Role of Increased Penile Expression of Transforming Growth Factor‐β1 and Activation of the Smad Signaling Pathway in Erectile Dysfunction in Streptozotocin‐Induced Diabetic Rats

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Role of Increased Penile Expression of Transforming Growth Factor-b1 and Activation of the Smad Signaling Pathway in Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats Lu Wei Zhang, MD,* Shuguang Piao, MD, PhD,* Min Ji Choi, MS,* Hwa-Yean Shin, MS,* Hai-Rong Jin, MD,* Woo Jean Kim, PhD,* Sun U. Song, PhD,† Jee-Young Han, MD, PhD,‡ Seok Hee Park, PhD,‡ Mizuko Mamura, MD, PhD,§ Seong-Jin Kim, PhD,§ Ji-Kan Ryu, MD, PhD,* and Jun-Kyu Suh, MD, PhD* *Department of Urology and Laboratory of Regenerative Sexual Medicine, Inha University School of Medicine, Incheon, Korea; †Clinical Research Center, Inha University School of Medicine, Incheon, Korea; ‡Department of Pathology, Inha University School of Medicine, Incheon, Korea; §Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon, Korea DOI: 10.1111/j.1743-6109.2008.00977.x

ABSTRACT

Introduction. It has been suggested that transforming growth factor-b1 (TGF-b1) plays an important role in the pathogenesis of diabetes-induced erectile dysfunction. Aim. To investigate the expression and activity of Smad transcriptional factors, the key molecules for the initiation of TGF-b-mediated fibrosis, in the penis of streptozotocin (STZ)-induced diabetic rats. Methods. Fifty-two 8-week-old Sprague–Dawley rats were used and divided into control and diabetic groups. Diabetes was induced by an intravenous injection of STZ. Main Outcome Measures. Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (N = 12 per group). The penis was harvested and stained with Masson trichrome or antibody to TGF-b1, phospho-Smad2 (P-Smad2), smooth muscle a-actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF-b1 enzyme-linked immunosorbent assay (ELISA), P-Smad2/ Smad2, phospho-Smad3 (P-Smad3)/Smad3, fibronectin, collagen I, and collagen IV western blot, or hydroxyproline determination. Results. Erectile function was significantly reduced in diabetic rats compared with that in controls. The expression of TGF-b1, P-Smad2, and P-Smad3 protein evaluated by ELISA or western blot was higher in diabetic rats than in controls. Compared with that in control rats, P-Smad2 expression was higher mainly in smooth muscle cells and fibroblasts of diabetic rats, whereas no significant differences were noted in endothelial cells or in the dorsal nerve bundle. Cavernous smooth muscle and endothelial cell contents were lower in diabetic rats than in controls. Cavernous fibronectin, collagen IV, and hydroxyproline content was significantly higher in diabetic rats than in controls. Conclusion. Upregulation of TGF-b1 and activation of the Smad signaling pathway in the penis of diabetic rats might play important roles in diabetes-induced structural changes and deterioration of erectile function. Zhang LW, Piao S, Choi MJ, Shin H-Y, Jin H-R, Kim WJ, Song SU, Han J-Y, Park SH, Mamura M, Kim S-J, Ryu J-K, and Suh J-K. Role of increased penile expression of transforming growth factor-b1 and activation of the Smad signaling pathway in erectile dysfunction in streptozotocin-induced diabetic rats. J Sex Med 2008;5:2318–2329. Key Words. Diabetes Mellitus; Erectile Dysfunction; Transforming Growth Factor-beta; Smad; Fibrosis

J Sex Med 2008;5:2318–2329

© 2008 International Society for Sexual Medicine

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction Introduction

E

rectile dysfunction (ED) is three times more prevalent in diabetic men than in nondiabetic men, affecting up to 75% of all men with diabetes mellitus, and occurs at an earlier age in diabetic men than in the general population [1]. The etiology of ED in diabetes is multifactorial. The proposed mechanisms of ED in diabetes include central or autonomic neuropathy, and functional or structural derangements of corpus cavernosum endothelial cells and smooth muscle cells, which are essential for penile erection [2–10]. It has been suggested that alterations in growth factors, such as vascular endothelial growth factor [11], insulin-like growth factor-1 and its binding proteins [12], and transforming growth factor-b (TGF-b) [5], play an important role in the pathogenesis of diabetes-induced ED. Of those growth factors, TGF-b1 has been identified as the most relevant fibrogenic cytokine and is known to be upregulated in the corpus cavernosum tissues of streptozotocin (STZ)-induced diabetic rats [5]. This protein increases collagen synthesis in human corpus cavernous smooth muscle cells by 2.5- to 4.5-fold [13]. Furthermore, in an animal model, TGF-b1 causes a dose-dependent decrease in the percentage of cavernous smooth muscle cells when administered intracavernously in a single alginate microsphere [14]. TGF-b is known to mediate its fibrotic effects by activating the receptor-associated Smads, including Smad2 and Smad3 [15]. TGF-b binds to a type-I receptor known as activin receptor-like kinase (ALK)5. The phosphorylation of serine/ threonine residues in ALK5 subsequently phosphorylates the major downstream signaling molecules Smad2 and Smad3. When phosphorylated, Smad2/3 form a heteromeric complex with Smad4, which translocates into the nucleus and regulates the transcription of TGF-b-responsive genes, thereby inducing fibrosis-related changes [15]. Activation of Smad3 or Smad2 was observed in kidneys of STZ-induced diabetic mice [16] and db/db mice [17], whereas the loss of Smad3 attenuated a fibrotic response in a STZ-induced renal fibrosis model [18]. These findings support a role of Smad2 and Smad3 in tissue fibrosis. Furthermore, TGF-b is known to inhibit endothelial cell proliferation and migration via the ALK5Smad2/3 pathway [19,20]. Therefore, study of the expression and activity of Smad2/3 in the penis of a diabetic animal model is crucial for expanding our understanding of diabetes-related structural

