Department of Urology, Tulane University Health Sciences Center, New Orleans, Lousiana, USA

Correspondence to: S C Sikka, Department of Urology, Tulane University Medical School, 1430 Tulane Avenue, SL-42, New Orleans, LA 70112-2699, USA. E-mail: ssikka@tulane.edu

International Journal of Impotence Research (2002) 14, 353-360. doi:10.1038/sj.ijir.3900880

Peyronie's; plaque; inflammation; collagen; tunica albuginea; free radicals; oxidative stress; antioxidants; NF-B

Introduction

Although Francois Gigot de la Peyronie first reported penile curvature in 1743 and named it as Peyronie's disease the etiology, pathophysiology, and treatment of this now well-recognized penile condition remains complex and undetermined. Peyronie's disease usually affects 1-3% of males between the ages of 40 and 70, and the number of patients with such lesions have increased since the advent of oral sildenafil (Viagra, Pfizer) either because more men are becoming manifest and/or not hesitant anymore to come to clinics for such evaluations.^1,2 Men with Peyronie's disease usually complain of palpable plaque, penile angulation with or without pain, decreased erectile function, some discomfort to their partners, and poor quality of their sexual life. Recent scientific developments in understanding the pathophysiology of this localized connective tissue disorder, which is characterized by the presence of one or more fibrous plaques has shown an excessive amount of collagen and elastin in the tunica albuginea of the penis.^3,4 Both collagen and elastin play a vital role in the penile elasticity, rigidity, vascular compliance, and veno-occlusion mechanisms. Several etiological factors have been proposed to explain fibrosis and plaque formation in Peyronie's disease.⁵ The most common factors are: (a) repetitive penile trauma/inflammation,⁶ (b) low-level autoimmune response,^7,8,9 (c) genetic predisposition, as witnessed by its association with Dupuytren's contracture and HLA-B7 antigens.¹⁰ The inflammatory response involves perivascular infiltration of lymphocytes, monocytes, and neutrophils at the site of injury.³ This is followed by excessive generation of free radicals called reactive oxygen species (ROS), and inflammatory cytokines (chemokines). During plaque formation, there is increased myofibroblast/fibroblast proliferation accompanied by overexpression of fibrin and collagen, the main extracellular matrix components of a Peyronie's plaque.^8,11 Despite these many etiological theories, the scientific understanding of pathophysiology and treatment of Peyronie's disease is still in its infancy. This article focuses on our understanding of the nature of ROS, oxidative stress, and inflammation as related to etiology and pathophysiology of plaque formation and the role of ROS scavengers (antioxidants) towards possible prevention and/or therapy of Peyronie's disease.

What are ROS and oxidative stress?

ROS belong to the class of free radicals derived from oxygen and are highly reactive oxidizing agents. A free radical contains one or more unpaired electrons ready to be shared by other molecules, and this makes them very reactive. Biologically important ROS include superoxide (O₂^-) anion, hydrogen peroxide (H₂O₂), and the very reactive hydroxyl (OH), and peroxyl (ROO) radicals.¹² In addition, some nitrogen-derived free radicals, eg nitric oxide (NO) and peroxynitrite anion (ONOO^-), referred to as reactive nitrogen species, also belong to the class of ROS. Elevated levels of ROS have been implicated in numerous disease states, eg cancer, arthritis, connective tissue disorders, toxin exposure, physical injury, infection, inflammation, infertility, acquired immunodeficiency syndrome and aging. It seems that collective action of these ROS play a significant role in development of these diseases.¹³ Knowledge about the role of ROS and oxidative stress in Peyronie's disease is still primitive and an approach to counteract their impact on penile tissue with antioxidants (eg vitamin E) has met with limited success and is under investigation.

Superoxide anion

During inflammatory response, infiltration of neutrophils, monocytes and lymphocytes results in activation of intracellular NADPH oxidase accompanied by generation of superoxide anion (O₂^-) from molecular oxygen (Equation 1). Superoxide anion, a negatively charged free radical, is also produced by one-electron reduction of molecular oxygen by auto-oxidation (Figure 1).

