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Keywords:

  • Erectile Dysfunction;
  • Radical Prostatectomy;
  • Cavernous Nerve Injury;
  • Endothelial Dysfunction;
  • Cavernous Fibrosis;
  • Transforming Growth Factor Beta;
  • Smad7

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

Introduction

Men with erectile dysfunction (ED) respond poorly to oral phosphodiesterase-5 inhibitors following radical prostatectomy. Recent studies have reported that up-regulation of transforming growth factor-β1 (TGF-β1) and activation of the Smad signaling pathway play important roles in cavernous fibrosis and in the deterioration of erectile function in a mouse model of cavernous nerve injury (CNI) and in patients with spinal cord injury. The mothers against decapentaplegic homolog 7 (Smad7) is known to inhibit the phosphorylation of Smad2 and Smad3.

Aim

To investigate the effectiveness of adenoviruses encoding Smad7 gene (Ad-Smad7) on erectile function in a mouse model of CNI.

Methods

Twelve-week-old C57BL/6J mice were used and distributed into 7 groups: sham operation group, untreated CNI group, and CNI groups receiving a single intracavernous injection of adenovirus encoding LacZ (1 × 108 virus particles [vp]/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 107, 1 × 108, 2 × 108, or 1 × 109 vp/20 μL).

Main Outcome Measures

Two weeks after bilateral cavernous nerve crushing and treatment, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis.

Results

The highest erectile response was noted in CNI mice treated with Ad-Smad7 at a dose of 1 × 108 vp, which reached up to 82–85% of sham control values. Local delivery of Ad-Smad7 significantly decreased endothelial cell apoptosis and the production of extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV, and induced endothelial nitric oxide synthase phosphorylation in the corpus cavernosum tissue of CNI mice.

Conclusion

The adenovirus-mediated gene transfer of Smad7 successfully restored erectile function by enhancing endothelial cell function and through antifibrotic effects. These findings suggest that inhibition of the TGF-β signaling pathway by use of Smad7 may represent a promising therapeutic strategy for ED induced by radical prostatectomy. Song KM, Chung J-S, Choi MJ, Jin H-R, Yin GN, Kwon M-H, Park J-M, Kim WJ, Lee S-J, Kim S-J, Ryu J-K, and Suh J-K. Effectiveness of intracavernous delivery of adenovirus encoding Smad7 gene on erectile function in a mouse model of cavernous nerve injury. J Sex Med 2014;11:51–63.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

Despite advances in surgical techniques for radical prostatectomy in the treatment of prostate cancer, such as nerve-sparing techniques and robotic procedures, a significant proportion of patients still suffer from erectile dysfunction (ED) following surgery [1,2]. Because of the close proximity of the cavernous nerves to the prostate gland and their microscopic nature, partial cavernous nerve injury (CNI) or neurapraxia is unavoidable even with bilateral nerve-sparing approaches [3,4]. Although oral phosphodiesterase-5 (PDE5) inhibitors are generally effective therapy for ED, men with ED resulting from radical prostatectomy respond poorly to these therapies [5,6].

The pathophysiologic mechanisms of radical prostatectomy-induced ED are not yet completely understood. The accumulating evidence suggests that a variety of etiologic factors, including increased reactive oxygen species, cavernous hypoxia, up-regulation of profibrotic factors and cavernous fibrosis from increased collagen synthesis, increases in cavernous endothelial and smooth muscle cell apoptosis, and a reduction of nitric oxide synthase nerve density, are responsible for the development of ED after CNI [7–16].

The transforming growth factor beta (TGF-β) pathway has been found to be involved in tissue fibrosis. We and other investigators reported that CNI in animals increases the expression of TGF-β1 [9–12] and induces the phosphorylation of the mothers against decapentaplegic homolog 2 (Smad2) transcriptional factor [12], the crucial step in the initiation of TGF-β-mediated tissue fibrosis, in the corpus cavernosum tissue, which promotes the deposition of extracellular matrix (ECM) proteins and tissue fibrosis. Moreover, the TGF-β and Smad2/3 signaling pathway is known to mediate apoptosis of smooth muscle and endothelial cells and to inhibit the proliferation of these cells [17–22]. We recently observed in mice that up-regulation of the TGF-β1–Smad2 signaling pathway in the corpus cavernosum tissue and apoptosis of cavernous smooth muscle and endothelial cells begin as early as 3 days after CNI [12]. These findings suggest that early intervention targeting the TGF-β signaling pathway might be efficacious in the treatment of radical prostatectomy-induced ED.