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and functional changes in erectile tissue and for developing new therapeutics for diabetic ED targeting this pathway. In the present study, we investigated for the first time the distribution and activity of the TGF-b1– Smad signaling pathway in the penis of STZinduced diabetic ED rats. We also quantified the key structural components of the corpus cavernosum, including smooth muscle cells, endothelial cells, and extracellular matrix (ECM). Methods

Animals and Study Design A total of 52 male, 8-week-old Sprague–Dawley rats (weighing 270–300 g) were used in this study, divided into control (N = 26) and diabetic (N = 26) groups. The experiments performed were approved by the Institutional Animal Care and Use Subcommittee of our university. Diabetes was induced by an intravenous injection of STZ (55 mg/kg in 2 mL saline). Blood glucose levels were measured with an Accu-check blood glucose meter (Roche Diagnostics, Mannheim, Germany) before and 8 weeks after intravenous injection of STZ. Animals were considered to be diabetic if their fasting glucose concentrations were greater than 300 mg/dL. At 8 weeks after the induction of diabetes, we evaluated erectile function by cavernous nerve electrical stimulation (N = 12 per group), and the penis was then harvested for histologic examination. Penis specimens or blood samples from a separate group of animals were used for enzymelinked immunosorbent assay (ELISA), western blot, or hydroxyproline determination. Measurement of Erectile Function Rats from each group were anesthetized with chloral hydrate (20 mg/kg) intraperitoneally, and a carotid artery was cannulated to measure systemic arterial pressure. Bipolar platinum wire electrodes were placed around the cavernous nerve. Stimulation parameters were 1 to 5 V at a frequency of 12 Hz, a pulse width of 1 ms, and a duration of 1 minute. During tumescence, the maximal intracavernous pressure (ICP) and slope for the ICP to reach 80% of maximal ICP (S80) were recorded. The total ICP was determined by the area under the curve from the beginning of cavernous nerve stimulation until ICP returned to baseline or prestimulation pressure. The ratio of maximal ICP, slope (S80), and total ICP to mean arterial J Sex Med 2008;5:2318–2329

2320 pressure (MAP) were calculated to normalize for variations in systemic blood pressure.

Measurement of Cavernous Tissue and Plasma TGF-b1 Levels Penile tissues and plasma samples were obtained (N = 6 per group), quick frozen in liquid nitrogen, and stored at -70°C until measurement of TGF-b1 levels by ELISA. The samples were then processed according to the instructions provided with the kit (Quantikine ELISA kit; R&D Systems, Inc., Minneapolis, MN, USA). Data are expressed as pmol/g of wet weight tissue for cavernous tissue levels or pg/mL for plasma levels. Western Blot Equal amounts of protein (80 mg/lane) were electrophoresed on 10% sodium dodecylsulfatepolyacrylamide gels, transferred to nitrocellulose membranes, and probed with polyclonal antibody against phospho-Smad2 (P-Smad2), total Smad2 (which also recognizes Smad3), P-Smad3 (which also recognizes P-Smad1; Cell Signaling, Beverly, MA, USA; 1:750, respectively), total Smad3 (Zymed Laboratories, South San Francisco, CA, USA; 1:750), fibronectin (Abcam, Cambridge, UK; 1:300), collagen I (Abcam; 1:6,000), collagen IV (Abcam; 1:6,000), or b-actin (Abcam). Results were quantified by densitometry (N = 4 per group). Histologic Examinations A midportion of each penile segment was harvested and immediately fixed in 10% formalin phosphate-buffered solution before paraffin embedding. The specimens were cut (4 mm) and stained with Masson trichrome (N = 12 per group). For immunohistochemistry (N = 12 per group), paraffin block sections (4 mm) were incubated with antibody to TGF-b1 (Chemicon, Temecula, CA, USA; 1:50), P-Smad2 (Cell Signaling; 1:100), and smooth muscle a-actin (Sigma-Aldrich, St. Louis, MO, USA; 1:100), followed by Histostain®-Plus Bulk Kit (Zymed Laboratories). Control sections were incubated without the primary antibody at this step. For fluorescence microscopy (N = 12 per group), frozen tissue sections (20 mm) were incubated with antibody to factor VIII (DakoCytomation, Glostrup, Denmark; 1:50) at 4°C overnight. After several washes with phosphate-buffered saline, the sections were incubated with rhodamine-conjugated rabbit antibody to immunoglobulin G for 1 hour at room temperature. J Sex Med 2008;5:2318–2329