Although, O₂^- reacts with itself to form H₂O₂ (rate constant of 8´10⁴ mol/l per s), superoxide dismutase (SOD) accelerates the removal of O₂^- with a rate constant of 2´10⁹ mol/l per s. Thus, high levels of cellular SOD will lower O₂^- concentrations into the picomolar range by its superoxide anion scavenging effects.¹² However, this generates another species (H₂O₂) in the high picomolar to low nanomolar range that is likely to interact with iron-sulfur (Fe-S) moieties at key cellular sites and many other cell-signaling systems.

Hydrogen peroxide (H₂O₂)

H₂O₂ is derived from O₂^- by SOD and/or directly by the action of certain oxidases through a 2-electron reduction of O₂. This ROS is relatively stable with high diffusion properties across biological membranes and can contribute to DNA adduct formation. Peroxide-derived ROS readily react with either transition metals such as free iron present in biological systems to form highly reactive hydroxyl (HO) radicals (Fenton reaction, Equation 2), or with superoxide anion (Haber-Weiss reaction, Equation 3) and alter signaling or tissue injury processes.

Removal of intracellular iron by specific chelators (eg deferoxamine, which prevents experimental ischemia-reperfusion injury of the heart) may represent a possible new therapeutic approach to ROS induced disease state.

Hydroxyl radicals (HO)

The hydroxyl radical is a very energetic, short-lived and most toxic ROS. The toxicity of H₂O₂ and superoxide is usually considered to be due to their conversion to the hydroxyl free radical via Equation (2) or Equation (3). Enzymes with peroxidase-like activities, eg catalase and lipoxygenase interact with oxidant-related signaling systems. Catalase metabolizes H₂O₂ to H₂O by donating two electrons from its ferric heme, forming a highly oxidized heme intermediate at the rate constant of 1 nmol/l per s.

Nitric oxide radical (NO)

Nitric oxide (NO), which regulates cavernosal smooth muscle relaxation in the penis, requires a number of cofactors (NADPH, flavin adenine nucleotides, tetrahydrobiopterin, and calmodulin) during its biosynthesis from L-arginine by NO-synthase (NOS) enzymes.¹⁴ This reaction requires molecular oxygen and proceeds via synthesis of an intermediate N-hydroxyarginine resulting in the formation of L-citrulline and NO (Equation 4).

Nitric oxide radical (NO), generated by the inducible form of NOS (iNOS) in response to inflammation, mediates many cytotoxic and pathological events and could contribute in part to Peyronie's plaque formation.¹⁵ The ultimate effects of (NO) depend upon its concentration and interactions with hydrogen peroxide.¹⁶ When the levels of NO increase, eg nanomolar range during inflammation, it is able to compete with SOD for the scavenging of O₂^-. This results in the generation of peroxynitrite (ONOO^-) in amounts that can potentially interact with the cell redox systems glutathione, cysteine, deoxyribose, and other thiols/thioethers complexes.^17,18

Superoxide ion reacts with NO at a rate constant of 7´10⁹ mol/l per s. This is almost three times the rate of reaction of O₂^- with SOD.¹⁹ This peroxynitrite anion, when protonated, forms an intermediate (HOONO), which then decomposes to form two cytotoxic OH and NO₂ radicals (Figure 1). Many important cellular redox systems have a major influence on the function of signaling processes activated by these radicals. Potential products derived from direct chemical reactions between ROS and lipids may generate lipid-derived species with biological activity.²⁰ The nitric oxide radical (NO), can also add to these lipid radicals (L, LO, and LOO) to form lipid NO-containing species (Equation 6).

Although the actual role of these species remains to be defined, they appear to have some potential diagnostic value, eg several of the metabolites including 8-isoprostanes²¹ and malonaldehydes²² are considered to be indicators of oxidative stress and lipid peroxidation, respectively.²³

Cellular antioxidants

Antioxidants, in general, are compounds and reactions that scavenge and/or suppress the formation of ROS, or oppose their actions. The most common biological/cellular antioxidants are superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase. SOD, though limited by its size to extracellular action, spontaneously dismutates superoxide anion to form O₂ and H₂O₂ (Equation 7), and if sufficient catalase is present, will convert H₂O₂ to O₂ and H₂O (Equation 8).

