New approaches to the design and discovery of therapies to prevent erectile dysfunction.

Review

New approaches to the design and discovery of therapies to prevent erectile dysfunction 1.

Introduction

2.

Physiology of penile erection

3.

Role of L-arginine and erectile

Haroldo A Toque† & Robert William Caldwell Georgia Regents University, Augusta, Medical College of Georgia, Department of Pharmacology and Toxicology, Augusta, GA, USA

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Laurentian University on 11/24/14 For personal use only.

function 4.

Literature of ED

5.

Current standard conservative treatment for ED

6.

Novel therapies for ED

7.

Role of arginase

8.

Conclusion

9.

Expert opinion

Introduction: Nitric oxide (NO) is critically involved in erectile function. Since NO synthase (NOS) and arginase compete for the same substrate L-arginine, limiting arginase activity may provide more NO and thus be a beneficial therapeutic approach to erectile dysfunction (ED). In the corpora cavernosa, excessive arginase activity/expression has been implicated through studies of preclinical and clinical models of ED. Further, the inhibition of arginase has shown to increase vascular system relaxation and enhance blood flow in penile circulation. Further studies, therefore, looking at therapies targeting arginase could prove to be clinically useful. Areas covered: The authors review gene- and cell-based therapies, the involvement of RhoA/Rho-kinase (ROCK), MAPK and arginase in ED. Expert opinion: Extensive literature supports the view that upregulated arginase activity in cavernosal tissue can reduce NOS function and NO production. Excessive arginase activity has been shown to contribute to the progression of aging-, hypertension- and diabetes-induced vascular dysfunction as well as ED. Earlier studies have shown that RhoA/ROCK and subsequent activation of p38 MAPK mediate elevation of arginase expression/activity in diabetic and hypertensive mice. Reducing corporal arginase activity by gene-based or pharmacological therapy and/or inhibition of upstream regulators of arginase expression may provide novel therapeutic approaches in the management of ED. Keywords: arginase, cavernosal smooth muscle relaxation, erectile dysfunction, nitric oxide, penile erection Expert Opin. Drug Discov. (2014) 9(12):1447-1469

1.

Introduction

Erectile function is an important component of quality of life in men. Impairment of erectile function can affect total health, relationships, overall quality of life and potentially procreative advancement [1]. Over the past 20 years, erectile dysfunction (ED) has been extensively studied because over 30 million men suffer with this disorder in the USA and the number is expected to rise considerably over the next 25 years, impacting > 300 million men by 2025 [2]. The fact that ED often coexists with obesity, hypertension, cigarette smoking, aging and diabetes has led to the conclusion that ED is predominantly a disease of vascular origin [3]. Beyond its association with vascular risk factors, ED is a strong predictor for coronary artery disease mainly in men > 40 years [4]. Nitric oxide (NO), synthesized from L-arginine by NO synthase (NOS), is the key physiological mediator of penile erection [1]. Given that NOS and arginase share and compete for their common substrate L-arginine, NO bioavailability or biosynthesis is likely linked to the regulation of arginase activity. Arginase is highly expressed in corpus cavernosum (CC) and is implicated in ED [3]. Pharmacological 10.1517/17460441.2014.949234 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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H. A. Toque & R. W. Caldwell

Article highlights. . .

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New therapies are needed for erectile dysfunction (ED). An overview of currently used and available conservative therapy options for ED is provided. In particular, pharmacotherapies such as melanocortin receptor agonist, testosterone replacement, supplementation of L-citrulline or L-arginine or their combination and stimulators of soluble guanylyl cyclase are described. Treatments that are not just palliative but restore erectile function, such as shockwave, gene and stem cell therapies are also addressed. Enhanced arginase activity has been strongly implicated in the pathogenesis of endothelial dysfunction and impaired erectile function. In models of ED such as diabetes, hypertension and aging, inhibition or deletion of arginase gene has shown to increase nitric oxide bioavailability and cGMP levels accompanied with enhanced erectile function. Strategies for limiting arginase activity and expression and/or inhibition of its upstream regulators promise new therapy for ED.

This box summarizes key points contained in the article.

therapies have focused essentially in NO/cGMP signaling pathway in the treatment of ED. The oral PDE5 inhibitors have become the first-line treatment for ED [5]. However, the efficacy of PDE5 inhibitors would be reduced when NO bioavailability is decreased. Inhibition of PDE5 has limitations in men with impaired penile vascular flow due to advanced diabetes or cardiovascular disease (CVD). Thus, either significant endothelial dysfunction or autonomic neuropathy may limit the efficacy of a PDE5 inhibitor. New therapeutic strategies including melanocortin receptor (MCR) targets, shockwave therapy, combined therapy of testosterone and a PDE5 inhibitor, supplementation of L-citrulline or L-arginine, gene and cell-based therapy, soluble guanylyl cyclase (sGC) stimulators and inhibition of arginase may provide better overall treatment for ED. 2.

nerves. These nerves are responsible for the release of NO to the trabecular smooth muscle, which results in relaxation. Because the CC is surrounded by the tunica albuginea, a tissue that does not distend easily, the increased blood flow to the penis enlarges the CC and enhances the intracavernosal pressure (ICP). This change causes mechanical compression of the emissary veins, which voids their ability to drain blood and thereby results in penile rigidity. In the flaccid state in which CC tissue is contracted, blood flow is limited and is used only for nutritional purposes. In this state, blood pressure within the CC is low. However, on arousal and erection, ICP increases to about 100 mmHg [7]. Therefore, penile erection includes sinusoidal relaxation, arterial dilation and venous compression [8]. Central neurotransmitters such as dopamine and oxytocin are also involved in a facilitatory effect on penile erection [9]. Increasing levels of dopamine in patients with Parkinson’s disease have shown to improve libido. Although both D1 and D2 dopamine receptors have been implicated in central erectile functions, the D2 receptor seems to have the predominant role [10]. Research on several compounds designed to act selectively on D2 receptors (ABT 724, PD 168077 and PIP 3EA) have been found to induce erectile activity when given systemically or into the lateral ventricles [11]. The mechanism and potency of these agents are similar to those observed for apomorphine [11,12]. Penile detumescence begins with cessation of neurotransmitter release, the breakdown of second messengers or activation of the a-adrenergic receptors on the cavernous arteries and trabecular smooth muscle, leading to a reduction in arterial inflow and a reduction of lacunar spaces. Contraction of the trabecular smooth muscle restores the venous outflow and the trapped blood is expelled and flaccidity returns [9]. In the pathophysiology context, several factors can disrupt the normal physiologic mechanisms involved in penile erection (Table 1). A mix of two or more factors, including those psychogenic and organic, is usually observed in patients with ED. The most important therapeutic targets in the pathophysiology of ED are addressed in Figure 1.

Physiology of penile erection 3.

Penile erection involves interaction between the central neural and peripheral pathways. Tumescence is a neurovascular event modulated by sensory stimulation (such as visual, tactile, olfactory and imaginative stimuli), psychological and hormonal factors. On sexual arousal, neurotransmitters are released from the cavernous nerve terminals and the penile endothelial cells, relaxing the cavernosal arteries and arterioles and increasing the penile blood flow. Evidences indicate that pudendal artery is the main supplier of blood (70%) to the CC of the penis [6]. Concomitantly, relaxation of the trabecular smooth muscle increases the compliance of the sinusoids, resulting in an engorgement of the penis with blood. Penile blood flow is mediated by the autonomic nervous system, which includes nonadrenergic noncholinergic (NANC) 1448

Role of L-arginine and erectile function

NO and erectile function It is well documented that NO is the main factor mediating penile erection. It is released from both endothelium and NANC nerves. When released, NO diffuses into adjacent smooth muscle cells of the CC, binds to sGC and converts GTP to 3¢,5¢- cGMP. In turns, cGMP activates protein kinase G (PKG) and exerts actions on ion channels, contractile regulatory proteins and PDE, which results in smooth muscle relaxation (Figure 2) [13-15]. Thus, at the onset of sexual stimulation, neuronal NOS (nNOS) induced by neuronal depolarization and endothelial NOS (eNOS) induced in response to shear forces brought on by dilation of arterial vessels and increased blood flow into the sinuses of the CC serve as the 3.1

Expert Opin. Drug Discov. (2014) 9(12)

New approaches to the design and discovery of therapies to prevent erectile dysfunction

Table 1. List of gene therapy in different models of ED.


Model of ED

Vector used

Gene target

Cell/tissue target

Aged rats Diabetic rats Diabetic rats Diabetic rats Aged rats

eNOS eNOS VEGF and Ang-1 EC-SOD EC-SOD

Adenovirus Adenovirus Adenovirus Adenovirus Adenovirus

Endothelial Endothelial Endothelial Endothelial Endothelial

Aged rat Aged rat Aged mice Castrated Hypercholesterolemic rats Aged rats Diabetic rats

nNOS PIN Anti-arginase VEGF VEGF

Adenovirus Cytomegalovirus Adenovirus Adenovirus Adenovirus

Neuronal Neuronal Endothelial Penile Penile

iNOS PKG1-a

Adenovirus Adenovirus

Penile Penile

Functional outcome

Ref.