Smad7 is an inhibitory Smad protein that blocks TGF-β signaling through a negative feedback loop. Smad7 binds to TGF-β type I receptor, which blocks the phosphorylation of the receptor-associated Smads, including Smad2 and Smad3, and eventually inhibits downstream signaling of TGF-β [23]. Thus, Smad7 can inhibit diverse cellular processes regulated by TGF-β, such as cell proliferation, apoptosis, and fibrosis [23]. A recent study reported that Smad7 gene transfer attenuated renal fibrosis in diabetic rats, whereas targeted deletion of the Smad7 gene accelerated renal fibrosis and inflammation [24].

In the present study, we examined the effectiveness of intracavernous delivery of adenoviruses encoding the Smad7 gene (Ad-Smad7) on erectile function in a mouse model of CNI induced by bilateral cavernous nerve crushing.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

Generation of Smad7 or LacZ Adenovirus

Replication-deficient adenoviruses expressing FLAG-tagged Smad7 or bacterial β-galactosidase (LacZ) were constructed. Viruses were propagated in HEK-293 cells and purified by cesium chloride gradient centrifugation. All cesium chloride-purified adenoviruses were titered by O.D. 260 (O.D. 260, Inc., Boise, ID, USA) and then stored at −80°C until used.

Animals and Treatments

Twelve-week-old C57BL/6J mice were used in this study. The experiments performed were approved by the Institutional Animal Care and Use Subcommittee of our university. The mice were distributed into seven groups: sham operation group, bilateral CNI group without treatment, bilateral CNI group receiving a single intracavernous injection of adenovirus encoding LacZ (Ad-LacZ; 1 × 108 virus particles [vp]/20 μL), and bilateral CNI groups receiving a single intracavernous injection of Ad-Smad7 (1 × 107, 1 × 108, 2 × 108, or 1 × 109 vp/20 μL, respectively). To determine the optimal dosage for erectile function recovery, we administered Ad-Smad7 at different doses. We compressed the penis at the base with a vascular clamp immediately before injection, and the clamp remained in place for 30 minutes to restrict blood flow out of the penis.

The sham operation group underwent exposure of the prostate to enable visualization of the cavernous nerves bilaterally without any direct cavernous nerve manipulation. In the CNI groups, the cavernous nerves were crushed with a nonserrated hemostat (Karl Stortz Co., Tuttlingen, Germany). The hemostat was applied with full tip closure to each cavernous nerve 1 mm distal to the ganglion for 2 minutes. After CNI, the penis was exposed by use of a sterile technique. A 30-gauge syringe was used to administer a single injection of Ad-LacZ or Ad-Smad7 into the midportion of the corpus cavernosum. The incision was closed with 6-O Vicryl (polyglactin 910) sutures. All procedures were done with the aid of a dissecting microscope (Zeiss, Gottingen, Germany).

We evaluated erectile function by electrical stimulation of the cavernous nerve 2 weeks after CNI and treatment (n = 6 per group), after which the penis was harvested for histologic examination. We also determined the effect of Ad-Smad7 on the production of ECM proteins by Western blot analysis (n = 4 per group) as previously described [25]. The number of animals used for this study is summarized in Table 1.

figure

Figure 1. In vivo expression of Smad7 protein. Immunofluorescent staining of cavernous tissue performed with antibodies to FLAG (red), platelet endothelial cell adhesion molecule 1 (PECAM-1; blue), and smooth muscle α-actin (green) in cavernous nerve injury mice 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). Scale bar = 50 μm. Results were similar from six independent samples. At least six different fields in the cavernous tissue were examined.

