ArticleLiterature Review

Gene Therapy for Erectile Dysfunction: Fact or Fiction?

Authors:
  • Istinye University Faculty of Medicine Liv Hospital Ulus, Istanbul, Turkey
  • Tulane University Medical Center
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Abstract

Erectile dysfunction (ED) is a major health problem that seriously affects the quality of life of patients and their partners. Although all three selective phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the majority of ED cases, PDE5-I therapy is less efficacious in some hard-to-treat populations (diabetics, men after radical prostatectomy), prompting the development of new approaches, including gene therapy strategies for ED. Gene therapy approaches are discussed in terms of the possible role of gene therapy for the treatment of ED, potential targets for gene transfer, vectors to carry targeted genes, and gene strategies for ED in certain disease states, such as diabetes, ageing, arterial and venogenic insufficiency, and cavernous nerve injury. The penis is a convenient tissue target for gene therapy because of its external location and accessibility, the ubiquity of endothelial-lined spaces, and low level of blood flow, especially in the flaccid state. Gene therapy approaches have focused on a number of signaling pathways that are crucial for penile erection, such as nitric oxide/cyclic guanosine monophosphate, RhoA/Rho-kinase, growth factors, ion channels, peptides, and control of oxidative stress. The need for effective ED therapies in difficult-to-treat patients has encouraged investigators to seek novel modalities for the treatment of ED. Recent preclinical and clinical trials have demonstrated that gene therapy strategies may be feasible for these purposes.

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... In other words, therapeutic nucleic acids [TNAs; plasmid DNA (pDNA) or short interfering RNA (siRNA)] need to be shuttled and successfully transferred into the defective cells by gene carriers (vectors) (Tang et al., 2016). Fig. 6.1 schematically shows the principles of gene therapy showing that therapeutic genes of interest or growth factors that influence cellular function can be placed in viral or nonviral vectors that enter a targeted cell to significantly alter its function (Kendirci et al., 2006). Vector-assisted DNA/gene delivery systems can be classified into two types based on their origin: biological (viral DNA delivery systems) and chemical (nonviral delivery systems) (Pushpendra et al., 2012). ...
... Adapted from Kendirci, M., Teloken, P., Champion, H., Hellstrom, W. and Bivalacqua, T. (2006). Gene therapy for erectile dysfunction: fact or fiction? ...
... Gene therapy holds great promise for the prevention or treatment of certain diseases and genetic disorders by delivering therapeutic nucleic acids into the defective cells, thereby adjusting and controlling the corresponding cellular processes and responses [393][394][395] ( Fig. 9). The delivery of therapeutic nucleic acids into cells is one of the major hurdles for the successful gene therapy [396,397]. ...
... Therapeutic genes of interest or growth factors that influence cellular function can be placed in viral or nonviral vectors that enter a targeted cell to significantly alter its function. [395], Copyright 2006. Reproduced with permission from Elsevier Ltd. poly(beta-amino esters), helical polypeptides and cationic aliphatic polyesters. ...
... Leonore Tiefer, for example, has traced how endocrinology quietly replaced vascular biology as the presumed explanatory paradigm for female sexual dysfunction, illustrating well how 'the effort to match up some drug with FSD moved freely among symptoms and labels' (Tiefer 2006b: 439). Treatments for sexual dysfunctions in ageing men have also seen shifting, and more precise justifications, as erectile function and libido become tied to different physiological anchors variously conceptualised as vascular or hormonal (Marshall 2007, Watkins 2008) and increasingly, genetic (Kendirci et al. 2006). All of these demonstrate the varying ways in which sexual dysfunctions and the sexual bodies that suffer from them are materialised and accorded facticity and explanatory power. ...
... Active sexuality and older men and women 219 Although preclinical studies have highlighted the application of local gene therapy as a viable treatment option for ED in diverse pathologic conditions including diabetes, ageing, hypercholesterolaemia, and cavernous nerve injury, this therapeutic approach still requires more clinical studies in humans (Kendirci et al. 2006: 1218, emphasis added) What is remarkable here is not the extension of gene therapy into the treatment of sexual performance problems – that has been on the horizon for at least a decade – but the explicit inclusion of 'ageing' itself as a generalisable 'pathologic condition'. As sexual medicine and biogerontology join forces, the remarkable disassembling and reconstitution of ageing, sexualised bodies that has occurred over the last 150 years seems headed for some new frontiers. ...
Article
While historically sex has been seen primarily as the prerogative of the young, more recently, the emphasis has been on the maintenance of active sexuality as a marker of successful ageing. A new cultural consensus appears to have emerged which not only emphasises the importance of continued sexual activity across the lifespan, but links sexual function with overall health and encourages increased self-surveillance of, and medical attention to, late-life sexuality. Drawing on historical accounts, clinical research, popular science reporting and health promotion literatures, I explore several key shifts in models of sexual ageing, culminating in the contemporary model of gender, sexuality and ageing that has made ageing populations a key market for biotechnologies aimed at enhancing sexual function. Two central concepts frame my analysis: 'virility surveillance', where age-related changes in sexual function are taken as indicative of decline, and the 'pharmaceutical imagination', where sexual lifecourses are reconstructed as drug effects revise standards of sexual function. After consideration of how narratives emerging from qualitative research with older adults challenge the narrow depiction of sexual functionality promoted by pharmaculture, conclusions call for continued critical inquiry into the biomedical construction of sex and age.
... Subsequently, hyperpolarization due to K þ efflux and a decrease in intracellular Ca 2þ levels lead to vasodilation. Galcanezumab targets CGRP and might impede this vasodilatory response, which occurs in response to sexual stimuli, leading to erectile dysfunction (25)(26)(27). ...
Article
Full-text available
Background Recently, antimigraine drugs targeting the calcitonin gene-related peptide pathway have been approved for clinical use as preventive migraine medication. Case report We present a case of a 54-year-old male migraine patient, who reported erectile dysfunction as a possible side effect of treatment with galcanezumab, a monoclonal antibody targeting calcitonin gene-related peptide. His potency recovered after treatment discontinuation. Discussion As calcitonin gene-related peptide is involved in mammalian penile erection, erectile dysfunction is a conceivable side effect associated with calcitonin gene-related peptide inhibition. Postmarketing surveillance will elucidate the actual incidence of erectile dysfunction in patients using these new antimigraine drugs, and determine whether a causal relationship between calcitonin gene-related peptide inhibition and erectile dysfunction exists. This would be relevant not only because of the direct sexual consequences of erectile dysfunction, but also considering the potential cardiovascular consequences of calcitonin gene-related peptide (receptor) blockade and the association of both migraine and erectile dysfunction with cardiovascular disease. Conclusion Erectile dysfunction might be an overlooked, but reversible side effect in male migraine patients using monoclonal antibodies that inhibit the calcitonin gene-related peptide pathway, including galcanezumab. This paper may raise clinical awareness and suggest that this potential side effect needs to be studied further.
... The penis is a suitable tissue target for gene therapy because of its external location and accessibility, the ubiquity of endothelial-lined spaced and low level of blood flow, especially in the flaccid state. Gene therapy approaches have focused on a number of signaling pathways, which are crucial for penile erection, including NO, cGMP, Rho-kinase, growth factors, ion channels, peptides and factors controlling oxidative stress [127, 128]. Local gene therapy, as shown in preclinical trials, may be considered a valid treatment option for ED in diverse pathological conditions, such as cavernous nerve injury [129]. ...
Article
Erection is a neurovascular event characterized by the tumescence of the cavernous bodies that relies upon integration of neural and humoral mechanisms requiring the participation of autonomic and somatic nerves and the integration of numerous spinal and supraspinal sites. Erectile dysfunction (ED) is a highly prevalent problem increasing with age, as well as the major men's sexual concern. Significant advances in the pharmacological treatment of ED have occurred in recent years, most notably after the introduction of sildenafil, the first oral selective phosphodiesterase type 5 inhibitor. Nevertheless, many other oral, local and surgical treatments are available and their efficacy and safety depend on the specific cases. This review provides a comprehensive description of both currently available neurogenic ED treatments and most promising future therapies, including assigned patents.
... Therefore, the acceleration of nerve repair is important in not only the context of regaining function as soon as possible; moreover, it might dramatically increase the likelihood that regeneration will occur in time to reinnervate an intact corpus cavernosum, a prerequisite for functional recovery. Preclinical and clinical trials have demonstrated that gene therapy strategies may be feasible for these purposes [17]. Recent findings have shown that GDNF and related compounds are neuroprotective after cavernous nerve injury when administered as a penile injection [18] or in a viral vector injected at the site of nerve injury [19] . ...
Article
Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats. Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves). The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p=0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls. These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.
... The NO/cyclic GMP pathway has been demonstrated to be the principal mediator of cavernous smooth relaxation and penile erection. 13,14,74 Recently, both arginase isoforms have been shown to exists in human corpus cavernosum, and the inhibition of this enzyme resulted in the facilitation of corporal smooth muscle relaxation. 75,76 It has been reported that basal NO production from the endothelium regulates intrinsic cavernous tone and endogenous arginase activity in the endothelium modulates tone by inhibiting NO production, presumably through competition with eNOS for the common substrate L-arginine 12 (Fig. 5). ...