Zhang et al. Signals were visualized and digital images were obtained with an Apotome microscope (Zeiss, Gottingen, Germany). Semiquantitative analysis was performed to evaluate the intensity of TGF-b1 and P-Smad2 staining by one highly experienced pathologist. The TGF-b1 immunostaining was considered positive if brown color was noted in the cytoplasm or ECM, whereas P-Smad2 immunostaining was considered positive if brown color was noted in the cytoplasm or nucleus. Immunoreactivity was scored as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong), and was measured in smooth muscle cells, fibroblasts, endothelial cells, nerve bundles, and ECM. Because it is difficult to discriminate between smooth muscle cells and fibroblasts by morphology alone, we also performed immunohistochemical staining with antibody to smooth muscle a-actin in consecutive penile tissue sections to determine cellular distribution of TGF-b1 or P-Smad2 protein. Quantitative analysis of endothelium, smooth muscle, and collagen in cavernous tissue was done with an image analyzer system (NIH Image J 1.34, National Institutes of Health, Bethesda, MD, USA, available at http:// rsb.info.nih.gov/ij/index.html.). At least six fields (for magnification ¥ 400) or two fields (for magnification ¥ 100) in the cavernous tissue were examined from each tissue section and we analyzed two sections per animal.

Hydroxyproline Assay Collagen protein levels were estimated by hydroxyproline determinations (N = 4 per group) as previously described [21]. Briefly, aliquots of standard hydroxyproline or penis samples were hydrolyzed in alkali. The hydrolyzed samples were then mixed with a buffered chloramine-T reagent, and the oxidation was allowed to proceed for 25 minutes at room temperature. The chromophore was then developed by the addition of Ehrlich’s reagent, and the absorbance of the reddish purple complex was measured at 550 nm with a spectrophotometer. Absorbance values were plotted against the concentration of standard hydroxyproline, and the presence of hydroxyproline in penis tissue extracts was determined from the standard curve. Statistical Analysis Results are expressed as means ⫾ standard deviation. Mann–Whitney U-tests were used to evaluate whether differences between groups were significant. Probability values of less than 5% were considered significant.

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction Table 1

Metabolic and physiologic variables

Variable

Control

DM

P value

Initial weight (g) Final weight (g) Initial fasting glucose (mg/dL) Initial postprandial glucose (mg/dL) Final fasting glucose (mg/dL) Final postprandial glucose (mg/dL) MAP (cm H2O) Maximal ICP/MAP* 1.0V 2.5V 5.0V Total ICP/MAP* 1.0V 2.5V 5.0V Slope (S80)/MAP* 1.0V 2.5V 5.0V

286 ⫾ 7.1 467.2 ⫾ 26.3 82.3 ⫾ 4.4

288.7 ⫾ 6.9 240.9 ⫾ 44.5 81.2 ⫾ 5.5

0.09 <0.0001 0.40

92.8 ⫾ 9.9

90 ⫾ 10.5

0.35

96 ⫾ 10.1

429.6 ⫾ 94.0

<0.0001

109.7 ⫾ 11.1

570.8 ⫾ 55.2

<0.0001

142.5 ⫾ 5.8

137.5 ⫾ 10.3

0.35

0.80 ⫾ 0.09 0.93 ⫾ 0.14 0.93 ⫾ 0.07

0.58 ⫾ 0.11 0.62 ⫾ 0.13 0.92 ⫾ 0.03

<0.0001 <0.0001 0.39

43.1 ⫾ 6.3 49.1 ⫾ 7.5 59.2 ⫾ 5.7

33.7 ⫾ 9.8 33.4 ⫾ 8.6 56.9 ⫾ 3.6

<0.0001 <0.0001 0.10

0.023 ⫾ 0.003 0.042 ⫾ 0.004 0.050 ⫾ 0.006

0.007 ⫾ 0.002 0.012 ⫾ 0.004 0.049 ⫾ 0.004

<0.0001 <0.0001 0.49

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protein was mainly expressed in the fibroblasts and ECM of diabetic or control rats, whereas weak or virtually no expression was noted in smooth muscle cells, endothelial cells, or dorsal nerve. The penis tissue from diabetic rats showed higher TGF-b1 immunoreactivity in fibroblasts, ECM, and smooth muscles cells of the corpus cavernosum than did penis tissue from control rats (Table 2 and Figure 3).

Increased Expression of P-Smad2 and P-Smad3 in Diabetic Rat Penis We performed western blots to evaluate the expression of P-Smad2, total Smad2, P-Smad3,

*Ratio of maximal ICP, total ICP, and slope (S80) to MAP were calculated to normalize for variations in systemic blood pressure. DM = diabetes mellitus; ICP = intracavernous pressure; MAP = mean arterial pressure; Slope (S80) = slope for the ICP to reach 80% of maximal ICP; Total ICP = area under the curve from the beginning of cavernous nerve stimulation until ICP returned to baseline or prestimulation pressure.