Thus, the activity of these ROS-scavenging systems appears to be highly regulated by physiological and environmental factors. Glutathione (GSH) peroxidase, a selenium containing antioxidant enzyme, metabolizes H₂O₂ and other peroxides through the use of electrons derived from the oxidation of GSH to GSSG (Equation 9). GSSG regulates cellular signaling systems by promoting S-thiolation (RSSG) and disulfide formation of thiol (RSH).²⁴ Glutathione reductase regenerates reduced GSH from its oxidized form (GSSG) as shown in Equation 10.

It is possible that during inflammation the enzyme myeloperoxidase (MPO) present in inflammatory cells metabolizes H₂O₂ to hypochlorous acid (HOCl) (Figure 1). This acid readily reacts with biological amines (RNH₂) to form chloramines (RNHCl) and interferes with S-thiolation.^24,25 It may be possible that many of these ROS scavenging mechanisms, some of which involve NADP(H) and affect thiol-redox status,²⁶ are turned off during the development of Peyronie's disease and thus are unable to control the levels of ROS generation leading to a state called oxidative stress.

Oxidative stress

Oxidative stress is a condition associated with an increased generation of reactive oxygen species (ROS) and/or impaired cellular antioxidant capacity leading to an imbalance between radical generating and scavenging potential. Under normal physiological conditions, there is an appropriate balance between cellular pro-oxidants and anti-oxidants. A shift in the levels of ROS towards pro-oxidants can induce oxidative stress. It is possible that an increased rate of ROS production may inhibit the action of many antioxidants or decrease in cellular antioxidant enzyme activities mainly SOD, catalase, glutathione peroxidase/reductase (Table 1). Alternatively, the inherent decreased expression of these antioxidant enzymes in association with increased ROS generation may also cause increased oxidative stress.¹³

Oxidative stress can modify more than one component of the signal transduction pathway. The type and extent of such modifications depend upon the nature, amount and duration of ROS exposure²⁷ and also on the status of a cell's redox potential.²⁶ Basic and clinical research on the involvement of oxidative stress and the role of cellular and synthetic antioxidants in maintaining normal penile integrity is still in its infancy.

Oxidative stress and pathophysiology of Peyronie's disease

The histopathology of Peyronie's disease reveals an inflammatory process, characterized by chronic lymphocytic and plasmacytic infiltration of the tunica albuginea and the surrounding erectile tissues.²⁸ The tunica albuginea is a thick fibroelastic sheath composed predominantly of thick collagenous bundles and elastic fibers surrounding the trabecular smooth muscle of the corpora cavernosa. It joins centrally to form the septum, which is located between the two corpora cavernosa. Any defect in tunica albuginea can deform penile fibroelastic framework, resulting in curvature or bend (dorsal, ventral or lateral), shortness, indentation, decreased elasticity, and also occasionally affecting hemodynamic function of the penis.²⁹

The origin of the initial inflammatory process that leads to fibrosis, calcification, and plaque formation in the tunica albuginea, is unknown. Devine et al have postulated that internal penile trauma can occur either during sexual intercourse, or externally whereby the corpora cavernosa bend and stretch.³ This injury causes delamination of the tunica albuginea predominantly at the dorsal, midline septum resulting in small hematoma. This process incites further inflammation, induration, and accumulation of leukocytes, neutrophils. These activated inflammatory cells produce ROS thereby initiating a chain of biochemical events leading to myofibroblast/fibroblast proliferation apparently as a wound healing process and resulting in collagen and fibrin biosynthesis/deposition⁸ between the layers of the tunica albuginea (Figure 2). It is postulated that elastic fibers located within the tunica albuginea of the penis form an irregular latticed framework upon which this collagen and fibrin rest. At this juncture, fibrin residues stimulate an amplification of histocytes, which infiltrate the tunica albuginea, resulting in increased collagen deposition. During this wound healing process the balance between extracellular matrix and scar tissue formation exceeds that of their degradation.¹¹

Although type I and III collagen is present in penile scar tissue, type III collagen is more abundant in tunica of men with Peyronie's plaques.⁴ The middle-aged and older men are more prone to such injury and plaque formation because of reduced penile elasticity, while this finding is rare in the tunica albuginea of young potent men.⁹ Cellular and physiological aging has been associated with increased oxidative stress and ROS production. It may be possible that even a minor trauma/injury triggers this localized inflammatory reaction in the susceptible aging male (with a tendency of high oxidative stress) resulting in excessive ROS generation that contributes to Peyronie's disease. Also some Peyronie's patients exhibit genetic predisposition to increased elastin biosynthesis suggesting that an autoimmune mechanism specifically affecting the elastin framework may be involved in the pathogenesis of Peyronie's disease.⁹ Whether ROS also contribute towards this hereditary autoimmune process leading to Peyronie's disease in these men is subject to further investigations.

