OXIDATIVE STRESS IN ARTERIOGENIC ERECTILE DYSFUNCTION: PROPHYLACTIC ROLE OF ANTIOXIDANTS

0022-5347/05/1741-0386/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 174, 386 –393, July 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000161209.39959.67

OXIDATIVE STRESS IN ARTERIOGENIC ERECTILE DYSFUNCTION: PROPHYLACTIC ROLE OF ANTIOXIDANTS KAZEM M. AZADZOI,*, † RISA N. SCHULMAN, MICHAEL AVIRAM

AND

MIKE B. SIROKY‡

From the Departments of Urology (KMA, MBS) and Pathology (KMA), Boston University School of Medicine and Veterans Affairs Boston Healthcare System, Boston, Massachusetts, Department of Medicine, University of California-Los Angeles, School of Medicine (RNS), Los Angeles, California, and Department of Biochemistry, Technion Faculty of Medicine, Rambam Medical Center (MA), Haifa, Israel

ABSTRACT

Purpose: We searched for markers of oxidative stress in cavernous ischemia and examined the effect of long-term antioxidant intake on arteriogenic erectile dysfunction (ED) in the rabbit. Materials and Methods: Antioxidant activity of known antioxidant beverages, such as pomegranate juice (PJ), red wine, blueberry juice, cranberry juice, orange juice and green tea, was examined spectrophotometrically. PJ demonstrated the highest free radical scavenging capacity. The effect of long-term PJ intake on intracavernous blood flow and penile erection was then examined in the rabbit model. Erectile tissues were processed to assess oxidative stress and smooth muscle relaxation, immunohistochemical staining of nitric oxide synthase (NOS) and histomorphometry. Results: On spectrophotometric analysis PJ showed the highest capacity to decrease low density lipoprotein oxidation and inhibit cellular oxidative stress in macrophages. The rabbit model of arteriogenic ED demonstrated decreased intracavernous blood flow, erectile dysfunction, loss of smooth muscle relaxation, decreased endothelial NOS and neuronal NOS, increased inducible NOS expression, diffused cavernous fibrosis and increased cavernous levels of the oxidative product isoprostane 8-epi-prostaglandin F2␣. Long-term PJ intake increased intracavernous blood flow, improved erectile response and smooth muscle relaxation in ED and control groups while having no significant effect on NOS expression. PJ intake prevented erectile tissue fibrosis in the ED group. Conclusions: Arteriogenic ED accumulates oxidative products in erectile tissue, possibly via an intrinsic mechanism. Oxidative stress may be of great importance in the pathophysiology of arteriogenic ED. Antioxidant therapy may be a useful prophylactic tool for preventing smooth muscle dysfunction and fibrosis in ED. KEY WORDS: penis; impotence; rabbits; muscle, smooth; antioxidants

Vascular risk factors, including hypercholesterolemia, atherosclerosis, hypertension and diabetes mellitus, can interfere with the intricate neurovascular mechanisms underlying normal erection.1⫺3 Hypoxemia, sleep apnea and respiratory failure are also increasingly recognized as causes of erectile dysfunction (ED).4, 5 These conditions are known to induce oxidative tissue injury due to accumulation of reactive oxygen species (ROS) such as superoxide, H2O2 and hydroxyl radicals.6 Oxidative injury occurs when the oxidative burden of the body exceeds its antioxidant capacity. The mechanism of oxidative injury is thought to involve lipid peroxidation, protein oxidation, DNA oxidation, decreased synthesis and bioavailability of endothelial (e) and neuronal (n) nitric oxide (NO), and the up-regulation of proinflammatory cytokines, growth factors and tissue specific receptors.6 Oxidative injury is known to alter tissue structure and function in many organs, including the heart,7 blood vessels,8 lung,9 kidney10 Submitted for publication September 15, 2004. Supported by a Department of Veterans Affairs Merit Review Grant. * Correspondence: Urology Research (151), Boston Veterans Affairs Medical Center, 150 South Huntington Ave., Boston, Massachusetts 02130 (telephone: 617-232-9500, extension 5602; FAX: 617278-4540; e-mail: [email protected]). † Financial interest and/or other relationship with Ely-Lilly and Roll International. ‡ Financial interest and/or other relationship with Ely-Lilly and Yamanouchi.