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Figure 2. Adenoviral Smad7 gene transfer restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. (A) Representative ICP responses for the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 107, 1 × 108, 2 × 108, or 1 × 109 vp/20 μL, respectively). The stimulus interval is indicated by the solid bar. (B, C) Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group. Each bar depicts the mean (±SE) values (n = 6 animals per group). *P < 0.01 compared with the sham operation group. #P < 0.01 compared with the untreated and Ad-LacZ-treated CNI groups. No Tx. = no treatment.

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Figure 3. Adenoviral Smad7 gene transfer decreases cavernous endothelial cell apoptosis. (A) Double labeling of cavernous tissue with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay (TUNEL) and antibody to platelet endothelial cell adhesion molecule 1 (PECAM-1) in the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). Scale bar = 50 μm. (B) Number of apoptotic cells per high power field (screen magnification ×400). Each bar depicts the mean (±SE) values (n = 6 animals per group). At least six different fields in the cavernous tissue were examined. *P < 0.01 compared with the sham operation group. #P < 0.01 compared with the untreated and Ad-LacZ-treated CNI groups. HPF = high-power field; No Tx. = no treatment.

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Figure 4. Adenoviral Smad7 gene transfer does not induce endothelial cell proliferation. (A) Anti-platelet endothelial cell adhesion molecule 1 (PECAM-1) staining of cavernous tissue from the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). Scale bar = 100 μm. (B) Immunofluorescent double staining of cavernous tissue with antibodies to PECAM-1 (red) and phosphohistone H3 (PH3; green) in the sham operation group, untreated CNI mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of Ad-LacZ (1 × 108 vp/20 μL) or Ad-Smad7 (1 × 108 vp/20 μL). Nuclei were labeled with 4,6-diamidino-2-phenylindole (blue). The mice receiving a single intracavernous injection of adenovirus encoding angiopoietin-1 (2 × 108 vp/20 μL) were used as a positive control for PH3 staining. Scale bar = 25 μm. (C) Quantitative analysis of endothelial cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean (±SE) values (n = 6 animals per group). At least two different fields in the cavernous tissue were examined. *P < 0.05 compared with the sham operation group. (D) Number of PH3-immunopositive endothelial cells per high-power field (HPF) (screen magnification ×400). Each bar depicts the mean (±SE) values (n = 6 animals per group). At least six different fields in the cavernous tissue were examined. *P < 0.05 compared with the sham operation group. No Tx. = no treatment.

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figure

Figure 5. Adenoviral Smad7 gene transfer restores cavernous endothelial nitric oxide synthase (eNOS) phosphorylation. (A) Immunofluorescent double staining of cavernous tissue performed with antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) and phospho-eNOS (P-eNOS; Ser1177) in the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). Scale bar = 100 μm. (B) Quantitative analysis of phospho-eNOS-positive area in cavernous tissue was performed with an image analyzer. Each bar depicts the mean (±SE) values (n = 6 animals per group). At least two different fields in the cavernous tissue were examined. *P < 0.05 compared with the sham operation group. #P < 0.05 compared with the untreated and Ad-LacZ-treated CNI groups. (C) Representative Western blot for phospho-eNOS (Ser1177, 140 kDa) and eNOS (140 kDa) in each group. Corpus cavernosum tissues were harvested immediately after electrical stimulation of the cavernous nerve (5 V at a frequency of 12 Hz and a pulse width of 1 ms) for 1 minute. (D) Data are presented as the relative density of phospho-eNOS to eNOS. The relative ratio measured in the control group is arbitrarily presented as 1. Each bar depicts the mean values (±SE) (n = 4 animals per group). *P < 0.01 compared with the sham operation group. #P < 0.05 compared with the sham operation group and the untreated and Ad-LacZ-treated CNI groups. No Tx. = no treatment.

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Figure 6. Adenoviral Smad7 gene transfer reduces cavernous extracellular matrix protein production. (A) Representative Western blot for plasminogen activator inhibitor-1 (PAI-1, 45 kDa), fibronectin (265 kDa), collagen I (139 kDa), and collagen IV (200 kDa) in the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding bacterial β-galactosidase (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). (B) Data are presented as the relative density of each protein compared with that of β-actin. Each bar depicts the mean values (±SE) (n = 4 animals per group). *P < 0.05 compared with the sham operation group. #P < 0.05 compared with the untreated and Ad-LacZ-treated CNI groups. No Tx. = no treatment.