Results

Metabolic and Physiologic Variables There were no significant differences in initial body weight or serum glucose concentrations between groups. At 8 weeks after diabetes was induced, the fasting and postprandial blood glucose concentrations of the diabetic rats were significantly higher than those of the control rats. Body weight was significantly lower in the diabetic rats than in the controls (Table 1). All erectile function variables, such as the ratio of maximal ICP, total ICP, and slope (S80) to MAP, which were recorded at 1 V and 2.5 V, were significantly lower in the diabetic rats than in the controls (Table 1 and Figure 1). Increase in the TGF-b1 Level in Diabetic Cavernous Tissue but not in Plasma TGF-b1 levels were measured in cavernous tissue and plasma by ELISA. The TGF-b1 level in the corpus cavernosum tissue of diabetic rats was 2.3fold that in the corpus cavernosum of controls (Figure 2A). However, the plasma TGF-b1 level did not differ significantly between groups (Figure 2B). We performed immunohistochemical staining to localize TGF-b1 protein in the penis. TGF-b1

Figure 1 Decrease in erectile function in diabetic rat. Representative intracavernous pressure (ICP) responses to electrical stimulation of cavernous nerve for age-matched control and diabetic group. The stimulus interval is indicated by a solid bar. DM = diabetes mellitus; SAP = systemic arterial pressure.

J Sex Med 2008;5:2318–2329

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Figure 2 TGF-b1 level in corpus cavernosum tissue and plasma. Each bar depicts the mean values (⫾standard deviation) for N = 6 animals per group. (A) Cavernous tissue TGF-b1 level. *P < 0.01 compared with the control group. (B) Plasma TGF-b1 level. DM = diabetes mellitus.

and total Smad3 in the corpus cavernosum. We quantified the ratios of P-Smad2 to total Smad2, and of P-Smad3 to total Smad3 to determine the activity of Smad2 and Smad3. The ratios of P-Smad2 to total Smad2 and of P-Smad3 to total Smad3 were significantly greater in the diabetic group than in the controls. Although the antibody used to detect total Smad2 protein cross-reacts with total Smad3, the expression of total Smad2 seemed to be higher in the diabetic rats than in the controls, because the expression of total Smad3 remained unchanged. However, the difference was not as great as that in P-Smad2 (Figure 4). To localize Smad protein expression in the penis, we performed immunohistochemical staining with antibody to P-Smad2, but not P-Smad3, because the P-Smad3 antibody used in this study can also recognize P-Smad1, an intracellular signal mediator of bone morphogenetic protein, and an antibody that specifically detects P-Smad3 protein is not yet available. Similar to the results of the western blot, P-Smad2 immunoreactivity in the penis was higher in the diabetic rats than in the controls. P-Smad2 immunoreactivity in smooth muscles cells of the corpus cavernosum and dorsal Table 2 Semiquantitative analysis of TGF-b1 immunoreactivity

Smooth muscle CC DBV Endothelium CC DBV ECM Fibroblasts Dorsal nerve

Control

DM

P value

0.13 ⫾ 0.35 0.12 ⫾ 0.33

0.75 ⫾ 0.71 0.5 ⫾ 0.53

0.042 0.12

0.63 ⫾ 0.52 0.38 ⫾ 0.52 1.5 ⫾ 0.53 1.38 ⫾ 0.52 0.13 ⫾ 0.35

1 ⫾ 0.53 0.5 ⫾ 0.53 2.63 ⫾ 0.52 2.25 ⫾ 0.71 0.25 ⫾ 0.46

0.176 0.642 <0.0001 0.014 0.554

CC = corpus cavernosum; DBV = dorsal blood vessel; DM = diabetes mellitus; ECM = extracellular matrix; TGF-b1 = transforming growth factor-b1.

J Sex Med 2008;5:2318–2329

blood vessels and in fibroblasts of the corpus cavernosum was higher in penis tissue from diabetic rats than in penis tissue from control rats. Although weak to moderate P-Smad2 expression was noted in endothelial cells of the corpus cavernosum and dorsal blood vessels, this expression did not differ significantly between groups. We observed weak or almost no P-Smad2 expression in the dorsal nerve bundles (Table 3 and Figure 5).

Determination of Fibronectin, Collagen I, Collagen IV, and Hydroxyproline Content We performed western blots to evaluate the expression of fibronectin, collagen I, and collagen IV in the corpus cavernosum. The fibronectin and collagen IV protein expression was significantly greater in the diabetic group than in the controls. No significant difference was found in collagen I protein expression between the groups (Figure 6). We also determined collagen content in the corpus cavernosum tissue by measuring the amount of hydroxyproline. Hydroxyproline contents in the penile tissue of diabetic rats increased to 126% of those of control rats (9.9 ⫾ 1.1 vs. 12.5 ⫾ 0.9 mg/g of tissue, P < 0.05). Quantification of Endothelium, Smooth Muscle, and Collagen by Use of an Automated Computer Morphometric Analysis System Cavernous endothelial content was significantly lower in the diabetic group than in the control group (Figure 7A, B). Also, there was significantly less smooth muscle content, as evaluated by both immunohistochemical staining with antibody against smooth muscle a-actin (brown) and Masson trichrome staining (red), in the diabetic rats than in the controls (Figure 7A, C). However, no significant difference was found in collagen content between groups (Figure 7A, D).