and brain.11 The role of oxidative stress in ED has not been thoroughly investigated. Antioxidants such as vitamin E and vitamin C have been used widely in clinical practice to protect the body from harmful free radicals.12, 13 Other families of antioxidants with more potent free radical scavenging capacities, such as polyphenols, might also be effective for protecting the cardiovascular system. Indeed, the consumption of red wine or pomegranate juice polyphenols by mice as well as by humans has significantly inhibited oxidative stress, atherogenesis and atherosclerotic lesion development.14⫺17 We searched for markers of oxidative stress in arteriogenic ED and examined the effect of the long-term intake of dietary antioxidants on penile erection in the rabbit. MATERIALS AND METHODS

The selection of an antioxidant. We examined the antioxidant potency of several known antioxidant beverages, such as 100% pomegranate juice (POM Wonderful, Los Angeles, California), red wine (French Chateau Larose Bordeaux, Chateau Larose Trintandon, Haut-Me´doc, France and Californian Beringer Founder’s Estate Cabernet Sauvignon, Beringer Blass Wine Estates, St. Helena, California), 100% blueberry juice, 100% cranberry juice, cranberry juice cocktail (30% cranberry juice), orange juice and green tea (tea bag steeped for 1 minute in 8 ounces of hot water). The antioxidant potency of these beverages was determined based on their free radical scavenging capacity.

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Spectrophotometric analysis. Free radical scavenging capacity, which is a measure of antioxidant potency, was measured in equal volume of the described antioxidant beverages using the DPPH (1,1-diphenyl–2-picryl-hydrazyl) assay as a decrease in free radical content. DPPH is a radical generating substance that is widely used to monitor the free radical scavenging abilities of various antioxidants. The DPPH radical has a deep violet color and radical scavenging can be followed spectrophotometrically by the loss of absorbance at 517 nm. Pomegranate juice (PJ) demonstrated the highest antioxidant activity among the noted beverages. The antioxidant effect of PJ was further examined. Effect of PJ on low density lipoprotein (LDL) oxidation. Pomegranate concentrate was used. The concentrate is prepared from fresh pomegranates crushed in a champagne press. Because whole fruit is crushed, beneficial antioxidants from the peel and membranes are added to those already found in the juice. After crushing the juice is enzymatically treated with pectinase, filtered, concentrated and stored at –18C. Low density lipoprotein was isolated from plasma derived from healthy normolipidemic volunteers by discontinuous density gradient ultracentrifugation. LDL was washed at d ⫽ 1.063 gm/ml and dialyzed against 150 mmol/l NaCl and 1 mmol/l Na2 ethylenediaminetetraacetic acid, pH 7.4, at 4C. LDL was then sterilized by filtration (0.45 ␮M), kept at 4C and used within 2 weeks. The LDL protein concentration was determined with Folin phenol reagent. Prior to oxidation LDL was dialyzed against ethylenediaminetetraacetic acidfree, phosphate buffered saline solution, pH 7.4, at 4C. LDL (100 ␮g protein/ml) was incubated for 10 minutes at room temperature with increasing concentrations of juices. Subsequently 5 ␮mol/l CuSO4 were added and the tubes were incubated for 2 hours at 37C. At the end of incubation the extent of LDL oxidation was determined by measuring the generated amount of thiobarbituric acid reactive substances (TBARS) and lipid peroxides. The extent of LDL oxidation was measured directly by TBARS assay at 532 nm using malondialdehyde for the standard curve. Lipoprotein oxidation was also determined by the lipid peroxide test, which analyzes lipid peroxide formation by the capacity to convert iodide to iodine, as measured spectrophotometrically at 365 nm. Effect of PJ on cellular oxidative stress and on macrophage uptake of oxidized LDL. Macrophages (J774.A1) were incubated with juices for 1 hour, followed by cell wash and assay performance (cellular oxidation and lipoproteins uptake). Cellular oxidative stress was examined in dichlorofluorescein (DCF) loaded J774.A1 macrophages by flow cytometry using the conversion of nonfluorescent DCF diacetate to its fluorescent counterpart DCF as an index. J774.A1 macrophages were incubated with 125I labeled oxidized LDL (10 ␮g protein per ml) and lipoprotein degradation by these cells was determined. Lipoprotein cellular degradation was measured as trichloroacetic acid soluble, nonlipid radioactivity, which was not due to free iodide. Lipoprotein degradation in a cell-free system was measured under identical conditions and subtracted from the total degradation. The remaining cells were washed 3 times with cold phosphate buffered saline and dissolved in 0.1 N NaOH for protein determination. The effect of PJ on erectile function and dysfunction was examined in an animal model of arteriogenic ED. The rabbit model of arteriogenic ED. New Zealand White male rabbits weighing 3 to 3.5 kg were assigned into 2 groups. The first group of 14 rabbits received a regular diet and served as the age matched control. In the second group of 14 treated rabbits arteriogenic ED was induced by balloon injury of the iliac arteries, as previously described.3, 18 After balloon injury the animals received a 0.5% cholesterol diet for 4 weeks and then a regular diet until studied. In vivo experimental protocol. The treated and control

