Medical Meeting Reports

Sexual Function: Basic Research

Authors: Trinity J. Bivalacqua M.D. Ph.D.; Johns Hopkins Hospital, Brady Urological Institute

The current advances in the field of sexual medicine have been robust in the last ten years. We now have a better understanding of the physiology and pathophysiology of male and female sexual function, which has allowed for improved pharmacological and gene therapies for sexual dysfunction. We have seen a direct connection between basic science research performed at the bench side translating into new therapies for sexual dysfunction. Once again at this year's AUA in San Antonio, we have seen elegant cellular and molecular studies presented on sexual dysfunction in both men and women. In this communication, the most novel and clinically relevant abstracts will be presented.

The role of the vascular endothelium in the erectile physiology and disease has become further delineated. As men age there is an age-dependent decline in erectile function. The mechanisms involved in the impairment in erectile function are multi-factorial but one common theme is impairments in smooth muscle cell biology. It is thought that age-related erectile dysfunction is associated with reduced nitric oxide synthase (NOS) activity and NO-bioavailability or biosynthesis. Therefore, one hypothesis is that there is reduced endothelial-derived NO which causes impaired penile smooth muscle relaxation. Musicki et al demonstrated (Abstract 1042) sought to evaluate whether age-related ED involves dysregulation of endothelial NOS (eNOS) phosphorylation. In aged rat penile tissue, there was an age-dependent decline in phosphorylation of eNOS (Ser-1177). Additionally, the vascular endothelial growth factor (VEGF) gene transfer significantly improved the phosphorylation of eNOS (Ser-1177) which improved cavernous nerve stimulated erectile responses in the age rat. Therefore, altered eNOS phosphorylation resulting in diminished endothelial-derived NO may contribute to compromised vascular homeostasis in the aged penis and directly impair penile smooth muscle relaxation and erection. The finding that VEGF partly corrects alterations in eNOS phosphorylation and improvements in erectile function offers a basis for the specific use of growth factor gene therapy for the treatment of age-associated erectile dysfunction.

Impaired endothelial NOS function is associated with erectile dysfunction in diabetes mellitus, however, the exact mechanisms associated with impaired eNOS regulation are unclear at this time. Musicki and colleagues (Abstract 1043) investigated whether hyperglycemia increases O-linked N-acetylglucosamine (O-GlcNAc) modification of eNOS at Ser-1177 in the diabetic rat penis. The hypothesis is that hyperglycemia associated with diabetes increases O-GlcNAc thus impairing phosphorylation of eNOS and thereby hampering endothelial-derived NO in the penis. Five weeks after the induction of diabetes by alloxan (140 mg/kg ip) there was a significant elevation in O-GlcNAc at a time when phosphorylation of eNOS (Ser-1177) and Akt (Ser-473) was significantly reduced both at baseline and after cavernous nerve stimulation. This study demonstrates that hyperglycemia causes an upregulation of O-GlcNAc thus impairing phosphorylation of eNOS in the penile vascular bed. Such eNOS impairments may contribute to ED associated with diabetes mellitus. Another study investigating the etiology of diabetes-associated ED was conducted from Hunter Wessells group in Seattle, Washington. Sullivan and colleagues (Abstract 1044) evaluated the molecular changes that occur in the diabetic corpus cavernosum of experimental rats after intraperitoneal (ip) injection of streptozotocin (STZ). Ten weeks after ip injection of STZ, penile total RNA was obtained and hybridized to Affymetrix 230A Rat GeneChips to determine differentially expressed genes and fold change. In diabetic rats, expression was increased in 323 genes and decreased in 206 genes compared to age-matched controls. There was a downregulation of genes involved in extracellular matrix (ECM) organization and ECM constituents conferring tensile strength, including collagen and elastin. Additionally, genes associated with endothelial dysfunction (lipoprotein lipase and ceruloplasmin) were upregulated in the diabetic rat penis. In summary, in an experimental model of type I diabetes, there is a diverse range of signaling pathways which are altered in the penis. Further studies are warranted to determine if the genes associated with ECM and endothelial function are active and directly related to the development of ED.

The RhoA/Rho-kinase signaling pathway influences corporal smooth muscle tone and has been shown to contribute to erectile dysfunction-associated with aging and diabetes. It is thought that endothelial-derived NO may influence the expression and activity of RhoA/Rho-kinase signaling in the systemic vasculature. Therefore, Bivalacqua and colleagues (Abstract 1041) sought to investigate the basal regulatory influence of endothelial-derived nitric oxide (NO) on the RhoA/Rho-kinase signaling cascade in the penis employing endothelial NOS synthase (eNOS) knockout mice at baseline and after eNOS transfection of the penis. eNOS -/- mice had no change in RhoA or Rho-kinase protein expression, however, there was a significant decrease in phosphorylation of MYPT-1 (marker of Rho-kinase activity). After gene transfer of eNOS to the penis of eNOS -/- mice, both Rho-kinase activity was restored to a level similar to WT mice. These data suggest that RhoA/Rho-kinase activity is directly related to the levels of endothelial-derived NO in the penis providing new insight into the regulatory basis involved in maintaining functional penile vascular homeostasis.