Article
Evidence indicates that nitric oxide (NO) deficiency contributes to micturition disorders, especially in the afferent pathway and erectile dysfunction (ED). Two possible causes of NO deficiency are substrate (L-arginine) limitation and increased levels of endogenous inhibitors of NO synthase (particularly asymmetric dimethylarginine: ADMA) in plasma and tissues. Elevated tissues of ADMA and N(G)-monomethyl-L-arginine (L-NMMA) have been reported to be associated with impaired NO-mediated urethral, trigonal and cavernosal relaxations by pelvic ischemia. Also, plasma ADMA may help to identify underlying cardiovascular disease in men with ED. Decreased l-arginine availability to NO synthase is due to the shunting of L-arginine into other pathways such as arginase. Interaction between NO synthase and arginase has been reported to be involved in NO-mediated urethral and prostatic relaxations. Also, increased arginase activity in cavernosal tissues likely contributes to the ED that accompanies diabetes mellitus and aging. Therefore, arginase inhibition has been reported to enhance the NO-dependent physiological process for erectile function.
... The incidence of ED increases with age, coronary artery disease, peripheral vascular disease, smoking, dyslipidemia, higher BMI, diabetes mellitus, and after radical prostatectomy. Oral therapy with PDE5 inhibitors are only partially effective in certain ED populations, including men with diabetes and those treated for prostate cancer (including prostatectomy and radiation therapy)234. Approximately 50% of diabetic men [5,6,1] NIH Public Access ...
Article
The role of sonic hedgehog (SHH) in maintaining corpora cavernosal morphology in the adult penis has been established; however, the mechanism of how SHH itself is regulated remains unclear. Since decreased SHH protein is a cause of smooth muscle apoptosis and erectile dysfunction (ED) in the penis, and SHH treatment can suppress cavernous nerve (CN) injury-induced apoptosis, the question of how SHH signaling is regulated is significant. It is likely that neural input is involved in this process since two models of neuropathy-induced ED exhibit decreased SHH protein and increased apoptosis in the penis. We propose the hypothesis that SHH abundance in the corpora cavernosa is regulated by SHH signaling in the pelvic ganglia, neural activity, or neural transport of a trophic factor from the pelvic ganglia to the corpora. We have examined each of these potential mechanisms. SHH inhibition in the penis shows a 12-fold increase in smooth muscle apoptosis. SHH inhibition in the pelvic ganglia causes significantly increased apoptosis (1.3-fold) and decreased SHH protein (1.1-fold) in the corpora cavernosa. SHH protein is not transported by the CN. Colchicine treatment of the CN resulted in significantly increased smooth muscle apoptosis (1.2-fold) and decreased SHH protein (1.3-fold) in the penis. Lidocaine treatment of the CN caused a similar increase in apoptosis (1.6-fold) and decrease in SHH protein (1.3-fold) in the penis. These results show that neural activity and a trophic factor from the pelvic ganglia/CN are necessary to regulate SHH protein and smooth muscle abundance in the penis.
... Another potential benefit of gene therapy for ED is the directed correction of any aberrant biochemical pathway that manifests as penile vascular dysfunction. A number of deviant biochemical pathways are acknowledged to lead to ED, and as long as a therapeutic gene exists that can enhance or replace deficient functions it is theoretically possible to introduce the reparative gene [13]. Currently, gene therapy for ED has emphasized the NO/ cGMP/protein kinase G (PKG) pathway, reflecting the principal role of NO bioavailability in the achievement and maintenance of erections [14,15]. ...
Article
Erectile dysfunction (ED) commonly results from endothelial dysfunction of the systemic vasculature. Although phosphodiesterase type 5 (PDE-5) inhibitors are effective at treating most cases of ED, they must be taken routinely and are ineffectual for a meaningful number of men. In recent years gene and stem cell-based therapies targeted at the penile endothelium have been gaining momentum in preclinical studies. These early studies reveal that gene and stem cell-based therapies may be both enduring and efficacious, and may eventually lead to a cure for ED. The following review will highlight our current understanding of endothelial-specific gene and stem cell-based therapies performed to date in a number of experimental animal models.
... A number of angiogenic growth factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), have been shown to be present in corporal tissue and can also act as protective factors in the setting of any vascular injury, such as ED [14][15][16][17]. The penis is a convenient tissue target for gene therapy because of its external location and accessibility, the ubiquity of endothelial lined spaces, and low level of blood flow, especially in the flaccid state [18]. This review will focus on the development of growth factors in hypercholesterolemic models of ED, the use of various growth factors being investigated for ED therapy, their routes of delivery, and the results in animal studies. ...
Article
The past decade has seen an explosion of new information on the physiology of penile erection, and pathophysiology of erectile dysfunction (ED). Hypercholesterolemia is a chronic condition that can lead to degeneration in the vasculature bed and can result in ED if the penile vasculature is involved. Angiogenesis is the growth of new blood vessels from preexisting vasculature. Therapeutic angiogenesis seeks to harness the mechanisms of vascular growth to treat disorders of inadequate tissue perfusion, such as coronary artery disease and ED. There have been tremendous changes in the field of therapeutic angiogenesis over the past decade, and there is much promise for the future. Initial preclinical work with cytokine growth factor delivery resulted in a great deal of enthusiasm for the treatment of ischemic heart and/or peripheral vascular disease, though clinical studies have not achieved similar success. With an increased understanding of the complex mechanisms involved in angiogenesis, novel therapies which target multiple different angiogenic pathways are also being developed and tested. The penis is a convenient tissue target for gene therapy because of its external location and accessibility, the ubiquity of endothelial lined spaces, and low level of blood flow, especially in the flaccid state. Therapeutic angiogenesis is an exciting field that continues to evolve. This review will focus on the development of growth factors for hypercholesterolemic ED, the use of various growth factors for ED therapy, their routes of delivery, and the results in animal studies.
... however, for use in humans, the product must be proven safe and practical to pass the muster of the US Food and Drug Administration (FDA) as well as the target population. Detailed reviews of these varied gene therapy approaches for ED were published recently [3] [5] [16] [17]. Our rationale for ion channel gene therapy that led to the first human trial of hMaxi-K for ED is based on the unique ultrastructural features of the smooth muscle of the genitourinary system and the role of ion channel function in the cavernous bodies. ...
Article
The potential clinical advantages of a gene transfer therapy-based approach to treatment of genitourinary smooth muscle-based disorders are several: potential single therapy for restoration of normal bladder or erectile function; eliminating the need for daily medication; use in combination with other therapies to reduce dose requirements and side effects; and development of mechanism-based, patient-specific treatment approaches. With the safe administration of hMax-K to men with erectile dysfunction in the first human phase 1 trial and the initiation of the phase 1 trial of hMaxi-K for patients who have detrusor overactivity, we have entered an exciting new era in the development of safe enduring therapies for genitourinary disorders.
... Previous work has shown that 8 wk of streptozotocin (STZ)-induced diabetes in Fischer-344 (F-344) rats produced demonstrable changes in erectile capacity[16]. The importance of the Slo gene in regulating corporal SM tone and its restorative effects after gene transfer in aged and STZ-diabetic animals has been recently established[6,15,17], as well as its potential use in human gene therapy[18]. The α-subunit of the Maxi-K channel is encoded by the Slo gene, which can undergo alternative splicing to generate several isoforms[19]. ...
Article
Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.
... The application of gene therapy for ED represents an exciting new field. Although preclinical studies have highlighted the application of local gene therapy as a viable treatment option for ED in diverse pathologic conditions including diabetes, ageing, hypercholesterolaemia and cavernous nerve injury, this therapeutic approach still requires more clinical studies in humans (Muammer, Patrick, Hunter, Wayne, & Trinity, 2006). ...
Article
Erectile dysfunction (ED) is defined as the inability of the male to attain and maintain erection of penis sufficient to permit satisfactory sexual intercourse. Prevalence of impotence in diabetic men is ≥50%. The pathophysiology of diabetes-induced erectile dysfunction (DIED) is multifactorial and no single etiology is at the forefront. The proposed mechanisms of erectile dysfunction in diabetic patients includes elevated advanced glycation end-products, increased levels of oxygen free radicals, impaired nitric oxide synthesis, increased endothelin B receptor binding sites and up-regulated RhoA/Rho-kinase pathway, neuropathic damage and impaired cyclic guanosine monophosphate (cGMP)-dependent protein kinase-1. The treatment of DIED is multimodal. Treatment of the underlying hyperglycemia and comorbidities is of utmost importance to prevent or halt the progression of disease. Oral medications are considered as the first line therapy for management of DIED. If oral agents cannot be used or have insufficient efficacy despite appropriate dosing and education, second-line treatments should be addressed. When there is lack of efficacy or when there is dissatisfaction with other modalities, penile prostheses are often the best alternative for ED and are considered as the third line therapy for DIED. Future strategies in the evolution of the treatment of DIED are aimed at correcting or treating the underlying mechanisms of DIED.
... The maxi-K channel (Ca2+sensitive potassium channel or KCa channel) is the most important in corporal smooth muscle physiology. Alterations in the function and regulation of the maxi-K channel are likely to be important in the pathogenesis of organic ED [42]. Activation of the maxi-K channel in corporal smooth muscle represents an important and attractive mechanism for control of corporal smooth muscle function. ...