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groups of animals were divided into 2 subgroups. The first subgroup (7 treated and 7 control rabbits) received PJ concentrate added to drinking water (8 ml concentrate per 0.992 l water for 8 weeks). The rabbits ingested an average of 3.87 ml daily of PJ concentrate, equivalent to 112 ␮mol polyphenols daily. The second subgroup of 7 treated and 7 control rabbits received placebo (drinking water without PJ) for 8 weeks. After 8 weeks in anesthetized animals the erectile response was examined and erectile tissues were processed for structural and functional evaluation. Hemodynamic measurements. Systemic arterial pressure and intracavernous blood flow were measured by laser Doppler flowmetry, as previously reported.3, 18 Penile erection was examined by electrical stimulation of the cavernous nerve with 10 V for 8 milliseconds at 16 Hz for 30 seconds. Erectile function and dysfunction was judged based on the ratio of mean intracavernous pressure (MICP) to mean arterial pressure (MAP). After hemodynamic studies the animals were sacrificed and cavernous tissues were processed for study. Measure of cavernous oxidative stress. Enzyme immunoassay of isoprostane 8-epi-prostaglandin (PG)F2␣, a leading oxidative modified product of oxidative stress, was performed as we have previously reported.19 All assays were done in triplicate with commercially available enzyme immunoassay kits. Microtiter assay plates were scanned with a microplate reader. The quantity of isoprostane 8-epi-PGF2␣ was standardized as pg/ml incubation medium per mg tissue wet weight per 30 minutes of incubation. Isometric tension measurement. Organ bath studies were performed, as previously described.18, 19 Briefly, corpus cavernosum tissues were submerged in 25 ml organ chambers containing physiological solution gassed with 95% air and 5% CO2 at 37C, pH 7.4. Tissue relaxation to electrical field stimulation (EFS) was studied after contraction with phenylephrine (2,000 nM). Histology and immunohistochemical staining. Cross sections of the iliac artery and penis were processed for hematoxylin and eosin, and Masson’s trichrome staining in accordance with the standard protocol. Cross sections (5 ␮m) of penile tissue were processed for immunostaining of endothelial NO synthase (eNOS), nNOS and iNOS, as we have previously reported.18 Histomorphometric analysis. Masson’s trichrome stained slides of erectile tissues were processed for histomorphometry. The technique involved randomly selecting 15 to 20 high power fields using a microscope and examining them with image analysis software. The sum of all representative red (smooth muscle) and blue (connective tissue) areas was calculated in all high power fields. Statistical analysis. Data are expressed as the mean ⫾ SEM. Statistically significant differences in treated groups compared with control groups were assessed by ANOVA or the unpaired Student t test, when applicable, at the 95% confidence level. RESULTS