Improved ICP and cGMP Improved ICP Improved ICP, cGMP, and eNOS levels Improved ICP and cGMP Reduced O2- formation and restored erection Improved ICP Improved ICP and cGMP Improved ICP and cGMP Preserved erectile function Enhanced CC angiogenesis and erectile function Ameliorated erectile function Restored PKG activity and improved ICP

[56] [58] [80] [72] [71] [54] [55] [88] [77] [80] [61] [83]

Ang-1: Angiopoietin-1; CC: Corpus cavernosum; EC-SOD: Extracellular superoxide dismutase; ED: Erectile dysfunction; eNOS: Endothelial nitric oxide synthase; ICP: Intracavernosal pressure; iNOS: Inducible nitric oxide synthase; nNOS: Neuronal nitric oxide synthase; O2-: Superoxide anion; PIN: Protein inhibitor of neuronal nitric oxide synthase; PKG1: cGMP-dependent protein kinase 1.

initiating factor in the erectile function process. PDE5 selectively degrades cGMP and attenuates the erectile process. This enzyme is highly expressed in the CC and controls the cGMP accumulation caused by NO signaling and consequently limits its relaxant actions [5]. Possible key causes of reduced formation of NO are: i) limitation of the substrate L-arginine; ii) increased levels of endogenous inhibitors of NOS in plasma and tissues; and iii) upregulation of vascular arginase levels. Endogenous NOS inhibitors and erectile function NO deficiency contributes to ED and is associated with reduced endothelial and nitrergic nerve functions. Elevated levels of endogenous NOS inhibitors such as asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), or N(G)-monomethyl-L-arginine have been reported in patients with adverse cardiovascular events for which endothelial dysfunction has been observed as a common etiology [16]. Increased ADMA has also been associated with impaired NO-mediated urethral, trigonal and cavernosal relaxation by pelvic ischemia [17]. ADMA is formed in endothelial cells by catabolism of proteins containing methylated arginine residues. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH), and reduced activity/expression of DDAH is reported to be responsible for elevation of ADMA [18-21]. Studies have indicated that increased ADMA levels are involved in reduced NOS activity and the pathogenesis of ED in animal and human models [21]. Concentrations of ADMA and SDMA are higher in those patients with arteriogenic ED compared with those of nonarteriogenic ED and controls. The authors concluded that increased ADMA and SDMA concentrations contribute to NO deficiency and impaired NO-mediated relaxation in the lower urinary tracts and CC. 3.2

4.

Literature of ED

ED is a common complaint in men over 40 years of age and becomes more frequent in those over the age of 60 [22-24]. A well-accepted definition of ED is the consistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance [14]. The Massachusetts Male Aging Study predicts that 300 million men in the USA will suffer from ED by 2025 [2,25]. Clearly, ED is now considered as a major health problem for the increasing healthy aging population. Physical, emotional and medical factors contribute to ED, and this condition also is usually associated with vascular risk factors. However, increased observation of young patients seeking clinical help for problems with erection has been reported. A recent study states that one of four men < 40 years have ED [26]. Although the sample number is limited, the authors suggest that lifestyle changes such as chronic use of cigarettes, alcohol or consumption of illicit drugs, which is more frequently observed in younger than older population, may influence the onset of ED in young men. Further studies with larger population samples are needed to confirm this report and to characterize the potential role of ED severity as a harbinger of medical disorders in men below the age of 40 years. Over the past 20 years, several studies have revealed the molecular mechanisms involved in the pathogenesis of ED and have provided convincing evidence that ED is a disease of psychogenic and organic etiologies. Organic causes include neurogenic, endocrinologic, and vasculogenic damage as well as drug-induced ED, which in total constitutes now > 80% of clinical presentations. Psychogenic causes are mental problems, traumatic past experiences, relationship problems and family or social pressures [4]. It is widely recognized that ED is associated with diseases related to decreased NO bioavailability such as arterial


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Central nervous system Sensory stimuli (visual, erotic thought, tactile, olfactory and imaginative)

Neurotransmitter release (serotonin, dopamine, NO) Peripheral nerves Dopamine agonists (apomorphine, ABT 724, PD168077) 3


Ca2+

NANC nerves Ca2+ nNOS

Parasympathetic nerves NO

Oral supplement Endogenous 2 NOS inhibitors

eNOS L-arginine arginase (L-citruline)

NO 1. Arginaseinhibitors : BEC, ABH

6 VIP

3. Oral supplement: L-arginine, L-citrulline, L-argilin

Arginase inhibitors

Urea + L-ornithine

Ca2+

Endothelial cell

PKG

Lower Ca2+

4

sGC activators

2. Endogenous NOS inhibitors: ADMA, SDMA,

4. sGC activators: (BAY 41-2272; BAY 60-4552; BAY 63-2521, etc.) 5. PDE5 inhibitors: sildenafil, tadalafil, vardenafil

1

7 PGE1

6. Vasoactive intestinal peptide

sGC

cGMP GTP PDE5 5 PDE5 inhibitors cGMP inactive cAMP G-protein AC

Relaxation

ATP

7. Prostaglandin E1 stimulator: alprostadil

Cavernosal smooth muscle cell

Ameliorates ED

Figure 1. Established and new therapeutic targets for the pathophysiology of ED are shown. CNS coordinates sensory stimuli from a variety of sources. Release of neurotransmitter such as dopamine also contributes to sexual stimuli. Peripheral excitation evokes stimulation of the NANC and parasympathetic nerves, increases Ca2+ influxes and promotes eNOS and nNOS activation. All these events enhance NO formation in the penis. NO binds to sGC and catalyzes conversion of GTP to cGMP in the smooth muscle cell. Arginase enzyme that competes with eNOS for the substrate L-arginine. Release of VIP from the NANC nerve or administration of PGE1 increases cAMP levels, which together with cGMP causes vasodilation of arteries and sinusoidal spaces of the CC. Reduction of intracellular CC calcium levels by protein kinase A and PKG leads to penile erection. Therapeutic targets for the treatment of ED are addressed in this review such as, endogenous NOS inhibitors, arginase inhibitors, sGC activators, oral supplementation of both L-citrulline and L-arginine or their combination. Increased reaction of 1 and 2 impairs cavernosal relaxation and penile erection. Agents 3, 4 and 5 enhances NO/sGC/cGMP pathway which contributes to cavernosal smooth muscle relaxation. Agents 6 and 7 enhance AC/cAMP pathway and contribute to cavernosal relaxation. ABH: 2(S)-amino-6-boronohexanoic acid; AC: Adenylate cyclase; BEC: S-(2-boronoethyl)-L-cysteine; CC: Corpus cavernosum; ED: Erectile dysfunction; eNOS: endothelial nitric oxide synthase; NANC: Nonadrenergic noncholinergic; nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; PGE1: Prostaglandin E1; PKG: Protein kinase G; sGC: Soluble guanylate cyclase; VIP: Vasoactive intestinal peptide.

hypertension, hypercholesterolemia and diabetes. Impaired NO/cGMP signaling pathways in the penile vasculature result in reduced cavernosal smooth muscle relaxation and/or increased smooth muscle contraction and ED. In 1998, the treatment of ED was revolutionized by effective oral therapy with PDE5 inhibitors such as sildenafil, tadalafil and vardenafil. Clinical efficacy of oral agents marked the beginning of noninvasive pharmacological treatment for ED. Although PDE5 inhibitors are effective in most of ED cases, these agents have been shown to be less effective in certain disease states, such as diabetes, post-radical prostatectomy, and severe veno-occlusive dysfunction [27]. 1450

Additionally, recent reports indicate that ED is an early marker of coronary artery disease, stroke and other adverse cardiac events, because it precedes by 2 -- 3 years in advance to the occurrence of a cardiovascular event [28,29]. Both cavernous and coronary arteries share the common property of being ‘end’ arteries without collateral circulation. However, penile arteries have a smaller diameter (1 -- 2 mm) than coronary vessels (3 -- 4 mm), thus making them more prone to fail to initiate erection, even with a minimal narrowing [28]. The importance of examining the relationship of ED to the onset of CVD has key relevance in public health and clinical issues as it may assist in reducing CVD complications [30].



Endothelial cells

ACh

Shear force

[Ca+2] eNOS

Urea L-ornithine

L-arginine

NO

Arginase NO NO

NO Nitrergic nerve nNOS

NO

NO NO

NO NO


NO

NO

NO

NO NO

NO NO

NO

NO NO

K–

sGC GTP

Ca+2

Corpora cavernosa smooth muscle

cGMP

PKG Smooth muscle relaxation

PDE5 5′GMP PDE5 inhibitor

Stimulation Inhibition

Penile erection

Figure 2. Regulation of cavernosal smooth muscle relaxation and penile erection is shown. Central or local excitation evoked by endothelial cells and nitrergic nerves in the penis causes calcium [Ca2+] influxes and promotes eNOS and nNOS activation, which in turn increases NO production. When released, NO diffuses into cavernosal smooth muscle cells, binds to sGC and catalyzes the conversion of GTP to cGMP. In turns, cGMP activates PKG and exerts actions on calcium and potassium ion channels, which results in smooth muscle relaxation and penile erection. PDE5 selectively degrades cGMP and attenuates erectile process. Inhibition of PDE5 enhances cGMP levels and cavernosal relaxation. Ach: Acetylcholine; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; nNOS: Neuronal nitric oxide synthase; PKG: Protein kinase G; sGC: Soluble guanylate cyclase.

