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Molecular pathophysiology and gene therapy of aging-related erectile dysfunction
Experimental Gerontology 39 (2004) 1705–1712 www.elsevier.com/locate/expgero
Review
Molecular pathophysiology and gene therapy of aging-related erectile dysfunction N.F. Gonzalez-Cadavida,b,*, J. Rajfera,b a
Harbor-UCLA Research and Education Institute, Urology, Bldg. F-6, 1124 West Carson Street, Torrance, CA, USA b Department of Urology, UCLA School of Medicine, Los Angeles, CA, USA Received 27 May 2004; accepted 12 June 2004 Available online 7 October 2004
Abstract Erectile dysfunction (ED) is a major public health problem that seriously affects the quality of life of patients and their partners. ED is mainly associated with vascular disease, diabetes, smoking, and radical prostatectomy, and its prevalence increases significantly with aging. Vasculogenic ED, specifically corporal veno-occlusive dysfunction (CVOD), is caused by the impairment of the relaxation of the smooth muscle in the penile corpora cavernosa and occurs in 2/3 of cases, whereas the less common neurogenic ED is due to a defective nitrergic neurotransmission triggered by the sexual stimulus, either at the central hypothalamic and spinal levels or at the penile nerves. Based on animal and cell studies, neurogenic ED is assumed to be caused mainly by: (a) an insufficient synthesis of nitric oxide (NO) due to a decrease in the levels of the penile neuronal nitric oxide synthase (PnNOS) or the impairment of its regulation by protein effectors (NMDA receptor, protein inhibitor of nNOS: PIN), occurring in the neuronal bodies or nerve terminals, or (b) a loss of the cells themselves by apoptosis caused by the induction of inducible NOS (iNOS) and the production of peroxynitrite. In contrast vasculogenic ED, although may involve endothelial damage and down-regulation of endothelial NOS (eNOS), appears to be mainly caused by the relative loss of smooth muscle cells and replacement by collagen fibers (fibrosis) that impairs tissue compliance. In this case, iNOS induction may not be deleterious, but a defense mechanism preventing excessive collagen deposition. Gene therapy to the penile corpora cavernosa of cDNAs expressing PnNOS or eNOS, or counteracting PIN, has been effective in ameliorating ED in the aging rat model that exhibits both neurogenic ED and CVOD. cDNA constructs for other genes involved in the control of penile erection have also been successfully tested. Gene transfer into the penis may soon translate to the clinic as a therapy aimed to cure the underlying conditions in ED, including fibrosis, as opposed to the facilitation of erection on demand offered by the current oral therapies. q 2004 Elsevier Inc. All rights reserved. Keywords: Penis; Corpora cavernosa; Hypothalamus; Neuronal nitric oxide synthase; Endothelial nitric oxide synthase; Protein inhibitor of nNOS; Nitric oxide; Fibrosis; Oxidative stress; Inducible nitric oxide synthase; Phosphodiesterase
1. Introduction Penile erection is a complex process involving a series of sequential and coordinating events between: (a) the central Abbreviations: ED, erectile dysfunction; ROS, reactive oxygen species; NO, nitric oxide; iNOS, inducible nitric oxide synthase, NOS II; nNOS, neuronal nitric oxide synthase, NOS I; eNOS, endothelial nitric oxide synthase, NOS III; CVOD, corporal veno-cclusive dysfunction; PDE5A, phosphodiesterase 5A; PIN, protein inhibitor of nNOS; PVN, paraventricular nucleus; MPOA, medial preoptic area; EFS, electrical field stimulation. * Corresponding author. Address: Harbor-UCLA Research and Education Institute, Urology, Bldg. F-6, 1124 West Carson Street, Torrance, CA 90502, USA. Tel.: C1 310 222 3824; fax: C1 310 222 1914. E-mail address: [email protected] (N.F. Gonzalez-Cadavid). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.06.022
nervous system, mainly at the cortical, hypothalamic and spinal levels, which have connections to the efferent and afferent nerves of the penis that trigger and conduct the neurotransmission signals to the organ; and (b) the vascular system, specifically the smooth muscle in the corpora cavernosa and within the media of the arteries that deliver blood to the penis (Allard and Giuliano, 2001; Andersson, 2003; Russell and Nehra, 2003). Indeed, the penis can be considered simply as an extension of the vascular system, and in actuality, it is the relaxation of the smooth muscle both in the helicine arteries and in the trabeculae that causes the blood to enter and pool into the cavernous sinusoids. This active process of smooth muscle relaxation is under the regulation of the cavernous nerves and is followed by
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the subsequent passive compression of the penile veins against the underside of the comparatively rigid tunica albuginea within the corporal bodies, thereby preventing the egress of blood out of the corpora. It is this latter process that allows penile erection to sustain. Erectile dysfunction (ED), the inability to achieve or maintain an erection of sufficient rigidity for vaginal penetration and completion of the sexual act, is a common condition affecting 20–30 million men in the USA. This disorder is highly associated with aging, particularly in men older than 50 years (Seftel, 2003; Montorsi et al., 2003). Major concurrent factors for organic, as opposed to psychogenic ED, are cardiovascular disease, in particular atherosclerosis and hypertension (Kloner and Speakman, 2002; Kloner, 2003), diabetes (Richardson and Vinik, 2002), and radical prostatectomy (Burnett, 2003). These disorders lead to two forms of ED, vasculogenic and neurogenic, which often may present together (Siroky and Azadzoi, 2003). Vasculogenic ED is the most prevalent condition, affecting nearly 80% of patients with organic impotence. In the majority of patients with vasculogenic ED, the main vasculopathy is an impairment in smooth muscle relaxation within the corporal tissue itself, and this clinically may lead to inadequate compression of the subtunical penile veins, causing corporal veno cclusive dysfunction (CVOD) or penile venous leak (Moreland, 2000; Wespes, 2002; Siroky and Azadzoi, 2003). Endothelial damage, both in the penile blood vessels and the lining of the trabecular cisternae may also contribute significantly. Neurogenic ED results from the impairment of neurotransmission to the smooth muscle either because of an injury or neuropathy subsequent to trauma, surgery, diabetes, or aging (Montorsi et al., 2004). In many instances, neurogenic ED is often associated with CVOD as a result of the relative atrophy of the trabecular smooth muscle (User et al., 2003). Our emphasis in this presentation will be on our recent work in the last 3 years which continued to be focused on the modulation of NO synthesis in the penile tissue and its role in penile erection and the prevention of penile fibrosis as an underlying cause of ED. A pertinent general background will be provided to discuss its implications, but the discussion is not intended as a comprehensive review of the area.
the erectile response (Gonza´lez-Cadavid et al., 1999; Melman and Gingell, 2003; Gonzalez-Cadavid and Rajfer, 2004b). The reaction leading to NO production is the conversion of L-arginine into citrulline, and this in the penis occurs in the nerve terminals of the corpora cavernosa, involving the neuronal NOS (nNOS or NOS I), and presumably as well in the endothelium of the cavernosal cisternae and arteries, involving endothelial NOS (eNOS or NOS III). The primary trigger for erection is the nitrergic neurotransmission through the non-adrenergic non-cholinergic nerves catalyzed by the stimulation of nNOS activity via Ca2C binding to the nNOS associated calmodulin. This process is in part modulated by Ca2C fluxes through another nNOS associated protein: the NMDA receptor (Gonza´lez-Cadavid et al., 2000b). The eNOS activation via sheer stress, Ca2C flux, and phosphorylation, appears to be ancillary and supports NO synthesis in the corpora cavernosa during erection (Hurt et al., 2002; Musicki et al., 2004). The NO liberated from penile nerves and endothelium activates guanilyl cyclase in the trabecular and penile arterial smooth muscle, respectively, causing the elevation of cGMP levels that stimulate a cyclic GMP phosphokinase (PKG) and the subsequent reduction in cytoplasmic Ca2C (Fig. 1 top). This leads to smooth muscle relaxation and penile erection, counteracting adrenergic and related agents that maintain its tone during the flaccid stage. In turn, cGMP breakdown is catalyzed in the smooth muscle by cGMPdependent phosphodiesterase 5A (PDE5A), the target of the PDE5 inhibitors used orally for the treatment of erectile dysfunction, such as sildenafil (VIAGRA). In addition, NO
2. The role of nitric oxide synthases in the central and peripheral control of erection Since, the first report by Ignarro et al. (1990) identifying nitric oxide (NO) as the mediator of corpora cavernosa relaxation in the rabbit, and subsequently in 1992 in the human (Rajfer et al., 1992), corpora cavernosa, a considerable number of studies, including those from our laboratory, have elucidated the respective roles of the nitric oxide synthase (NOS) isoforms in synthesizing NO involved in
Fig. 1. Schematic representation of the nitric oxide (NO) signaling pathway responsible for penile erection, and its metabolism that affects the smooth muscle, in the penile corpora cavernosa. Top panel: signaling pathway triggered by PnNOS activation upon sexual stimulation, leading to NO release and the elevation of cGMP that causes smooth muscle relaxation. The endothelium contributes to relaxation through NO produced by eNOS. Bottom panel: NO metabolism by reaction with OK 2 and other radicals (ROS), that is considerably stimulated by iNOS induction, a NOS isoform that does not appear to mediate erection.