Comparative antioxidant activity of antioxidant rich beverages. PJ had the highest free radical scavenging activity (p ⬍0.01). It decreased free radical content by 71% after 5 minutes of incubation with DPPH. The next most potent beverages were French and California red wines, followed by blueberry juice, cranberry juice, cranberry juice cocktail, orange juice and green tea (fig. 1, A). Macrophage oxidative status. The oxidative status of J774.A1 macrophages decreased by 56% in the presence of PJ (fig. 1, B). As measured by lipoprotein degradation, the macrophage uptake of oxidized LDL was inhibited by 52%. There was no significant effect of PJ on the macrophage uptake of native, nonoxidized LDL, as measured by cellular LDL degradation (fig. 2, E and F).

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ROLE OF OXIDATIVE STRESS AND ANTIOXIDANTS IN ERECTILE DYSFUNCTION

FIG. 1. A, percent of free radicals neutralized by PJ and several beverages known for antioxidant capacity. Asterisk indicates significance for each beverage vs PJ (p ⬍0.01). B, effect of PJ on J-774.A1 cell line macrophage oxidative status. Inhibition of cellular oxidative stress was measured by DCF diacetate fluorescence assay using flow cytometry analysis system. Asterisk indicates significance for PJ vs control (p ⬍0.01).

Antioxidant capacity of PJ. The total amount of polyphenols measured in PJ was 4.20 ⫾ 0.25 mM. PJ showed strong free radical scavenging capacity on DPPH assay, decreasing absorbance at 517 nm by almost 50% after 5 minutes of incubation. PJ inhibited the oxidation of LDL cholesterol markedly at the highest dose used, as shown by TBARS and by lipid peroxides assays. IC50 in these assays was measured at 0.60 and 0.48 ␮l/ml for TBARS and lipid peroxides, respectively (fig. 2, A to D). Cavernous ischemia and ED in the rabbit model. Balloon arterial injury caused diffuse atherosclerotic occlusive disease and arteriogenic ED in treated animals. Electrical nerve stimulation caused a full erectile response in control animals. Impaired erectile function, characterized by significant decreases in intracavernous blood flow and the MICP-to-MAP ratio, was evident in animals with arterial atherosclerosis (see table, fig. 3). Oxidative stress in the model. The product of oxidative stress isoprostane 8-epi-PGF2␣ was detected in ischemic and control cavernous tissues. Mean levels of isoprostane 8-epiPGF2␣ ⫾ SE in medium from ischemic tissues was approximately 3-fold greater than levels in control tissues (3.94 ⫾ 0.88 vs 1.36 ⫾ 0.16 pg/ml/mg tissue per 30 minutes, p ⬍0.001). Effect of PJ on erection. PJ had no significant effect on intracavernous blood flow in the flaccid state of the penis,

