Michael Pan1 and Jason R. Kovac2
(1)
Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA
(2)
Urology of Indiana, 12188-A North Meridian Street, Suite 200, Carmel, IN 46032, USA
Jason R. Kovac
Email: jason.r.kovac@gmail.com
Keywords
Erectile dysfunctionShockwave therapyNanoparticlesStem cellsTissue engineering
11.1 Introduction
Erectile dysfunction (ED) , defined as the inability to develop and maintain a penile erection satisfactory for sexual intercourse, is a widespread clinical problem. The prevalence of ED increases with age, and up to 77.5 % of men aged 75 and older are affected [1]. Current pharmacological options such as phosphodiesterase 5 inhibitors (PDE5i’s), while effective, do not produce satisfactory results for many men [2]. Indeed, the success rates for sildenafil, the first and prototypical PDE5i, are up to 82 % [2]. For the many men with ED that do not respond to PDE5i’s, including those who have undergone non-nerve-sparing radical prostatectomy, there remain few nonsurgical or minimally invasive treatment options for their ED.
Current practice involves the use of intracavernosal injections (ICI) and intraurethral suppositories using vasodilator medications as second-line therapies and surgical implantation of inflatable penile prostheses (IPP) to treat ED refractory to pharmacotherapy as third-line treatment. Understanding the various biochemical pathways involved in erectile function is critically important for the development of novel therapies, as deficient and/or malfunctioning pathways represent targets for therapeutic intervention. Currently, available treatment modalities often address only the symptoms of ED but fail to definitively and durably correct the underlying pathophysiology.
Several novel therapies target the reversal and/or prevention of the underlying endothelial and vascular dysfunction and nerve injury that are major components of ED pathophysiology. While many physicians understand the need for more options for the effective management of ED, these have yet to be put into practice. This chapter focuses on several promising potential and novel treatment modalities including nanoparticles, shockwave therapy, stem cells, tissue engineering, and gene therapy.
11.2 Nanoparticles
Nanoparticles represent a novel and exciting field in the treatment of ED. Friedman et al. [3] in 2008 described a novel nanoparticle delivery platform using a hydrogel- and nitrite-containing glass composites for delivery of small peptides and small molecules, including nitric oxide (NO) , to tissues [3, 4]. Physiologically, the NO and cyclic GMP (cGMP) pathway is one of the primary pathways regulating corporal smooth muscle relaxation and subsequent erection.
On a molecular scale, such a nitrite-containing platform consists of a stable amorphous solid with strong hydrogen bonds creating lattices to entrap dry NO and subsequently release it on exposure to moisture. This platform is extremely effective at generating, storing, and releasing NO [3]. Initial work has found nanoparticles constructed from sugar-derived glassy matrices to have smaller pores than those constructed from silica “sol-gel”-based matrices [5] and that by mixing glassy and “sol-gel” components together, matrix pore size can be modified to dramatically alter and fine-tune the rate of release of the particles’ contents [3].
Nitric oxide is an important mediator of vasodilation throughout the body and is essential for penile erection, inducing the production of cGMP and subsequent activation of protein kinase G followed by smooth muscle relaxation in the penile vasculature. Previous studies suggest that decreases in NO production are associated with ED secondary to aging, diabetes mellitus, and cavernous nerve injury [6]. By increasing cGMP levels within the corporal cavernosal endothelial cells, administration of NO has the potential to work synergistically with PDE5i to further inhibit cGMP breakdown. Such an approach, via administration of NO-releasing nanoparticles, has the potential to improve erectile function and responsiveness to PDE5i in patients with ED.
The ability of NO-releasing nanoparticles to improve ED in animal studies has been promising. Han et al. [4] were able to construct nanoparticles capable of carrying drugs including tadalafil and sialorphin, a neutral endopeptidase inhibitor that can cause erections by potentially prolonging the activity of signaling molecules at their receptors [4, 7, 8]. Recent work used nanoparticles containing NO, sialorphin, tadalafil, and placebo that were applied as a gel to the glans penis and penile shaft of diabetic rats [4]. Rats treated with NO and sialorphin nanoparticle microspheres had spontaneous erections, and those treated with tadalafil-containing nanoparticles exhibited increased mean intracavernosal pressures (ICP) with visibly improved erectile response following cavernous nerve stimulation (CNS) [4]. Taken together, the study by Han et al. [4] proved the feasibility of creating a nanoparticle-based delivery system to transport NO and other drugs directly to the corpora cavernosa.