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Table 1. Animals and evaluations
EvaluationNo. animals/groupNo. animals (total)
  1. *The penis was harvested for histologic examination after evaluation of erectile function by electrical stimulation of the cavernous nerve

  2. No. = number; PECAM-1 = platelet endothelial cell adhesion molecule 1; TUNEL = terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; phospho-eNOS = phosphorylated endothelial nitric oxide synthase; ECM = extracellular matrix

Erectile function (Figure 2)642
Histologic examination*  
FLAG staining (Figure 1)612
PECAM-1 and TUNEL staining (Figure 3)624
PECAM-1 and phosphohistone H3 staining (Figure 4)624
PECAM-1 and phospho-eNOS staining (Figure 5A, B)624
α-actin and TUNEL staining (Supporting Information Figure S1)624
α-actin and phosphohistone H3 staining (Supporting Information Figure S1)624
Western blot for phospho-eNOS (Figure 5C, D)416
Western blot for ECM proteins (Figure 6)416

Measurement of Erectile Function

The mice from each group were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscularly. Bipolar platinum wire electrodes were placed around the cavernous nerve. Stimulation parameters were 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) was recorded. The total ICP was determined by the area under the curve from the beginning of cavernous nerve stimulation to a point 20 seconds after stimulus termination. Systemic blood pressure was measured with a noninvasive tail-cuff system (Visitech Systems, Apex, NC, USA). The ratios of maximal ICP and total ICP to mean systolic blood pressure (MSBP) were calculated to normalize for variations in systemic blood pressure as previously described [12].

Histological Examinations

A midportion of each penile tissue sample was fixed in 4% paraformaldehyde for 24 hours at 4°C. For fluorescence microscopy, frozen tissue sections (12 μm thick) were incubated with antibodies to platelet/endothelial cell adhesion molecule (PECAM)-1 (an endothelial cell marker; Chemicon, Temecula, CA, USA; 1:50; Cat #MAB1398Z), phosphohistone H3 (Abcam, Cambridge, UK; 1:50; Cat #Ab5168), phosphorylated endothelial nitric oxide synthase (phospho-eNOS; Ser1177, Cell Signaling, Beverly, MA, USA; 1:25; Cat #9571L), fluorescein isothiocyanate (FITC)- or Cy3-conjugated antibody to smooth muscle α-actin (a smooth muscle cell marker; Sigma-Aldrich, St. Louis, MO, USA; 1:200; cat. #F3777 and cat. #C6198, respectively), or FLAG (Sigma-Aldrich; 1:100; cat. #F7425) at 4°C overnight. Control sections were incubated without the primary antibody at this step. After several washes with phosphate buffered saline, the sections were incubated with tetramethyl rhodamine isothiocyanate- or FITC-conjugated secondary antibodies for 2 hours at room temperature. Mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) was applied to the samples, and nuclei were labeled when appropriate. Signals were visualized, and digital images were obtained by use of a confocal microscope (FV1000, Olympus, Tokyo, Japan).

The numbers of phosphohistone H3-immunopositive endothelial cells were counted at a screen magnification of ×400 in six different regions. Values were expressed per high-power field. The areas covered by cavernous endothelial and smooth muscle cells and the phospho-eNOS-positive area were expressed as percentages of the total cavernous area ([positive area/total cavernous area] × 100).

TUNEL Assay

The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed by using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon; cat. #S7160) as previously described [12]. The number of apoptotic cells in cavernous endothelium was counted at a screen magnification of ×400 in 6 different regions. Values were expressed per high-power field.