while it caused a significant increase during nerve stimulated erection in the control and arteriogenic ED groups (see table). However, PJ did not normalize intracavernous blood flow in the arteriogenic ED group to the level in the control group. Time to maximum intracavernosal pressure (ICP) was significantly shorter in the PJ treated groups than in controls (see table). Effect of PJ on smooth muscle relaxation. EFS induced relaxation of cavernous tissue from the arteriogenic ED group was significantly decreased compared with that in control group tissue (p ⱕ0.05, fig. 4). PJ caused a significant increase in the relaxation response to low frequencies EFS (0.5, 1 and 2 Hz) in each group but it had no significant effect on the relaxation response to high frequency EFS (5, 15 and 40 Hz) (fig. 4). PJ did not normalize relaxation differences between the arteriogenic ED and control groups. NOS expression in arteriogenic ED. Immunohistochemical staining showed sporadic nNOS on cavernous nerves, endothelium and smooth muscle, eNOS on endothelium and sporadic iNOS throughout the cavernous tissue (figs. 5 and 6). Ischemia did not alter NOS localization, while it caused a dramatic decrease in nNOS and eNOS, and increase in iNOS expression. PJ did not have a significant effect on nNOS, eNOS or iNOS expression (figs. 5 and 6). PJ prevented erectile tissue fibrosis. On histology the severity of erectile tissue fibrosis correlated with the severity of

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389

FIG. 2. PJ antioxidant characteristics and effect of PJ on J-774.A1 macrophage uptake of oxidized and native LDL. A, total polyphenol concentration of PJ. B, DPPH assay showed that free radical scavenging capacity of PJ was 2.0 ␮l/ml. C, PJ inhibited LDL oxidation. D, PJ inhibited LDL oxidation. E, oxidized LDL (Ox-LDL) degradation. F, native LDL degradation.

Hemodynamic measurements in placebo and PJ treated control and ED groups Mean Control ⫾ SEM Placebo MAP (mm Hg) 95.7 ⫾ 6.1 Intracavernous blood flow (ml/min/100 gm): Baseline 2.7 ⫾ 0.4 Erection 26.3 ⫾ 2.1 MICP/MAP 88.4 ⫾ 5.0 Time to max ICP (mins) 14.6 ⫾ 2.8 * ED placebo vs control placebo significantly different. † Control PJ vs control placebo significantly different.

Mean ED ⫾ SEM

PJ

Placebo

PJ

86.4 ⫾ 4.2

92.5 ⫾ 7.1

84.4 ⫾ 5.5

3.4 ⫾ 0.4 32.8 ⫾ 1.4† 95.4 ⫾ 6.5 8.6 ⫾ 3.1†

1.6 ⫾ 0.3* 8.2 ⫾ 1.3* 37.4 ⫾ 7.1* 27 ⫾ 5.0*

1.7 ⫾ 0.3 11.8 ⫾ 2.0 49.0 ⫾ 5.1 19 ⫾ 4.1

arterial occlusive disease (fig. 7). Histomorphometry revealed a decreased percent of smooth muscle in the arteriogenic ED group compared with that in controls (33% ⫾ 1.4% vs 42% ⫾ 1.0%). PJ prevented erectile tissue fibrosis in the arteriogenic ED group (fig. 8). The percent of cavernous smooth muscle in the ED group receiving PJ was significantly greater than that in the ED group receiving placebo (38.4% ⫾ 1.8% vs 33% ⫾ 1.4%, p ⫽ 0.002).

ED PJ Significant Difference

Vs Vs Vs Vs

control PJ ED placebo ⫹ control PJ control PJ ED placebo ⫹ control PJ

DISCUSSION

This study suggests that oxidative stress may have an important role in arteriogenic ED. Antioxidants may improve intracavernous blood flow, smooth muscle relaxation and erectile function in arteriogenic ED and nonpathogenic conditions, and prevent ischemia induced fibrosis. In addition to hemodynamic impairment, the mechanism of arteriogenic ED involves chronic exposure of erectile tissue

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FIG. 3. Erectile response to nerve stimulation. PJ significantly increased intracavernous blood flow and decreased time to maximum ICP in each group but it did not normalize differences that existed between 2 groups. Arrow indicates start of EFS.