The mechanisms governing abnormally prolonged erections associated with priapism are unknown. Recently, it has been shown that mice lacking the gene for endothelial NOS exhibit priapic activity. These abnormally prolonged erections are associated with a downregulation in phosphodiesterase type 5 (PDE5). The group from Johns Hopkins sought to evaluate PDE5 expression in a mouse model of sickle cell disease in order to support the hypothesis that downregulation of PDE5 serves as a mechanism of priapism. Bivalacqua and colleagues (Abstract 1040) demonstrated that mice expressing human sickle cell hemoglobin (SS -/-) have significant reduction in penile constitutive NOS activity and PDE5 expression/activity. Additionally, the SS -/- mice had significant increases in cavernous nerve stimulated erectile responses as well as pre- and post-stimulated increases in intracavernous pressure suggesting they had enhanced erectile responses or priapic activity. All results were compared to heterozygotic sickle cell mice (SS +/-) and wild type mice. These data demonstrate that PDE5 downregulation occurs in sickle cell mice penes and provide support for this biochemical effect as a novel mechanism in the pathogenesis of priapism.

Recently, the central nervous system has become a pharmacological target for the treatment of erectile dysfunction. After intracerebroventricular or hypothalamic periventricular injection into various animal models, the adrenocorticotropic hormone (ACTH) and the melanocyte-stimulating hormone (a-MSHs) have been shown to induce penile erection. Therefore, Yath and colleagues (Abstract 1056) sought to determine if melanocortin neurons undergo increased activation during sexual behavior. Double labeled immunohistochemistry was employed to identify activation of POMC neurons by c-FOS and alpha-melanocyte stimulating hormone (MSH) co-staining in the arcuate nucleus. After Long Evans rats were exposed to an estrous female there was a significant increase in colocalization of MSH with c-FOS in the rostral arcuate nucleus. These data demonstrate that melanocortinergic neurons in the arcuate nucleus are activated during sexual behavior in the rat and provide support for this signaling pathway as a molecular and pharmacological target for the treatment of sexual dysfunction.

In women, sexual dysfunction is highly prevalent and associated with chronic disease and menopause. Recently, it is thought that the melanocortin signaling pathway may be involved in the control of female sexual behavior, in particular sexual arousal. MT-II is a cyclic peptide analogue of alpha-MSH with affinity for the five known melanocortin receptors. MT-II can improve erectile function in men with ED. Therefore, Rossler and colleagues (Abstract 1078) investigated the effect of MT-II on proceptive behavior in female rats in order to determine the role of Melanocortins in the physiology and pathophysiology of female sexual arousal. Ovariectomized Long-Evans rats underwent sexual behavior testing to define baseline characteristics of proceptive behavior. They were then administered MT-II (1 and 3 mg/kg iv) and subsequent behavioral tests were performed. Ovariectomized rats supplemented with hormones who were administered MT-II had improvements in sexual proceptive behavior but no psychological behavior changes suggesting that melanocortin receptor agonists may be useful in the treatment of women with hyposexual desire dysfunction. Further studies are warranted to validate these results obtained in an experimental model of female sexual dysfunction before use of this agent in women with female sexual dysfunction.

Oral pharmacotherapies are first line treatment for ED. However, there are a subset of patients that the three highly selective PDE5 inhibitors (sildenafil, vardenafil, and tadalafil) are less efficacious, have adverse events, or contraindicated. Therefore, new centrally acting agents which may have less side-effects and work via different mechanisms may prove efficacious for the treatment of ED. PT-141, a cyclic heptapeptide melanocortin analog, is a centrally acting erectogenic agent which has been shown to improve erectile function in healthy men and ED patients. Diamond and colleagues (Abstract 1057) investigated the co-administration of sub-therapeutic doeses of either sildenafil or vardenafil with intranasal PT-141 in order to determine if there is an additive or synthergistic effect of these two erectogenic agents working through two distinct mechanisms. The erectile responses induced by co-administration of PT-141 and sildenafil were significantly enhanced from the response elicited by administration of sildenafil alone. This novel pharmacological combination of sildenafil and PT-141 was well-tolerated and did not result in any adverse events. The authors conclude that co-administration of sildenafil and PT-141 may constitute a treatment alternative for patients who are PDE5 inhibitor non-responders. However, the patient population chosen had adequate response to sildenafil in sub-therapeutic doses. Therefore, further clinical trials targeting PDE5 inhibitor non-responders are warranted at this time before any absolute conclusions can be drawn from this study.