Western Blot

Equal amounts of protein (80 μg/lane) were electrophoresed on 10% sodium dodecylsulfate–polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against eNOS (Transduction Laboratories, Inc., Lexington, KY, USA; 1:300; cat. #610296), phospho-eNOS (Ser1177, Cell Signaling; 1:300; cat. #9571L), plasminogen activator inhibitor-1 (PAI-1; Abcam; 1:600; cat #Ab28207), fibronectin (Abcam; 1:300; cat. #Ab23750), collagen I (Abcam; 1:300; cat. #Ab34710), collagen IV (Abcam; 1:300; cat. #Ab6586), or β-actin (Abcam; 1:6000; cat. #Ab8226). Results were quantified by densitometry (n = 4 per group).

Statistical Analysis

Results are expressed as means ± standard errors. Group comparisons of parametric data were made by one-way anova followed by Newman–Keuls post hoc tests. We used the Kruskal–Wallis test for nonparametric data. We performed statistical analysis with SigmaStat 3.5 software (Systat Software Inc., Richmond, CA, USA). P values less than 5% were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

In Vivo Protein Expression in Mouse Corpora Cavernosa

To localize Smad7 protein, immunohistochemical staining for FLAG-tagged Smad7 was performed 2 weeks after intracavernous injection of Ad-LacZ or Ad-Smad7. FLAG-tagged protein was mainly expressed in cavernous endothelial cells, but rarely overlapped with smooth muscle cells (Figure 1).

Restoration of Erectile Function

A representative intracavernous tracing after stimulation of the cavernous nerve (5 V, 12 Hz, 1 ms) for 1 minute in sham control or CNI mice 2 weeks after treatment is shown in Figure 2A. The ratios of maximal ICP and total ICP to MSBP were significantly lower in the untreated and Ad-LacZ-treated CNI mice than in the sham controls (P < 0.01). A single intracavernous injection of Ad-Smad7 induced significant recovery of erectile function at all doses tested compared with erectile function in the untreated and Ad-LacZ-treated CNI mice (P < 0.01). The highest erectile response was recorded in CNI mice that received Ad-Smad7 at a dose of 1 × 108 vp/20 μL, which reached values up to 82–85% of sham control values (Figure 2B, C).

Decrease in Apoptosis in Cavernous Endothelial Cells

Double labeling of cavernous tissue with TUNEL and antibody to PECAM-1 showed that the number of apoptotic cells in cavernous endothelium was significantly greater in the untreated and Ad-LacZ-treated CNI groups than in the sham control group (P < 0.01). A single intracavernous injection of Ad-Smad7 significantly decreased apoptosis in the cavernous endothelium of the CNI mice (P < 0.01 vs. untreated and Ad-LacZ-treated CNI groups), although not to the level found in the sham controls (Figure 3).

Failure to Induce Cavernous Endothelial Cell Proliferation

We found significantly fewer endothelial cells in untreated and Ad-LacZ-treated CNI mice than in the sham group (P < 0.05). To test whether Ad-Smad7 could induce endothelial cell proliferation, we assessed the number of endothelial cells staining positive for phosphohistone H3 (a nuclear protein indicative of cell proliferation). We observed no significant increases in phosphohistone H3-positive endothelial cells or in cavernous endothelial cell content in the cavernous sinusoids of CNI mice 2 weeks after intracavernous injection of Ad-Smad7. In all experimental groups, only a few or virtually no phosphohistone H3-positive cells were noted (Figure 4).

Induction of Cavernous eNOS Phosphorylation

Cavernous phospho-eNOS (Ser1177) expression as assessed by immunohistochemical staining and Western blot analysis was significantly lower in the untreated and Ad-LacZ-treated CNI mice than in the sham controls at 2 weeks after treatment (P < 0.05 for immunohistochemistry and P < 0.01 for Western blot analysis). A single intracavernous injection of Ad-Smad7 significantly increased endogenous eNOS phosphorylation in the CNI mice compared with that in the untreated and Ad-LacZ-treated CNI mice (P < 0.05 for immunohistochemistry and Western blot analysis), although not to the level found in the sham controls ( Figure 5).