Current standard conservative treatment for ED

5.

Despite the broad efficacy, tolerability and safety of PDE5 inhibitors for treatment of ED, an array of other treatments and combination therapies have been examined and developed. A wide variety of ED treatments exist and are publicized in the market. Each patient may choose a treatment according to his attitude and experience and the treatment’s cost or availability. Porst et al. [31] have proposed five levels for ED treatment. Level 1 includes PDE5 inhibitors, intracavernosal injection of prostaglandin E1 (PGE1, alprostadil) or vasoactive intestinal peptide/phentolamine and transurethral PGE1 therapy. Also, treatment of hypogonadism with testosterone treatment, adopting an active lifestyle with resultant weight loss and optimal treatment of concomitant CVDs can either improve ED or add to the efficacy of PDE5 inhibitors. Level 2 includes vacuum-erection therapy, oral L-arginine, topical PGE1, intracavernosal injection therapy with papaverine/phentolamine (Bimix) or papaverine/phentolamine/PGE1 (Trimix)

combination mixtures. Level 3 includes oral treatment with yohimbine in non-organic ED, and combination therapies of PDE5 inhibitors and either transurethral or intracavernosal injection which generate better efficacy rates than either therapy alone. Level 4 includes the combination of vacuumerection therapy and either a PDE5 inhibitor or transurethral PGE1 or intracavernosal injection therapy. Level 5 includes combination therapy of PDE5 inhibitors and L-arginine or daily dosing of tadalafil and short-acting PDE5 inhibitors. Mechanical therapy as vacuum-erection devices (VED) can be successfully applied nearly in all etiologies of ED. Vacuum therapy can also be used in combination with other therapies such as PDE5 inhibitors or others [32]. However, some contraindications of VEDs are reported in patients with prolonged erections, in patients with severe penile curvature, and those with severe bleeding disorders [33]. It is documented that about 30% do not respond to either monotherapy. Combined medical therapies have been reported for patients who fail monotherapy to produce satisfactory erection. Thus, basic research and medical findings together with sexual medical experts are still needed to obtain a higher efficacy for the management of ED.


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6.

Novel therapies for ED

Current advances in sexual medicine field have led to an increased understanding of the underlying molecular mechanisms involved in erectile function. Overall, PDE5 inhibitors represent the treatment of choice in ED. However, targeting MCRs, shockwave therapy and testosterone replacement in combination with PDE5 inhibitors have been reported to be alternative therapies for ED [27,34-36]. Additionally, gene- and cell-based therapies seem to act at the cellular level to improve specific cellular and enzymatic functions which delay/reverse ED. Further, since NOS and arginase share the same substrate L-arginine, limiting arginase activity may provide a beneficial therapeutic approach for preventing impaired erectile function. The current review addresses the major cellular and molecular targets of biological systems responsible for erectile function and the new therapeutic approaches in the treatment of ED. Current literature and experimental data support the prospect of enhanced arginase activity playing a key role in endothelium impairment and ED. MCR agonist therapy Melanocortins, peptides derived from a larger precursor molecule known as the pro-opiomelanocortin protein, have been shown to cause penile erection following intracerebroventricular administration in animal models. This includes a-melanocyte-stimulating hormone (a-MSH) and adrenocorticotropic hormone (ACTH) [37]. The authors concluded that the proerectile effects of a-MSH and ACTH were mediated by cAMP and occur only in the presence of testosterone. However, the specific MCR subtype(s) through which these peptides produce erectogenic effects is not known [38]. Targeting MCR is an alternative therapy for ED. Administration of PT-141, a nonselective MCR agonist, has been shown to initiate erectile responses in a dose-dependent manner in men with ED [39]. However, side effects such as flushing and nausea, but not hypotension, were observed.

applied into the target tissues or organs, creating a focal zone during its treatment. The goal of LESWT in ED treatment is to reverse the pathological changes in CC to regain spontaneous sexual activity with few side effects [35,46]. Recent studies have reported that LESWT treatment improves erectile function of diabetic rats by increasing the expression levels of asmooth muscle actin, von willebrand factor, nNOS and VEGF, and by reducing receptor of advanced glycation endproducts levels in CC tissues [47,48]. Additionally, LESWT has been shown to improve erectile responses in patients with ED [49]. Also, clinical studies have reported that LESWT markedly enhances the duration of erection, penile rigidity and penile endothelial function in patients who did not respond to oral treatment of PDE5 inhibitors [50]. Further, combined treatment of tadalafil and LESWT markedly increases erectile responses in patients with Peyronie’s disease and ED [51]. These findings suggest that LESWT could represent an effective approach for patients with severe ED who respond poorly to PDE5 inhibitors therapy. However, further studies are necessary to support these findings.

6.1

Shockwave therapy Low-energy shockwave therapy (LESWT) was initially used to breakup/dissolve and treat renal calculi without open surgery [40]. More recently, the technology has been applied for treatment of gall stones, pancreatic stones, parotid gland stones and pseudarthrosis [41-43]. However, the mechanism underlying the effects of LESWT is not well understood. Previous reports have indicated that LESWT may exert its effect by increasing angiogenic factors and promoting neovascularization, thus improving blood supply [43]. Additionally, LESWT is reported to improve recruitment of circulating endothelial progenitor cells in patients with chronic ischemic disease [44]. At present, this technique is widely used for treating myocardial ischemia, diabetic foot, wound healing, Peyronie’s disease, weight loss and ED [45]. Shockwave therapy is a transmitted sonic wave with frequencies around 16 to 20 MHz, duration < 10 µs and is often 6.2

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Gene therapy Gene therapy involves introduction of new genetic material into an appropriate cell type in vivo or in vitro to produce a positive effect by restoring or correcting a defective or deficient gene in the nucleus of the cell. This therapy may provide an alternate approach to that of PDE5 inhibitors which treat only the symptoms but do not aim to cure the underlying condition [52]. Because the penis is one of the few organs with easy access and homogeneous parenchymatous content that provides an ideal location for gene delivery, this approach offers the possibility in theory of curing ED after a single injection of the cDNA construct into the penis. Alternatively, the combination of local gene therapy with any of the currently available oral medications (such as PDE5 inhibitors) could synergistically improve the efficacy of both therapies. Gene delivery for treatment of ED represents a novel and promising approach. Figure 3 shows the scheme of gene-based therapy. The sequential order of gene therapeutic approach include: i) administration of the vector to the specific cell/organ; ii) cellular uptake; iii) nuclear translocation; iv) chromosomal integration; and v) transcriptional activity and expression of the transgene product [53]. Several gene therapy strategies have investigated the improvement of ED in animal models (19). Since NO pathway regulates corporal cavernosal smooth muscle relaxation, resulting in penile erection, gene therapeutic approach to restore NO production is highly considered in patients with ED. Since nNOS may initiate cavernosal tissue relaxation, whereas activated eNOS may facilitate attainment and maintenance of penile erection, gene delivery involved with NO production in CC has been examined for potential therapies of ED. The following section summarizes the available findings of preclinical animal studies for gene therapy of ED in NO/cGMP/PKG signaling pathway. 6.3



Gene DNA vector Promoter

Viral vector

Transcription


Target cell

Nucleus

p p

Cytoplasm

Translation

p p

p p p

p p

p

p

p

Protein

Figure 3. Schematic representation of gene-based therapy. Therapeutic gene of interest is placed in viral vector that enters into the nucleus of a targeted cell to alter its function. This process of transcription occurs only in the nucleus and requires a promoter sequence of DNA to drive the process. The mRNA is then translated, which results in the new protein. The translation process occurs only within the cytoplasm of the cell.