Article
Erectile dysfunction (ED) is a neurovascular phenomenon modulated by hormonal, local biochemical, and biomechanical/structural factors of the penis. The success of the orally active phosphodiesterase inhibitors for the treatment of ED has boosted research activities into the physiology of the erectile mechanism. Peripheral intracellular signal transduction in the penis as well as central brain and spinal cord pathways controlling penile erection have been investigated and are now better understood. The results of this ongoing research have provided the basis for the development and introduction of several novel therapeutic modalities into the management of ED. Many novel pharmacotherapeutic approaches under development including the use of melanocortins and Rho-kinase inhibitors as well as the introduction of gene therapy and tissue engineering have demonstrated efficacy in animal as well as early human trials. This review describes the major new and evolving pharmacological advances in the field of oral pharmacotherapy for the treatment of male ED.
... This damage leads to a temporary blockade of the signal function of the nerves (neurapraxia) and may also cause degeneration of nerve fibers and structural and functional alterations of penile erectile tissue [3,4]. The rat model of cavernous nerve crush injury is widely used to simulate the neurapraxia associated with NSRP, and various approaches, such as pharmacotherapy, gene therapy, or stem cell therapy, have been investigated in this model to overcome neurapraxia, reduce nerve damage, and improve functional autonomic nerve recovery [5][6][7]. Similar models are also used for somatosensory nerves, and crush of the rat pudendal nerve may be used for the study of the external urethral sphincter [8,9]. ...
Article
Background: Information on autonomic neurapraxia in female urogenital surgery is scarce, and a model to study it is not available. Objective: To develop a model to study the impact of autonomic neurapraxia on bladder function in female rats, as well as to assess the effects of corticosteroid therapy on the recovery of bladder function in this model. Design, setting, and participants: Female Sprague-Dawley rats were subjected to bilateral pelvic nerve crush (PNC) and perioperatively treated with betamethasone or vehicle. Bladder function and morphology of bladder tissue were evaluated and compared with sham-operated rats. Outcome measurements and statistical analysis: Western blot, immunohistochemistry, organ bath experiments, and cystometry. Results and limitations: Sham-operated rats exhibited regular micturitions without nonvoiding contractions (NVCs). Crush of all nerve branches of the pelvic plexus or PNC resulted in overflow incontinence and/or NVCs. Betamethasone treatment improved recovery of regular micturitions (87.5% compared with 27% for vehicle; p<0.05), reduced lowest bladder pressure (8 ± 2 cm H(2)O compared with 21 ± 5 cm H(2)O for vehicle; p<0.05), and reduced the amplitude of NVCs but had no effect on NVC frequency in PNC rats. Compared with vehicle, betamethasone-treated PNC rats had less CD68 (a macrophage marker) in the pelvic plexus and bladder tissue. Isolated bladder from betamethasone-treated PNC rats exhibited better nerve-induced contractions, contained more cholinergic and sensory nerves, and expressed lower amounts of collagen III than bladder tissue from vehicle-treated rats. Conclusions: PNC causes autonomic neurapraxia and functional and morphologic changes of isolated bladder tissue that can be recorded as bladder dysfunction during awake cystometry in female rats. Perioperative systemic betamethasone treatment reduced macrophage contents of the pelvic plexus and bladder, partially counteracted changes in the bladder tissue, and had protective effects on micturition function.
... -Gene therapy ensures the lasting production of a stable quantity protein, -Gene therapy localizes transgene-expression, which leads to avoiding the unwanted effects caused by the systematic presence of a protein. (Kendirci et al., 2006). ...
Article
Gene therapy is a promising new technique for treating many serious incurable diseases, such as cancer and genetic disorders. The main problem limiting the application of this strategy in vivo is the difficulty of transporting large, fragile and negatively charged molecules like DNA into the nucleus of the cell without degradation. The key to success of gene therapy is to create safe and efficient gene delivery vehicles. Ideally, the vehicle must be able to remain in the bloodstream for a long time and avoid uptake by the mononuclear phagocyte system, in order to ensure its arrival at the desired targets. Moreover, this carrier must also be able to transport the DNA efficiently into the cell cytoplasm, avoiding lysosomal degradation. Viral vehicles are the most commonly used carriers for delivering DNA and have long been used for their high efficiency. However, these vehicles can trigger dangerous immunological responses. Scientists need to find safer and cheaper alternatives. Consequently, the non-viral carriers are being prepared and developed until techniques for encapsulating DNA can be found. This review highlights gene therapy as a new promising technique used to treat many incurable diseases and the different strategies used to transfer DNA, taking into account that introducing DNA into the cell nucleus without degradation is essential for the success of this therapeutic technique.
... 9,10 All 3 isoforms have been investigated as potential gene therapy targets to modulate the erectile response. 10 Of the 3 isoforms iNOS does not directly intervene with physiological penile erection but it is Ca 2ϩ independent and can produce NO in a sustained manner at a high concentration. 11 Thus, iNOS over expression in the corpus cavernosum of the penis would potentially reverse ED. ...
Article
Purpose: Promoter targeted saRNAs mediate sequence specific up-regulation of gene expression. We explored the therapeutic effect of RNA activation mediated iNOS gene activation on improving erectile function in a rat model of diabetes mellitus. Materials and methods: An optimal saRNA sequence specific for iNOS promoter was cloned into an adenoviral vector, resulting in AdU6/shiNOS and AdU6/shControl. The corresponding viruses were used to transduce cultured rat cavernous smooth muscle cells. Streptozotocin induced diabetes models were established in rats and used to test the effects of intracavernous delivery of iNOS saRNA viruses on erectile function. iNOS expression in the cavernous smooth muscle cells or penile tissue of treated rats was assessed by reverse transcriptase-polymerase chain reaction and Western blot. Cyclic guanosine monophosphate was analyzed by enzyme-linked immunosorbent assay. Intracavernous pressure in response to cavernous nerve stimulation was measured using a data acquisition system on post-injection days 1, 3, 5, 7, 10 and 14. Results: Adenovirus mediated expression of iNOS saRNA caused sustained up-regulation of iNOS in cavernous smooth muscle cells. Intracavernous injection of AdU6/shiNOS activated iNOS expression in vivo and significantly increased peak intracavernous pressure in streptozotocin induced diabetic rats via nitric oxide/intracellular cyclic guanosine monophosphate activation. Conclusions: Results show that saRNA mediated iNOS over expression in the penis can restore erectile function in streptozocin diabetic rats via the nitric oxide-cyclic guanosine monophosphate pathway.
Article
Full-text available
While erectile dysfunction (ED) is highly prevalent worldwide, unrevealed cavernous smooth muscles (CSM) defects can confound the diagnosis of vascular ED and lead to failure of treatments. Currently, the first-line oral treatment for ED is phosphodiesterase type 5 inhibitors (PDE5Is). Patients with diabetes mellitus (DM), those who have undergone a radical prostatectomy (RP), and the elderly population are difficult to treat by the PDE5Is; unrevealed CSM defects can result in corporo veno-occlusive dysfunction (CVOD); and penile veno-ligation surgeries are currently abandoned due to high failure rates. It has been found that gene and stem cell therapies, among others, reduce cavernous tissue apoptosis and fibrosis and can specifically target CSM defects such as the nitric oxide (NO)-mediated signaling pathway, Rho–ROCK system, and transformation growth factor (TGF)-β1/angiotensin II (Ang II) pathway, in several laboratory animals. Current data clarify the need of diagnostic techniques that can provide an initial assessment of CSM. This assessment should be essential before giving a diagnosis of vascular ED and before applying several tests searching for a specific CSM defect to guide the specific therapy. Moreover, while patients with corporal fibrosis would fail the current medical therapies, these patients can benefit from the stem cell-based therapies that induce the internal mechanisms of tissue repair. However, penile elastography can determine the stiffness of tissues and corpus cavernosum electromyography (CC-EMG) can assess the integrated activity of CSM bulk, further refinements are required for these techniques before being considered in the evaluation of patients with ED. In conclusion, on the basis of the current scientific research, it may be possible to formulate new therapies and achieve the appropriate selection of patients who can benefit from these therapies.
Article
The treatment of erectile dysfunction (ED) has been revolutionized during the last 2 decades with several treatment options now available. Most of these treatments are associated with high efficacy rates and favourable safety profiles. A MEDLINE search was undertaken to evaluate all currently available data on treatment modalities for ED. Phosphodiesterase type 5 (PDE5) inhibitors (sildenafil, tadalafil, vardenafil) are currently the first-choice treatment option for ED by most physicians and patients. In addition, several new PDE5 inhibitors are candidates to enter the market in forthcoming years (avanafil, udenafil, SLx-2101, mirodenafil [SK3530]). However, obvious pharmacokinetic differences that result in a faster time-to-onset, longer half-life time and better safety profile are required for these drugs to be considered a truly better option for patients. Other molecules in development include selective dopamine, glutamate, serotonin and melanocortin receptor agonists, guanylate cyclase activators, rho-kinase inhibitors and hexarelin analogues, while the first trials on gene therapy and tissue engineering for reconstruction of corporal tissue are under way. Patients must be aware of all treatment options since no ideal treatment exists. It is expected that the availability of drugs with different mechanisms of action will allow physicians to offer more personalized medicine to their patients in the future. The development and adaptation of a patient-centred care model in sexual medicine will increase the efficacy and safety of current and future treatments.