Oxidative stress and TGF- in pathophysiology of Peyronie's disease

Collagen biosynthesis in adult tissues is regulated by a variety of endogenous and exogenous factors. Many biologically active peptides, such as interleukin-1, tumor necrosis factor, epidermal growth factor, and transforming growth factor beta (TGF-), have been implicated in normal collagen biosynthesis and fibrosis.^30,31 TGF-, a cytokine that is vital to tissue repair, when produced in excess especially during tissue injury and inflammatory response, may lead to fibrosis and plaque formation.³² Recently, El-Sakka and colleagues have demonstrated an up-regulation of TGF- mRNA and protein in the tunica albuginea of Peyronie's disease patients when compared with that of men without Peyronie's disease.³¹ The expression of TGF- in the tunica albuginea of man and animal penis as well as the induction of collagen biosynthesis in a cell culture model suggests a role for TGF- in fibrosis and plaque formation.^32,33 TGF-³¹ and surgical trauma³⁴ have been shown to induce a Peyronie's-like condition in the rat penis. Histological and ultrastructural alterations in the rat penis after induction of a Peyronie's state suggest infiltration of inflammatory molecules; focal and diffuse elastosis; appearance of dense collagen bundles, and thickening, disorganization, and clumping of the tunica albuginea.³⁴

Oxidative stress and nuclear transcription factor NF-kappa B

Activation of nuclear factor kappa B (NF-B), a transcription factor, which regulates the expression of several genes that encode adhesion molecules, has recently been demonstrated in the rat during the first 3 weeks after TGF- injection and injury to the rat penis.³⁵ Our recent in vitro studies have further established the role of NF-B activation under hypoxia and oxidative stress conditions in order to understand the mechanism of ROS induced Peyronie's condition (Figure 2). Our recent studies demonstrate role of hypoxia and hypoxia inducing factor (HIF) leading to NF-B activation in pathophysiology of Peyronie's disease (unpublished data).

The nuclear transcription factor NF-B is a heterodimeric, sequence-specific transcription factor found in many cell types. Several forms of NF-B proteins have been characterized. The activated form of NF-B usually consists of two proteins, a p65 (also called relA) subunit, and a p50 subunit. In unstimulated cells, NF-B is found in the cytoplasm and is bound to its inhibitor B, which prevent it from entering nuclei. In stimulated cells, however, specific kinases phosphorylate B, leading to rapid degradation by proteasomes and subsequent nuclear translocation and binding of NF-B to specific sequences in the promoter regions of target genes. A number of stimuli have been shown to activate NF-B, including cytokines, activators of protein kinase C, viruses, and oxidants.³⁶ Inhibition by antioxidants, such as pyrrolidine dithiocarbamate and acetylcysteine, suggests that ROS have an intermediary role in NF-B activation. In different cell types, NF-B could perform many opposing functions by activating distinct subset of genes in conjunction with cell-type-specific transcription factors. Therefore, this nuclear factor appears to be a 'genetic switch' whose biological effect is determined by the set of genes it can transactivate in a given cell type. However, many putative genes that are activated by NF-B in response to inflammation (eg by TNF) and/or by ROS remain to be indentified.³⁷ How potential chemopreventive agents might affect this gene expression by NF-B activation and regulate apoptosis is not known. Further studies may demonstrate the role of ROS and antioxidants in the pathophysiology and prevention of Peyronie's disease, and usefulness of certain signal transduction pathways as potential early target sites for establishing various treatment modalities.