NOS gene therapy 6.3.1.1 nNOS gene therapy 6.3.1

Adenovirus (AdV) vector-mediated overexpression of nNOS has been examined in aged rats [54]. When recombinant nNOS AdV was injected in the CC of aged rats, penile nNOS was effective in ameliorating age-related ED at 18 days after administration by electroporation, without inducing the expression of cytotoxic genes [54]. The authors suggest that a significant portion of the nNOS gene is taken up by the CC and corpus spongiosum smooth muscles, and to a much lesser degree by the cavernosal sinusoids space and vascular endothelium, with only a minor fraction going into the sparse nerve terminals of the penis. Additionally, the role of protein inhibitor of NOS (PIN), a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis was investigated [55]. Treatment with short hairpin RNA construct targeting PIN in the aged rat penis significantly decreased PIN messenger RNA and protein levels, increased nNOS activity and ameliorated aging-related ED [55]. Although PIN inhibition restores erectile function in aged rats, further studies are necessary to determine the effect of PIN ablation in molecular and cellular targets before it would be applied in human gene therapy. eNOS gene therapy The role of eNOS has been examined by gene therapy approach in vascular pathologies leading to ED with 6.3.1.2

promising findings [56-59]. Adenoviral vector-mediated overexpression of eNOS has been reported in age- and diabeticrelated rat models of ED [56,58]. After delivery of recombinant AdV containing eNOS gene into the CC of aged rats, increased expression of eNOS transgene accompanied with enhancement of cGMP levels and ICP were observed in penile tissue compared to control rats. Similarly, in a rat model of diabetes-induced ED, intracavernous transduction of eNOS enhanced erectile responses to cavernous nerve stimulation and increased corporal NO bioavailability [58]. Additionally, combination therapy of AdV-mediated eNOS gene and acute systemic administration of PDE5 inhibitor sildenafil in streptozotocin (STZ)-induced diabetic rats resulted in a synergistic erectile function accompanied with enhanced cGMP levels that was greater than that of either therapy alone [60]. Delivery of eNOS gene to the penis may offer therapeutic utility by restoring regulatory balance associated with NO signaling of penile erection. Although most studies have examined vascular disease models and implicated eNOS gene therapy with promising results, the next goal is to translate the benefit of NOS gene therapy in animal models into clinical practice. Inducible NOS gene therapy In addition to nNOS and eNOS gene therapies, plasmid DNA containing inducible NOS (iNOS) has been shown to increase ICP by cavernosal nerve stimulation and enhance NOS activity in a rat model of age-induced ED [61]. Further, 6.3.1.3


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modest delivery of iNOS gene into the CC of aged rats was compared using three different techniques: AdV, AdVtransduced myoblast cells or plasmid vector alone [62]. Myoblast-mediated gene therapy markedly increased erectile responses to cavernosal nerve stimulation over that of AdV or plasmid injection. Transduction of iNOS gene into the penis in low abundance may provide important advantages over eNOS and nNOS as a therapeutic tool for ED from neurogenic and vascular causes. 6.4

Additional gene therapies Superoxide dismutase gene therapy


6.4.1

Superoxide dismutase (SOD) is an endogenous antioxidant, which catalyzes conversion of super oxide to hydrogen peroxide. Several studies have considered the therapeutic effect of AdV gene delivery of SOD in models of heart failure, hypercholesterolemia, hypertension and aging [63,64]. Gene therapy of SOD represents a potential target in the treatment of ED because it is the major endogenous antioxidant that protects against upregulation of the reactive oxygen species (ROS) as superoxide (O2-). Vascular diseases, including hypertension [65], hypercholesterolemia [66], atherosclerosis [67], aging and diabetes mellitus [68] can interfere with the intricate vascular mechanisms underlying normal erection, and they are often associated with increased O2- formation in the penile vasculature. O2- reacts with NO to form peroxynitrite, resulting in a diminished NO bioavailability [69]. SOD catalyzes the dismutation of the O2- into hydrogen peroxide and water and is expressed in a number of cells including the vascular endothelium. Three SOD isozymes have been identified: mitochondrial MnSOD, cytosolic CuZn-SOD and extracellular (EC)-SOD (EC-SOD) [70]. In a model of age-related ED in rats, AdV gene transfer of EC-SOD into the penis resulted in enhanced SOD activity, cGMP levels and higher expression of EC-SOD mRNAs and proteins. These alterations were accompanied with increased erectile response to cavernosal nerve stimulation similar to those of young rats [71]. Further, transduction of AdV EC-SOD into the penis of aged rats decreased oxidative stress as measured by nitrotyrosine formation. Additionally, in a STZ-diabetic rat model, protein levels of EC-SOD were markedly decreased, contributing to elevation of ROS formation. Further, delivery of AdV EC-SOD into the penis of STZ-diabetic rats decreased O2- levels and increased NO bioavailability and cGMP levels [72]. These studies suggest that transfer of AdV EC-SOD into the penis represent a viable gene therapeutic target in diabetic- and age-associated ED involving increased ROS formation into the penile endothelium. VEGF gene therapy Growth factor therapy has emerged as a novel therapeutic tool for the treatment of ED because it mediates endothelial and smooth muscle physiology and regulates biological process of erectile function. VEGF stimulates angiogenic activity in vivo and in vitro, and its mRNA isoforms are extensively 6.4.2

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expressed in CC of humans and rats [73]. Decreased VEGF has been associated with a number of pathophysiological changes in the penis [74]. Several studies have indicated the potential benefits of VEGF in the treatment of ED. In a model of atherosclerosis-associated ED in rabbits, intracavernosal injection of VEGF prevented impairment of endothelialdependent CC smooth muscle relaxation [75]. Similarly, VEGF and adeno-associated virus (AAV) carrying brainderived neurotrophic factor seemed to alleviate the neurogenic and vasculogenic ED associated with hypercholesterolemia [74,76]. Additionally, intracavernosal VEGF injection and AAV-mediated VEGF gene therapy prevented and reversed venogenic ED in castrated rats [77]. Further studies have shown a protective effect of intracavernosal VEGF injection in traumatic arteriogenic and diabetic models [78]. Results of these studies suggested that VEGF protein exerts its effect on vascular endothelium by causing hyperplasia and hypertrophy of endothelial cells which may counteract the endothelial apoptosis common to some manifestation of ED [79]. In a model of diabetes-induced ED in rats, combination of an AdV-VEGF construct and injection of angiogenic factor-1 (Ang-1) into the CC prevented ED accompanied with increased cavernous angiogenesis, eNOS phosphorylation and cGMP levels, whereas either AdV-VEGF or Ang-1 alone elicited partial improvement [80]. All these basic research studies have clarified the role of VEGF and its effect on vascular endothelium in the penis. Translation of these findings into clinical treatments of vasculogenic ED needs to be explored. Future clinical trials will promise whether this form of therapy will be useful in restoring normal erectile function in patients with ED. cGMP-dependent PKG1 gene therapy When NO is released from the endothelium or nitrergic nerves on to the smooth muscle cells, it activates sGC and consequently cGMP levels, which results in cavernosal smooth muscle relaxation. PKG1 is one of the most important effector targets for cGMP to increase vascular compliance in the erectile process [81]. It is reported that PKG1 also activates large conductance Ca2+-activated potassium channels [82], which hyperpolarize smooth muscle cell membranes, causing muscle relaxation. Mice lacking PKG1 have been shown to exhibit ED [81]. Reduced levels of cavernosal cGMP have been observed in models of ED [82,83]. In a model of STZ-diabetic rat, reduced activity of PKG1-a and -b has been observed in cavernosal tissues. However, adenoviral-mediated gene delivery of PKG1-a into the penis of diabetic rats restores PKG activity to levels similar to those observed in control rats accompanied with improved erectile function during cavernosal nerve stimulation [83]. 6.4.3

Anti-arginase gene therapy Recent evidence indicates that elevated arginase activity contributes to impaired nitrergic nerve and endotheliummediated relaxation of cavernosal smooth muscle in aging, 6.4.4




hypertension and diabetes [65,68,84]. Given that NOS and arginase share L-arginine as their common substrate, elevation of arginase activity can limit availability of L-arginine for NOS, thereby reducing NO production and impairing vascular function. Previous studies have shown that human diabetic CC with ED exhibits higher arginase activity and diminished NO synthesis with reduced cavernosal relaxation [85]. Supplementation of the amino acid L-arginine, either through the diet or direct infusion, has been shown to increase NO formation in the vasculature and restore endothelium-dependent vasorelaxation in the penis of aged rats [3]. Additionally, in vascular tissues, inhibition of arginase has been shown to enhance NO production and reduce vascular dysfunction in hypertensive, high-fat diet and diabetic states [86,87]. Considering that increased arginase impairs vascular function, Bivalacqua et al. examined the physiological role of arginase in modulating erectile function in vivo using AAV gene transfer of anti-arginase-1 into the penis of aged mouse [88]. The data showed that AAV anti-arginase-1: i) decreases arginase-1 protein and mRNA; ii) restores endothelial and erectile function in vivo; iii) increases NOS activity; and iv) elevates penile cGMP levels in aged mice penis. These findings in vivo demonstrated that arginase is involved in ED through attenuation of endothelial-derived NO in the aged mice penis. Considerations of gene therapy in ED Although delivery of viral genes into the penis has shown beneficial effects to restore erectile function, a number of potential issues need to be considered. Viral vectors for local delivery could enter the systemic circulation, causing random transgene expression in undesired tissues or organs. Risk of immune responses induced by viral vectors also represents another concern, as this could hinder use of the therapy in either acute or repeated clinical treatments. To limit undesired immune and inflammatory responses, it would be beneficial to decrease the virus load required to transduce cells in the penis. The possibility of increasing efficiency and specificity of viral vectors to target specific cells could allow for injection systemically rather than intracavernously. Experimental techniques to increase the specificity of viral vectors have been examined such as transcriptional and transductional targeting. Transcriptional targeting works through a specific cell promoter, whereas transductional targeting utilizes cellspecific membrane markers to induce delivery of the viral gene. Several potential risks of viral vectors have limited the use in humans, such as endogenous viral recombination, cancer development and immunological reactions [89]. Although preclinical animal findings of gene therapy are highly promising for treatment of ED, attention is needed in regard to specificity and long-term safety of viral therapy before the translation to clinical application is attempted. 6.5