to ischemia and hypoxia, nutrient deficiency and a lack of metabolic waste clearance. This condition creates a cytotoxic environment, leading to impaired NO synthesis and smooth muscle relaxation, ultimately resulting in erectile tissue fibrosis. It is suggested that the accumulation of endogenous NOS inhibitors in the corpus cavernosum may be involved in ischemic tissue dysfunction and structural damage. To our knowledge the nature of the endogenous cytotoxic factors in arteriogenic ED has not been reported. In normal tissue ROS such as superoxide are tightly regulated by physiological homeostatic mechanisms. ROS that are formed are detoxified by biological antioxidants. In pathological conditions such as ischemia ROS levels exceed the antioxidant capacity of a cell, leading to a deleterious state known as oxidative stress. When unchecked, these ROS can initiate a chain reaction of redox sensitive signaling events involving cytokines, kinases and transcription factors that regulate the expression of genes and proteins influencing vasoconstriction, modifying the extracellular matrix and inflammatory processes.20 They also lead to destructive changes in cellular components, including lipids, protein and DNA, ultimately leading to smooth muscle degeneration.20 It is known that aspects of the atherogenic process stimulate ROS production, including vascular endothelial injury, penetration and plaque accumulation in the arterial wall, macrophage activation, and the release of cytokines and growth factors that stimulate ROS producing enzymes.20 This may produce an excess of ROS in arteriogenic ED. In addition, arterial insufficiency may limit antioxidant availability and alteration in mitochondrial function due to chronic ischemia, while hypoxia may generate excessive ROS. The current study shows that ischemia related smooth muscle dysfunction and fibrosis are associated with the marked accumulation of isoprostane 8-epi-PGF2␣. Isoprostanes are known as unique markers of oxidative stress.19 They are the end products of arachidonate peroxidation and are produced by a free radical catalyzed mechanism involv-

ing ROS. Of the several subtypes isoprostane 8-epi-PGF2␣ has been extensively investigated in several organs because of its important role in oxidative stress related smooth muscle dysfunction and structural damage. Our data show that the levels of isoprostane 8-epi-PGF2␣ in ischemic tissues were approximately 3-fold greater than in controls. Oxidative stress has a role in NO metabolism and, thereby, it affects the relaxation of erectile tissue smooth muscle and intracavernous arteries. It has been shown that ROS such as superoxide destroy NO faster than its natural degradation rate, thus, limiting its bioavailability.13 The presence of antioxidants can neutralize superoxides, allowing NO to endure long enough to exert its biological effects. In the rabbit atherosclerotic model it was shown that the loss of vascular relaxation was due to the destruction of NO by excessive endothelial superoxide production, rather than to decreased synthesis. In the current study prolonged antioxidant therapy with PJ increased NO mediated smooth muscle relaxation in ischemic and control tissues without altering NOS expression. PJ significantly increased NO dependent smooth muscle relaxation at low frequency EFS but not at high frequencies. This may have occurred because high frequencies induce a huge release of NO in control tissue that surpasses the levels stimulated by PJ. In previous studies we found that during the course of cavernous ischemia nNOS and eNOS gene and protein expression decreased, while iNOS expression significantly increased.3, 18 In this study PJ did not affect these changes. The main active ingredients in PJ are polyphenol antioxidants, including hydrolyzable tannins, anthocyanins, ellagic acid and gallic acid. It is thought that a combination of these antioxidants is maximally active, rather than any individual component. Other natural polyphenol sources have been shown to affect NO mediated processes. Red wine polyphenols improved vasorelaxation with a concurrent increase in NO in hypercholesterolemic rabbits. However, in rat aortas it was shown that this red wine effect could be abolished by the addi-

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391

FIG. 4. Smooth muscle relaxation response to EFS was significantly decreased in ED group vs control (p ⱕ0.05). PJ enhanced smooth muscle relaxation to low frequency stimulation but had no significant effect on high frequencies. Asterisk indicates significant differences between PJ and placebo treated animals in same group (p ⱕ0.05).