Role of antioxidants in Peyronie's disease

Peyronie's disease can be simplistically classified into two phases based upon symptoms: (1) an acute inflammatory phase which persists for approximately 6-18 months, in which patients present with pain, slight penile curvature, and nodule formation; and (2) a chronic phase in which patients present with stable plaque size, penile curvature, and in some instances, impaired erectile function.²⁹ The fact that approximately 13% of patients with Peyronie's disease demonstrate complete resolution of their plaques especially in early stages of inflammation and collagen deposition,¹ these cellular antioxidants may play significant role in preventing the progression of plaques. Numerous studies have shown that the Peyronie's patients most likely to benefit from vitamin E and other antioxidant therapy for their symptoms are mainly those patients with early-stage disease.³

Although oral therapy with vitamin E (-tocopherol) remains a popular treatment modality in part because of its safety even at pharmacological doses and low cost, it has not proven to be an established treatment because most patients are usually in the advanced stages. Moreover such studies did not include proper control groups, and had limited and short-term objective evaluation. Vitamin E is an efficient free-radical trap, which interacts with ROS especially the more damaging hydroxyl ion (OH), donating a hydrogen atom to restore the molecule to normal inert state. It has been found to prevent membrane lipid peroxidation, elevate cellular GSH levels, lower protein glycosylation, and decrease blood triglyceride and thromboxane levels.²⁷ Thus, regular intake of vitamin E after middle-age may help prevent the development of Peyronie's plaques especially in susceptible individuals.

The oral potassium aminobenzoate (Potaba) in the treatment of Peyronie's disease has shown a reduction in penile pain, curvature, and resolution of plaque size.^37,38 The mechanism of its action is considered to be due to an increase in monoamine oxidase, decreased serotonin, and/or an increased utilization of oxygen, which may decrease the generation of potential damaging free radical species.³⁸

Tamoxifen, another nonsteroidal anti-estrogenic anti-inflammatory agent has been shown to have a beneficial effect in the treatment of desmoid tumors, a condition with histologic properties similar to Peyronie's disease.^39,40 Tamoxifen facilitates the release of TGF- from human fibroblasts in vitro, and thus can inhibit the inflammatory response and decreased fibroblast production. The TGF- in lower doses may regulate inflammation, the immune response, and help in tissue repair by deactivating macrophages and T-lymphocytes.⁷ However, large amounts of this growth factor can augment tissue fibrosis.³⁰

Colchicine is an agent with anti-inflammatory activity, which can decrease collagen biosynthesis and stimulate collagenase activity.⁴¹ It interferes with the transcellular movement of collagen and diminishes the activity of the enzymes responsible for collagen processing. Purified clostridial collagenase in vitro could dissolve surgically excised Peyronie's plaques. Also verapamil, a calcium channel blocker, has been used with limited success for the treatment of Peyronie's disease and has gained considerable attention.⁴²

The interferons are a group of naturally occurring, low molecular weight proteins and glycoproteins that play an integral role in the immune system. The interferons inhibited fibroblast proliferation, which diminished collagen production, and additionally that interferon alpha-2b stimulated collagenase activity.^43,44 Our in vitro studies using human cavernosal cell culture demonstrated stimulation of collagen production on exposure to oxygen-free radicals and diminished collagen production in the presence of interferon alpha.^45,46 Furthermore, interferon alpha 2b is recognized to effectively decrease keloid scars and scleroderma.⁴⁷ Based on the success of intralesional collagenase and verapamil and the recognized in vitro effects of interferon alpha, the first report with intralesional interferon alpha 2b demonstrated decreased penile pain, penile curvature, and plaque size.^43,47,48

In summary, the antioxidants are both natural and synthetic compounds (Table 1), and have recently gained considerable attention by the nutritional, cosmetic, and pharmaceutical industries. However, their usefulness in prevention and/or management of Peyronie's disease has not yet been established. Vitamins E, C, beta-carotene, selenium, and ubiquinols may protect against ROS induced oxidative damage to DNA, protein, and membrane lipids by quenching singlet oxygen. High doses of vitamin E have been used in Peyronie's patients but without much success.⁴⁹ Selenium is an important component of glutathione peroxidase and may show additional benefit when combined with vitamin E in preventing/treating certain cases of Peyronie's disease. Although therapeutic use of such antoxidants appears attractive, further controlled clinical studies will determine if many of these putative antioxidants can prevent plaque formation in selected groups of patients.