Stem cell therapy Therapy with stem cells constitutes a novel approach for the treatment of ED because of their ability to self-renew and 6.6

differentiate into several different types of cells. As a result of degenerative diseases such as diabetes and heart failure, cells become dysfunctional or succumb to apoptosis or necrosis [90]. Stem cells have the capacity for functional regeneration of damaged tissues, depending on the stimuli or signals that they received. Differing from other pharmacotherapies, the strategy of stem cells in the field of ED is to replace the lost or damaged cells, providing normal penile tissue and function. Three types of stem cells have been reported: totipotent, pluripotent and multipotent [91]. Totipotent cells are formed from the fusion of an egg and sperm cell [91], and they can turn into any cell in the body but do not reproduce themselves indefinitely. Pluripotent stem cells are derived from the inner cell mass of the embryo and can form any of the > 200 different cell types in the human body. Embryonic stem cell isolated from the inner cell mass of blastocysts is an example of pluripotent cells [92]. Multipotent stem cells are derived from fetal tissue, cord blood and adult tissues. These cells are capable of self-renewal and can form all cell types within the embryonic lineage to which these cells belong. Several laboratories have induced some multipotent cells to reprogram and become pluripotent cells. These cells are called induced pluripotent stem cells. Since endothelial dysfunction is one of the primary causes of ED, transplanted stem cell approaches to restore the penile endothelium and cavernosal smooth muscle cell function are highly needed for patients with ED. Wessells and Williams [93] were the first to demonstrate endothelial cell transplantation into the rat CC for cell-based therapy. Among the various types of adult stem cells, bone marrowderived stem cells (BM-SCs), adipose-derived stem cells (ADSC) and umbilical cord blood stem cells represent the most attractive adult stem cells for their use in practical, clinical and cell-based therapeutic purposes. BM-SCs have been evaluated for their potential to treat vasculogenic ED [92]. Umbilical cord blood stem cells is also an attractive stem cell type because they can be readily and noninvasively collected from donors, and their collection and use raise no problematic ethical implications. The principal routes of stem cell transplant are intravascular infusion, via lumbar puncture, and during surgery when it is used to treat cardiovascular or vascular lesions, internal organ lesions and nervous lesions, including lesions in the brain or spinal cord. Mesenchymal stem cells therapy Mesenchymal stem cells (MSCs) are multipotent adult stem cells formed in the BM with the capacity to differentiate into endothelial and smooth muscle cells in vivo, making them a viable option for the treatment of vasculogenic ED [94]. Transplantation of MSCs showed effective therapy against vascular diseases in vivo, suggesting the possibility that this stem cell population may be effective in replacing or rejuvenating the dysfunctional analogous tissues within the penis [95]. In a age-associated ED rat model, transplanted 6.6.1


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MSCs alone or eNOS-enhanced MSCs into the penis demonstrated improvement of erectile function through mechanisms involving enhanced endothelium-derived NO biosynthesis [3,96]. Similarly, transplantation of MSCs into the penis markedly increased dilation of sinusoidal vascular spaces in the CC and restored erectile function in age-associated ED [97]. In addition, the efficacy of MSCs alone or in combination with VEGF gene therapy is observed in a model of diabetesassociated ED [98]. While the survival of engrafted stem cells in the target tissue is a concern during the treatment, MSCs survival increased when combined with VEGF treatment, as noted by increased pro-survival factors -- phosphorylated Akt and Bcl-xL. Further, combination of MSCs and VEGF gene therapy in the rat penis increased endothelial and smooth muscle cells and enhanced erectile responses compared with those of MSCs alone [98]. These findings suggest that MSCs are cell-based therapy of ED, especially when combined with gene therapeutic treatments. Similarly, administration of MSCs into major pelvic ganglion showed the preservation of erectile function after bilateral cavernous nerve injury (CNI). However, the use of collagen-based biocompatible polymer matrix enhanced the implantation of MSCs and improved the therapeutic effect of MSCs against CNI [95]. This latter study suggested that use of polymers may promote the retention of stem cells in the target tissues as observed previously [99]. In patients with type 2 diabetes-associated ED, stem cell therapy produced improvements in the libido, erectile function and blood glucose levels after intracavernous transplant of human umbilical cord blood stem cells without immune suppression [100]. Despite the positive effects on diabetic ED, the exact mechanisms underlying these effects are missing. ADSC therapy ADSCs are found in fat tissue and have the ability of selfrenewal and differentiation into multiple cell phenotypes. In terms of differentiation and therapeutic potentials, ADSCs are similar to MSCs but are easier and safer to harvest in large quantities. ADSCs have been shown to be vascular precursor stem cells, making them an appropriate choice for stem cell therapy of ED. Earlier studies demonstrated that ADSCs can differentiate into endothelial cells in many tissues [101] including the penis. Fibroblast growth factor 2 is reported to promote ADSCs differentiation into endothelial cell, and intracavernous injection of ADSCs can improve erectile function in rats with ED [102]. In a rat model of bilateral CNI-induced ED, intracavernous injection of ADSCs restored erectile function by repopulating endothelial and smooth muscle cells in the penis [103]. Also, intracavernosal injection of ADSCs showed enhanced erectile function in obese type 2 diabetic zucker diet fat (ZDF) rats compared with untreated animals [104]. The increased erectile response by ADSCs observed in ZDF rats 6.6.2

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resulted from an increased population of endothelial cells, elevated nNOS protein levels and decreased intracorporal tissue apoptosis compared with untreated rats. These findings indicate that there may have been a restorative impact on nitrergic neuron axons and ganglia leading to improved erectile function. However, it is also possible that this increase in nNOS was a result of increased oxygenated blood flow supplying more dorsal nerve nNOS following erectile function improvement. VEGF is a cytokine with strong angiogenic properties that improves survival of transplanted MSCs in a myocardial infarction model [105]. Application of stem cell together with VEGF gene therapy would result in higher stem cell survival and increased efficiency for tissue repair [106]. Cell-based therapy with ADSCs expressing VEGF accelerates the recovery of erectile function by increasing endothelial function. It also improves smooth muscle growth through its paracrine effects and VEGF expression, compared with ADSCs injection alone in STZ-diabetic rats [107]. Further, in a rat model of CNI ED, combined therapeutic effect of PDE5 inhibitor and ADSCs expressing brain-derived neurotrophic factor-membrane system protected cavernous nerve and improved angiogenesis in the penis compared to untreated controls [108]. In conclusion, MSCs and ADSCs are creating an impressive record of high efficacy in various preclinical disease models. Their success in clinical trials of various CVDs promises extension of the applicability of these cells for other vascular diseases such as ED. Currently, many studies have demonstrated that transplantation of either MSCs or ADSCs sources results in improvement of erectile function in models of ED. We believe that stem cell therapy for ED has unveiled new horizons for the future of medicine and clinical treatments. The use of gene and stem cell therapies alone or in combination with each other and other treatments such as pharmacological therapy is highly promising for the management of patients concerned with ED. Testosterone replacement therapy Androgens play a key role in male sexual function and the physiological mechanisms of penile erection [109]. Evidence suggests that testosterone deficiency is an essential factor observed in hypogonadism- and aging-induced ED [110]. Testosterone plays a critical role in maintaining the structure and function of the vascular smooth muscle components of the penis and mediates the penile vasculature tone [111]. It has been shown that testosterone regulates not only cGMP formation, through NOS stimulation, but also its catabolism, through PDE5 activity [112]. Deficiency of testosterone levels may have an indirect effect to elevate arginase. However, it is currently unknown. It has been shown that hypogonadism reduces number and quality of erections, and reports indicate that testosterone replacement therapy improve erectile function in > 50% of men with ED [113,114]. Testosterone replacement therapy in hypogonadal men with sexual disorders has been 6.7




recommended as an initial treatment by several investigators, followed by specific ED treatment such as PDE5 inhibitors [115,116]. The prevalence of hypogonadism is higher in men with diabetes, and low testosterone is associated with sexual dysfunction and reduced response to oral therapy for ED [117]. In human studies, long-acting testosterone undecanoate therapy improved sexual function and quality of life but not the metabolic disorders in men with type 2 diabetes [118]. Recent studies have proposed the syndrome of late-onset hypogonadism as a clinical and biochemical state in men with advancing age. It is characterized by both low testosterone levels and sexual symptoms (as decreased sexual interest and reduced erections) independently. Indication of low testosterone in men having sexual symptoms offers an opportunity to identify a small subgroup of aging men at particularly high risk of dying [119]. 6.8 L-arginine