FIG. 5. Immunohistochemical staining of eNOS. Cavernous eNOS (arrows) was dramatically decreased in ED group vs controls. PJ had no effect on eNOS expression. Reduced from ⫻400.

FIG. 6. Immunohistochemical staining of iNOS. Cavernous iNOS (arrows) was dramatically increased in ED group vs controls. PJ had no effect on iNOS expression. Reduced from ⫻400.

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FIG. 7. Relationship between arterial disease and cavernous fibrosis. Severity of cavernous fibrosis correlated with degree of arterial occlusive disease. A, iliac artery and erectile tissue structure in control animals. B, moderate cavernous fibrosis in animal with moderate arterial occlusive disease. C, severe cavernous fibrosis in animal with severe arterial occlusive disease. Reduced from ⫻400.

FIG. 8. Effect of antioxidant treatment on cavernous fibrosis in samples of tissues that underwent histomorphometric analysis. Erectile tissue smooth muscle content (red areas) was dramatically decreased and connective tissue (blue areas) was increased in ED group. Antioxidant treatment prevented fibrosis development in ED group. Reduced from ⫻400.

tion of the NO synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester), suggesting increased NO synthesis. Polyphenols from cocoa also showed a similar mechanism in humans. In addition to effects on NO, antioxidants may also act via angiotensin II, a key intermediary in vasoconstriction. Angiotensin II is formed from angiotensin I via angiotensin converting enzyme. This process is regulated by ROS and blocked by the addition of antioxidants. Pomegranate polyphenols have been shown to significantly inhibit angiotensin converting enzyme in humans. Antioxidant treatment with PJ prevented ischemia induced cavernous fibrosis in the arteriogenic ED group. These findings are consistent with the reported efficacy of antioxi-

dants in attenuating tubulointerstitial disease in rats with nephrosis. Therefore, oxidative stress and the formation of oxidative products in arteriogenic ED are presumed to have significant roles in erectile tissue fibrosis. Oxidative products are known to stimulate transforming growth factor-␤1 production and up-regulate the gene expression of fibronectin. It is thought that the transforming growth factor-␤1 that is newly produced in response to oxidative stress stimulates fibronectin expression. It has also been shown that collagen degradation is affected by ROS. In atherosclerotic rabbits metalloproteinases secreted from macrophage foam cells degrade the cellular matrix. The antioxidant N-acetylcysteine prevents this metalloproteinase release. These observations

ROLE OF OXIDATIVE STRESS AND ANTIOXIDANTS IN ERECTILE DYSFUNCTION

suggest that antioxidant therapy may be a promising option for preventing or reversing erectile tissue fibrosis. CONCLUSIONS

Our findings suggest that oxidative products accumulate in erectile tissue after prolonged ischemia, possibly through an intrinsic mechanism, and they appear to have a great role in the impairment of NO mediated smooth muscle relaxation and cavernous fibrosis in arteriogenic ED. Chronic administration of PJ caused significant increases in intracavernous blood flow and smooth muscle cell relaxation, possibly via increasing NO bioavailability. Antioxidant therapy with dietary products such as PJ may be a useful prophylactic tool for preserving erectile tissue function and preventing cavernous fibrosis in arteriogenic ED. Pomegranate concentrate was provided by POM Wonderful L. L. C., Los Angeles, California. REFERENCES