Reduction in Cavernous ECM Protein Production

We performed Western blot to evaluate the cavernous tissue production of ECM proteins, including PAI-1, fibronectin, collagen I, and collagen IV. Cavernous ECM protein production was significantly higher in the untreated and Ad-LacZ-treated CNI mice than in the sham controls (P < 0.05). A single intracavernous injection of Ad-Smad7 significantly reduced cavernous ECM protein production in the CNI mice (P < 0.05 vs. untreated and Ad-LacZ-treated CNI groups) (Figure 6).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

We showed here that a single injection of an adenovirus encoding the Smad7 gene into the corpus cavernosum of CNI mice significantly decreased endothelial cell apoptosis; significantly decreased the production of ECM proteins, including PAI-1, fibronectin, collagen I, and collagen IV; and induced eNOS phosphorylation—and that these changes were associated with physiologically relevant changes in erectile function.

We recently found in mice that phosphorylation of the Smad2 transcriptional factor is significantly increased in cavernous endothelial cells and smooth muscle cells after CNI [12]. Previous studies reported that Smad7 gene transfer inhibits the activation of Smad2 and Smad3 in bleomycin-induced lung fibrosis [26] and in an obstructive renal fibrosis model [27]. TGF-β is known to inhibit endothelial cell migration and proliferation via the TGF-β type I receptor-Smad2/3 pathway [20,21], whereas inhibition of TGF-β type I receptor facilitates endothelial cell proliferation [21,22]. TGF-β has also been shown to induce apoptosis in pulmonary microvascular endothelial cells and in human umbilical vein endothelial cells [28,29]. In the present study, intracavernous administration of Ad-Smad7 did not induce endothelial cell proliferation, but did significantly reduce endothelial cell apoptosis in the CNI mice. In agreement with the results of previous studies showing a decrease in cavernous endothelial cell content after CNI [30,31], we observed a significant decrease in endothelial content after CNI. The decrease in cavernous endothelial cell apoptosis by Ad-Smad7 did not quantitatively restore endothelial cell content but did enhance cavernous endothelial function qualitatively, as shown by the increase in eNOS activity.

TGF-β1 is also known to inhibit the proliferation of smooth muscle cells through modification of the cell cycle [17,18], and the TGF-β1-Smad2 pathway is involved in apoptosis of vascular smooth muscle cells [19]. In the present study, however, Ad-Smad7 did not affect the proliferation or apoptosis of cavernous smooth muscle cells in CNI mice (Supporting Information Figure S1). Previous studies reported that chronic administration of PDE5 inhibitors prevents smooth muscle cell loss and fibrosis in the corpus cavernosum tissue of rats with CNI [9,32] and of men with radical prostatectomy [33]. Therefore, it would be quite interesting to determine whether dual inhibition of the TGF-β pathway and PDE5 enzyme could induce a synergic effect on the recovery of erectile function in radical prostatectomy-induced ED by exerting protective effects on both cavernous endothelial and smooth muscle cells.

Nitric oxide is a principal mediator of penile erection [34] and plays important roles in angiogenesis and endothelial cell protection [35]. In the present study, eNOS phosphorylation (Ser1177) was significantly decreased in both untreated and Ad-LacZ-treated CNI mice compared with that in the sham control group. Previous studies reported a decrease in eNOS protein expression in the corpus cavernosum tissue of CNI rats [30,31]. Intracavernous administration of Ad-Smad7 significantly restored endogenous eNOS phosphorylation in the CNI mice, although not to the level found in the sham controls. Previous studies reported that TGF-β decreases nitric oxide production in renal tubular epithelial cells. Moreover, anti-TGF-β antibody and antisense oligonucleotides have been shown to ameliorate renal interstitial fibrosis and to restore constitutional NOS activity in a unilateral ureteral obstruction model [36,37]. Therefore, we believe that inhibition of the TGF-β pathway as well as the prevention of endothelial cell apoptosis by Ad-Smad7 may account for the partial restoration of eNOS activity.

It has been demonstrated in animal models that CNI leads to increased expression of TGF-β1 and promotes cavernous fibrosis [10,11]. These studies suggest that a high level of hypoxia-inducible factor-1α, a transcription factor related to hypoxia, induced by cavernous hypoxia after CNI may facilitate the activation of the TGF-β signaling pathway [10,11]. Increases in the protein expression of collagen I and III and in cavernous collagen content have been noted in the corpus cavernosum tissue of CNI rats [10] and in men who underwent radical prostatectomy [38]. Similarly, we observed a significant increase in the production of ECM protein in the corpus cavernosum tissue of CNI mice as evaluated by Western blot analysis. Local delivery of Ad-Smad7 significantly decreased this production of ECM protein in the corpus cavernosum tissue of CNI mice.