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Fig. 2. Schematic representation of the functional domains encoded in the cDNA for penile neuronal nitric oxide synthase (PnNOS), the enzyme that generates NO as the main neurotransmitter causing penile erection. The 102 bp insert, encoding a 34 amino acid stretch, is present in PnNOS and absent in the brain nNOS counterpart. PDZ: region that binds PnNOS modulators important for the control of erection; Heme: heme binding region (PnNOS and nNOS are hemeproteins); CaM: calmodulin binding site where Ca2C binding activates the enzyme; FMN, FAD, NADPH: sites for binding of the respective coenzymes.
metabolism leads to other products (bottom), discussed in another section below. A few years ago, our group cloned from a penile cDNA library a variant of the brain type nNOS having a 34 amino acid insert between exons 16 and 17, that was named penile nNOS (PnNOS) (Fig. 2) and showed that it was the nNOS variant expressed in the penis and prostate, whereas the nNOS brain variant was expressed in the bladder and other urogenital organs (Magee et al., 1996). PnNOS seems to be identical to nNOSu expressed in the skeletal muscle (Silvagno et al., 1996). In a more recent work (Gonza´lez-Cadavid et al., 2000a), we have shown that PnNOS is expressed in penile nerves as both the fulllength a-form and the truncated splicing b variant lacking a portion of the N-terminal region named PDZ (Fig. 3). This is important because this is the region where at least in the case of the brain nNOS, the NMDA receptor binds (Gonza´lez-Cadavid et al., 2000b), as well other nNOS modulators, the protein inhibitor of nNOS (PIN), and CAPON. The nNOS knockout mouse, engineered by the blockade of the expression of the nNOS gene, lacks as expected in the a-form, but by an unknown mechanism the b form is expressed. This explains the retention of erectile function in this knockout animal, since the residual PnNOS-b retains NOS activity for NO synthesis, and in addition it is not able to bind PIN, and therefore is insensitive to this inhibition (Gonza´lez-Cadavid et al., 2000a). By applying a combination of immunohistochemistry, dual immunofluorescence, DNA cloning, and western blot/RT-PCR procedures, we have been able to show that in the cavernosal and dorsal nerves of the penis, there is co-localization of PnNOS with both PIN and the NMDA receptor, suggesting the interaction of these three proteins in the modulation of NO synthesis during nitrergic neurotransmission to and from the penis, although we could not show a direct inhibition of PnNOS by PIN (Magee et al., 2003). As a natural corollary, we have found that essentially the same PnNOS/PIN/NMDA receptor co-localization occurs in the hypothalamic regions known to control penile erection, ejaculation, and other sexual responses (paraventricular nucleus: PVN,
and medial preoptic area: MPOA), and in the spinal cord at the S1–S4 levels which is also involved in the control of penile erection (Ferrini et al., 2003). This suggests that PnNOS is an essential component in the nitrergic neurotransmission controlling erection, along its entire regulatory axis from the brain via the spinal cord to the smooth muscle of the penis itself. We also found that PnNOS in the hypothalamus is also expressed in oxitocinergic neurons (Ferrini et al., 2003), previously demonstrated by other groups to be involved in NO release in the same regions, that mediate the central nitrergic control of penile erection. It is therefore likely that PIN and the NMDA receptor, and possibly other proteins that have recently been identified in the penis (Qian et al., 2003), regulate PnNOS activation to trigger and transduce nitrergic responses to sexual stimulation.
Fig. 3. Interaction of nNOS variants with protein modulators of PnNOS enzyme activity, that is postulated to contribute to the control of penile erection. Top: N-terminal exons in the cDNA encoding the 160 kDa fulllength nNOS or PnNOS proteins (a-form) encompassing the PDZ proper, and neighbor, sequences where the protein modulators (NMDA receptor (NMDAR), PIN and CAPON) bind. ATG: translation initiation for nNOS or PnNOS protein synthesis. Bottom: exon 2, containing the binding regions, is missing in the 130 kDa b-form of nNOS or PnNOS protein that is expressed in the nNOSK/K mouse and maintains penile erection in this mouse.
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3. Molecular pathophysiology of aging-related erectile dysfunction Thanks to the intensive research conducted in animal (McKenna et al., 2004; Wang et al., 2002) and cell models (Moreland, 2000; Gonzalez-Cadavid et al., 2004), it is now clear that at the molecular and cellular level, ED is due to four main causes: (a) an insufficient production of NO during nitrergic neurotransmission elicited by sexual stimulation, mostly at the penile nerve terminals or possibly at the brain and spinal cord; this impairment can de due to the loss of neuronal bodies and neural connections to and from the penis, or to a down-regulation of NOS activity and/ or expression in the respective organs (Garban et al., 1995; Carrier et al., 1997; Vernet et al., 1998; Gonza´lez-Cadavid et al., 2000a; Ferrini et al., 2001a,b; Magee et al., 2003), and also occuring in diabetes-related ED (Vernet et al., 1995; McVary et al., 1997); (b) an ancillary reduction in NO levels from eNOS down-regulation due to endothelial damage, in diabetes and vascular disease (Vernet et al., 1995; McVary et al., 1997; Bivalacqua et al., 2000, 2003, 2004); (c) a putative excessive release of adrenergic compounds that increase the tone of the corpora cavernosal smooth muscle, mainly through endothelin and rho kinase activation (Khan et al., 2000; Mills et al., 2003); and (d) an impairment of the relaxation of this target smooth muscle by endogenous factors, resulting mainly from either a putative excessive cGMP degradation by PDE5A, or from a relative loss of smooth muscle cells in the penis and their replacement by collagen fibers (fibrosis) (Ferrini et al., 2001a; User et al., 2003; Yaman et al., 2003; Magee et al., 2003). Aging in otherwise healthy patients is assumed to be associated with the three processes described above, and this is even compounded by the additive effects of cardiovascular disease, and other co-morbidities, e.g. diabetes, that are prevalent in old age. It is well accepted that the rat is an excellent model for aging-related ED, since aged rats are known to exhibit two main features of this disorder: (a) a reduction in erectile function and a decreased mounting and copulatory behavior (Sato et al., 1998); and (b) a reduced response to electrical field stimulation (EFS) of the cavernosal nerve as measured by the reduction in the ratio between the maximal intracavernosal pressure and the mean arterial pressure (Burnett et al., 1992; Garban et al., 1995; McKenna et al., 2004). This is associated with a reduction in the content of nitrergic penile nerves (Carrier et al., 1997), and in the down-regulation of NOS activity (Garban et al., 1995), thus fitting into the assumption that aging in the rat leads to a neurogenic type of ED. However, we have recently shown that in the aged rat when compared to the young rat, there is an altered response to dynamic infusion cavernosometry as measured by the erection achieved pharmacologically with intracorporeal papaverine injection or mechanically by the rate of infusion with saline into the corpora, or the rate of detumescence (Davila et al., 2004a). We believe this finding is important