and L-citrulline supplemental therapy is the substrate required by eNOS to produce NO, and adequate supply of L-arginine is essential for normal vascular function. Reduced levels of L-arginine have been reported in diabetic animals and patients [120,121]. Oral administration of L-arginine has been reported to prevent impairment of endothelium-dependent vasorelaxation in CVD models [122,123]. However, several studies in experimental animal models and in human clinical trials have reported no beneficial effect on chronic administration of L-arginine [124]. It is because a large portion of L-arginine passes through the gastrointestinal tract and hepatic system where it is catabolized by arginase I to ornithine and urea [125]. Chronic L-arginine treatment may produce undesirable effects on cardiovascular function by enhancing arginase expression. L-citrulline supplementation may become an important substitute for L-arginine supply where it is decreased under pathologic conditions. Supplementation of L-citrulline, a production of NOS and the precursor of L-arginine, can also increase formation of NO. L-citrulline is recycled into L-arginine sequentially by argininosuccinate synthetase and argininosuccinate lyase [126]. Unlike L-arginine, L-citrulline is not largely affected by hepatic metabolism and is not a substrate for arginase. Systemic administration of L-citrulline appears to be a more efficient way to increase blood NO levels than L-arginine [127]. Previous studies have revealed that supplemental L-citrulline does not increase arginase activity in blood vessels or liver and is able to enhance endothelium-dependent NO production and hypotension in rabbits [126]. Interestingly, the relevance of Lcitrulline on ED models has also been reported. Studies have revealed that oral administration of L-citrulline improves erectile function in arteriogenic and castrated ED rat model by increasing blood NO levels and by attenuating damage of cavernosal smooth muscle cells [128]. Additionally, oral L-citrulline supplementation for 1 month increased erection hardness enough to restore normal erectile L-arginine

function in patients with mild ED [129]. Although less effective than PDE5 inhibitors, the authors concluded that in the short term, L-citrulline treatment is safe and well accepted and may become an alternative and less-expensive treatment for mildto-moderate ED patients. We believe that long-term oral supplemental therapy of L-citrulline may be effective in supplying L-arginine and NO formation in the cavernosal vasculature with enhancement of endothelial and nitrergic function in patients with ED. Combination of L-arginine and L-citrulline, named Argilinin in the European market, has shown protective effect in CVDs. However, its efficacy for ED is still unknown and further studies are needed. sGC stimulator therapy On its release, NO diffuses into adjacent cavernosal smooth muscle cells and binds with its physiologic receptor, sGC. NO promotes enzyme sGC activation to generate cGMP, which in turn promotes cavernosal relaxation leading to penile erection [14,15]. Decreased formation of NO is observed in patients with diabetes-induced ED [85]. In support of this, STZ-induced diabetes or spontaneously diabetic mice have reduced endothelial and nitrergic responses [65,130]. Pharmacological agents that inhibit cGMP degradation such as the PDE inhibitors and/or agents that enhance cGMP levels have attracted much interest as potential therapeutic strategy in the management of ED. However, studies reported that > 30% of patients with ED do not respond to treatment with PDE5 inhibitors, indicating that endogenous levels of NO are reduced to such extent that PDE5 inhibitors can no longer increase cGMP levels to a sufficient extent [131]. Pharmacological stimulators of sGC may be beneficial for those patients suffering with ED. The benzylindazole derivate YC-1 was identified to be the first NO-independent sGC stimulator to cause erectile responses [132]. This pharmacological agent formed a lead structure for the development of optimized sGC stimulators with improved potency and specificity for heme-dependent sGC stimulators (including CFM-1571, BAY 41-2272, BAY 41-8543, BAY 60-4552 and BAY 63-2521) and hemeindependent sGC activators (e.g., BAY 58-2667, BAY 60-2770, A-778935) [133]. Results from animal and human studies have reported that BAY 41-2272 relaxes CC and causes penile erection in vivo [134,135]. The relaxing effect of BAY 41-2272 is uniquely mediated by sGC stimulation and not by PDE5 inhibition [136]. Additionally, BAY 41-2272 has been shown to act in synergy with the NO donor, sodium nitroprusside, to produce penile erection in rabbits, implying that these compounds will enhance the response to endogenous NO released during sexual stimulation and thus facilitate a natural penile erection [134]. Further, BAY 41-2272 also reduces O2- formation and NADPH oxidase expression in the CC, thereby increasing the bioavailability of NO [135,137]. Recent studies have also observed synergistic effects of combined administration of a sGC stimulator, BAY 6.9


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60-4552 and a PDE5 inhibitor, vardenafil, in CC relaxation with increased proerectile effect in a CNI model [138]. Additionally, BAY 60-2770 has been shown to increase intracavernosal responses under cavernosal nerve injury [139]. Thus, the development of pharmacological agents capable of directly stimulating the NO receptor, sGC, may represent a potential therapeutic strategy in the management of ED. RhoA/Rho-kinase pathway and ED Rho-kinase (ROCK), a serine/threonine protein kinase, has been identified as the major downstream effector of RhoA which mediates calcium (Ca2+) sensitization [140]. The ROCK isoforms found in the vasculature are ROCK-a/ ROCK2 and ROCK-b/ROCK1. ROCK is necessary for diverse functions such as blood flow, arterial blood pressure, airway resistance and intestinal peristalsis. ROCK activation permits actin--myosin interactions and smooth muscle cells contraction by maintaining the activity of myosin light-chain kinase, independently of the free cytosolic Ca2+ levels. Activated RhoA/ROCK pathway has been implicated in many pathological conditions including arterial hypertension [141], atherosclerosis [142], heart attack [143], stroke [144], coronary vasospasm [145], myocardial hypertrophy [146], myocardial ischemia reperfusion injury [147], aging [148], diabetes [149] and ED [150]. Past studies have indicated that the use of ROCK inhibitors like Y-27632, H1152 and fasudil offers clinical benefits regarding the treatment of these diseases, as well as contributes pharmacological tools for vascular studies [151]. The effect of ROCK inhibitors in preclinical models of ED will be described below. The release of NO from eNOS and nNOS into the penile vasculature is fundamental for cavernosal smooth muscle relaxation and normal erectile function [15]. Since the main function of ROCK is the regulation of smooth muscle tone, the upregulation of the ROCK pathway increases cavernosal smooth muscle contraction, leading to ED [140,148]. One of the first studies demonstrated that the ROCK inhibitor Y-27632 caused a dramatic increase in CC pressure and erection independent of NO in an in vivo rat model [152]. Also, higher expression of active RhoA contributes to flaccid state of CC, and oral treatment with ROCK inhibitor reversed impaired ED [153]. Additionally, a study combined an NO donor and a ROCK inhibitor in the rat CC and observed a synergistic effect of the erectile response to electrical field stimulation compared to each therapy alone [152]. This study demonstrated that inhibition of the RhoA/ROCK pathway enhances the action of NO. In experimental preclinical models of ED, several studies have reported elevations in ROCK activity/expression in association with impaired erectile function [141,150,154]. The interaction of NO and ROCK pathway has been studied in the vasculature, focusing on disease states. Accumulating evidence indicates that eNOS is regulated by the ROCK pathway [155]. Inhibition of ROCK upregulates eNOS in vivo via mechanisms that involve stabilization of eNOS mRNA and increase in NO synthesis. Inhibition of


6.10

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RhoA in CC of STZ-induced diabetic rats has been shown to increase eNOS activity/expression, restoring erectile function [155]. These observations suggest that RhoA/ROCK signaling suppresses NO synthesis. Delivery of gene AAV encoding dominant negative RhoA into the penis of aged and young rats for 7 days reduced ROCK activity and markedly improved erectile function in aged rats when compared with those of young rats [148]. Similarly, in age-associated ED, ROCK inhibitor Y-27632 significantly improved erectile function compared to younger rats [156]. Also, decreased expression of nNOS accompanied with impaired erectile function in aged rats is reversed by treatment with ROCK inhibitor, restoring the imbalance between nNOS and ROCK activity [157]. Further, chronic administration of fasudil, an oral ROCK inhibitor, prevented the development of both vasculogenic ED and pelvic atherosclerosis [158]. Implication of RhoA/ROCK and arginase in ED Growing evidence indicate that enhanced RhoA/ROCK pathway function is linked to upregulation of arginase in endothelial cells exposed to oxidative species [159], angiotensin II [160], thrombin [161] and in inflammatory bowel disease [162]. Two isoforms of ROCK (1 and 2) have been identified in mammalian tissues and both isoforms are expressed in vascular smooth muscle and endothelial cells. To circumvent potential nonspecific effects of ROCK inhibitors and their identical actions on the two ROCK isoforms, haploinsufficient mouse models with partial deletion of ROCK 1+/- and ROCK 2+/genes have been used to address the specific role of each ROCK isoform in activation of corporal arginase. Recently, our study in CC tissues confirmed that diabetes increases ROCK activity, protein expression of ROCK 2, active p38 MAPK, arginase 2 (ARG2) and vascular arginase activity as well as decreases endothelial and nitrergic nervedependent relaxation responses. Diabetic heterozygous ROCK 2+/- knockout mice evoked less ROCK activity, lower levels of CC arginase activity/expression, less p38 MAPK activation and less impairment of endothelium-dependent and nitrergic nerve-mediated relaxation than diabetic wild-type mice [150]. Thus, our findings indicate that increased corporal arginase activity caused by diabetes involves the ROCK and p38 MAPK pathways, which contribute to penile vascular dysfunction. 6.11