Another recent study of fluorescently tagged NO-releasing nanoparticle microspheres showed persistent release of NO for 4 weeks in vitro [9]. Measurement of various parameters of erectile function after CNS also found significantly increased peak penile ICP to mean arterial pressure (MAP) ratios in the microsphere and combined microsphere and sildenafil treatment groups when compared with control and sildenafil only groups [9]. The microspheres were also seen to enhance the effect of sildenafil for 3 weeks. Lastly, after injection into the corpus cavernosum of adult diabetic rats, NO microspheres did not migrate into adjacent tissues, providing further evidence for the safety of the technique [9].
In summary, nanoparticles provide a promising platform for the treatment of ED. Several characteristics of these particles lend themselves particularly well for treatment, including their stability at room temperature and minimal toxicity [3]. The ability to fine-tune the rate of release of nanoparticle contents anywhere from an initial burst to a protracted, slow release makes this platform particularly effective for drug delivery [3]. The particles also have the ability to carry various pharmacologic agents, including NO or PDE5i’s, directly into desired tissue [3, 4]. Application of a gel or injection containing these nanoparticles to a specific site may also help to prevent systemic side effects, such as the headache, flushing, and congestion associated with PDE5i’s [4, 10]. In addition, absorption of PDE5i’s can be heavily affected by diet, and the drug is subject to first-pass metabolism by the liver, creating individual differences in serum levels among patients [4, 11]. Local application of nanoparticles can help to mitigate many of these concerns. Indeed, the glans penis may be an excellent site for such local transdermal applications given its rich venous supply and the absence of tunica albuginea that may act as a physical barrier to the corpus cavernosum [4]. Further studies are necessary, but the premise bears promise.
11.3 Stem Cells
The use of stem cell therapy ) has attracted significant attention, particularly regarding its potential applications in men with post-prostatectomy ED. Currently, few options exist for patients with post-radical prostatectomy ED as a result of cavernosal nerve damage. Stem cells have the ability to divide and renew themselves over a protracted time frame while retaining the ability to differentiate into specialized cells (including endothelial, smooth muscle, and neuronal cells) and replace damaged tissues [12–23]. Stem cells can also be combined with other treatment approaches, such as tissue engineering and gene therapy, to provide increased efficacy.
The best-studied stem cells in the treatment of ED are adipose tissue-derived stem cells (ADSCs) . However, stems cells obtained from muscle and bone marrow have also been evaluated [21, 24–26]. ADSCs are isolated from the stromal vascular fraction of adipose tissue. Advantages of using ADSCs in stem cell therapy include easy accessibility and plentiful supply [21]. Previous studies have documented success in rats when ADSCs were injected directly into the corpus cavernosum with improvements in ICP/MAP and increased NOS expression [27–29]. Other studies demonstrated the ability of ADSCs to partially regenerate damaged cavernous nerves [28, 30].
Recent studies have found that injection of ADSCs along with brain-derived neurotrophic factor (BDNF) and FGF2-hydrogel into the corpus cavernosum of rats with bilateral cavernosal nerve crush injury induced incrementally greater responses in ICP/MAP and increased smooth muscle/collagen ratios, neuronal NOS content, α-SMA expression, and cGMP compared to rats who received treatments with ADSCs alone [31]. Interestingly, no significant differences were observed between rats treated with FGF2-hydrogel and ADSC/BDNF compared to control rats without cavernous nerve crush injury [31]. Addition of the PDE5i udenafil to ADSCs/BDNF resulted in marked improvements in erectile function and preservation of corpus cavernosum architecture [32]. Udenafil also increased expression of VEGF, but not neuronal NOS (nNOS), indicating that inclusion of udenafil with ADSC treatment may have a synergistic effect [32].
Treatment of ED using stem cells derived from other tissues has also shown promising results. Urine-derived stem cells express mesenchymal stem cell markers, and transfection of these cells with FGF2 induces expression of the endothelial markers CD31 and von Willebrand factor (vWF) [26]. Intracavernous injection of these stem cells with and without FGF2 has shown increased expression of endothelial and smooth muscle cell markers [26]. When injected into the corpus cavernosum of rats, muscle-derived stem cells exhibited smooth muscle morphology and increased expression of α-SMA while bone marrow-derived stem cells increased smooth muscle content as well as penile nNOS, neurofilament, and cavernous endothelial content [24, 25].
In summary, stem cells appear potentially effective at improving ED, primarily in the experimental animal. More studies are required, especially in man, prior to wide-scale acceptance of the technique.
11.4 Tissue Engineering
Tissue engineering aims) to generate new tissues by using an artificial “support system” that serves as a scaffold for and stimulates tissue growth [6]. The newly formed tissue should resemble the function and structure of native tissues as closely as possible. The scaffold can be created using various materials such as decellularized xenografts, allografts, autografts, or synthetic materials that can then be seeded using stem cells and differentiated cell lines or used to stimulate growth of native tissue [6].