Article
Insulin-like growth factor-1 (IGF-1) is one of the growth factors that have a wide range of biologic effects. We have confirmed that gene transfer of IGF-1 to the penis could improve erectile capacity. However, there are some limitations in gene therapies, such as toxicity or a risk of insertional mutagenesis. Protein treatment may be another choice for decreasing these risks. To investigate whether intracavernosal injection of IGF-1 protein can restore erectile function in the aging rat. Erectile responses, morphological changes, and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathways-related marker were determined. Ten young (4 months) and 30 old (24 months) Sprague-Dawley male rats were enrolled in this study. The old rats were divided into three groups: vehicle-only (N = 10), IGF-1 1 microg/kg (N = 10) and IGF-1 10 microg/kg treatment group (N = 10). After 4 and 8 weeks of single IGF-1 injection treatment, intracavernous pressure (ICP) responses with electrical stimulation to the cavernous nerve were evaluated. The percent of smooth muscle in corpus cavernosum tissue, the expression of mRNA and protein of endothelial nitric oxide synthase (eNOS) were also evaluated. The activity of nitric oxide synthase (NOS) and concentration of guanosine 3',5'-cyclic-monophosphate (cGMP) that act upon the major NO-cGMP signaling pathways in penile tissue were also analyzed. After IGF-1 treatment, the ICP responses was significantly increased as the young control group in both the IGF-1 1 microg/kg and the IGF-1 10 microg/kg group compared with the vehicle-only group at 4 and 8 weeks (P < 0.05). Masson's trichrom staining showed the percentage of cavernosal smooth muscle was increased in IGF-1 treatment group. IGF-1 increased e-NOS expression. NOS activities and cGMP concentrations were also significantly increased in IGF-1 treatment rats. IGF-1 improved erectile function in aged rats via restoration the integrity of smooth muscle of corpus cavernosum and modulation of NO-cGMP pathways.
Article
Oxidative stress dependent-decrease in nitric oxide (NO) bioavailability plays an integral role in hypercholesterolemia-induced erectile dysfunction (ED). Resveratrol has been demonstrated to exert beneficial effects against oxidative stress and improve NO bioavailability.
Article
Erectile dysfunction (ED) is a major epidemiological problem that affects many patients' quality of life. Scientific advancements during the last 20 years have brought about a revolution in the understanding of the physiology of erection, which has changed the way ED is understood and treated.
Article
Sexual dysfunction, a common disease among the general population, strongly compromises the quality of life of those affected and their partners. In the past it was mainly attributed to psychological causes, but recent progress in pathophysiology has indicated that endothelial dysfunction also appears to play a major role. Pharmacological and non-pharmacological therapies for erectile dysfunction (ED), their indications, contra-indications, adverse effects and literature evidence are reviewed in this paper. There is an increased awareness regarding the close association between cardiovascular disease and ED: both conditions share common risk factors such as diabetes mellitus, hypertension, smoking, hyperlipidemia, a sedentary lifestyle and cardiological therapies, while certain anti-hypertensive agents are also associated with ED. Additionally, since phosphodiesterase-5 inhibitors, the most common drugs used to treat ED, can affect blood pressure and interact with anti-ischemic drugs, ED is an emerging topic in internal medicine and for hypertensiologists in particular. If sexual dysfunction in females is not clearly understood and poorly treated, a sequential therapeutical approach is proposed in males.
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As the maintenance of a satisfactory quality-of-life is recognized as the principle concern for men and sexual dysfunction is an independent determinant of a worse general health-related quality-of-life, practicing urologists have focused on the understanding of the pathophysiology of erectile dysfunction (ED) following radical prostatectomy (RP) with the concepts of instituting prophylactic measures for prevention and early recovery from ED. ED following RP is often the result of inadvertent injury to the cavernous nerves that course along the prostate capsule and innervates the corpora cavernosa of the penis. The introduction of the nerve-sparing procedures to preserve erectile function has been embraced globally and has made for greater acceptance. However, as unassisted nerve regeneration is a slow process, denervation-induced damage, including cavernosal fibrosis and cavernosal smooth muscle apoptosis frequently develops following RP. Pharmacological penile rehabilitation postoperatively with oral or intracavernosal vasoactive drugs is theorized to be of benefit in most patients, allowing for faster and more complete recovery of erectile function. The current belief is that penile rehabilitation programs with available measures maintains erectile tissue integrity and prevents corporal smooth muscle atrophy and collagen production. Studies on neuroprotection and neuroregeneration will help to preserve erectile function following RP.
Article
In recent years male sexual research has increasingly centered on molecular mechanisms operating from the central nervous system to peripheral end-organ levels involved in the penile erectile response. Major progress has been made in the field, and currently a whole host of neurotransmitters, chemical effectors, growth factors, second-messenger molecules, ions, intercellular proteins, and hormones have been characterized as components of the complex physiology of erectile function. Foremost among these mediators is nitric oxide (NO), which was initially characterized as a locally released physiologic mediator of the erectile response. Impaired formation and action of NO is closely associated with erectile dysfunction (ED), which may be caused by a variety of pathogenic factors. The impact of this knowledge has been substantial, leading to the development of several NO-based medical approaches for the treatment of ED. This review will focus on recent patents and current clinical trials involving innovative pharmacological and gene therapies in the field of male ED, particularly targeting the NO/intracellular cyclic GMP pathway, which still represents the most promising therapeutic approach to treat patients with ED.
Article
Erectile dysfunction (ED) is one of the most common diseases in male urology that greatly affects the quality of life in senior people. Relaxation of corpus cavernosum smooth muscle is the key to penile erection. To explore effects of human telomerase reverse transcriptase (hTERT) gene transfection on biological behaviors of human penile smooth muscle cells. Human penile smooth muscle cells were grown in primary culture. A fluorescent eukaryotic expression vector, hTERT-internal ribosome entry site 2 (IRES2)-enhanced green fluorescent protein (EGFP), was constructed and transfected into human penile smooth muscle cells using Lipofectin reagent. The telomerase activity, mitotic index, cell apoptosis, and cell growth curves of transfected smooth muscle cells were determined; the potential formation of malignant phenotypes in these transfected cells was investigated. Telomerase activity, mitotic index, and cell growth of hTERT-transfected cells were significantly higher than those of nontransfected cells and cells transfected with the empty EGFP vector, while apoptosis rates were the lowest in hTERT-transfected cells. No changes in cell morphology, chromosome number, and tumorigenicity were observed between hTERT-transfected cells and control cells. In this study, for the first time, the hTERT gene was transfected into human penile smooth muscle cells, and the gene increased telomerase activity in cells, reduced cell apoptosis, and slowed down cell aging. We believe that this finding is of potential clinical value in the prevention and treatment of organic ED.
Article
The management of erectile dysfunction (ED) has been revolutionized by the discovery of phosphodiesterase 5 (PDE5) inhibitors, which have been commercially available for more than a decade and are the first-line therapeutic option for men with ED. Sildenafil, vardenafil and tadalafil were approved by the European Medicine Agency and the US FDA for the treatment of ED on the back of their high efficacy rates and favorable safety profiles. However, despite the fact that more than 50 million patients with ED worldwide have been successfully treated with one of these PDE5 inhibitors, some men--most notably those with severe neurologic damage, diabetes mellitus or severe vascular disease--are resistant to the currently available drugs and require more-invasive treatments, such as intracavernosal injection therapy. Partly as a consequence of this, research into alternative therapeutic approaches continues, including the development of new PDE5 inhibitors, centrally acting pharmaceutical agents, and application of molecular technologies such as gene therapy and stem cell therapy.
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Prostate cancer is the most common cancer among men, representing approximately 25% of all new cancer diagnoses in the USA. For clinically localized prostate cancer, the gold standard for therapy remains radical prostatectomy. One of the main adverse effects of this procedure is erectile dysfunction, which can have a significant impact on the patient's quality of life. There are several mechanisms of erectile dysfunction postprostatectomy, including arteriogenic, venogenic and neurogenic types, as well as the potentially heightened risk of postprostatectomy patients to develop Peyronie's disease. The purpose of this review is to explain the various treatment options available, including phosphodiesterase type 5 inhibitors, intracavernosal injections, intraurethral alprostadil suppositories, vacuum erection devices, and penile prostheses. The role of these therapies in an erectile-dysfunction-treatment function, as well as in penile rehabilitation, will be discussed. Finally, a review of research on novel therapies will also be presented. A comprehensive literature review was performed using the PubMed database. Articles were chosen based on topical relevance and assessed for methodology and major findings. There are data to support the use of each of the therapeutic options in both treatment and rehabilitative roles. More study is needed, however, specifically in regard to penile rehabilitation, to confirm its benefits, as well as to determine optimal rehabilitation protocols.