MAPKs MAPK constitute a class of serine/threonine protein kinases that evoke an intracellular signaling cascade in response to EC stimuli such as proliferation, stresses, proinflammatory cytokines, ROS, growth factors and apoptosis [163]. Several groups of MAPK have been so far identified including EC signal-regulated kinases (ERK1/2), p38 MAPK, c-Jun N-terminal kinases (JNKs), ERK5 (MAPK7), ERK3 (MAPK6), ERK4 (MAPK4) and ERK7/8. The major MAPK signaling pathways associated with various vascular 6.12



diseases including hypertension, diabetes cancer, inflammation and neurodegeneration are p38 MAPK, ERK1/2 and the JNK. However, inhibitors of p38 MAPK and ERK1/2 are been tested in preclinical models of ED, and their outcomes will be discussed in this review. p38 MAPK and ED Activation of p38 MAPK is a stress-sensitive mechanism triggered by angiotensin II, diabetes, hyperglycemia and oxidative stress that has been linked to the pathogenesis of vasculopathy via increased endothelial cell proliferation with lesion formation and impairment of endothelial function [160,164]. At the present time, few published studies are known to evaluate the role of p38 MAPK on ED. In the first of these studies, Nangle et al. [164] examined the effects of the p38 MAPK inhibitor, LY2161793, on penile neurovascular function in STZ-induced diabetic mice. This study demonstrated that inhibition of p38 MAPK corrects nitrergic neurovascular dysfunction in diabetic mice CC. Subsequently, Toque et al. [65] showed that p38 MAPK is involved in increased arginase activity and contributes to endothelial dysfunction in CC of mice treated for 2 weeks with angiotensin II. This study showed that in vivo treatment with the p38 MAPK inhibitor, SB 203580, attenuates angiotensin II-induced activation of p38 MAPK, suppresses arginase activity and arginase II expression and blocks angiotensin II-induced endothelial dysfunction in mice CC. Also, decrease in eNOS phosphorylation at Ser1177 observed in CC of angiotensin II-treated mice is prevented by p38 inhibitor. Additionally, Lysiak et al. [165] reported that ED and cell death observed after bilateral cavernous nerve resection in the penile tissue is due to an increase in apoptotic endothelial cells and cavernous smooth muscle accompanied with increased activation of p38 MAPK and JNK. Evidence indicates that RhoA/ROCK pathway is an upstream regulator of MAPK activity such as p38 MAPK [160]. Recently, Toque et al. [150] demonstrated that p38 MAPK activation is a downstream target of Rho/ROCK pathway, which is involved in increased arginase activity and impaired endothelial and nitrergic relaxation in diabetic mice CC.


6.12.1

ERK1/2 and ED ERK1/2 can be triggered by cellular stresses such as oxidative stress and hyperglycemia, which play an important role in the development of diabetic complications, a disease associated with ED. Currently few studies have focused on the role of ERK1/2 in ED. In the first of these studies, Sommer et al. [166] demonstrated that ERK1/2 is present and active in human CC, and the expression of ERK1/2 is more pronounced in endothelial rather than smooth muscle cells. Higher levels of active ERK1/2 are also observed in diabetic patients with ED. Recently, Nunes et al. [167] showed that inhibition of ERK1/2 corrects penile arginase activity and protects against ED caused by STZ-induced diabetic mice. It seems that the MAPKs are indirectly associated with NOS 6.12.2

regulation, which affects NO availability. Earlier study showed that ERK plays a key role in eNOS regulation. Phosphorylation of eNOS catalyzed by ERK can lead to a reduction in enzyme activity [168]. Additionally, increased activation of RhoA/ROCK and ERK1/2 are associated with mineralocorticoid hypertensioninduced ED [169]. Although further studies are needed to better clarify the exact role of both p38 MAPK and ERK1/2 pathway in ED, new insights point to these pathways as novel therapeutic targets worthy of consideration for clinical trials. 7.

Role of arginase

Arginase is the hydrolytic enzyme that catalyzes the conversion of L-arginine into urea and L-ornithine. Arginase competes with NOS for their common substrate L-arginine and can alter NOS function when elevated during disease or aging [125]. When the supply of L-arginine is insufficient, NOS produce less NO and becomes ‘uncoupled’ and uses more molecular oxygen as substrate to produce O2- which reacts rapidly with any available NO to generate the toxic oxidant peroxynitrite. Competition between NOS and arginase for L-arginine within the cell to produce either NO or ornithine and urea is quite feasible given their individual enzymatic properties. Although the affinity of L-arginine is much higher for NOS (Km ~ 6 µM) than for arginase (Km ~ 5 mM), the maximum activity (Vmax) of arginase is > 1000 times than that of NOS, indicating similar rates of substrate utilization at physiological L-arginine levels [170]. Two isoforms of arginase exist: the cytosolic isozyme arginase I that is mainly expressed in the liver where it is a central player in the urea cycle, and the mitochondrial isozyme arginase II that is highly expressed in the kidney. Each is encoded by a separate gene and is found in vascular tissues, endothelial and smooth muscle cells, but their distribution appears to be vessel- and species-dependent [171]. Both arginase isoforms have been identified in vascular tissue and endothelial cells and have important roles in endothelial dysfunction in diabetes and other diseases. Arginase provides substrate for the ornithine decarboxylase pathway, producing polyamines. Arginase also plays a role in the production of proline, a critical component of collagen, through the ornithine aminotransferase/pyrroline-5-carboxylate reductase pathway [125]. Therefore, increased arginase activity is associated with cell growth and collagen formation and fibrosis. Arginase and ED Enhanced arginase activity has been strongly implicated in the pathogenesis of vascular inflammatory reactions [172] and endothelial and ED [85,87]. Penile erection and flaccidity are regulated mainly by neurophysiological process involving the relaxation and contraction of cavernosal smooth muscle. Strong evidence indicates NO as the principal mediator of penile erection [14]. NO released from sinusoidal endothelial 7.1


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Diabetes

Smoking Arginase (activity/expression)

Hypertension

Angiotensin II Atherosclerosis

Aging

Vascular endothelial dysfunction Erectile dysfunction


NO Urea

ONOO– Arginase

L

L

-arginine

NOS

-ornithine

O2– -citrulline

L

Figure 4. Enhanced arginase activity and expression is linked to many cardiovascular risk factors. Loss of functional endothelium and subsequent endothelial dysfunction play a key role in the occurrence of erectile dysfunction. Increased arginase competes with NOS for their common substrate L-arginine and can alter NOS function during disease. When L-arginine is insufficient, NOS produces less NO and becomes uncoupled and uses molecular oxygen to produce superoxide, which reacts rapidly with any available NO to generate peroxynitrate. NO: Nitric oxide; NOS: Nitric oxide synthase.

cells (eNOS) or from nitrergic nerves (nNOS) causes CC smooth muscle relaxation [14,15]. The pathological process is characterized by impaired eNOS and nNOS relaxation due to decreased production and bioavailability of NO. Reduced availability of the substrate L-arginine to eNOS has been implicated in vascular dysfunction in diabetes and a variety of other disease conditions (Figure 4). Cavernosal tissues from human diabetic patients with ED exhibit elevated arginase activity and expression, diminished NO synthesis and reduced cavernosal relaxation [85]. Conversely, inhibition of arginase with 2(S)-amino-6-boronohexanoic acid (ABH) or S-(2-boronoethyl)-L-cysteine maintains cellular L-arginine concentrations, which in turn enhance NOS activity and NO-dependent cavernosal smooth muscle relaxation in human and rabbit penile CC and penile erection in live rabbits. Additionally, deletion of arginase II gene in mouse can enhance CC relaxation and prevent vascular dysfunction in diabetic mice [68]. The most common factors/pathologies involved with excessive arginase-associated ED are described below. Arginase and aging Evidences from epidemiological studies confirm that age is the primary risk factor for ED, and the prevalence and severity of ED augments with age. A recent summary reported by the International Consultation Committee for Sexual Medicine reported that 39% of men have some degree of ED by the age of 40 years, reaching 67% by the age of 70 years [173]. These findings are in agreement with large-scale studies as 7.1.1

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reported previously [174]. Aging is associated with marked changes in the penile endothelium and smooth muscle cells. Therefore, age-related vascular endothelial dysfunction has been associated with increased arginase activity/expression [175], reduced expression and activity of eNOS and increased oxidative stress [176]. Moreover, aging contributes to the development of ED through the occurrence of endothelial dysfunction [3]. Because arginase participates in the regulation of NO production by modulating the availability of L-arginine for NOS, inhibition of arginase can have a potential therapeutic effect in treating impaired NO-dependent cavernosal smooth muscle relaxation in ED. In the past years, few in vitro and in vivo studies have reported the use of arginase inhibitors in the treatment of age-associated ED. Bivalacqua et al. [88] demonstrated that penile endothelial cells isolated from the aged mouse penis over expressed arginase, which results in decreased eNOS activity and impaired vascular function. The use of an arginase inhibitor, ABH, or delivery of AVV anti-arginase gene into the penis of aged mice increased the penile eNOS activity and cGMP levels, restoring endothelial-derived NO vasodilatation and erectile function. Additionally, attenuated nNOS protein levels and increased arginase activity contributed to impaired neurogenic relaxation in aged rabbit CC [177]. Further, oral inhibition of arginase with ABH results in improved erectile function in aged rats [178]. All these findings support that inhibition of arginase may represent a novel molecular therapeutic target for the treatment of age-associated vasculogenic ED.