The use of gene therapy for the treatment of ED has been proposed by a number of investigators. Molecular targets include endothelial and neuronal NOS, growth factors (VEGF, fibroblast growth factor), and the alpha-subunit of the Maxi-K channel. Intracorporal injection of the alpha-subunit of the human Maxi-K channel (hSlo) can restore erectile function in diabetic and aging rat models of erectile dysfunction. Drs. Arnold Melman and George Christ group in New York have been pioneers in the use of a gene therapy approach for the treatment of male ED. Of note, pVAX-hSlo is currently being evaluated in Clinical Phase I trials. At this year's AUA, their group presents new data on the use of a smooth muscle specific promoter driving hSlo for the restoration of penile vascular function. Biggs et al (Abstract 1076) developed a smooth muscle specific promoter in a plasmid (pSMAA-hSlo) expressing hSlo (Maxi-k channel) to demonstrate that the restoration of erectile function can be mediated specifically through expression in smooth muscle cells. After gene transfer of both the pVAX-hSlo and pSMAA-hSlo improved cavernous nerve mediated erectile function in aged and diabetic rats was observed. There was no difference in efficacy between the two plasmids. This study demonstrates that smooth muscle tissue specific-overexpression of hSlo in corporal tissue is sufficient to restore erectile function. Because expression of hSlo from the SMAA promoter is restricted to specific cell types, the plasmid pSMAA-hSlo may represent an improved safety profile compared to pVAX-hSlo.

ED is a known complication after radical prostatectomy for prostate cancer. The etiologies of ED after nerve-sparing radical prostatectomy have not been fully elucidated. Therefore, Kuk Yang et al (Abstract 1048) investigated whether bilateral cavernous nerve (CN) transection induces apoptosis in the penis. More importantly, they sought to delineate whether specific cell signaling pathways involved in either cell death or survival are affected. Adult male mice underwent bilateral cavernous nerve transection, 4 weeks after transection the penile shaft and crura were separately harvested and apoptotic cells and phosphorylation of eight different protein kinases were measured. There was a significant increase in apoptotic cells in both the penile shaft and crura was evident by 4 weeks after cavernous nerve transection. Interstitial smooth muscle cells and endothelial cells were the major cell types undergoing apoptosis. Additionally, an increase in phosphorylation of p38 and JNK, two stress-related kinases, were upregulated. In another study by McVary et al (Abstract 1046) using a similar animal model of cavernous nerve injury showed that both caspase 8 and 9 (markers of apoptosis) were upregulated in the rat penis. In summary, cavernous nerve transaction stimulates apoptotic cell death in the penis. Both the intrinsic and extrinsic apoptotic pathways are activated in a model of neuropathic ED. The mechanism by which apoptosis occurs may involve activation of the stress-related signaling pathway including the kinases p38 and JNK. Future studies are warranted to determine if inhibition of apoptosis will improve penile vascular homeostasis and erectile function.

ED following radical prostatectomy occurs as a result of neural and arterial injury with subsequent postoperative corporal smooth muscle alterations. Therefore, the development of a new pharmacological agents with neuromodulatory properties and at the same time protects the penile vasculature is warranted. In clinical studies, there is recent evidence that daily PDE5 inhibitor therapy may improve erectile function post-radical prostatectomy. However, the mechanisms have not been fully delineated. Therefore, the Mulhall group at Cornell sought to define the effect of daily sildenafil administration on erectile function as measured by cavernous nerve stimulation, apoptosis (TUNEL assay), CD31 and GAP43 immunohistochemistry for endothelial cell integrity and neural regeneration respectively, as well as electron microscopy. Donohue and colleagues (Abstracts 1050 and 1051) demonstrated that daily administration of sildenafil improved erectile function in vivo to cavernous nerve stimulation in a time and dose-dependent manner. This improvement in erectile function was associated with a reduction in apoptotic indices and preservation of smooth muscle and endothelial cell expression as measured by immunohistochemistry. There was significant degeneration of neural cell bodies. Of note, the neural regeneration marker GAP43 was unchanged between the control and treatment arms. Based on these data, the mode of action of short-term administration of sildenafil appears to be mediated via protection of the penile vascular smooth muscle and endothelium and not through neuroprotection. Future studies are warranted to delineate the mechanism by which sildenafil preserves the penile vascular bed.

Davila and colleagues (Abstract 1061) also used a similar animal model of cavernous nerve injury to investigate the mechanisms of penile erectile dysfunction after injury. In this study, the cavernous nerve was resected bilaterally and the penis was stained for determination of smooth muscle to collagen ration, collagen III/I ratio, and TUNEL assay for apoptosis. Forty-five days after cavernous nerve transection, the media of the penile dorsal artery showed a 70% decrease in smooth muscle to collagen ratio, a 30% decrease in smooth muscle and collagen III/I ratio, and a 2 fold increase in apoptosis. These data collaborate the clinical findings of arterial dysfunction post-radical prostatectomy.

In summary, the basic science ED session highlighted important signaling pathways that are involved in the pathophysiology of female sexual dysfunction and ED-associated with diabetes and post-cavernous nerve injury. Importantly, these molecular mechanisms may be explored in order to develop new pharmacological agents with fewer side-effects as well as gene therapies that target disease-specific genetic approaches for the treatment of ED.