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The endothelium has an important regulatory role in the maintenance of vascular homeostasis, vascular tone, blood flow, and in preserving a non-thrombogenic blood-tissue interface. Injury to the vascular wall with subsequent endothelial dysfunction alters these important regulatory functions leading to a state of abnormal endothelial function. In this paper, we review the pathophysiology of endothelial dysfunction and how this disorder is common to the development of erectile dysfunction and of pulmonary arterial hypertension. Current medical therapies for these two disorders are discussed followed by a review of the preclinical studies involving currently available strategies for gene and stem cell therapy and their potential for the clinical treatment of these two disorders of endothelial dysfunction.
Article
Introduction: The past 25 years of basic science research on erectile physiology has been devoted to investigating the pathogenesis of erectile dysfunction. Research has led to a better understanding of the biochemical factors and intracellular mechanisms responsible for corporal smooth muscle contraction and relaxation, as well as the influence of endothelial-derived relaxing factors. Aim: In this essay, we propose the use of gene transfer technology to study mechanisms of disease involved in penile vascular dysfunction. Methods: The development of methods to deliver therapeutic genes to the penis has kindled a keen interest in treating ED with gene- and cell-based therapies. Results: Gene therapy has delineated putative mechanisms of disease in animal models of erectile dysfunction. Conclusion: Investigation of animal models using gene therapy may ultimately lead to mechanism-based therapies for the treatment of erectile dysfunction.
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Erectile dysfunction is a common problem affecting sexual function in men. Approximately one in 10 men over the age of 40 is affected by this condition and the incidence is age related. Erectile dysfunction is a sentinel marker for several reversible conditions including peripheral and coronary vascular disease, hypertension and diabetes mellitus. Endothelial dysfunction is a common factor between the disease states. Concurrent conditions such as depression, late-onset hypogonadism, Peyronie's disease and lower urinary tract symptoms may significantly worsen erectile function, other sexual and relationship issues and penis dysmorphophobia. A focused physical examination and baseline laboratory investigations are mandatory. Management consists of initiating modifiable lifestyle changes, psychological and psychosexual/couples interventions and pharmacological and other interventions. In combination and with treatment of concurrent comorbid states, these interventions will often bring about successful resolution of symptoms and avoid the need for surgical interventions.
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Signal transduction pathways are involved in responses of the cells to different environmental cues and hence, the regulation of these pathways is highly significant. Proteins are the major players in the signaling pathways that are regulated at different stages during their span- from synthesis to degradation. Transcriptional and translational control of the protein synthesis, regulation by protein folding machinery, post-translational modifications, protein localization, protein-protein interactions, and regulated proteolysis- all these regulations work in concert to maintain the specificity and efficiency of the signaling networks. The need for a tight regulation is cemented by the fact that defects in signaling networks can cause severe cellular changes. This chapter is an overview of different modes of protein regulation in signal transduction and focuses on different regulators that function during signaling cascades. The chapter also throws light upon the manner in which covalent and non-covalent modifications affect the signaling mechanism and how irregularities in signaling networks result in deleterious effects.
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The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.
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RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.
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Previous reports have shown that retrovirus infection is inhibited in nonreplicating (stationary-phase [hereafter called stationary]) cells. Infection of stationary cells was shown to occur when the cells were allowed to replicate at times up to a week after infection, suggesting that an unintegrated retrovirus could persist in a form that was competent to integrate after release of the block to replication. However, those studies were complicated by the use of replication-competent virus, which can spread in the infected cells. We have used a replication-defective retrovirus vector to compare the efficiency of gene transfer in stationary and replicating rat embryo fibroblasts. In agreement with previous results, gene transfer was inhibited 100-fold in stationary versus replicating cells. In contrast to previously reported results, the block to infection could not be relieved by stimulating stationary cells to divide at times from 6 h to 10 days after infection. Thus, for successful retroviral infection, the infected cells must be replicating at the time of infection. These results have important implications for the use of retroviral vectors for gene transfer.
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Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L-arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L-citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS)
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We report the purification from pig brain of a factor supporting the survival of, and fibre outgrowth from, cultured embryonic chick sensory neurons. The purified factor migrates as one single band, mol. wt. 12 300, on gel electrophoresis in the presence of sodium dodecylsulphate (SDS) and is a basic molecule (pI greater than or equal to 10.1). Approximately 1 microgram factor was isolated from 1.5 kg brain. The final degree of purification was estimated to be 1.4 X 10(6)‐fold, and the specific activity 0.4 ng/ml/unit, which is similar to that of nerve growth factor (NGF) using the same assay system. This factor is the first neurotrophic factor to be purified since NGF, from which it is clearly distinguished because it has different antigenic and functional properties.
Article
Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and β-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-β-gal) and “gutless” AdV (AdV-CMV-PnNOS; AdV-CMV-β-gal) vectors, and injected into the penis of adult (β-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of β-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-β-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10⁶ viral particles (vp) of AdV-CMV-β-gal, and with 10⁷ vp β-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10⁷ vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.
Chapter
During the development of many parts of the vertebrate nervous system, more neurons are produced than are found in the adult, the quantitative adjustment of neuronal numbers taking place largely by their elimination (Jacobson, 1978). Evidence from transplantation and ablation experiments indicates that this process is not genetically pre-programmed, but can be modulated. If, for example, the eyeball of a chick embryo is removed early in development, most of the ciliary ganglion neurons which would have projected to it die (Landmesser and Pilar, 1974). Conversely, if an additional optic cup is grafted, more neurons than normal are found in the ciliary ganglia (Narayanan and Narayanan, 1978; Boydston and Sohal, 1979). These examples, together with many others (for review, see T. J. Cunningham, 1982) indicate that neuronal death is a general phenomenon under epigenetic control. However, little is known about the nature of the molecules (with one exception discussed below) and the mechanisms which are responsible for this phenomenon.
Article
Stimulation of adenylate cyclase appears to activate ATP-sensitive K+ channels in the basilar artery. We tested the hypothesis that calcitonin gene-related peptide (CGRP), which increases intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels, activates ATP-sensitive K+ channels and thereby causes vasodilatation. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to CGRP in vivo. We also examined responses of the artery to another vasoactive peptide, vasoactive intestinal peptide (VIP). Topical application of CGRP (10(-11) to 10(-8) M) increased diameter of the basilar artery. Responses of the basilar artery to CGRP were almost abolished by a CGRP, receptor antagonist, CGRP-(8-37). Vasodilatation in response to VIP was much smaller than that produced by CGRP. Dilator responses of the basilar artery to 10(-9) and 10(-8) M CGRP were inhibited by glibenclamide (10(-6) M), a selective inhibitor of ATP-sensitive K+ channels, by 69 +/- 19 and 41 +/- 9%, respectively. N(G)-nitro-L-arginine methyl ester (10(-5) M), an inhibitor of nitric oxide synthase, did not attenuate dilator response to 10(-8) M CGRP but inhibited responses to 10(-9) M CGRP by 34 +/- 12%. Indomethacin did not alter dilator responses to CGRP. These findings suggest that a minor component of CGRP-induced dilatation of the basilar artery is mediated by endothelium-derived relaxing factor. Vasodilatation in response to CGRP appears to be mediated primarily by direct activation of CGRP1 receptors on vascular muscle. Activation of ATP-sensitive K+ channels appears to be the major mechanism of dilatation of the basilar artery in response to CGRP in vivo.
Article
The Ca2+-sensitive K+ channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+ channel, and 2) to determine whether transfection of the maxi-K+ channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVβ/Lac Z DNA in 10- wk-old rats resulted in significant incorporation and expression of β- galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals (N = 12), intracavernous injection of naked pcDNA/hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5-10 mA) at both 1 mo (N = 5) and 2 mo (N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo (N = 2) and 4 mo (N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.
Article
Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the aging rat model, erectile dysfunction is accompanied by a reduction of penile smooth muscle compliance and, in very old animals, by a decrease in penile nitric oxide synthase (NOS), which is responsible for the synthesis of the mediator of penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by local induction or gene therapy can mitigate erectile dysfunction in the aged rat. A mix of iNOS (inducible NOS) inducers was continuously delivered to the penises of 5- ("adult"), 20- ("old"), and 30- ("very old") mo-old rats for 3-6 days, and the erectile response to electrical field stimulation of the cavernosal nerve was measured. The erectile dysfunction observed in old and very old rats as compared to adult animals was ameliorated by treatment with iNOS inducers. Penile iNOS was detectable in the penis of these rats by Western blot, NADPH diaphorase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indicating that the increased NOS levels were under physiological control. The iNOS cDNA was cloned from induced RPSMC mRNA and generated by reverse transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions from both the rat (RPiNOS) and human (HPiNOS) penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissues. RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal tissue by PCR, and its expression by RT-PCR and Western blots. These results open the way for the possible use of NOS isoforms in the management of erectile dysfunction.