7.1.2

Arginase and diabetes

Both type 1 and type 2 diabetes represent a common risk factor of ED, which in turns develops in 50 -- 75% of male diabetics [173,179]. ED also occurs three times more frequently in diabetics than non-diabetics patients [180]. ED was the first sign of diabetes mellitus in 12% of patients [181]. Vascular changes such as endothelial impairment, reduced nitrergic innervation, smooth muscle changes and reduced corporal compliance are observed in diabetes [173]. Decreases in plasma L-arginine have been reported in diabetic patients [120,121] and in vascular tissue of STZ-diabetic rats [120]. Increased arginase activity seems to be involved in these conditions. Our group has demonstrated that increases in arginase activity and arginase I expression in diabetes and high glucose-induced endothelial dysfunction of aorta, coronary and retinal arteries [182,183]. Researchers in the sexual medicine field have examined effects of arginase inhibitor administration on these pathophysiological disturbances. Cox et al. [184] showed arginase activity in human CC, and that inhibition of arginase significantly enhanced nitrergic nervemediated relaxation of cavernosal smooth muscle cells. Increased expression of arginase II in human diabetic CC accompanied with diminished NO production contributes to ED [85]. Additionally, enhanced arginase activity is observed in the reproductive system of diabetic dogs, which contributes to ED and low fertility in diabetics [185]. Further, in a genetic spontaneously non-obese type 1 diabetes mouse model, cavernosal tissues exhibited increased arginase activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production [130]. Inhibitors of arginase are not isoform-selective and their specificity may be species-dependent [125]. Thus, it is not possible to identify the role and function of a specific arginase isoform using pharmacological tools. Development of transgenic mice lacking specific arginase isoforms has provided a more complete and precise view of arginase function in a living system. Our group has recently reported that deletion of arginase II in mice markedly attenuated endothelial-dependent and nitrergic nerve relaxation responses in CC of diabetic mice [68]. Arginase II appears to modulate decreased NO production in the cavernosal vasculature in diabetes. Therefore, arginase is a potential target for therapeutic intervention in the treatment of ED. Arginase and hypertension Hypertension is a major risk factor of CVDs by decreasing NO bioavailability, increasing O2- production and decreasing endothelial levels of eNOS cofactor tetrahydrobiopterin or substrate L-arginine [186-188]. Acute administration of L-arginine has been shown to restore endotheliumdependent vasodilator function in patients with essential hypertension [189]. Recent clinical and basic science findings have confirmed a higher incidence and prevalence of ED in patients with hypertension (> 68%) in which endothelial

dysfunction is the major contributing factor of penile vascular pathology [190-192]. Elevated arginase activity has been reported in aorta, heart and lung of spontaneously hypertensive rats (SHR). Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine reduced systemic blood pressure and cardiac fibrosis and improved vascular function in SHR [84]. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, expression and activity of arginase I protein in the aorta are elevated accompanied with increased blood pressure. These findings suggest that arginase is involved in the pathophysiology of arterial hypertension [193]. Additionally, the detrimental role of arginase I in mediating elevation of blood pressure and endothelial dysfunction is reported in the DOCA-salt hypertensive mice [194]. Our group has determined that elevated angiotensin II is a key component of endothelial dysfunction in CVDs, including hypertension and diabetes and has been linked to upregulation of arginase activity in mice [65,160]. Angiotensin II, which is found in human CC endothelial and smooth muscle cells [195], appears to play a significant role in the regulation of the erection process since higher concentrations of it are associated with penile detumescence [196]. Our group has demonstrated that angiotensin II-treated mice have increased systolic blood pressure accompanied with elevation of aortic and cavernosal arginase activity/expression and impaired vascular and CC endothelial function [65]. These findings suggest that increased arginase activity and decreased phospho-eNOS expression in the positive regulatory site may contribute to impaired vascular function in hypertensionassociated ED. Arginase and smoking Cigarette smoking is an independent risk factor of vasculogenic ED. When adults rabbits are given subcutaneous injection of cigarette smoke extract daily for 5 weeks, a marked increase in cavernosal arginase activity/expression (arginase I) has been observed. Additionally, increased levels of endogenous NOS inhibitors (monomethylarginine and ADMA) decreased nNOS expression, attenuated NOS activity and reduced cGMP levels were observed. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue [197]. 7.1.4

7.1.3

8.

Conclusion

Study findings that inhibition of arginase or deletion of arginase gene enhances NO production and reduces vascular endothelial dysfunction in CVD models, including ED appear to be highly relevant for future management of ED. Elevation of arginase activity can limit availability of L-arginine for NOS, resulting in reduced NO production accompanied by impaired CC smooth muscle relaxation. Evidence


1461


Diabetes Extracellular

Intracellular

G-protein

RhoA Rho-kinase


SB-203580 p38 MAPK

Arginase

Impaired cavernosal relaxation

BEC Stimulation Inhibition

p38 MAPK inhibitor: SB203580 Arginase inhibitor: BEC

Figure 5. Excessive arginase activity by diabetes-induced RhoA/Rho-kinase and p38 MAPK activation leads to impaired cavernosal relaxation. BEC: S-(2-boronoethyl)-L-cysteine.

that increased vascular system relaxation and enhanced blood flow in the penile circulation through inhibition of arginase advances the possibility that therapies targeting arginase could indeed be clinically useful. Identification of signal transduction steps involved in enhancing arginase expression and activity could provide novel therapeutic targets for development of pharmacological treatments that limit its activities in pathological conditions. 9.

Expert opinion

Over the past several years, there have been a large number of preclinical and clinical studies reporting significant advances in our understanding of the physiology and the pathophysiology of penile erection. The exact etiological mechanisms responsible for impaired erectile function have not yet been determined. The main target in the mechanisms associated with erectile function is still NO, and the deep understanding of NO/cGMP pathway has added significantly to the treatment of ED. Decreased NO bioavailability in the penile endothelium observed in preclinical models of ED may be caused by lack of substrate (L-arginine), reduction of eNOS, diminished cofactors of eNOS, uncoupled NOS or increased reaction of NO with O2- anion. Extensive literature supports the concept that upregulated arginase activity in cavernosal 1462

tissue can alter NOS function, producing less NO. Excessive arginase activity has been shown to contribute to the progression of aging-, hypertension- and diabetes-induced vascular dysfunction and ED [65,68,85,88]. Additionally, significant advances in basic research have shown the usefulness of pharmacotherapy such as supplementation of L-citrulline, MCR agonists, testosterone replacement, sGC stimulators and shockwave, stem cell and gene therapies in the treatment of ED. Administration of antiarginase genes into the penis of aged mice has been shown to restore endothelial function and to enhance cGMP levels followed by improved erectile function. Upstream mediators of arginase activation are being investigated in various disease conditions. Studies by our group and others have shown that RhoA/ROCK and subsequent activation of p38 MAPK mediate elevation of arginase expression and activity in STZdiabetic mice [150], angiotensin II-induced hypertensive mice [65,160] and endothelial cell treated with thrombin [161] or ROS [159]. Activated RhoA/ROCK pathway has been shown to be involved in penile detumescence and ED, and partial deletion of ROCK II gene in diabetic mice evoked less ROCK activity, lower levels of CC arginase activity, less p38 MAPK activity and less impairment of endotheliumdependent and nitrergic nerve-mediated relaxation than diabetic control mice [150]. Our findings indicate that increased




corporal arginase activity caused by diabetes involves the ROCK and p38 MAPK pathways, which contribute to impaired CC relaxation and ED. Thus, reduction of corporal arginase activity and/or inhibition of upstream regulators of arginase expression represent important targets for the treatment of ED (Figure 5). Despite the efficacy of PDE5 inhibitors and their position as the primary choice for treating ED, the search for new drugs for treatment of ED has been extensive. The efficacy of sildenafil in the relaxation of the CC is decreased when NOS is blocked [198]. Moderate reduction of arginase function represents a promising new strategy since it can drive greater NO production at the endothelial levels and can enhance the effectiveness of PDE5 inhibitors in patients with ED. Indeed, targeting arginase by gene-based or pharmacological Bibliography

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Declaration of interest HA Toque is funded by a Scientist Development Grant (13SDG17410007) from the American Heart Association (National Affiliate) and by a research grant from Sexual Medicine Society of North America. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Affiliation

Haroldo A Toque† PhD & Robert William Caldwell † Author for correspondence Georgia Regents University, Medical College of Georgia, Department of Pharmacology and Toxicology, 1459 Laney Walker Blvd, Augusta, GA 30912-2308, USA Tel: +1 706 721 6351; Fax: +1 706 721 2347; E-mail: [email protected]

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