Assessment of oxidative stress status

The measurement of ROS generation

The most common methods for quantitating ROS include: (a) measuring the rate of ROS generation using lumonol-induced chemiluminescence; (b) reactions involving nitroblue tetrazolium (NBT) or cytochrome c-Fe³⁺ complexes measure ROS on the cell membrane surface; (c) electron spin resonance (EPR) methods, which are more sensitive and can characterize the type of ROS generated inside the cell; (d) ROS can be artificially generated under defined experimental conditions. For this, the reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O₂^-) anion and hydrogen peroxide (H₂O₂), respectively (Equation 11).

This rate of ROS measurement is dynamic and may represent a more physiological assessment of oxidative stress. Selective modifications of these defined conditions can identify: (a) the type of free radicals involved, (b) their mode of action, and (c) the type of selective protective mechanisms. EPR measurement requires skillful operation, accurate interpretations, and expensive instrumentation.

Measurement of antioxidant levels

In general, an evaluation of the rate of ROS generation using specific probes can be a dynamic measure of oxidative stress.¹³ However, due to a very short half-life of these free radicals, clinical evaluation of ROS generation is very limited and needs immediate attention under specific controlled conditions. The potential methods that can be developed for evaluation of OSS may utilize measurement of various antioxidant levels and an oxidized component that remains in the body fluids (eg TBA reactive substances; GSH/GSSG balance; the levels of unaltered tocopherol or ascorbate). There have been recent concerns about the specificity, interference, and reliability of measuring TBA-MDA activity as an indicator of LPO. Although this test remains one of the most efficacious methods for assessing the oxidative damage to cell, more recent methods employ measurement of 8-Iso-PGF2_alpha as an indicator of oxidative stress. Many other assays are being developed to measure the rate of ROS production and cellular antioxidant level for the overall assessment of oxidative stress status (OSS) in a tissue as a reflection of disease state. Since oxidative stress has been demonstrated to play a key role in Peyronie's disease, it is imperative to identify measures that would help predict, with accuracy, if OSS is a significant contributor of the disease state. ROS and total antioxidant capacity, as described above, can provide accurate information about oxidative stress status.¹³

Concluding remarks

Oxygen toxicity is an inherent challenge to aerobic life forms. How this toxicity affects the interaction of cavernosa and tunica albuginea in penis is unknown. Although increased oxidative damage to membrane, proteins, and DNA is associated with alterations in signal transduction mechanisms, a large number of rather poorly understood signaling pathways seem to exist. More in vitro research, especially using organ culture or matrix models, is needed in order to understand the role of oxidative stress related mechanisms of calcification and plaque formation.^33,50,51 Also no proper animal model that can mimic this clinical condition is yet available in which these interactions could be evaluated. A variety of defense mechanisms encompassing antioxidant enzymes (SOD, catalase, glutathione peroxidase and reductase), vitamins (E, C, and carotenoids), and biomolecules (glutathione and ubiquinol) are involved in biological systems. A balance between the benefits of antioxidants and risks from ROS appears to be necessary for the normal penile function. A rapid assay system for the evaluation of oxidative stress status (OSS) may aid the clinician in the assessment and identification of sub-group of potential responders and non-responders to any putative antioxidant therapy. Although the therapeutic use of such antioxidants appears attractive with minimal side effects, the proper multi-center clinical trials are required to determine efficacy and any toxicity before clinicians could recommend such therapeutic alternatives.

Acknowledgements

Thanks to numerous Peyronie's patients for making us understand their problems, and also to Samar Sikka for his help in making the schematic diagrams.

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Figure 1 Scheme suggesting generation of reactive oxygen species (ROS) and interactions with nitric oxide and myeloperoxidase. SOD, superoxide dismutase; MPO, myeloperoxidase; NO, nitric oxide; H₂O₂ hydrogen peroxide; OH, hydroxy radical; HOCL, hypochlorus acid; NO₂, nitrogen dioxide radical; OONO, peroxynitrite anion; ¹O₂, singlet oxygen.

Figure 2 Scheme suggesting role of oxidative stress and antioxidants involving NF-B activation pathway leading to over expression of specific target genes and formation of Peyronie's plaques. The shaded boxes represent various penile compartments where these major cellular events are predominantly taking place. The potential sites of inhibitory effects of alpha-interferon and ROS scavengers are also shown in circles.

Table 1 Potential cellular antioxidants and their role in management of oxidative stress and Peyronie's disease