Current investigations using tissue engineering principles have produced exciting results with the regeneration of nerve, cavernosal, and tunica albuginea tissues [6]. Regeneration of cavernosal and tunica albuginea tissue may play a larger role in reconstruction of penile tissues after trauma, burns, or other injuries; however, tissue engineering of nerves represents a significant innovation in the treatment of ED due to cavernosal nerve injury during radical prostatectomy.
Application of tissue engineering techniques via neural tissue grafting may promote nerve regeneration and recovery of erectile function. End-to-end suturing of nerves and primary repair of the nerve are limited to bridging gaps of 5 mm [33]. Distances greater than 5 mm typically require interposed bridging materials that serve as a guide for growing axons. These engineered materials can extend the repairable gap distance to ~3 cm [33]. The current gold standard of nerve repair is autologous nerve grafting, but this approach is fraught with disadvantages including requirement of a second surgical site for nerve harvesting, damage to the harvested nerve, infection of the harvest site, and size and internal structure mismatches between the graft and target nerves [33].
With regard to the role of tissue engineering in ED, early animal studies in rats whose cavernous nerves were excised found that reconstruction of the cavernous nerve by interposition of silicone tubes seeded with Schwann cells resulted in greater erectile responses to neurostimulation compared to rats treated with Schwann cell grafts or silicone tubes alone [34]. Schwann cells are important in that they are able to stimulate regeneration of damaged nerves through the production of extracellular matrix as well as through remyelination and regeneration of axons while secreting neurotrophic factors such as the Sonic hedgehog (SHH) protein [34, 35].
SHH is essential in the maintenance of cavernous nerve architecture and likely acts through regulation of BDNF [36, 37], which enhances nerve growth through activation of the JAK/STAT pathway [38, 39]. Treatment of rats with bilateral cavernosal nerve crush injuries using linear peptide amphiphile nanofibers, a platform for extended release of SHH, promoted cavernosal nerve regeneration, suppressed penile apoptosis, and resulted in a 58 % improvement in erectile function compared to controls [36]. Peptide amphiphile nanofibers are biodegradable and are advantageous for their ability to provide directional guidance for growing axons as well as the ability to continually release proteins for an extended period [36].
Implanted tubes constructed of polycaprolactone fumarate (PCLF) have demonstrated significantly greater myelin thickness in recovering nerves compared to other materials such as poly-1-lactide acid, poly-lactic-co-glycolic acid, and oligo(polyethylene glycol) [33]. However, nerve autografts generally showed greater electrophysiological recovery and number of regenerated axons than the biomaterials studied [33].
When applied to decellularized corporal collagen scaffolds in animal studies, autologous and muscle-derived stem cell (MDSC)-derived smooth muscle and endothelial cells, as well as umbilical artery smooth muscle cells, have shown progressive regeneration of smooth muscle similar to that of native tissues [40–42]. Indeed, rabbits with their entire pendular penile corpora replaced with tissue generated using autologous smooth muscle and endothelial cell-seeded scaffolds had the ability to achieve erections rigid enough to copulate and impregnate females compared to negative controls [41].
Other studies have found that tissue engineering can regenerate the tunica albuginea as well [43]. For example, rats with Peyronie’s disease underwent plaque excision and grafting using tunica albuginea grafts of human umbilical cells seeded onto sheets of human fibroblasts [43], with resulting endothelial cells forming capillary-like structures in addition to extracellular matrix produced entirely by fibroblasts [43]. Furthermore, in a model of tunica albuginea excision, grafts of porcine small intestinal mucosa seeded with ADSCs showed significant restoration of erectile function and increased endothelial and neural NOS expression compared to rats that received graft alone [44].
11.5 Gene Therapy
Gene therapy has) many exciting potential applications, from the correction of genetic defects to the repair of genetic material due to oxidative damage or other DNA injuries. Gene therapy focuses on the introduction of foreign genetic material into cells with the aim of restoring defective cellular function or suppressing aberrant cellular processes [45]. Genetic material can be delivered to the patient through a variety of methods: introduction of cells containing the desired genetic information to host tissues; insertion of genetic material into the nuclei of host cells via viruses, naked DNA transfer, cDNA liposomes, and polyethylenimine; or transfection of mesenchymal stem cells that are then implanted and allowed to divide and differentiate [6]. The penis is an excellent target for gene therapy for several reasons: its external location provides easy access to penile tissue and it can be readily isolated from the systemic circulation using a constriction band at its base, preventing systemic spread of foreign genetic material [46]. Moreover, gap junctions between cavernosal smooth muscle cells enable signal transduction even if only a small number of cells are modified [46]. Lastly, the slow turnover rate of smooth muscle cells enables the effects of any genetic modulation to persist for an extended period [46].
