency is thought to induce venous leak by generating smooth muscle atrophy in the corpus cavernosum. It seems, therefore, that in addition to decreasing libido, androgen deprivation induces ED at the cellular level. • The chronic detumescence theory and the more traditional tumescence inhibition theory of diabetes-related ED are not mutually exclusive. Indeed, both mechanisms likely contribute to diabetes-induced ED. • An evolving understanding of the molecular basis of ED has led to the development of new therapies for sexual dysfunction. Strategies being investigated include efforts to increase circulating levels of penile nitric oxide, such as using viral vectors to transfect genes and correct for deficiencies. VOL. 7 NO. 3 2005 REVIEWS IN UROLOGY 133 Molecular Basis of Erectile Dysfunction continued 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 134 El-Sakka AI, Morsy AM, Fagih BI, Nassar AH. Coronary artery risk factors in patients with erectile dysfunction. J Urol. 2004;172:251-254. User HM, Zelner DJ, McKenna KE, McVary KT. Microarray analysis and description of SMR1 gene in rat penis in a post-radical prostatectomy model of erectile dysfunction. J Urol. 2003;170: 298-301. Corona G, Mannucci E, Mansani R, et al. Aging and pathogenesis of erectile dysfunction. Int J Impot Res. 2004;16:395-402. Aversa A, Isidori AM, De Martino MU, et al. Androgens and penile erection: evidence for a direct relationship between free testosterone and cavernous vasodilation in men with erectile dysfunction. Clin Endocrinol (Oxf ). 2000;53: 517-522. Marin R, Escrig A, Abreu P, Mas M. Androgendependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes. Biol Reprod. 1999;61: 1012-1016. Baba K, Yajima M, Carrier S, et al. Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis. BJU Int. 2000;85:953-958. Traish AM, Munarriz R, O’Connell L, et al. Effects of medical or surgical castration on erectile function in an animal model. J Androl. 2003;24: 381-387. Aversa A, Isidori AM, Greco EA, et al. Hormonal supplementation and erectile dysfunction. Eur Urol. 2004;45:535-538. Yoshino O, Matsuno H, Nakamura H, et al. The role of Fas-mediated apoptosis after traumatic spinal cord injury. Spine. 2004;29:1394-1404. Fraiman MC, Lepor H, McCullough AR. Changes in penile morphometrics in men with erectile VOL. 7 NO. 3 2005 13. 14. 15. 16. 17. 18. 19. 20. 21. REVIEWS IN UROLOGY dysfunction after nerve-sparing radical retropubic prostatectomy. Mol Urol. 1999;3:109-115. Siroky MB, Azadzoi KM. Vasculogenic erectile dysfunction: newer therapeutic strategies. J Urol. 2003;170:S24-S29; discussion S29-S30. Gongora Castillo C, Alvarez Gomez de Segura I, Bonet Furgeri H, de Miguel del Campo E. [Impotence of arterial origin: experimental models and evaluation of sexual behavior in rats.] J Urol (Paris). 1993;99:122-126. Lue TF. Penile venous surgery. Urol Clin North Am. 1989;16:607-611. Muller SC, Hsieh JT, Lue TF, Tanagho EA. Castration and erection; an animal study. Eur Urol. 1988;15:118-124. Cartledge JJ, Eardley I, Morrison JF. Advanced glycation end-products are responsible for the impairment of corpus cavernosal smooth muscle relaxation seen in diabetes. BJU Int. 2001;87: 402-407. Yamagishi S, Takeuchi M, Inagaki Y, Nakamura K, Imaizumi T. Role of advanced glycation end products (AGEs) and their receptor (RAGE) in the pathogenesis of diabetic microangiopathy. Int J Clin Pharmacol Res. 2003;23:129-134. Bivalacqua TJ, Champion HC, Usta MF, et al. RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. Proc Natl Acad Sci U S A. 2004;101: 9121-9126. Alici B, Gumustas MK, Ozkara H, et al. Apoptosis in the erectile tissues of diabetic and healthy rats. BJU Int. 2000;85:326-329. Yamanaka M, Shirai M, Shiina H, et al. Diabetes induced erectile dysfunction and apoptosis in penile crura are recovered by insulin treatment in rats. J Urol. 2003;170:291-297. 22. 23. 24. 25. 26. 27. 28. 29. Ferber D. Gene therapy: repair kits for faulty genes. Science. 2001;294:1639. Magee TR, Ferrini M, Garban HJ, et al. Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase. Biol Reprod. 2002;67:1033-1041. Magee TR, Ferrini MG, Davila HH, et al. Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS. Biol Reprod. 2003;68: 478-488. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation. 2003;107:2677-2683. Lin CS, Ho HC, Chen KC, et al. Intracavernosal injection of vascular endothelial growth factor induces nitric oxide synthase isoforms. BJU Int. 2002;89:955-960. Rogers RS, Graziottin TM, Lin CS, et al. Intracavernosal vascular endothelial growth factor (VEGF) injection and adeno-associated virusmediated VEGF gene therapy prevent and reverse venogenic erectile dysfunction in rats. Int J Impot Res. 2003;15:26-37. Yamanaka M, Shirai M, Shiina H, et al. Vascular endothelial growth factor restores erectile function through inhibition of apoptosis in diabetic rat penile crura. J Urol. 2005;173: 318-323. Christ GJ. Frontiers in gene therapy for erectile dysfunction. Int J Impot Res. 2003;15(suppl 5): S33-S40.