because it demonstrates that at least one of the common wild type laboratory rat strains, the Fisher 344 rat, exhibits, in addition to the neuropathic component, a marked venous leak (CVOD) which as mentioned, is the most prevalent type of ED in men. Therefore, the rat is an excellent model to study the molecular and cellular pathophysiology of both neurogenic and vasculogenic aging-related ED. The mechanism for the neurogenic component of the age-related deterioration of the erectile response involves, in addition to the already described decrease in nitrergic penile nerves, a marked increase in the old rats in the apoptotic index in the hypothalamic PVN and MPOA, the regions that control erection (Ferrini et al., 2001b). These changes occurred in the glia and in oxytocinergic and GnRH neurons associated with erection and testosterone production, two of the processes affected during reproductive decay with aging. In turn, the vasculogenic component of ED in the aged rat is probably due to a similar increase in the apoptotic index in the penile trabecular (Ferrini et al., 2001a) and arterial smooth muscle (Ferrini et al., 2004). This, combined with an increase in fibrosis in these tissues, explains the loss in corpora cavernosal compliance with aging which is responsible for its impaired response to EFS and/or intracorporeal papaverine. These studies confirmed our previous observations that in the aged rat both the hypothalamus as a whole (Vernet et al., 1998) and the penis (Ferrini et al., 2001a) harbored a considerable ‘spontaneous’ expression of the mRNA and protein corresponding to the third NOS isoform, the inducible NOS (iNOS or NOS II). This expression of iNOS that was seen in both organs in the aged animals was markedly increased from the negligible basal levels found in the young rats. It should be stressed that iNOS is not known to participate in the physiological control of penile erection at any level, be it central or peripheral, since in contrast to nNOS or eNOS, iNOS does not have the type of enzyme activity regulation required for the fast erectile response to sexual stimulus (Gonzalez-Cadavid and Rajfer, 2004a). iNOS is only controlled at the transcriptional level (leading to changes in protein levels) which is not fast enough to either turn on or turn off NO synthesis in a time mode appropriate to sexual response. When iNOS expression was studied in several hypothalamic regions (Ferrini et al., 2001b), it was detected in the PVN and MPOA, in both glia and neurons, specifically in those expressing oxytocin and GnRH, in parallel to the increase in the apoptotic index. Since the sustained levels of NO produced from iNOS give rise to the pro-apoptotic compound peroxynitrite, or ONOOK (Fig. 1 bottom), it follows that aging-related iNOS induction in the brain is deleterious since it leads to a considerable loss of neurons in those regions related to the control of penile erection. The cumulative production of NO, and hence of peroxynitrite, can be detected in tissue sections by immunohistochemistry for its metabolite, 3-nitrotyrosine, bound to different proteins, mainly
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collagen, and its co-localization with apoptosis may be assayed with the TUNEL procedure. In contrast, in the corporal smooth muscle, both in the trabecular tissue and the arterial media, aging-related induction of iNOS, although may contribute to an increased apoptotic rate, appears on the whole to be a defense mechanism against fibrosis. Evidence for this hypothesis is based on the exacerbation of the already excessive deposition and disorganization of collagen fibers in the aged penis that leads to loss of compliance in both tissues when iNOS activity is blocked long-term by L-NIL, an iNOS inhibitor that does not affect nNOS or eNOS (Ferrini et al., 2004). This resembles the situation observed in a rat model for a localized fibrotic plaque in the tunica albuginea of the penis, Peyronie’s disease, where chronic L-NIL administration considerably worsens the fibrosis (Ferrini et al., 2002; Vernet et al., 2002; Gholami et al., 2002; Gonzalez-Cadavid et al., 2002; Gonzalez-Cadavid and Rajfer, 2004c). Conversely, chronic administration of L-arginine as NOS substrate (which should increase NO from all NOS isoforms) counteracts plaque development (Valente et al., 2003). This L-arginine inhibition of fibrosis may also explain the amelioration of ED in aged rats subjected to a similar long-term treatment with the amino acid (Moody et al., 1997), since in addition to the expected direct improvement in the relaxation of the trabecular smooth muscle by the release of higher levels of NO, it may be speculated that the smooth muscle/collagen ratio may have been increased in this tissue. In all these conditions associated with aging and impotence, i.e. neuronal cytotoxicity in critical hypothalamic regions, fibrosis of either the tunica albuginea or the trabecular smooth muscle, or arteriosclerosis or stiffness of the penile arteries, oxidative stress appears to be an underlying factor (Jones et al., 2002). The production of reactive oxygen species (ROS) may be pro-fibrotic by releasing TGFb and other cytokines, or also cause cell death by apoptosis. This agrees with the long held theory that aging may be the result of an accumulation of oxidative compounds (Poon et al., 2004; Junqueira et al., 2004). The high levels of NO produced from the spontaneous induction of iNOS are known to: (a) quench ROS by forming peroxynitrite, and (b) also inhibit collagen synthesis and promote its breakdown (Gholami et al., 2002; Gonzalez-Cadavid et al., 2002). However, peroxynitrite per se induces apoptosis, and therefore iNOS induction during aging has a yin-yang effect on the different levels of control of penile erection. The overall result would depend on the tissue environment and cell types: cytotoxic in the hypothalamic neurons, and antifibrotic in the penile smooth muscle. Although in the penis, it is certainly possible that the pro-apoptotic effect of NO may contribute to some smooth muscle loss, is the NO/ROS balance, the main factor that will ultimately determine if the smooth muscle/collagen ratio, and hence tissue compliance, is reduced or not by oxidative stress Gonzalez-Cadavid and Rajfer (2004c).
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4. Gene therapy of ED and penile fibrosis Seven years ago, the first use of gene therapy for the treatment of ED was reported by our group who demonstrated that the pharmacological transfer of a plasmid expressing iNOS was effective in correcting ED in the aged rat (Garban et al., 1997; Gonzalez-Cadavid et al., 2004). This proof of principle was based on the fact that since NO is the chemical mediator of penile erection, higher NOS levels would lead to a higher NO output and overcome the defective cavernosal smooth muscle compliance and/or a putative excessive contractile tone present in the aged animal. By cloning iNOS from the rat and human penis and injecting the iNOS cDNA into the corpora cavernosa of aged rats, it was demonstrated that the expression of the recombinant iNOS protein occurred in the corpora cavernosa and corrected the defective erectile response for at least 10 days, without causing priapism or other side effects. Why would there be a need for gene therapy for ED when the oral PDE5 inhibitors for the treatment of ED are available today? Simply because even the most effective PDE inhibitor trials indicate only a 50–65% success rate, and these drugs are palliative in that they treat only the symptoms (erectile failure) but do not aim to cure the underlying condition (Gonzalez-Cadavid et al., 2004). Therefore, gene therapy offers the possibility that in theory a single injection of the cDNA construct into the penis may ameliorate ED for months or even years, and lead to a partial or total cure of the defective neurotransmission or the underlying impairment of its target vascular smooth muscle. More recently, we showed that the cDNA for the PnNOS variant cloned from both the rat and human penis corrected ED for at least 17 days in the aged rat (Magee et al., 2002). Rather than plasmid/liposome preparations, in this study the combination of a helper-dependent adenoviral vector (AdV) and a new procedure (in vivo electroporation) to increase uptake of the cDNA construct in the corpora cavernosa was used. This type of vector reduced the risk of immune reactions, a risk with first generation AdV, and combined with electroporation allowed a comparatively low viral load to increase PnNOS levels in the penis, and to elevate the MIP/MAP ratio to virtually the values observed in young rats. In as yet unpublished experiments, we applied a different approach based on the reverse concept, i.e. instead of elevating NOS levels we would attempt to block inhibition of PnNOS activity by one of its known protein inhibitors, specifically PIN, that, as stated above, co-localizes with PnNOS and the NMDA receptor in the rat hypothalamus, spinal cord and penis. A plasmid construct encoding the antisense cDNA for PIN, that inhibits the mRNA translation into protein by forming a duplex RNA, was prepared, and when injected and electroporated into the penis, it corrected the ED in the aged rat for at least 30 days (Davila et al., 2003a). An alternative procedure was to prepare the siRNA