Article
Erectile dysfunction (ED) is commonly experienced in men with diabetes mellitus. Vascular endothelial growth factor (VEGF) has been extensively documented for its pathogenic significance in different complications of diabetes. We hypothesized that expressions of VEGF, its receptors and its signaling pathway Akt may be drastically altered in diabetic penile tIssues and their alterations may modulate penile expression of the molecules that are believed to play a role in diabetic ED. Otsuka Long-Evans Fatty (OLETF) rats, a type II (non-insulin-dependent) diabetes mellitus, were used at the insulin-resistant stage of type II diabetes (20 weeks of age). We determined protein and mRNA expressions of VEGF, its receptors, Akt, nitric oxide synthase isoforms, and apoptosis-related molecules in the penis using immunohistochemistry, Western blotting, in situ hybridization, and real-time quantitative PCR analyses. The penile sections were also submitted to the Tdt-mediated dUTP nick end labeling assay for apoptosis. OLETF rats showed marked reductions in penile expression of VEGF, its two receptors and Akt. In OLETF rat penises, endothelial and neuronal nitric oxide synthase isoforms were expressed less abundantly. Furthermore, while anti-apoptotic markers, Bcl-2 and phosphorylated Bad, were down-regulated, pro-apoptotic markers, active caspase-3 and Bax, were up-regulated, resulting in the appearance of apoptotic cells in the penile tIssues of OLETF rats. The VEGF signaling system would work less well in diabetic penile tIssues as a result of the reduced expression, leading to diminished endothelial production of nitric oxide and apoptosis-related erectile tIssue damage. We propose that the abnormalities of the VEGF signaling system in the penis may play a role in the pathophysiology of diabetic ED.
Article
Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSα, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSß, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSα and β are expressed in the rat penis and are located in the nerves and whether the β form persists in the potent nNOS knock-out mouse (nNOS△△). A PnNOS antibody against the insert common to both PnNOSα and β detected the expected 155-kDa protein in PnNOSα-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSα mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSβ mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS△△ mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the β form. In conclusion, both PnNOSα and β are expressed in the rat penis at all ages and are located in the nerves. The β form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS△△ mouse occurs through transcription of the β mRNA, and this may explain the retention of erectile function when the expression of nNOSα is disrupted.
Article
This paper presents the evaluation of LPG (Liquefied petroleum gas) regulators for home use. For the evaluation, several properties of the regulators were experimentally analyzed, such as the operation of safety device, the adjusting and lock-up pressure, the adjusting spring and the diaphragm, with respect to the used time of the regulators. Experimental results showed that the initial operation performances of regulators were degraded with increase of the service time and also showed that the degradation of the performance and material property could become serious after about six-year-use of the regulators.
Article
The endothelium modulates vascular tone by producing vasodilator andvasoconstrictor substances. Of these, the best characterized and potentially most important are nitric oxide (NO•) and O2−•. These small molecules exhibit opposing effects on vascular tone and chemically react with each other in a fashion that negates their individual effects and leads to the production of potentially toxic substances, such as peroxynitrite (ONOO−). These dynamic interactions may likely have important implications, altering not only tissue perfusion but also contributing to the process of atherosclerosis. The precise O2−• source within vascular tissue remains to be determined. Recent work demonstrated that in endothelial cells as well as in vascular smooth muscle cells, a membrane-associated NAD(P)H-dependent oxidase represents the most significant O2−• source. Interestingly, this oxidase is activated upon stimulation with angiotensin II, suggesting that under all conditions of an activated circulating and/or local renin-angiotensin system endothelial dysfunction secondary to increased vascular O2−• production is expected.
Article
Objective To examine the effect of HbA1c, an isoform of glycosylated haemoglobin (GHb, a product of non-enzymatic reactions between elevated blood glucose and haemoglobin), on nitric oxide-mediated corpus cavernosal smooth muscle relaxation, and to categorize the mechanisms involved. Materials and methods Corpus cavernosal tissue from Wistar rats (300–350 g body weight) was prepared for the measurement of isometric tension. After equilibration in Krebs solution gassed with 95% O2/5% CO2 at 37 °C for 90 min, optimal resting tension was applied. Tissue was precontracted with 1 µmol/L noradrenaline (NAd) and either relaxed with incremental doses of acetylcholine (ACh) or sodium nitroprusside (SNP). After washout, strips were again precontracted with NAd and then incubated with pyrogallol (100 µmol/L), 100 µL of haemoglobin or 100 µL of GHb in the presence of either l-arginine (100 µmol/L), indomethacin (10 µmol/L), allopurinol (100 µmol/L), deferoxamine (100 µmol/L), catalase (600 IU/mL), or superoxide dismutase (SOD) (120 IU/mL) before ACh- or SNP-induced relaxation responses were repeated. Results Haemoglobin and GHb significantly impaired the relaxation of rat corpus cavernosum to ACh in a dose-dependent manner. l-arginine reversed the impairment caused by Hb, but not GHb. A donor of superoxide anions, pyrogallol, mimicked this impairment to ACh when added to control strips. Catalase, deferoxamine, indomethacin and allopurinol had no significant effect on the impaired relaxation response to ACh, whilst l-arginine partially reversed it. SOD completely reversed the GHb-induced impaired relaxation; GHb did not alter the relaxation response to SNP. Conclusion GHb significantly impairs endothelial NO-mediated corpus cavernosal relaxation in the rat, in vitro. This effect is caused partly by the generation of superoxide anions and the extracellular inactivation of NO.
Article
We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non-muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non-muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non-muscle. The active, GTP-bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho-kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G-protein-coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho-kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI-17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho-kinase pathway and plays only a minor, transient role in the G-protein-coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho-kinase pathway contributes to the tonic phase of agonist-induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis.
Article
OBJECTIVE To test the hypothesis that an intracavernosal injection with brain-derived neurotrophin factor (BDNF) and vascular endothelial growth factor (VEGF) can facilitate nerve regeneration and recovery of erectile function after cavernosal nerve injury.MATERIALS AND METHODS The study included 25 Sprague-Dawley rats; four had a sham operation, seven bilateral nerve crushing with no further intervention, and 14 bilateral nerve crushing with either an immediate (seven) or delayed for 1 month (seven) intracavernosal injection with BDNF+VEGF. Erectile function was assessed by cavernosal nerve electrostimulation at 3 months, and neural regeneration by NADPH-diaphorase staining and tyrosine hydroxylase (TH) staining of penile tissue and major pelvic ganglia (MPG).RESULTSAfter nerve crushing, the functional evaluation at 3 months showed a lower mean (sd) intracavernosal pressure (ICP) with cavernosal nerve stimulation, at 33.9 (15.3) cmH2O, than in the sham group, at 107.8 (18.1) cmH2O. With an immediate injection with BDNF+VEGF the ICP was significantly higher than in the controls, at 67.8 (38.5) cmH2O. Even delayed injection with BDNF+VEGF improved the ICP, to 78.0 (21.8) cmH2O. Histological analysis of specimens stained for NADPH and TH showed a significant change in the morphology of terminal branches of the cavernosal and dorsal nerves, and the staining quality of the neurones in the MPG. The number of positively stained nerve fibres tended to revert to normal after treatment with BDNF+VEGF.CONCLUSION An intracavernosal injection with BDNF+VEGF appears to both prevent degeneration and facilitate regeneration of neurones containing neuronal nitric oxide synthase in the MPG, dorsal nerve and intracavernosal tissue. Therefore it might have therapeutic potential for enhancing the recovery of erectile function after radical pelvic surgery.
Article
Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure/activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.
Article
Nitric oxide has been identified as an endothelium-derived relaxing factor in blood vessels. We tried to determine whether it is involved in the relaxation of the corpus cavernosum that allows penile erection. The relaxation of this smooth muscle is known to occur in response to stimulation by nonadrenergic, noncholinergic neurons. We studied strips of corpus cavernosum tissue obtained from 21 men in whom penile prostheses were inserted because of impotence. The mounted smooth-muscle specimens were pretreated with guanethidine and atropine and submaximally contracted with phenylephrine. We then studied the smooth-muscle relaxant responses to stimulation by an electrical field and to nitric oxide. Electrical-field stimulation caused a marked, transient, frequency-dependent relaxation of the corpus cavernosum that was inhibited in the presence of N-nitro-L-arginine and N-amino-L-arginine, which selectively inhibit the biosynthesis of nitric oxide from L-arginine. The addition of excess L-arginine, but not D-arginine, largely reversed these inhibitory effects. The specific liberation of nitric oxide (by S-nitroso-N-acetylpenicillamine) caused rapid, complete, and concentration-dependent relaxation of the corpus cavernosum. The relaxation caused by either electrical stimulation or nitric oxide was enhanced by a selective inhibitor of cyclic guanosine monophosphate (GMP) phosphodiesterase (M&B 22,948). Relaxation was inhibited by methylene blue, which inhibits cyclic GMP synthesis. Our findings support the hypothesis that nitric oxide is involved in the nonadrenergic, noncholinergic neurotransmission that leads to the smooth-muscle relaxation in the corpus cavernosum that permits penile erection. Defects in this pathway may cause some forms of impotence.
Article
In this report we review meeting highlights. Clearly there has been substantial progress in the laboratory and clinic with regard to blood stem cells. Having proven that these transplants work, it is now time to investigate biologic features in greater detail. Also needed are randomized trials evaluating several of the issues we raise such as whether the better results of blood cell autotransplants are due to more effective treatment or subject selection and whether in vitro removal of tumor cells is necessary or effective.
Article
Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.