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction

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Figure 3 Increase in TGF-b1 expression in diabetic rat penis. (A, B) Immunohistochemical staining of penile tissue performed with antibody to TGF-b1. (C) Immunohistochemical staining of consecutive penile tissue sections performed with antibody to TGF-b1 or smooth muscle a-actin. Arrows denote positive smooth muscle cells (SM), endothelial cells (E), fibroblasts (F), extracellular matrix (ECM), and nerve bundle (N). 2nd Ab = secondary antibody control; A = artery; DM = diabetes mellitus; V = vein. Magnification ¥ 100 (A) or ¥400 (B, C). Bars indicate 60 mm (A) or 30 mm (B, C).

Discussion

In the present study, cavernous tissue TGF-b1 expression was significantly higher in diabetic rats

than in controls, as determined by both ELISA and immunohistochemical staining. This result further supports the previous findings of Ahn et al. [5], who showed an increase in TGF-b1 expression in the J Sex Med 2008;5:2318–2329

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Figure 4 Increase in Smad2 and Smad3 phosphorylation in diabetic rat corpus cavernosum tissue. (A) Western blot analysis showing the protein expression of P-Smad2, total Smad2, P-Smad3, and total Smad3 in rat corpus cavernosum tissues. (B, C) Data are presented as the ratio of phosphorylated protein to total protein. The relative ratio measured in the control group is arbitrarily presented as 1. Each bar depicts the mean values (⫾standard deviation) from four experiments per group. *P < 0.01 compared with the control group. DM = diabetes mellitus; P-Smad2 = phospho-Smad2; P-Smad3 = phospho-Smad3.

corpus cavernosum tissue of STZ-induced diabetic rats by immunohistochemical staining. Similar to our previous study in patients with diabetes and ED [22], there was no difference in plasma TGF-b1 levels between groups, although there is clear evidence that glucose stimulates the production of TGF-b1 by several cell types in vitro. TGF-b1 most likely exerts this effect by stimulating the production of protein kinase C via the formation of Table 3 Semiquantitative analysis of P-Smad2 immunoreactivity

Smooth muscle CC DBV Endothelium CC DBV Fibroblasts Dorsal nerve

Control

DM

P value

1.63 ⫾ 0.74 1.88 ⫾ 0.64

2.75 ⫾ 0.46 2.63 ⫾ 0.52

0.0027 0.023

1.5 ⫾ 0.53 1.5 ⫾ 0.76 1.13 ⫾ 0.64 0.63 ⫾ 0.52

1.63 ⫾ 0.52 1.75 ⫾ 0.71 2.13 ⫾ 0.83 0.75 ⫾ 0.71

0.64 0.51 0.018 0.69

CC = corpus cavernosum; DBV = dorsal blood vessel; DM = diabetes mellitus; P-Smad2 = phospho-Smad2.

J Sex Med 2008;5:2318–2329

diacylglycerol [23]. On the contrary, other investigators reported that plasma and serum TGF-b1 levels are elevated in persons with type I or type II diabetes [24,25]. A possible reason for this discrepancy may be differences in the severity or duration of diabetes. Therefore, additional study is required to investigate whether the severity or duration of diabetes affects circulating TGF-b1 concentrations. We showed that phosphorylation of Smad2 and Smad3, the crucial step in the initiation of TGF-b signal transduction, was significantly higher in diabetic rats than in controls, as evaluated by immunoblot analysis. We also performed immunohistochemical staining to identify which subpopulation of corpus cavernosum cells or dorsal neurovascular bundle cells expressed P-Smad2. P-Smad2 immunoreactivity in smooth muscle cells and fibroblasts was higher in penis tissue from diabetic rats than in penis tissue from control rats. It has been reported that TGF-b1 inhibits smooth muscle cell proliferation via extension of the G2 phase or arrest in the late G1 phase of the cell cycle

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction

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Figure 5 Increase in P-Smad2 expression in diabetic rat penis. (A) Immunohistochemical staining of penile tissue performed with antibody to P-Smad2. (B) Immunohistochemical staining of consecutive penile tissue sections performed with antibody to P-Smad2 or smooth muscle a-actin. Arrows denote positive smooth muscle cells (SM), endothelial cells (E), fibroblasts (F), and nerve bundle (N). 2nd Ab = secondary antibody control; A = artery; DM = diabetes mellitus; P-Smad2 = phospho-Smad2; V = vein. Magnification ¥ 400. Bars indicate 30 mm.