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(small interference RNA) for PIN, a very novel and more effective approach than using antisense sequences that directly breaks down the specific mRNA. In preliminary ongoing studies, which need confirmation, the construct was administered to rats and we observed, as with the antisense vector, a stimulation of the erectile response (Davila et al., unpublished). Our data, together with studies from other groups showing that eNOS is also effective (GonzalezCadavid and Rajfer, 2004b; Bivalacqua et al., 2000, 2003, 2004), support the hypothesis that NOS gene therapy, irrespective of the isoform, or the use of constructs targeted to NOS regulators, is effective in improving erectile function, and that both viruses and plasmids may be adequate vectors. What started a few years ago as a proof of concept has now been translated into an active field of research. Additional genes have proven to be as or even more effective than those for the NOS isoforms, and some of them are being considered for future clinical trials. These genes are related to either cavernosal relaxation, such as maxi KC channels (hSlo) or calcitonin gene-related peptide, growth factors, such as VEGF, BDNF, and neurotrophic factor, counteraction of oxidative stress such as superoxide dismutase, or inhibition of contractile factors such as rho kinase (see Christ, 2003; Gonzalez-Cadavid and Rajfer, 2004b). Gene therapy for ED is not limited to approaches aimed to treat the functional impairment of cavernosal relaxation or the defective neurotransmission associated with ED. Studies are now attempting to reverse the histological changes that impair compliance during aging, e.g. trabecular tissue fibrosis. The first example of this kind was recently reported by our laboratory (Davila et al., 2003b, 2004b), by revisiting the use of iNOS cDNA, but this time as an antifibrotic agent, a concept gleaned from our previous studies in the aging rat and in the rat model of Peyronie’s disease. In the latter model, often associated with ED, we induced a fibrotic plaque by injection of fibrin into the tunica albuginea of the rat, as the target to demonstrate the effectiveness of iNOS in reducing ROS levels and collagen deposition. By giving the plasmid cDNA for iNOS directly into a well-formed plaque, we could induce its nearly complete regression, while simultaneously reducing oxidative stress. We are now preparing the AdV construct of iNOS in order to obtain longer effects and be able to correct or prevent smooth muscle fibrosis present in CVOD, although a stable chemical NO donor may be more effective. This approach needs still assessment by further studies of potential deleterious effects. The field of gene therapy for ED is in its initial phase of development, but it may be considerably expanded in the near future by its combination with ex vivo gene therapy, using either smooth muscle or stem cells from a patient that could be engineered to express a particular protein and followed by allograft cell re-implantation into the corpora cavernosa (Cao et al., 2002; Peng and Huard, 2003;
Gonzalez-Cadavid and Rajfer, 2004b). Our approach is based on the premise that adult stem cells from different sources can evolve into a specific cell lineage in vivo, according to the type of paracrine factors and cell-to-cell interactions in the host tissue, decreasing the risk of poor proliferation and immunorejection associated with fully differentiated cells, in this case smooth muscle (Singh et al., 2003; Artaza et al., 2004; Vernet et al., 2004). Other promising approaches are the use of DNA vectors allowing more prolonged expression of the inserted sequence, such as the adeno-associated virus, herpesvirus, lentivirus, or hybrid AAV/AdV virus. The utilization of tissue-specific gene promoters for preferential expression of the recombinant DNA in a given tissue, irrespective of disseminated uptake, or the design of novel promoter casettes where the recombinant cDNA is placed under a promoter controlled by very low, non-hazardous doses of a drug, in order to get a time-specific and dose-dependent regulation, is also under active investigation by different investigators (Gonzalez-Cadavid and Rajfer, 2004b). Depending on the outcome of the first clinical trials with plasmid constructs in men, these other modalities may serve to improve the efficacy of gene therapy for ED. It is not too speculative to assume that this approach may eventually help achieve a cure, or at least a long-term amelioration, of tissue and biochemical alterations underlying aging-related ED and penile fibrosis. This would replace dependence on the palliative pharmacological therapies currently available, or reduce dosages if biological (e.g. gene therapy) with chemical (e.g. PDE inhibitors) combination regimens are devised (Bivalacqua et al., 2004).
Acknowledgements Experimental studies by the authors quoted in this review were funded mainly by NIH grants R01DK-53069 and G12RR-03026, and by a grant from the Eli and Edythe L. Broad Foundation and the Peter Morton Foundation.
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Gonzalez-Cadavid, N.F., Rajfer, J., 2004a. The pleiotropic effects of inducible nitric oxide synthase (iNOS) on the physiology and pathology of penile erection. Curr. Pharm. Des. 2004; (in press). Gonzalez-Cadavid, N.F., Rajfer, J., 2004b. Therapy of erectile function: potential future treatments. Endocrine 23, 167–176. Gonzalez-Cadavid, N.F., Rajfer, J., 2004c. Molecular and cellular aspects of the pathophysiology of Peyronie’s disease. Drug Discov. Today Dis. Mech. 2004; (in press). Gonza´lez-Cadavid, N.F., Ignarro, L., Rajfer, J., 1999. Nitric oxide and cyclic GMP in the penis. Mol. Urol. 3, 51–59. Gonza´lez-Cadavid, N.F., Burnett, A.L., Magee, T., Zeller, C.B., Vernet, D., Gitter, J., Smith, N., Rajfer, J., 2000a. Expression of penile neuronal nitric oxide synthase variants in the rat and mouse penile nerves. Biol. Reprod. 63, 704–714. Gonza´lez-Cadavid, N.F., Ryndin, I., Vernet, D., Magee, T.R., Rajfer, J., 2000b. Presence of NMDA receptor subunits in the male lower urogenital tract. J. Androl. 21, 566–578. Gonzalez-Cadavid, N.F., Magee, T.R., Ferrini, M., Qian, A., Vernet, D., Rajfer, J., 2002. Gene expression in Peyronie’s disease. Int. J. Impot. Res. 14, 361–374. Gonzalez-Cadavid, N.F., Ignarro, L., Rajfer, J., 2004. Gene therapy of erectile dysfunction. In: Mulcahy, J.J. (Ed.), Male Sexual Dysfunction: a Guide in Clinical Management (in press). Hurt, K.J., Musicki, B., Palese, M.A., Crone, J.K., Becker, R.E., Moriarity, J.L., Snyder, S.H., Burnett, A.L., 2002. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc. Natl Acad. Sci. USA 99, 4061–4066. Ignarro, L.J., Bush, P.A., Buga, G.M., Wood, K.S., Fukuto, J.M., Rajfer, J., 1990. Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem. Biophys. Res. Commun. 170, 843–850. Jones, R.W., Rees, R.W., Minhas, S., Ralph, D., Persad, R.A., Jeremy, J.Y., 2002. Oxygen free radicals and the penis. Expert Opin. Pharmacother. 3, 889–897. Junqueira, V.B., Barros, S.B., Chan, S.S., Rodrigues, L., Giavarotti, L., Abud, R.L., Deucher, G.P., 2004. Aging and oxidative stress. Mol. Aspects Med. 25, 5–16. Khan, M.A., Calvert, R.C., Sullivan, M.E., Thompson, C.S., Mumtaz, F.H., Morgan, R.J., Mikhailidis, D.P., 2000. Normal and pathological erectile function: the potential clinical role of endothelin-1 antagonists. Curr. Drug Targets 1, 247–260. Kloner, R.A., 2003. Erectile dysfunction in the cardiac patient. Curr. Urol. Rep. 4, 466–471. Kloner, R.A., Speakman, M., 2002. Erectile dysfunction and atherosclerosis. Curr. Atheroscler. Rep. 4, 397–401. Magee, T., Fuentes, A.M., Garban, H., Rajavashish, T., Marquez, D., Rodriguez, J.A., Rajfer, J., Gonza´lez-Cadavid, N.F., 1996. Cloning of a novel neuronal nitric oxide synthase expressed in penis and lower urinary tract. Biochem. Biophys. Res. Commun. 226, 145–151. Magee, T.R., Ferrini, M., Garban, H., Vernet, D., Mitani, K., Rajfer, J., Gonzalez-Cadavid, N.F., 2002. Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase cDNA. Biol. Reprod. 67, 1033–1041. Magee, T., Zeller, C.B., Ferrini, M., Davila, H., Vernet, D., Burnett, A.L., Rajfer, J., Gonza´lez-Cadavid, N.F., 2003. A protein inhibitor of NOS (PIN) is expressed in the rat and mouse penile nerves and co-localizes with penile neuronal NOS (PnNOS). Biol. Reprod. 68, 478–488. McKenna, K., Adam, M.A., Bivalacqua, T., Coolen, L., Gonzalez-Cadavid, N.F., Hedlund, P., Park, K., Pescatori, E., Rajfer, J., Sato, Y., 2004. Experimental models for the study of male sexual dysfunction. In: Proceedings of the Second World Health Organization Workshop on Erectile Dysfunction (in press). McVary, K.T., Rathnau, C.H., McKenna, K.E., 1997. Sexual dysfunction in the diabetic BB/WOR rat: a role of central neuropathy. Am. J. Physiol. 272, R259–R267. Melman, A., Gingell, J.C., 2003. The epidemiology and pathophysiology of erectile dysfunction. J. Urol. 161, 5–11.