Article
Nitric oxide (NO) is a cytotoxic agent of macrophages, a messenger molecule of neurons, and a vasodilator produced by endothelial cells. NO synthase, the synthetic enzyme for NO, was localized to rat penile neurons innervating the corpora cavernosa and to neuronal plexuses in the adventitial layer of penile arteries. Small doses of NO synthase inhibitors abolished electrophysiologically induced penile erections. These results establish NO as a physiologic mediator of erectile function.
Article
Age-related changes in calcitonin gene-related peptide (CGRP) content were found in a number of rat tissues, and most can be explained by the physiological and pathological changes associated with advancing age. In some specific areas of the brain, and some cardiovascular tissues such as atria and arteries, the CGRP content declined with age (p < 0.01), while the contents were increased in the thyroid gland, plasma and in veins (p < 0.01). In the substantia nigra and striatum, the immunoreactive-CGRP (i-CGRP) levels found in older rats were about 50% of those found in adolescent rats (p < 0.01). The i-CGRP levels found in the penis, bladder, kidney, testis and adrenal gland gradually increased up to maturity, and then rapidly declined in the aging rats. However, in the lungs and sensory ganglia (dorsal root and trigeminal), the highest content of i-CGRP was found in immature 3-week-old rats, and then declined with age. Changes of i-CGRP observed with age may support multiple physiological roles for CGRP at various stages of development. The changes described here, particularly in the lungs, substantia nigra, striatum and the cardiovascular system, should help in further understanding the physiological and pathophysiological role of CGRP in health and in age-related disorders.
Article
We have evaluated the use of Adenovirus as a new vector for gene transfer in vivo. The capacity of this virus to express a foreign gene even in the absence of viral and cellular replication makes it attractive to deliver a gene to quiescent cells. We show here that organs like lung, liver and muscles are relevant to this strategy.
Article
A functional study was done to examine a possible role of calcitonin-gene-related peptide in human penile erection and its possible therapeutic applications for patients with erectile dysfunction. In the determination of an effective dosage, 5 ng. (2 patients), 50 ng. (2 patients), 500 ng. (4 patients), 5 μg. (4 patients) and 25 μg. (7 patients) were injected intracavernously, and pulse and blood pressure were monitored. Arterial inflow was measured by Doppler sonography, smooth muscle relaxation was determined by the analysis of cavernous electrical activity and cavernous outflow occlusion was recorded by cavernosometry. In 12 patients the erectile response of prostaglandin E1 was compared to the response of an equal (6 patients) or decreased dose of prostaglandin E1 combined with an equal weight of calcitonin-gene-related peptide. In 14 patients the erectile response to the combination of calcitonin-gene-related peptide and prostaglandin E1 was compared to the response of prostaglandin E1 alone, and with a combination of 15 mg./ml. papaverine and 0.5 mg./ml. phentolamine.
Article
A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.
Article
In the presence of functional adrenergic and cholinergic blockade, electrical field stimulation relaxes corpus cavernosum smooth muscle by unknown mechanisms. We report here that electrical field stimulation of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of nitric oxide (NO), nitrite, and cyclic GMP. Corporal smooth muscle relaxation in response to electrical field stimulation, in the presence of guanethidine and atropine, was abolished by tetrodotoxin and potassium-induced depolarization, and was markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but was unaffected by indomethacin. The inhibitory effects of NG-substituted analogs of L-arginine were nearly completely reversed by addition of excess L-arginine but not D-arginine. Corporal smooth muscle relaxation elicited by electrical field stimulation was accompanied by rapid and marked increases in tissue levels of nitrite and cyclic GMP, and all responses were nearly abolished by NG-nitro-L-arginine. These observations indicate that penile erection may be mediated by NO generated in response to nonadrenergic-noncholinergic neurotransmission.
Article
Penile tissue (consisting of corpus cavernosum and tunica albuginea) was obtained from 19 patients undergoing surgery for the implantation of penile prostheses. The tissue was examined for vasoactive intestinal polypeptide-like immunoreactivity in nerves, acetylcholinesterase-positive staining in nerves and noradrenaline content. Impotence was due to a variety of causes; 11 patients were classified as a 'non-neuropathic' group on the basis of their clinical history which included Peyronie's disease, vascular disease, hypertension and psychogenic impotence. Vasoactive intestinal polypeptide-like immunoreactive and acetylcholinesterase-positive nerves were present and the pattern and distribution were similar in each patient in this group. The noradrenaline content of the tunica albuginea was significantly lower than the corpus cavernosum (p less than 0.02), although there was a linear relationship between the noradrenaline contents of the two regions (r = 0.95, p less than 0.01). By comparison, a complete absence of vasoactive intestinal polypeptide-like immunoreactivity in nerves was observed in a patient with a cauda equina lesion. Five out of six diabetic patients studied revealed a marked reduction in vasoactive intestinal polypeptide-like immunoreactivity in nerves associated with the cavernous smooth muscle, while acetylcholinesterase-positive staining was reduced in three out of five diabetic patients studied. The noradrenaline content of the corpus cavernosum from diabetic patients was significantly lower (p less than 0.02) than that of the 'non-neuropathic' group. The noradrenaline content of the tunica albuginea, however, was similar in both groups. The results provide evidence that VIPergic, cholinergic and adrenergic nerves in the penis are affected in diabetes mellitus and thus may contribute to the development of impotence in diabetic patients.
Article
During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.
Article
High concentrations of vasoactive intestinal polypeptide (VIP) were detected by immunocytochemistry and radioimmunoassay in thirty surgical specimens of male external genitalia. VIP was found exclusively in fine autonomic nerves. VIPergic nerves were most densely concentrated in the penis around the pudendal arteries and in the erectile tissue of the corpus cavernosum. Considerable numbers of VIP nerve fibres were also seen in the vas deferens and epididymis. VIP is known to exert regulatory actions on blood-flow, secretion, and muscle tone. Its presence in considerable amounts in the male genital tract suggests that this newly discovered peptide neurotransmitter may be important in the nervous control of male external genitalia.
Article
Vasoactive intestinal polypeptide (VIP) has been demonstrated by immunofluorescence histochemistry in nerves in human and rat penile tissue. A reduction in VIP-like immunoreactivity in nerves was revealed in tissue from streptozotocin-diabetic rats and a human diabetic with impotence. These results suggest that an impairment in the VIP-ergic innervation in penile tissue may be an important factor in the development of impotence in diabetes. They also support the view that the streptozotocin-treated rat is a useful experimental model for diabetic autonomic neuropathy.
Article
Plasma levels of vasoactive intestinal polypeptide (VIP) in the corpora cavernosa penis and dorsal penile veins greatly exceeded those measured in the limb or caudal veins during anaesthesia in various mammals (Bennett's wallaby, Barbary sheep, cheetah, puma, sooty mangabey, pigtail macaque and chimpanzee). Tactile stimulation of the penis immediately before or during collection of blood samples resulted in an increase. In the wallaby, VIP levels (mean ± s.e.m. ) in blood samples collected from the flaccid penis in the absence of tactile stimulation were very low (0·6 ± 0·5 pmol/l). A 36-fold increase in VIP occurred after manual extension of the flaccid penis (24·8 ± 3·2 pmol/l) or during manually stimulated erections (25· 1 ± 1·7 pmol/l). Electrical stimulation of erection produced no significant increase in VIP levels (2·3±0·9 pmol/l) unless accompanied by tactile stimulation (17·5±1·4 pmol/l). These studies provide the first demonstration that sensory feedback from the penis plays an important role in regulating vasoactive intestinal polypeptidergic activity. Since VIP is a potent vasodilator its release due to tactile stimuli during copulation may play a role in the maintenance of penile erection. J. Endocr. (1984) 100 , 249–252
Article
The haemodynamics of erection were elucidated in the anaesthetized dog by analysing in quantitative terms the changes of penile arterial inflow, venous outflow and tissue volume during graded pelvic nerve stimulation. The study also provides information on possible neurotransmitter mechanisms of the erectile response. Erection evoked by pelvic nerve stimulation appeared to result from two main circulatory events: first, there was a prompt dilatation of the penile 'resistance vessels', causing a greatly increased arterial inflow which in the early phase bypassed the cavernous bodies and, hence, increased venous outflow to the same extent. Secondly, the erectile response proper began after a distinct delay (approximately equal to 20 s). This was apparently caused by sudden opening of low resistance 'shunt vessels' diverting part of the arterial inflow into the cavernous bodies, leading to rapid filling. During the filling phase arterial inflow greatly exceeded venous outflow, and returned to the venous outflow level again in the steady state of full erection. The initial dilator response seemed to ensure rapid erection by establishing a high pressure head from the arterial microvessels to the cavernous spaces. The threshold frequency for the penile vasodilator response to pelvic nerve stimulation was 1-2 Hz and was always higher for the erectile volume response, viz. 2-4 Hz. Maximal effects for both were obtained at 16 Hz, causing on the average a 25-fold increase in peak arterial inflow, a 17-fold increase in venous outflow and a 107% increase in penile volume. Muscarinic blockade by atropine caused no significant decrease in the blood flow response induced by pelvic nerve stimulation, but clearly curtailed the erectile response. This indicates that the dilatation of the penile 'resistance vessels' is mainly non-cholinergic in nature, whereas a cholinergic mechanism seems to contribute to the erectile volume response proper. Pelvic nerve stimulation caused a substantial output of vasoactive intestinal polypeptide (VIP) from the penis which was correlated in onset and duration to the vasodilator response. Intra-arterial (I.A.) infusion of VIP elicited moderate erection and a penile vasodilator response which resembled the neural response. Similar effects were evoked by I.A. infusion of substance P, but the output of this peptide from the penis during stimulation was poorly correlated to the vascular events. These in vivo observations indicate that VIP might be the neurotransmitter responsible for the non-cholinergic pelvic nerve induced penile vasodilatation.