[26,27]. Furthermore, Redondo et al. [28] reported that apoptosis of vascular smooth muscle cells induced by pioglitazone, a peroxisome proliferatoractivated receptor g ligand, was mediated by the TGF-b1-Smad2 pathway. TGF-b is also known to inhibit endothelial cell proliferation and migration via the ALK5Smad2/3 pathway [19,20] or ALK5/Smad3 pathway [29], whereas inhibition of ALK5 facilitates endothelial cell proliferation [20,29]. However, P-Smad2 expression in endothelial cells did not differ significantly between groups. Normal penile erection is a predominantly vascular event that involves interaction between endothelial and smooth muscle cells in the corpus cavernosum. Loss or dysfunction of these cells in

diabetes is thought to play a central role in the pathophysiology of ED [4–10]. In agreement with the result of previous studies [4,5], quantitative damage to the smooth muscle cells and endothelial cells of the corpus cavernosum was seen in the diabetic rats. These results suggest that diseasespecific or cell type-specific overexpression of P-Smad2 may play an important role in the diabetes-induced structural changes, i.e., loss of smooth muscle cell content, in the penis. Other factors, such as reactive oxygen species, lipid peroxidation, and advanced glycation end products, may result in a loss of endothelial cells content [30,31], because no significant difference in P-Smad2 expression was found in endothelial cells between diabetic and control rats in this study. J Sex Med 2008;5:2318–2329

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Figure 6 Increase in fibronectin and collagen IV expression in diabetic rat corpus cavernosum tissue. (A) Western blot analysis showing the protein expression of fibronectin, collagen I, and collagen IV in rat corpus cavernosum tissues. (B–D) Data are presented as the relative density of each protein compared with that of b-actin. The relative ratio measured in the control group is arbitrarily presented as 1. Each bar depicts the mean values (⫾standard deviation) from four experiments per group. *P < 0.01 compared with the control group. DM = diabetes mellitus.

The content of cavernous ECM is also important for normal penile erection. Previously, we found increases in cavernous ECM content in patients with vasculogenic ED by histologic study [32]. In the present study, the fibronectin and collagen IV protein expression was significantly greater in the corpus cavernosum tissue of diabetic rats than in the controls by western blot analysis. Previous studies reported that TGF-b increases ECM accumulation through the stimulation of fibronectin and collagen IV production in glomerular epithelial cells, resulting in interstitial fibrosis and glomerular sclerosis [33], whereas neutralizing anti-TGF-b antibody reduces renal expression of fibronectin and collagen IV [34]. However, our study revealed that collagen I protein expression did not differ significantly between groups. This finding is similar to those of Lin et al. [35], who showed an increase in collagen IV but not collagen I protein expression in the corpus cavernosum tissue of aging rats. We also determined collagen content by measuring the J Sex Med 2008;5:2318–2329

amount of hydroxyproline. A modest but significant increase in hydroxyproline content was noted in the corpus cavernosum tissue of diabetic rats compared with that in controls. Hydroxyproline content has been reported to be increased in vaginal and kidney tissues of diabetic animals [36,37]. However, no detectable difference in cavernous collagen area was found between groups by histologic study, i.e., Masson trichrome staining. One possible reason for this discrepancy may be due the use of relatively short-term (8-week period) diabetic rats in this study. Thus, it is possible that long-term diabetic animals may show increases in collagen content by both biochemical and histological evaluation. Although oral phosphodiesterase (PDE) type 5 inhibitors are effective and well-tolerated modalities for ED, men with diabetes tend to respond less positively to these agents [38]. Severe endothelial dysfunction or autonomic neuropathy may be responsible for this poor response, because PDE5 inhibitors would be ineffective if the endogenous

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction

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Figure 7 Quantification of endothelium, smooth muscle, and collagen area. (A) Masson trichrome staining and immunohistochemical staining of cavernous tissue performed with antibody to factor VIII and smooth muscle a-actin in control or diabetic rats (magnification ¥ 100). Bars indicate 100 mm. (B–D) An image analyzer was used to quantitate endothelium, smooth muscle, and collagen content in cavernous tissues. Each bar depicts the mean values (⫾standard deviation) for N = 12 animals per group. *P < 0.01 compared with the control group. DM = diabetes mellitus; MT = Masson trichrome staining.

bioavailable nitric oxide (NO) is insufficient. Therefore, additional therapeutic strategies are needed to overcome this problem. We think therapies aimed at blocking the TGF-b signal pathway, which would increase the smooth muscle content and relaxation response to endogenous NO by inhibiting smooth muscle apoptosis, might be efficacious in the treatment or prevention of diabetic ED when administered either alone or in combination with PDE5 inhibitors. To our knowledge, this is the first report documenting the activation and differential distribution of Smad2 and Smad3, the key intracellular signal molecules for the initiation of TGF-b-mediated fibrosis, in diabetic rat penis. However, further studies are needed to determine whether the inhibition of Smad2 and Smad3—using transgenic mice or antisense genes for Smad2 and Smad3—or inhibition of TGF-b type I receptor (ALK5) can restore diabetes-induced structural and functional derangements in the penis to clarify the precise roles and specific functions of Smad2 and Smad3.

rats may play important roles in diabetes-induced structural changes and deterioration of erectile function. Acknowledgments

This study was supported by Grant No. A060147 from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Korea (Ji-Kan Ryu) and by the Korea Science and Engineering Foundation through the National Research Lab. Program funded by the Ministry of Science and Technology (No. R0A-2007-00020018-0, Jun-Kyu Suh). The authors thank Jennifer Scales for help in preparing the manuscript.