Pentoxifylline, a nonselective PDE inhibitor, is known to antagonize the effects of TGF-β1 in tunica albuginea-derived fibroblasts [39]. Recent study reported in a rat model of CNI that daily oral administration of pentoxifylline increased neuronal NOS expression [40]. Therefore, it is necessary to determine whether intracavernous administration of Smad7 can induce neural regeneration.

To our knowledge, this is the first preclinical study showing that inhibition of the TGF-β pathway by using inhibitory Smad (Smad7) in the penis is useful for the treatment of ED resulting from CNI when given at the time of injury. The present study adds new evidence for a protective role of Smad7, a negative regulator of TGF-β/Smad signaling, in CNI-induced ED and provides evidence for the biological activity of Smad7 in ED in terms of protection against endothelial cell damage and fibrosis in the penis. Further studies are needed to evaluate the longevity of the effect of Smad7 gene therapy on penile erection. Future studies should also investigate whether inhibition of the TGF-β pathway can reduce endothelial cell damage and cavernous fibrosis if Smad7 is administered later after CNI when the structural changes in the corpus cavernosum tissue have already progressed. The limitation of our study is that we used adenovirus as a gene delivery vector. New vector systems that offer little or no immunogenic reaction and high gene-transfer efficiency are required for safe clinical application of Smad7.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

The adenovirus-mediated gene transfer of Smad7 successfully restored erectile function by preventing endothelial cell apoptosis and enhancing endothelial function and by antifibrotic effects. These findings suggest that inhibition of the TGF-β signaling pathway may represent a promising therapeutic strategy for radical prostatectomy-induced ED.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Ji-Kan Ryu, 2010-0023026), and by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs (Jun-Kyu Suh and Ji-Kan Ryu, A110076), Republic of Korea. We thank Jennifer Holmes for help in preparing the manuscript.

Conflict of Interest: The authors report no conflicts of interest.

Statement of Authorship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information

Category 1

  • (a)
    Conception and Design
    Kang Moon Song; Jae-Seung Chung; Ji-Kan Ryu; Jun-Kyu Suh
  • (b)
    Acquisition of Data
    Kang Moon Song; Jae-Seung Chung; Min Ji Choi; Guo Nan Yin; Woo Jean Kim
  • (c)
    Analysis and Interpretation of Data
    Kang Moon Song; Jae-Seung Chung; Hai-Rong Jin; Mi-Hye Kwon; Jin-Mi Park

Category 2

  • (a)
    Drafting the Article
    Kang Moon Song; Jae-Seung Chung
  • (b)
    Revising It for Intellectual Content
    Sang-Jin Lee; Seong-Jin Kim

Category 3

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

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Statement of Authorship
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
jsm12329-sup-0001-fig_s1.doc2255KFigure S1 Adenoviral Smad7 gene transfer does not affect the apoptosis or proliferation of cavernous smooth muscle cells. (A) Anti-smooth muscle α-actin staining of cavernous tissue from the sham operation group, untreated cavernous nerve injury (CNI) mice, or CNI mice stimulated at 2 weeks after a single intracavernous injection of adenovirus encoding LacZ (Ad-LacZ; 1 × 108 vp/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 108 vp/20 μL). Scale bar = 100 μm. (B) Quantitative analysis of smooth muscle cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean (±SE) values (n = 6 animals per group). At least two different fields in the cavernous tissue were examined. *P < 0.05 compared with the sham operation group. (C) Double labeling of cavernous tissue with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay (TUNEL; green) and antibody to smooth muscle α-actin (red) in each group. Scale bar = 25 μm. (D) Immunofluorescent double staining of cavernous tissue with antibodies to smooth muscle α-actin (red) and phosphohistone H3 (PH3; green) in each group. Scale bar = 25 μm. No Tx. = no treatment.

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