Article
We report the purification from pig brain of a factor supporting the survival of, and fibre outgrowth from, cultured embryonic chick sensory neurons. The purified factor migrates as one single band, mol. wt. 12 300, on gel electrophoresis in the presence of sodium dodecylsulphate (SDS) and is a basic molecule (pI greater than or equal to 10.1). Approximately 1 microgram factor was isolated from 1.5 kg brain. The final degree of purification was estimated to be 1.4 X 10(6)-fold, and the specific activity 0.4 ng/ml/unit, which is similar to that of nerve growth factor (NGF) using the same assay system. This factor is the first neurotrophic factor to be purified since NGF, from which it is clearly distinguished because it has different antigenic and functional properties.
Article
Filling of the sinusoidal spaces with blood due to smooth muscle relaxation results from parasympathetic neural pathway activation and probably simultaneous inhibition of sympathetic outflow. The final common pathway for proerectile fibers is represented by the cavernous nerves and fibers controlling detumescence and flaccidity originating in the sympathetic chain. The hypogastric nerve could represent an accessory proerectile pathway unmasked by a sacral spinal cord lesion. Nitric oxide, which can be colocalized with VIP and acetylcholine, is the main proerectile neurotransmitter and noradrenaline is considered to be the major antierectile agent. Reflexive erection elicited by recruitment of penile afferents involves both autonomic and somatic efferents. This reflex is mediated at the spinal cord level and modulated by supraspinal influences. Serotonergic pathways originating in the raphe nuclei may mediate inhibitory control on reflexive erections. The hypothalamic medial preoptic area is an important integrating center and dopamine may regulate penile erection at this level. Neuroendocrine regulation may vary depending on the context in which erection occurs, for example, coitus, in response to extrinsic or psychogenic stimuli, and rapid eye movement sleep.
Article
The intrinsic biological complexity of penile erection and the multifaceted nature of erectile dysfunction are just beginning to be fully appreciated. This article describes how mechanistic studies of the local control of penile erection, with specific emphasis on the primary role of the corporal smooth muscle, contribute to the improved understanding, diagnosis, and treatment of erectile dysfunction.
Article
Adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) mediate penile erection. We have previously established that adenylate and guanylate cyclase activity is elevated in the diabetic rat penis and aorta. This study investigates the action of papaverine and vasoactive intestinal polypeptide (VIP) on these cyclases. The aortae and penes of Sprague Dawley rats (n = 7) were stimulated with VIP and papaverine. Diabetes mellitus (DM) was induced in Sprague Dawley rats (n = 7) with streptozotocin and the penile and aortic tissues were treated with VIP. The penes, aortae and carotid arteries of New Zealand White rabbits were similarly processed. cAMP and cGMP generation was measured by radioimmunoassay. In all tissues: VIP stimulated cAMP synthesis; VIP did not increase cGMP levels; papaverine was without effect on either cAMP or cGMP synthesis. VIP-stimulated cAMP was significantly enhanced in the diabetic rat penis and aorta; there was also a significant elevation in the basal levels of cGMP in these tissues. These data: (1) consolidate that cAMP is a mediator of penile erection, (2) indicate that papaverine and VIP elicit erection by different mechanisms, (3) suggest that an enhanced penile capacity to generate cAMP in DM may constitute an adaptive response to counteract the previously reported reduction in VIP content and VIP receptors, and (4) indicate that the penile and vascular tissues of the rabbit respond in a similar manner to VIP and papaverine.
Article
By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus.
Article
In order to gain more mechanistic insight into the regulation of corporal smooth muscle tone, we conducted electrophysiological studies on homogeneous explant cell cultures of human corpus cavernosum smooth muscle. Patch clamp analyses in the whole cell mode revealed a mean resting potential of -43 +/- 4.9 m V (n = 12 cells). Large whole cell outward K currents were very prominent in these cells, and ranged from 0.5 to 1.5 nA. In some cells, a transient, voltage-dependent A current accounted for a significant portion of the observed whole cell currents. Furthermore, stimulation with the calcium channel agonist BAY K 8644 or the K channel agonist pinacidil doubled the magnitude of the whole cell K current, as would be expected for maxi-K (KCa) and metabolically gated K channels (KATP), respectively. Single channel recordings in the detached patch mode consistently revealed the presence of at least two K channels: 1) a KCa channel, with a conductance of approximately 190 pS; and 2) a putative delayed rectifier channel with a conductance of approximately 50 pS. Furthermore, all channel types showed some degree of voltage and/or calcium sensitivity. In conclusion, the large magnitude of the whole cell K currents and the observed K channel heterogeneity indicate a potentially important role for these channels in modulating corporal smooth muscle tone.
Article
Aging is an important risk factor for impotence in men. Because nitric oxide (NO) appears to be the mediator of corpora cavernosal smooth muscle relaxation, we have examined in 5-, 20-, and 30-mo-old rats, designated "adult," "old," and "senescent," respectively, whether aging causes a decrease of erectile response that may correlate with lower NO synthase (NOS) in the penis. Electric field stimulation (EFS) of the cavernosal nerve showed that the maximum intracavernosal pressure (MIP) declined in the old and senescent rats to 80 and 51% of the adult value, respectively. A low systemic dose of the NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME; 2 mg/kg), reduced the MIP by only 38% in the adult rats but decreased it in the old and senescent rats by 72 and 80%, respectively. In the absence of EFS, intracavernosal papaverine (phosphodiesterase inhibitor), or nitroglycerin (NO donor), caused a lower erectile response in the old and senescent rats compared with the adult animals (MIP: 41 and 14%, respectively; duration of the erection 46 and 21%, respectively). Tissue sections from old and senescent penises showed increasing degrees of sclerotic degeneration. In comparison with the adult rats, the penile soluble NOS activity per gram of tissue that is sensitive to L-NAME decreased significantly by 63% in the senescent rats but was elevated in the old rats. These results indicate that aging causes an erectile failure due to factors initially independent from an impairment of penile NO synthesis but which are compounded in the very old rats by the decrease of penile NOS activity.
Article
Whether old uteri that have undergone involution do so by an apoptotic mechanism was examined by the presence of known biochemical and morphological markers for programmed cell death. Terminin, a protein identified by an unique monoclonal antibody, has three forms, Tp-90, Tp-60, and Tp-30: Tp-90 (the 90 kDa form) is only present in growing and quiescent non-growing cells; Tp-60 (the 60 kDa form) is found in senescent cells; and finally, Tp-30 (the 30 kDa form) is found in cells committed to apoptotic death. Biochemical analysis of a protein, Tp30, previously identified as a marker for the commitment to programmed cell death, was performed with both young (5-month-old) and old (24-month-old) C57BL/6J mouse uteri. In addition to biochemical analysis of Tp30 presence in uterine tissue, propidium iodide (PI) staining and DNA framentation by nick-end labelling with fluorescence-conjugated UTP were used to characterize apoptosis-related changes in the chromatin organization of the nucleus. Results indicate that within the old uterus Tp-30 is indeed detected in the tissue extracts and was the major terminin band, while Tp-90 and Tp-60 were the major bands observed in extracts of the younger mouse uterus. The presence of Tp30 in the older uterine tissue suggests that the tissue regression which has occurred in the uterus of older mice may be apoptotic in nature. This suggestion is further supported by the demonstration of increases in the number of cells showing apoptotic morphology, i.e. positive staining with UTP reflecting the presence of nuclei with nicked DNA, localized exclusively in the uterine stroma of older females. The presence of DNA fragmentation, as reflected by UTP staining, was virtually absent from the young uteri. These data suggest that apoptosis may be a part of the cellular mechanism contributing to the regression of uterine tissue in the older female during involution, appearing as an age-dependent event.
Article
An improved understanding of the physiology of penile erection has resulted from recent evidence that implicates nitric oxide as the principal mediator of erectile function. Previously, the neuroanatomy of erection in man was established with descriptions of the autonomic innervation of the pelvic organs and external genitalia. The basis upon which novel physiological concepts of erection relate to earlier neuroanatomical principles remains to be determined. In the present study these relationships were explored with nitric oxide synthase immunohistochemistry and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry of select pelvic tissue specimens obtained from 4 men (3 at radical prostatectomy and 1 at autopsy). Nitric oxide synthase, the enzyme that catalyzes nitric oxide production, was identified in discrete neuronal locations, including the pelvic plexus, cavernous nerves and their terminal endings within the corporeal erectile tissue, branches of the dorsal penile nerves and nerve plexuses in the adventitia of the deep cavernous arteries. This distribution of nitric oxide synthase-containing nerves suggests that nitric oxide neuronally modulates local vascular smooth musculature of the penis. On this basis, nitric oxide is identified as a neuronal mediator of penile erection in man.