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Mills, T.M., Lewis, R.W., Wingard, C.J., Linder, A.E., Jin, L., Webb, R.C., 2003. Vasoconstriction, RhoA/Rho-kinase and the erectile response. Int. J. Impot. Res. 15 (Suppl 5), S20–S24. Montorsi, F., Briganti, A., Salonia, A., Deho’, F., Zanni, G., Cestari, A., Guazzoni, G., Rigatti, P., Stief, C., 2003. The ageing male and erectile dysfunction. BJU Int. 92, 516–520. Montorsi, F., Briganti, A., Salonia, A., Rigatti, P., Burnett, A.L., 2004. Current and future strategies for preventing and managing erectile dysfunction following radical prostatectomy. Eur. Urol. 45, 123–133. Moody, J., Vernet, D., Laidlaw, S., Rajfer, J., Gonza´lez-Cadavid, N.F., 1997. Effect of long-term administration of L-arginine on penile erection and nitric oxide synthase in the rat. J. Urol. 158, 942–947. Moreland, R.B., 2000. Pathophysiology of erectile dysfunction: the contributions of trabecular structure to function and the role of functional antagonism. Int. J. Impot. Res. 12 (Suppl 4), S39–S46. Musicki, B., Palese, M.A., Crone, J.K., Burnett, A.L., 2004. Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection. Biol. Reprod. 70, 282–289. Peng, H., Huard, J., 2003. Stem cells in the treatment of muscle and connective tissue diseases. Curr. Opin. Pharmacol. 3, 329–333. Poon, H.F., Calabrese, V., Scapagnini, G., Butterfield, D.A., 2004. Free radicals: key to brain aging and heme oxygenase as a cellular response to oxidative stress. J. Gerontol. A Biol. Sci. Med. Sci. 59, M478–M493. Qian, A., Rajfer, J., Gonzalez-Cadavid, N.F., 2003. Interactions of neuronal nitric oxide synthase with potential regulatory proteins expressed in the rat penis. J. Urol. 169, 304. Rajfer, J., Aronson, W.J., Bush, P.A., Dorey, F.J., Ignarro, L.J., 1992. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neuro-transmission. N. Engl. J. Med. 326, 90–94. Richardson, D., Vinik, A., 2002. Etiology and treatment of erectile failure in diabetes mellitus. Curr. Diab. Rep. 2, 501–509. Russell, S., Nehra, A., 2003. The physiology of erectile dysfunction. Herz 28, 277–283. Sato, Y., Shibuya, A., Adachi, H., Kato, R., Horita, H., Tsukamoto, T., 1998. Restoration of sexual behavior and dopaminergic neurotransmission by long term exogenous testosterone replacement in aged male rats. J. Urol. 160, 1572–1575. Seftel, A.D., 2003. Erectile dysfunction in the elderly: epidemiology, etiology and approaches to treatment. J. Urol. 169, 1999–2007.
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Review
Molecular pathophysiology and gene therapy of aging-related erectile dysfunction N.F. Gonzalez-Cadavida,b,*, J. Rajfera,b a
Harbor-UCLA Research and Education Institute, Urology, Bldg. F-6, 1124 West Carson Street, Torrance, CA, USA b Department of Urology, UCLA School of Medicine, Los Angeles, CA, USA Received 27 May 2004; accepted 12 June 2004 Available online 7 October 2004
Abstract Erectile dysfunction (ED) is a major public health problem that seriously affects the quality of life of patients and their partners. ED is mainly associated with vascular disease, diabetes, smoking, and radical prostatectomy, and its prevalence increases significantly with aging. Vasculogenic ED, specifically corporal veno-occlusive dysfunction (CVOD), is caused by the impairment of the relaxation of the smooth muscle in the penile corpora cavernosa and occurs in 2/3 of cases, whereas the less common neurogenic ED is due to a defective nitrergic neurotransmission triggered by the sexual stimulus, either at the central hypothalamic and spinal levels or at the penile nerves. Based on animal and cell studies, neurogenic ED is assumed to be caused mainly by: (a) an insufficient synthesis of nitric oxide (NO) due to a decrease in the levels of the penile neuronal nitric oxide synthase (PnNOS) or the impairment of its regulation by protein effectors (NMDA receptor, protein inhibitor of nNOS: PIN), occurring in the neuronal bodies or nerve terminals, or (b) a loss of the cells themselves by apoptosis caused by the induction of inducible NOS (iNOS) and the production of peroxynitrite. In contrast vasculogenic ED, although may involve endothelial damage and down-regulation of endothelial NOS (eNOS), appears to be mainly caused by the relative loss of smooth muscle cells and replacement by collagen fibers (fibrosis) that impairs tissue compliance. In this case, iNOS induction may not be deleterious, but a defense mechanism preventing excessive collagen deposition. Gene therapy to the penile corpora cavernosa of cDNAs expressing PnNOS or eNOS, or counteracting PIN, has been effective in ameliorating ED in the aging rat model that exhibits both neurogenic ED and CVOD. cDNA constructs for other genes involved in the control of penile erection have also been successfully tested. Gene transfer into the penis may soon translate to the clinic as a therapy aimed to cure the underlying conditions in ED, including fibrosis, as opposed to the facilitation of erection on demand offered by the current oral therapies. q 2004 Elsevier Inc. All rights reserved. Keywords: Penis; Corpora cavernosa; Hypothalamus; Neuronal nitric oxide synthase; Endothelial nitric oxide synthase; Protein inhibitor of nNOS; Nitric oxide; Fibrosis; Oxidative stress; Inducible nitric oxide synthase; Phosphodiesterase
1. Introduction Penile erection is a complex process involving a series of sequential and coordinating events between: (a) the central Abbreviations: ED, erectile dysfunction; ROS, reactive oxygen species; NO, nitric oxide; iNOS, inducible nitric oxide synthase, NOS II; nNOS, neuronal nitric oxide synthase, NOS I; eNOS, endothelial nitric oxide synthase, NOS III; CVOD, corporal veno-cclusive dysfunction; PDE5A, phosphodiesterase 5A; PIN, protein inhibitor of nNOS; PVN, paraventricular nucleus; MPOA, medial preoptic area; EFS, electrical field stimulation. * Corresponding author. Address: Harbor-UCLA Research and Education Institute, Urology, Bldg. F-6, 1124 West Carson Street, Torrance, CA 90502, USA. Tel.: C1 310 222 3824; fax: C1 310 222 1914. E-mail address: [email protected] (N.F. Gonzalez-Cadavid). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.06.022
nervous system, mainly at the cortical, hypothalamic and spinal levels, which have connections to the efferent and afferent nerves of the penis that trigger and conduct the neurotransmission signals to the organ; and (b) the vascular system, specifically the smooth muscle in the corpora cavernosa and within the media of the arteries that deliver blood to the penis (Allard and Giuliano, 2001; Andersson, 2003; Russell and Nehra, 2003). Indeed, the penis can be considered simply as an extension of the vascular system, and in actuality, it is the relaxation of the smooth muscle both in the helicine arteries and in the trabeculae that causes the blood to enter and pool into the cavernous sinusoids. This active process of smooth muscle relaxation is under the regulation of the cavernous nerves and is followed by
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the subsequent passive compression of the penile veins against the underside of the comparatively rigid tunica albuginea within the corporal bodies, thereby preventing the egress of blood out of the corpora. It is this latter process that allows penile erection to sustain. Erectile dysfunction (ED), the inability to achieve or maintain an erection of sufficient rigidity for vaginal penetration and completion of the sexual act, is a common condition affecting 20–30 million men in the USA. This disorder is highly associated with aging, particularly in men older than 50 years (Seftel, 2003; Montorsi et al., 2003). Major concurrent factors for organic, as opposed to psychogenic ED, are cardiovascular disease, in particular atherosclerosis and hypertension (Kloner and Speakman, 2002; Kloner, 2003), diabetes (Richardson and Vinik, 2002), and radical prostatectomy (Burnett, 2003). These disorders lead to two forms of ED, vasculogenic and neurogenic, which often may present together (Siroky and Azadzoi, 2003). Vasculogenic ED is the most prevalent condition, affecting nearly 80% of patients with organic impotence. In the majority of patients with vasculogenic ED, the main vasculopathy is an impairment in smooth muscle relaxation within the corporal tissue itself, and this clinically may lead to inadequate compression of the subtunical penile veins, causing corporal veno cclusive dysfunction (CVOD) or penile venous leak (Moreland, 2000; Wespes, 2002; Siroky and Azadzoi, 2003). Endothelial damage, both in the penile blood vessels and the lining of the trabecular cisternae may also contribute significantly. Neurogenic ED results from the impairment of neurotransmission to the smooth muscle either because of an injury or neuropathy subsequent to trauma, surgery, diabetes, or aging (Montorsi et al., 2004). In many instances, neurogenic ED is often associated with CVOD as a result of the relative atrophy of the trabecular smooth muscle (User et al., 2003). Our emphasis in this presentation will be on our recent work in the last 3 years which continued to be focused on the modulation of NO synthesis in the penile tissue and its role in penile erection and the prevention of penile fibrosis as an underlying cause of ED. A pertinent general background will be provided to discuss its implications, but the discussion is not intended as a comprehensive review of the area.