1. Krane, R. J., Goldstein, I. and Saenz de Tejada, I.: Impotence. N Engl J Med, 321: 1648, 1989 2. Siroky, M. B. and Azadzoi, K. M.: Male and female sexual dysfunction. In: Pelvic Floor Disorders. Edited by A. P. Bourcier, E. J. McGuire and P. Abrams. Philadelphia: W. B. Saunders Co., part II, chapt. 13, p. 89, 2004 3. Wang, T., Soker, S., Atala, A., Siroky, M. B. and Azadzoi, K. M.: Alterations in angiogenic growth factors and neuronal nitric oxide synthase expression in chronic cavernosal ischemia. Int J Impot Res, 16: 403, 2004 4. Brow, S. L., Seftel, A. D., Strohl, K. P. and Herbener, T..: Vasculogenic impotence and cavernosal oxygen tension. Int J Impot Res, 12: 19, 2000 5. Fanfulla, F., Malaguti, S., Montagna, T., Salvini, S., Bruschi, C., Crotti, P. et al: Erectile dysfunction in men with obstructive sleep apnea: an early sign of nerve involvement. Sleep, 23: 775, 2000 6. Slater, T. F.: Free-radical mechanisms in tissue injury. Biochem J, 222: 1, 1984 7. Bello-Klein, A., Bock, P. M., Travacio, M., Senna, S. M., Llesuy, S., de Bittencourt, P. I., Jr. et al: Myocardial oxidative stress and antioxidants in hypertension as a result of nitric oxide synthase inhibition. Cardiovasc Toxicol, 1: 43, 2001 8. Shi, Y., Patel, S., Davenpeck, K. L., Niculescu, R., Rodriguez, E. Magno, M. G., Ormont, M. L. et al: Oxidative stress and lipid retention in vascular grafts: comparison between venous and

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arterial conduits. Circulation, 103: 2408, 2001 9. Bowers, R., Cool, C., Murphy, R. C., Tuder, R. M., Hopken, M. W., Flores, S. C. et al: Oxidative stress in severe pulmonary hypertension. Am J Respir Crit Care Med, 169: 764, 2004 10. Aragno, M., Cutrin, J. C., Mastrocola, R., Perrelli, M. G., Restivo, F., Poli, G. et al: Oxidative stress and kidney dysfunction due to ischemia/reperfusion in rat: attenuation by dehydroepiandrosterone. Kidney Int, 64: 836, 2003 11. Hayashi, T., Saito, A., Okuno, S., Ferrand-Drake, M., Dodd, R. L. and Chan, P. H.: Oxidative injury to the endoplasmic reticulum in mouse brains after transient focal ischemia. Neurobiol Dis, 15: 229, 2004 12. Kinlay, S., Libby, P. and Ganz, P.: Endothelial function and coronary artery disease. Curr Opin Lipidol, 12: 383, 2001 13. Kinlay, S., Behrendt, D., Fang, J. C., Delagrange, D., Morrow, J., Witztum, J. L. et al: Long-term effect of combined vitamins E and C on coronary and peripheral endothelial function. J Am Coll Cardiol, 43: 629, 2004 14. Fuhrman, B., Lavy, A. and Aviram, M.: Consumption of red wine with meals reduces the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation. Am J Clin Nutr, 61: 549, 1995 15. Hayek, T., Fuhrman, B., Vaya, J., Rosenblat, M., Belinky, P., Coleman, R. et al: Reduced progression of atherosclerosis in apolipoprotein E-deficient mice following consumption of red wine, or its polyphenols quercetin, or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler Thromb Vasc Biol, 17: 2744, 1997 16. Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R. et al: Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr, 71: 1062, 2000 17. Aviram, M., Rosenbalt, M., Gaitini, D., Nitecki, S., Hoffman, A., Dorenfeld L. et al: Pomegranate juice consumption for 3 years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and LDL oxidation. Clin Nutr, 23: 423, 2004 18. Azadzoi, K. M., Master, T. A. and Siroky, M. B.: Effect of chronic ischemia on constitutive and inducible nitric oxide synthase expression in erectile tissue. J Androl, 25: 382, 2004 19. Tarcan, T., Siroky, M. B., Krane, R. J. and Azadzoi, K. M.: Isoprostane 8-epi PGF2alpha, a product of oxidative stress, is synthesized in the bladder and causes detrusor smooth muscle contraction. Neurourol Urodyn, 19: 43, 2000 20. Sayre, L. M., Smith, M. A. and Perry, G.: Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem, 8: 721, 2001