Conclusion

Corresponding Author: Ji-Kan Ryu, MD, PhD, Department of Urology and Laboratory of Regenerative Sexual Medicine, Inha University School of Medicine, 7-206, 3rd ST, Shinheung-Dong, Jung-Gu, Incheon 400711, Republic of Korea. Tel: +82-32-890-3505; Fax: +82-32-890-3099; E-mail: [email protected] Jun-Kyu Suh, MD, PhD, Department of Urology and Laboratory of Regenerative Sexual Medicine, Inha University School of Medicine, 7-206, 3rd ST, ShinheungDong, Jung-Gu, Incheon 400-711, Republic of Korea. Tel: +82-32-890-3441; Fax: +82-32-890-3097; E-mail: [email protected]

Upregulation of TGF-b1 and activation of the Smad signaling pathway in the penis of diabetic

Conflict of Interest: All authors declare no conflict of interest. J Sex Med 2008;5:2318–2329

2328 Statement of Authorship

Category 1 (a) Conception and Design Lu Wei Zhang; Woo Jean Kim; Mizuko Mamura; Seong-Jin Kim; Ji-Kan Ryu; Jun-Kyu Suh (b) Acquisition of Data Lu Wei Zhang; Shuguang Piao; Min Ji Choi; HwaYean Shin; Hai-Rong Jin (c) Analysis and Interpretation of Data Lu Wei Zhang; Sun U. Song; Jee-Young Han; Seok Hee Park

Category 2 (a) Drafting the Article Lu Wei Zhang; Ji-Kan Ryu; Jun-Kyu Suh (b) Revising It for Intellectual Content Mizuko Mamura; Seong-Jin Kim; Ji-Kan Ryu; JunKyu Suh

Category 3 (a) Final Approval of the Completed Article Ji-Kan Ryu; Jun-Kyu Suh

References

1 Hakim LS, Goldstein I. Diabetic sexual dysfunction. Endocrinol Metab Clin North Am 1996;25:379– 400. 2 McVary KT, Rathnau CH, McKenna KE. Sexual dysfunction in the diabetic BB/WOR rat: A role of central neuropathy. Am J Physiol 1997;272:R259– 67. 3 El-Sakka AI, Lin CS, Chui RM, Dahiya R, Lue TF. Effects of diabetes on nitric oxide synthase and growth factor genes and protein expression in an animal model. Int J Impot Res 1999;11:123–32. 4 Burchardt T, Burchardt M, Karden J, Buttyan R, Shabsigh A, de la Taille A, Ng PY, Anastasiadis AG, Shabsigh R. Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat. J Urol 2000;164: 1807–11. 5 Ahn GJ, Sohn YS, Kang KK, Ahn BO, Kwon JW, Kang SK, Lee BC, Hwang WS. The effect of PDE5 inhibition on the erectile function in streptozotocininduced diabetic rats. Int J Impot Res 2005;17:134– 41. 6 Chitaley K, Luttrell I. Strain differences in susceptibility to in vivo erectile dysfunction following 6 weeks of induced hyperglycemia in the mouse. J Sex Med 2008;5:1149–55. 7 Esposito K, Ciotola M, Giugliano F, Sardelli L, Giugliano F, Maiorino MI, Beneduce F, De Sio M, Giugliano D. Phenotypic assessment of endothelial microparticles in diabetic and nondiabetic men with erectile dysfunction. J Sex Med 2008;5:1436–42. J Sex Med 2008;5:2318–2329

Zhang et al. 8 Wingard C, Fulton D, Husain S. Altered penile vascular reactivity and erection in the Zucker obesediabetic rat. J Sex Med 2007;4:348–62. 9 Vignozzi L, Morelli A, Filippi S, Ambrosini S, Mancina R, Luconi M, Mungai S, Vannelli GB, Zhang XH, Forti G, Maggi M. Testosterone regulates RhoA/Rho-kinase signaling in two distinct animal models of chemical diabetes. J Sex Med 2007;4:620–30. 10 Carneiro FS, Giachini FR, Lima VV, Carneiro ZN, Leite R, Inscho EW, Tostes RC, Webb RC. Adenosine actions are preserved in corpus cavernosum from obese and type II diabetic db/db mouse. J Sex Med 2008;5:1156–66. 11 Jesmin S, Sakuma I, Salah-Eldin A, Nonomura K, Hattori Y, Kitabatake A. Diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type II diabetic rat model: A possible cause for erectile dysfunction in diabetes. J Mol Endocrinol 2003;31: 401–18. 12 Abdelbaky TM, Brock GB, Huynh H. Improvement of erectile function in diabetic rats by insulin: Possible role of the insulin-like growth factor system. Endocrinology 1998;139:3143–7. 13 Moreland RB, Traish AM, McMillin MA, Smith B, Goldstein I, Saenz de Tejada I. PGE1 suppresses the induction of collagen synthesis by transforming growth factor-beta 1 in human corpus cavernosum smooth muscle. J Urol 1995;153:826–34. 14 Nehra A, Gettman MT, Nugent M, Bostwick DG, Barrett DM, Goldstein I, Krane RJ, Moreland RB. Transforming growth factor-beta1 (TGF-beta1) is sufficient to induce fibrosis of rabbit corpus cavernosum in vivo. J Urol 1999;162:910–5. 15 Massague J, Chen YG. Controlling TGF-beta signaling. Genes Dev 2000;14:627–44. 16 Isono M, Chen S, Won HS, Carmen Iglesias-De La Cruz MC, Ziyadeh F. Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells. Biochem Biophys Res Commun 2002;296:1356– 65. 17 Hong SW, Isono M, Chen S, Iglesias-De La Cruz MC, Han DC, Ziyadeh FN. Increased glomerular and tubular expression of transforming growth factor-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 2001;158:1653–63. 18 Fujimoto M, Maezawa Y, Yokote K, Joh K, Kobayashi K, Kawamura H, Nishimura M, Roberts AB, Saito Y, Mori S. Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun 2003;305: 1002–7. 19 Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P. Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J 2002;21:1743–53.