the erectile response (Gonza´lez-Cadavid et al., 1999; Melman and Gingell, 2003; Gonzalez-Cadavid and Rajfer, 2004b). The reaction leading to NO production is the conversion of L-arginine into citrulline, and this in the penis occurs in the nerve terminals of the corpora cavernosa, involving the neuronal NOS (nNOS or NOS I), and presumably as well in the endothelium of the cavernosal cisternae and arteries, involving endothelial NOS (eNOS or NOS III). The primary trigger for erection is the nitrergic neurotransmission through the non-adrenergic non-cholinergic nerves catalyzed by the stimulation of nNOS activity via Ca2C binding to the nNOS associated calmodulin. This process is in part modulated by Ca2C fluxes through another nNOS associated protein: the NMDA receptor (Gonza´lez-Cadavid et al., 2000b). The eNOS activation via sheer stress, Ca2C flux, and phosphorylation, appears to be ancillary and supports NO synthesis in the corpora cavernosa during erection (Hurt et al., 2002; Musicki et al., 2004). The NO liberated from penile nerves and endothelium activates guanilyl cyclase in the trabecular and penile arterial smooth muscle, respectively, causing the elevation of cGMP levels that stimulate a cyclic GMP phosphokinase (PKG) and the subsequent reduction in cytoplasmic Ca2C (Fig. 1 top). This leads to smooth muscle relaxation and penile erection, counteracting adrenergic and related agents that maintain its tone during the flaccid stage. In turn, cGMP breakdown is catalyzed in the smooth muscle by cGMPdependent phosphodiesterase 5A (PDE5A), the target of the PDE5 inhibitors used orally for the treatment of erectile dysfunction, such as sildenafil (VIAGRA). In addition, NO
2. The role of nitric oxide synthases in the central and peripheral control of erection Since, the first report by Ignarro et al. (1990) identifying nitric oxide (NO) as the mediator of corpora cavernosa relaxation in the rabbit, and subsequently in 1992 in the human (Rajfer et al., 1992), corpora cavernosa, a considerable number of studies, including those from our laboratory, have elucidated the respective roles of the nitric oxide synthase (NOS) isoforms in synthesizing NO involved in
Fig. 1. Schematic representation of the nitric oxide (NO) signaling pathway responsible for penile erection, and its metabolism that affects the smooth muscle, in the penile corpora cavernosa. Top panel: signaling pathway triggered by PnNOS activation upon sexual stimulation, leading to NO release and the elevation of cGMP that causes smooth muscle relaxation. The endothelium contributes to relaxation through NO produced by eNOS. Bottom panel: NO metabolism by reaction with OK 2 and other radicals (ROS), that is considerably stimulated by iNOS induction, a NOS isoform that does not appear to mediate erection.
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Fig. 2. Schematic representation of the functional domains encoded in the cDNA for penile neuronal nitric oxide synthase (PnNOS), the enzyme that generates NO as the main neurotransmitter causing penile erection. The 102 bp insert, encoding a 34 amino acid stretch, is present in PnNOS and absent in the brain nNOS counterpart. PDZ: region that binds PnNOS modulators important for the control of erection; Heme: heme binding region (PnNOS and nNOS are hemeproteins); CaM: calmodulin binding site where Ca2C binding activates the enzyme; FMN, FAD, NADPH: sites for binding of the respective coenzymes.
metabolism leads to other products (bottom), discussed in another section below. A few years ago, our group cloned from a penile cDNA library a variant of the brain type nNOS having a 34 amino acid insert between exons 16 and 17, that was named penile nNOS (PnNOS) (Fig. 2) and showed that it was the nNOS variant expressed in the penis and prostate, whereas the nNOS brain variant was expressed in the bladder and other urogenital organs (Magee et al., 1996). PnNOS seems to be identical to nNOSu expressed in the skeletal muscle (Silvagno et al., 1996). In a more recent work (Gonza´lez-Cadavid et al., 2000a), we have shown that PnNOS is expressed in penile nerves as both the fulllength a-form and the truncated splicing b variant lacking a portion of the N-terminal region named PDZ (Fig. 3). This is important because this is the region where at least in the case of the brain nNOS, the NMDA receptor binds (Gonza´lez-Cadavid et al., 2000b), as well other nNOS modulators, the protein inhibitor of nNOS (PIN), and CAPON. The nNOS knockout mouse, engineered by the blockade of the expression of the nNOS gene, lacks as expected in the a-form, but by an unknown mechanism the b form is expressed. This explains the retention of erectile function in this knockout animal, since the residual PnNOS-b retains NOS activity for NO synthesis, and in addition it is not able to bind PIN, and therefore is insensitive to this inhibition (Gonza´lez-Cadavid et al., 2000a). By applying a combination of immunohistochemistry, dual immunofluorescence, DNA cloning, and western blot/RT-PCR procedures, we have been able to show that in the cavernosal and dorsal nerves of the penis, there is co-localization of PnNOS with both PIN and the NMDA receptor, suggesting the interaction of these three proteins in the modulation of NO synthesis during nitrergic neurotransmission to and from the penis, although we could not show a direct inhibition of PnNOS by PIN (Magee et al., 2003). As a natural corollary, we have found that essentially the same PnNOS/PIN/NMDA receptor co-localization occurs in the hypothalamic regions known to control penile erection, ejaculation, and other sexual responses (paraventricular nucleus: PVN,
and medial preoptic area: MPOA), and in the spinal cord at the S1–S4 levels which is also involved in the control of penile erection (Ferrini et al., 2003). This suggests that PnNOS is an essential component in the nitrergic neurotransmission controlling erection, along its entire regulatory axis from the brain via the spinal cord to the smooth muscle of the penis itself. We also found that PnNOS in the hypothalamus is also expressed in oxitocinergic neurons (Ferrini et al., 2003), previously demonstrated by other groups to be involved in NO release in the same regions, that mediate the central nitrergic control of penile erection. It is therefore likely that PIN and the NMDA receptor, and possibly other proteins that have recently been identified in the penis (Qian et al., 2003), regulate PnNOS activation to trigger and transduce nitrergic responses to sexual stimulation.