Major targets for gene therapy include the pathways involved in the normal erectile response including the NO-cGMP-PKG pathway (described above) as well as the modulation of endothelial and neural growth factors. Potential targets of the NO-cGMP-PKG pathway that can be upregulated include the actions of NOS, the activity of cGMP-dependent protein kinase, and suppression of the protein inhibitor of NOS [47–51]. Previous studies have shown that stem cells and myoblasts can be transfected using adenoviruses containing genetic information and implanted into the corpora cavernosa [52–54], resulting in significant increases in ICP/MAP and inducible NOS and highlighting the potential for this type of approach [52–54].
Modulation of growth factors such as BDNF and VEGF can also improve erectile function. In rats fed a high-cholesterol diet, transfection of BDNF into host cells via adenovirus resulted in increased nNOS-stained nerve fibers and higher ICP than in controls [55, 56]. Kato et al. [57, 58] have also studied a neurotrophic factor called neurturin, a member of the glial cell line-derived neurotrophic factors (GDNFs), and found that rats with cavernous nerve injury treated with neurturin had significant recovery of ICP and improved survival of major pelvic ganglion neurons [57, 58]. Transfection of VEGF-encoding genes into penile corpus cavernosal cells resulted in regeneration of smooth muscle and nerves in addition to endothelial cell hypertrophy and overall improvement in maximal ICP [59, 60].
Smad7, a protein that inhibits Smad2 and Smad3 and the TGF-β pathway, is also a potential gene therapy target for ED treatment. After intracavernosal injection of adenovirus encoding Smad7, cavernosal tissues exhibited decreased endothelial cell apoptosis and increased endothelial NOS phosphorylation, as well as increased production of extracellular matrix proteins like plasminogen activator inhibitor-1, fibronectin, collagen type I, and collagen type IV [61]. Lastly, the modulation of other targets such as superoxide dismutase and RhoA/Rho kinase and the expression of various potassium channels are promising as potential ED treatments as well [62–66]. Given this information, gene therapy appears to be poised to develop worthwhile treatments in the future, with the primary difficulty resting with selection of the most appropriate gene targets.
11.6 Alternative Therapies
A variety of other novel pharmacologic treatments are being studied as potential treatments for ED. Melatonin, an endogenous hormone produced by the pineal gland, affects sleep, direction orientation, sexual maturation, and sperm motility [67] but is also a potent antioxidant and scavenger of reactive oxygen species [68]. Melatonin is also ) produced by the testes, and chronic administration of the hormone inhibits reproductive behavior in male rats [69, 70]. In addition, recent studies indicate that melatonin supplementation may prevent the development of ED in certain cases. Indeed, rats injected with 100 μg/kg of melatonin demonstrated significantly increased mating behavior frequency and decreased time to mounting and intromission compared to control rats [67]. Studies of melatonin administration in rats at a dose of 10 mg/kg injected intraperitoneally showed increased ICP and reversal of oxidative changes caused by diabetes mellitus and spinal cord injury [68, 71]. The results from these preliminary studies suggest that melatonin may play a role in preventing oxidative damage to the cavernous nerves and may have a role in ED prevention.
Other novel pharmacologic therapies include rho kinase inhibitors and valproic acid. RhoA and ROCK, the major downstream target of RhoA,) enhance calcium sensitivity and lead to smooth muscle contraction [72]. Bilateral cavernous nerve injury results in upregulation of ROCK signaling in the penis, suggesting that increased smooth muscle contraction resulting from this increased signaling may contribute to the development of ED [72]. A ROCK inhibitor administered to a bilateral cavernous nerve injury rat model showed improvement in peak ICP and ICP/MAP with restoration of NO pathway signaling and significantly fewer apoptotic cells compared to injured controls. Valproic acid , widely used in the treatment of seizure disorders and bipolar mania, can also prevent penile fibrosis and development of ED in rats with bilateral cavernous nerve injuries, suggesting another potential role for this drug [73].
11.7 Summary
This chapter has examined the role of nanoparticles, stem cells, tissue engineering, and gene therapy for the treatment of ED. While several modalities have made the advance to human studies, much work still needs to be conducted for these novel ED treatments to become part of the treatment paradigm for ED.
Acknowledgments
JRK is an NIH K12 Scholar supported by a Male Reproductive Health Research Career (MRHR) Development Physician-Scientist Award (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program.
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