Activation of Smad Signaling Pathway in Diabetic Erectile Dysfunction 20 Watabe T, Nishihara A, Mishima K, Yamashita J, Shimizu K, Miyazawa K, Nishikawa S, Miyazono K. TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cellderived endothelial cells. J Cell Biol 2003;163: 1303–11. 21 Kesave Reddy G, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 1996;29:225–9. 22 Ryu JK, Song SU, Choi HK, Seong DH, Yoon SM, Kim SJ, Suh JK. Plasma transforming growth factor-beta1 levels in patients with erectile dysfunction. Asian J Androl 2004;6:349–53. 23 Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta. J Clin Invest 1994;93:536–42. 24 Pfeiffer A, Drewes C, Middelberg-Bisping K, Schatz H. Elevated plasma levels of transforming growth factor-beta 1 in NIDDM. Diabetes Care 1996;19:1113–7. 25 Esmatjes E, Flores L, Lario S, Clària J, Cases A, Iñigo P, Campistol JM. Smoking increases serum levels of transforming growth factor-beta in diabetic patients. Diabetes Care 1999;22:1915–6. 26 Grainger DJ, Kemp PR, Witchell CM, Weissberg PL, Metcalfe JC. Transforming growth factor beta decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase. Biochem J 1994;299: 227–35. 27 Reddy KB, Howe PH. Transforming growth factor beta 1-mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest. J Cell Physiol 1993;156:48–55. 28 Redondo S, Ruiz E, Santos-Gallego CG, Padilla E, Tejerina T. Pioglitazone induces vascular smooth muscle cell apoptosis through a peroxisome proliferator-activated receptor-gamma, transforming growth factor-beta1, and a Smad2-dependent mechanism. Diabetes 2005;54:811–7. 29 Castañares C, Redondo-Horcajo M, MagánMarchal N, ten Dijke P, Lamas S, RodríguezPascual F. Signaling by ALK5 mediates

30

31

32

33

34

35

36

37

38

2329

TGF-beta-induced ET-1 expression in endothelial cells: A role for migration and proliferation. J Cell Sci 2007;120:1256–66. Arduini A, Stern A, Storto S, Belfiglio M, Mancinelli G, Scurti R, Federici G. Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes. Arch Biochem Biophys 1989;273:112–20. Renard C, Chappey O, Wautier MP, Nagashima M, Lundh E, Morser J, Zhao L, Schmidt AM, Scherrmann JM, Wautier JL. Recombinant advanced glycation end product receptor pharmacokinetics in normal and diabetic rats. Mol Pharmacol 1997; 52:54–62. Ryu JK, Han JY, Chu YC, Song SU, Lee KH, Yoon SM, Suh JK, Kim SJ. Expression of cavernous transforming growth factor-beta1 and its type II receptor in patients with erectile dysfunction. Int J Androl 2004;27:42–9. Nakamura T, Miller D, Ruoslahti E, Border WA. Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-beta 1. Kidney Int 1992;41:1213–21. Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization of TGF-beta by anti-TGF-beta antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZinduced diabetic mice. Diabetes 1996;45:522–30. Lin JS, Tsai YS, Lin YM, Lin CS, Chow NH. Ageassociated changes in collagen content and its subtypes within rat corpora cavernosa with computerized histomorphometric analysis. Urology 2001; 57:837–42. Ferrini MG, Nolazco G, Vernet D, GonzalezCadavid NF, Berman J. Increased vaginal oxidative stress, apoptosis, and inducible nitric oxide synthase in a diabetic rat model: Implications for vaginal fibrosis. Fertil Steril 2006;86(4 suppl):1152–63. Klein L, Butcher DL, Sudilovsky O, Kikkawa R, Miller M. Quantification of collagen in renal glomeruli isolated from human nondiabetic and diabetic kidneys. Diabetes 1975;24:1057–65. Carson CC, Burnett AL, Levine LA, Nehra A. The efficacy of sildenafil citrate (Viagra) in clinical populations: An update. Urology 2002;60(2 suppl):12– 27.

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