Fig. 3. Interaction of nNOS variants with protein modulators of PnNOS enzyme activity, that is postulated to contribute to the control of penile erection. Top: N-terminal exons in the cDNA encoding the 160 kDa fulllength nNOS or PnNOS proteins (a-form) encompassing the PDZ proper, and neighbor, sequences where the protein modulators (NMDA receptor (NMDAR), PIN and CAPON) bind. ATG: translation initiation for nNOS or PnNOS protein synthesis. Bottom: exon 2, containing the binding regions, is missing in the 130 kDa b-form of nNOS or PnNOS protein that is expressed in the nNOSK/K mouse and maintains penile erection in this mouse.
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3. Molecular pathophysiology of aging-related erectile dysfunction Thanks to the intensive research conducted in animal (McKenna et al., 2004; Wang et al., 2002) and cell models (Moreland, 2000; Gonzalez-Cadavid et al., 2004), it is now clear that at the molecular and cellular level, ED is due to four main causes: (a) an insufficient production of NO during nitrergic neurotransmission elicited by sexual stimulation, mostly at the penile nerve terminals or possibly at the brain and spinal cord; this impairment can de due to the loss of neuronal bodies and neural connections to and from the penis, or to a down-regulation of NOS activity and/ or expression in the respective organs (Garban et al., 1995; Carrier et al., 1997; Vernet et al., 1998; Gonza´lez-Cadavid et al., 2000a; Ferrini et al., 2001a,b; Magee et al., 2003), and also occuring in diabetes-related ED (Vernet et al., 1995; McVary et al., 1997); (b) an ancillary reduction in NO levels from eNOS down-regulation due to endothelial damage, in diabetes and vascular disease (Vernet et al., 1995; McVary et al., 1997; Bivalacqua et al., 2000, 2003, 2004); (c) a putative excessive release of adrenergic compounds that increase the tone of the corpora cavernosal smooth muscle, mainly through endothelin and rho kinase activation (Khan et al., 2000; Mills et al., 2003); and (d) an impairment of the relaxation of this target smooth muscle by endogenous factors, resulting mainly from either a putative excessive cGMP degradation by PDE5A, or from a relative loss of smooth muscle cells in the penis and their replacement by collagen fibers (fibrosis) (Ferrini et al., 2001a; User et al., 2003; Yaman et al., 2003; Magee et al., 2003). Aging in otherwise healthy patients is assumed to be associated with the three processes described above, and this is even compounded by the additive effects of cardiovascular disease, and other co-morbidities, e.g. diabetes, that are prevalent in old age. It is well accepted that the rat is an excellent model for aging-related ED, since aged rats are known to exhibit two main features of this disorder: (a) a reduction in erectile function and a decreased mounting and copulatory behavior (Sato et al., 1998); and (b) a reduced response to electrical field stimulation (EFS) of the cavernosal nerve as measured by the reduction in the ratio between the maximal intracavernosal pressure and the mean arterial pressure (Burnett et al., 1992; Garban et al., 1995; McKenna et al., 2004). This is associated with a reduction in the content of nitrergic penile nerves (Carrier et al., 1997), and in the down-regulation of NOS activity (Garban et al., 1995), thus fitting into the assumption that aging in the rat leads to a neurogenic type of ED. However, we have recently shown that in the aged rat when compared to the young rat, there is an altered response to dynamic infusion cavernosometry as measured by the erection achieved pharmacologically with intracorporeal papaverine injection or mechanically by the rate of infusion with saline into the corpora, or the rate of detumescence (Davila et al., 2004a). We believe this finding is important
because it demonstrates that at least one of the common wild type laboratory rat strains, the Fisher 344 rat, exhibits, in addition to the neuropathic component, a marked venous leak (CVOD) which as mentioned, is the most prevalent type of ED in men. Therefore, the rat is an excellent model to study the molecular and cellular pathophysiology of both neurogenic and vasculogenic aging-related ED. The mechanism for the neurogenic component of the age-related deterioration of the erectile response involves, in addition to the already described decrease in nitrergic penile nerves, a marked increase in the old rats in the apoptotic index in the hypothalamic PVN and MPOA, the regions that control erection (Ferrini et al., 2001b). These changes occurred in the glia and in oxytocinergic and GnRH neurons associated with erection and testosterone production, two of the processes affected during reproductive decay with aging. In turn, the vasculogenic component of ED in the aged rat is probably due to a similar increase in the apoptotic index in the penile trabecular (Ferrini et al., 2001a) and arterial smooth muscle (Ferrini et al., 2004). This, combined with an increase in fibrosis in these tissues, explains the loss in corpora cavernosal compliance with aging which is responsible for its impaired response to EFS and/or intracorporeal papaverine. These studies confirmed our previous observations that in the aged rat both the hypothalamus as a whole (Vernet et al., 1998) and the penis (Ferrini et al., 2001a) harbored a considerable ‘spontaneous’ expression of the mRNA and protein corresponding to the third NOS isoform, the inducible NOS (iNOS or NOS II). This expression of iNOS that was seen in both organs in the aged animals was markedly increased from the negligible basal levels found in the young rats. It should be stressed that iNOS is not known to participate in the physiological control of penile erection at any level, be it central or peripheral, since in contrast to nNOS or eNOS, iNOS does not have the type of enzyme activity regulation required for the fast erectile response to sexual stimulus (Gonzalez-Cadavid and Rajfer, 2004a). iNOS is only controlled at the transcriptional level (leading to changes in protein levels) which is not fast enough to either turn on or turn off NO synthesis in a time mode appropriate to sexual response. When iNOS expression was studied in several hypothalamic regions (Ferrini et al., 2001b), it was detected in the PVN and MPOA, in both glia and neurons, specifically in those expressing oxytocin and GnRH, in parallel to the increase in the apoptotic index. Since the sustained levels of NO produced from iNOS give rise to the pro-apoptotic compound peroxynitrite, or ONOOK (Fig. 1 bottom), it follows that aging-related iNOS induction in the brain is deleterious since it leads to a considerable loss of neurons in those regions related to the control of penile erection. The cumulative production of NO, and hence of peroxynitrite, can be detected in tissue sections by immunohistochemistry for its metabolite, 3-nitrotyrosine, bound to different proteins, mainly
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collagen, and its co-localization with apoptosis may be assayed with the TUNEL procedure. In contrast, in the corporal smooth muscle, both in the trabecular tissue and the arterial media, aging-related induction of iNOS, although may contribute to an increased apoptotic rate, appears on the whole to be a defense mechanism against fibrosis. Evidence for this hypothesis is based on the exacerbation of the already excessive deposition and disorganization of collagen fibers in the aged penis that leads to loss of compliance in both tissues when iNOS activity is blocked long-term by L-NIL, an iNOS inhibitor that does not affect nNOS or eNOS (Ferrini et al., 2004). This resembles the situation observed in a rat model for a localized fibrotic plaque in the tunica albuginea of the penis, Peyronie’s disease, where chronic L-NIL administration considerably worsens the fibrosis (Ferrini et al., 2002; Vernet et al., 2002; Gholami et al., 2002; Gonzalez-Cadavid et al., 2002; Gonzalez-Cadavid and Rajfer, 2004c). Conversely, chronic administration of L-arginine as NOS substrate (which should increase NO from all NOS isoforms) counteracts plaque development (Valente et al., 2003). This L-arginine inhibition of fibrosis may also explain the amelioration of ED in aged rats subjected to a similar long-term treatment with the amino acid (Moody et al., 1997), since in addition to the expected direct improvement in the relaxation of the trabecular smooth muscle by the release of higher levels of NO, it may be speculated that the smooth muscle/collagen ratio may have been increased in this tissue. In all these conditions associated with aging and impotence, i.e. neuronal cytotoxicity in critical hypothalamic regions, fibrosis of either the tunica albuginea or the trabecular smooth muscle, or arteriosclerosis or stiffness of the penile arteries, oxidative stress appears to be an underlying factor (Jones et al., 2002). The production of reactive oxygen species (ROS) may be pro-fibrotic by releasing TGFb and other cytokines, or also cause cell death by apoptosis. This agrees with the long held theory that aging may be the result of an accumulation of oxidative compounds (Poon et al., 2004; Junqueira et al., 2004). The high levels of NO produced from the spontaneous induction of iNOS are known to: (a) quench ROS by forming peroxynitrite, and (b) also inhibit collagen synthesis and promote its breakdown (Gholami et al., 2002; Gonzalez-Cadavid et al., 2002). However, peroxynitrite per se induces apoptosis, and therefore iNOS induction during aging has a yin-yang effect on the different levels of control of penile erection. The overall result would depend on the tissue environment and cell types: cytotoxic in the hypothalamic neurons, and antifibrotic in the penile smooth muscle. Although in the penis, it is certainly possible that the pro-apoptotic effect of NO may contribute to some smooth muscle loss, is the NO/ROS balance, the main factor that will ultimately determine if the smooth muscle/collagen ratio, and hence tissue compliance, is reduced or not by oxidative stress Gonzalez-Cadavid and Rajfer (2004c).
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4. Gene therapy of ED and penile fibrosis Seven years ago, the first use of gene therapy for the treatment of ED was reported by our group who demonstrated that the pharmacological transfer of a plasmid expressing iNOS was effective in correcting ED in the aged rat (Garban et al., 1997; Gonzalez-Cadavid et al., 2004). This proof of principle was based on the fact that since NO is the chemical mediator of penile erection, higher NOS levels would lead to a higher NO output and overcome the defective cavernosal smooth muscle compliance and/or a putative excessive contractile tone present in the aged animal. By cloning iNOS from the rat and human penis and injecting the iNOS cDNA into the corpora cavernosa of aged rats, it was demonstrated that the expression of the recombinant iNOS protein occurred in the corpora cavernosa and corrected the defective erectile response for at least 10 days, without causing priapism or other side effects. Why would there be a need for gene therapy for ED when the oral PDE5 inhibitors for the treatment of ED are available today? Simply because even the most effective PDE inhibitor trials indicate only a 50–65% success rate, and these drugs are palliative in that they treat only the symptoms (erectile failure) but do not aim to cure the underlying condition (Gonzalez-Cadavid et al., 2004). Therefore, gene therapy offers the possibility that in theory a single injection of the cDNA construct into the penis may ameliorate ED for months or even years, and lead to a partial or total cure of the defective neurotransmission or the underlying impairment of its target vascular smooth muscle. More recently, we showed that the cDNA for the PnNOS variant cloned from both the rat and human penis corrected ED for at least 17 days in the aged rat (Magee et al., 2002). Rather than plasmid/liposome preparations, in this study the combination of a helper-dependent adenoviral vector (AdV) and a new procedure (in vivo electroporation) to increase uptake of the cDNA construct in the corpora cavernosa was used. This type of vector reduced the risk of immune reactions, a risk with first generation AdV, and combined with electroporation allowed a comparatively low viral load to increase PnNOS levels in the penis, and to elevate the MIP/MAP ratio to virtually the values observed in young rats. In as yet unpublished experiments, we applied a different approach based on the reverse concept, i.e. instead of elevating NOS levels we would attempt to block inhibition of PnNOS activity by one of its known protein inhibitors, specifically PIN, that, as stated above, co-localizes with PnNOS and the NMDA receptor in the rat hypothalamus, spinal cord and penis. A plasmid construct encoding the antisense cDNA for PIN, that inhibits the mRNA translation into protein by forming a duplex RNA, was prepared, and when injected and electroporated into the penis, it corrected the ED in the aged rat for at least 30 days (Davila et al., 2003a). An alternative procedure was to prepare the siRNA
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(small interference RNA) for PIN, a very novel and more effective approach than using antisense sequences that directly breaks down the specific mRNA. In preliminary ongoing studies, which need confirmation, the construct was administered to rats and we observed, as with the antisense vector, a stimulation of the erectile response (Davila et al., unpublished). Our data, together with studies from other groups showing that eNOS is also effective (GonzalezCadavid and Rajfer, 2004b; Bivalacqua et al., 2000, 2003, 2004), support the hypothesis that NOS gene therapy, irrespective of the isoform, or the use of constructs targeted to NOS regulators, is effective in improving erectile function, and that both viruses and plasmids may be adequate vectors. What started a few years ago as a proof of concept has now been translated into an active field of research. Additional genes have proven to be as or even more effective than those for the NOS isoforms, and some of them are being considered for future clinical trials. These genes are related to either cavernosal relaxation, such as maxi KC channels (hSlo) or calcitonin gene-related peptide, growth factors, such as VEGF, BDNF, and neurotrophic factor, counteraction of oxidative stress such as superoxide dismutase, or inhibition of contractile factors such as rho kinase (see Christ, 2003; Gonzalez-Cadavid and Rajfer, 2004b). Gene therapy for ED is not limited to approaches aimed to treat the functional impairment of cavernosal relaxation or the defective neurotransmission associated with ED. Studies are now attempting to reverse the histological changes that impair compliance during aging, e.g. trabecular tissue fibrosis. The first example of this kind was recently reported by our laboratory (Davila et al., 2003b, 2004b), by revisiting the use of iNOS cDNA, but this time as an antifibrotic agent, a concept gleaned from our previous studies in the aging rat and in the rat model of Peyronie’s disease. In the latter model, often associated with ED, we induced a fibrotic plaque by injection of fibrin into the tunica albuginea of the rat, as the target to demonstrate the effectiveness of iNOS in reducing ROS levels and collagen deposition. By giving the plasmid cDNA for iNOS directly into a well-formed plaque, we could induce its nearly complete regression, while simultaneously reducing oxidative stress. We are now preparing the AdV construct of iNOS in order to obtain longer effects and be able to correct or prevent smooth muscle fibrosis present in CVOD, although a stable chemical NO donor may be more effective. This approach needs still assessment by further studies of potential deleterious effects. The field of gene therapy for ED is in its initial phase of development, but it may be considerably expanded in the near future by its combination with ex vivo gene therapy, using either smooth muscle or stem cells from a patient that could be engineered to express a particular protein and followed by allograft cell re-implantation into the corpora cavernosa (Cao et al., 2002; Peng and Huard, 2003;
Gonzalez-Cadavid and Rajfer, 2004b). Our approach is based on the premise that adult stem cells from different sources can evolve into a specific cell lineage in vivo, according to the type of paracrine factors and cell-to-cell interactions in the host tissue, decreasing the risk of poor proliferation and immunorejection associated with fully differentiated cells, in this case smooth muscle (Singh et al., 2003; Artaza et al., 2004; Vernet et al., 2004). Other promising approaches are the use of DNA vectors allowing more prolonged expression of the inserted sequence, such as the adeno-associated virus, herpesvirus, lentivirus, or hybrid AAV/AdV virus. The utilization of tissue-specific gene promoters for preferential expression of the recombinant DNA in a given tissue, irrespective of disseminated uptake, or the design of novel promoter casettes where the recombinant cDNA is placed under a promoter controlled by very low, non-hazardous doses of a drug, in order to get a time-specific and dose-dependent regulation, is also under active investigation by different investigators (Gonzalez-Cadavid and Rajfer, 2004b). Depending on the outcome of the first clinical trials with plasmid constructs in men, these other modalities may serve to improve the efficacy of gene therapy for ED. It is not too speculative to assume that this approach may eventually help achieve a cure, or at least a long-term amelioration, of tissue and biochemical alterations underlying aging-related ED and penile fibrosis. This would replace dependence on the palliative pharmacological therapies currently available, or reduce dosages if biological (e.g. gene therapy) with chemical (e.g. PDE inhibitors) combination regimens are devised (Bivalacqua et al., 2004).
Acknowledgements Experimental studies by the authors quoted in this review were funded mainly by NIH grants R01DK-53069 and G12RR-03026, and by a grant from the Eli and Edythe L. Broad Foundation and the Peter Morton Foundation.
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