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COX-2-10aa-PGIS Gene Therapy Improves Erectile Function in Rats after Cavernous Nerve Injury
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COX-2-10aa-PGIS Gene Therapy Improves Erectile Function in Rats after Cavernous Nerve Injury Haocheng Lin, MD,*† Jiuhong Yuan, MD,‡ Ke-He Ruan, MD, PhD,§ Wenli Yang, PhD,¶ Junlan Zhang, MD, PhD,¶ Yutian Dai, MD, PhD,* and Run Wang, MD, FACS†** *Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, China; † Division of Urology, University of Texas Medical School at Houston, Houston, TX, USA; ‡Department of Urology, West China Hospital, Sichuan University, Chengdu, China; §Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, Houston, TX, USA; ¶Division of Gastroenterology, Hepatology & Nutrition, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX, USA; **Department of Urology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA DOI: 10.1111/jsm.12147
ABSTRACT
Introduction. Erectile dysfunction (ED) is a very common complication after radical prostatectomy. COX-2-10aaPGIS is a newly engineered protein with COX-2 and prostacyclin synthase activities that converts arachidonic acid directly to prostacyclin (prostaglandin I2 [PGI2]). PGI2 is a potent smooth muscle relaxant. Aim. The purpose of this study was to explore the effect and mechanism of COX-2-10aa-PGIS gene therapy in penile rehabilitation. Methods. Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley rats was used to mimic radical prostatectomy-induced ED. Sprague-Dawley rats were randomly assigned into four groups: 1. sham surgery; 2. BCNC; 3. BCNC + null control recombinant adenovirus intracavernous injection; and 4. BCNC + Ad-COX2-10aaPGIS intracavernous injection. Twenty-eight days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the mean arterial pressure (MAP) was monitored. At the end of the measurement, the penis was harvested and processed for (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (a-SMA), and transforming growth factor beta-1 (TGF-b1); (ii) Masson’s trichrome stain for smooth muscle/collagen ratios; (iii) Western blot of eNOS, a-SMA, TGF-b1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. Main Outcome Measures. Erectile function was evaluated by ICP/MAP. Smooth muscle and endothelium functions in corpora cavernosum were assessed by Masson’s trichrome stain, immunohistochemistry, and Western blot. Apoptosis was identified by TUNEL assay. Results. The results were the following: 1. COX2-10aa-PGIS gene therapy improved erectile function (82%, compared with control) in the BCNC rat model; 2. COX2-10aa-PGIS gene therapy increased eNOS (121%) and a-SMA (118%) expression and decreased TGF-b1 (45%) expression; 3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury (64%); and 4. COX2-10aa-PGIS gene therapy improved smooth muscle/ collagen ratios (81%). Conclusion. Our data demonstrated that COX2-10aa-PGIS improved erectile function after cavernous nerve injury through antifibrotic and anti-apoptotic mechanisms. Lin H, Yuan J, Ruan K-H, Yang W, Zhang J, Dai Y, and Wang R. COX-2-10aa-PGIS gene therapy improves erectile function in rats after cavernous nerve injury. J Sex Med 2013;10:1476–1487. Key Words. Cavernous Nerve Injury; COX2-10aa-PGIS; Erectile Dysfunction; Penile Rehabilitation; Radical Prostatectomy
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© 2013 University of Texas Medical School at Houston Journal of Sexual Medicine © 2013 International Society for Sexual Medicine
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COX-2-10aa-PGIS Gene Therapy for Penile Rehabilitation Introduction
C
ancer is one of the leading causes of death in the United States, with prostate cancer as the most common cancer in men and second as a cause of death [1]. As techniques improve, such as prostate-specific antigen screening, laparoscopic, and robotic surgery, radical prostatectomy (RP) is the primary option for treatment of clinically localized prostate cancer with excellent long-term results [2,3]. However, erectile dysfunction (ED) as a result of RP remains a significant challenge. Recent clinical study showed that the use of robotic-assisted surgery has not improved erectile function outcomes [4]. Currently, post-RP pharmacological sexual rehabilitation is widely practiced. It is commenced early and based primarily on phosphodiesterase type 5 inhibitors, intracavernosal or transurethral administration of vasodilators, or with the vacuum erection device. However, the reported recovery rate is varied and experiences low patient compliance due to cumbersome situation and ineffectiveness [3]. Although animal studies with nerve regeneration regimens showed promising results, their clinical applications were disappointing [3]. Therefore, there is a great need to explore novel rehabilitation modalities after RP. Prostacyclin (prostaglandin I2 [PGI2]) is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase (PGIS). PGH2 is converted from arachidonic acid (AA) by cyclooxygenase (COX) enzymes: COX-1 and/or COX-2. AA released from the membrane phosphoglycerides is converted to the prostaglandin G2 (catalytic step 1) and then to PGH2 (catalytic step 2) by COX-1 or COX-2. PGH2 is further isomerized to the biologically active end products of prostaglandin D2, prostaglandin E2 (PGE2), prostaglandin F2, and PGI2 or thromboxane A2 (TXA2) by individual synthases (catalytic step 3) in tissue-specific processes (Figure 1) [5,6]. Prostanoids act as local hormones in the vicinity of their production site to regulate hemostasis and smooth muscle functions [7]. TXA2 produced from PGH2 by TXA2 synthase has been implicated as a proaggregatory and vasoconstricting mediator in various pathophysiological conditions [8]. PGI2 is the primary AA metabolite in vascular walls, has opposing biological properties to TXA2, and therefore represents the most potent endogenous vascular protector acting as an inhibitor of platelet aggregation [9] and a strong vasodilator on vascular beds [10–12].
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2 activities to reduce the production of all prostanoids, which leads to the thinning of blood by reducing TXA2 production and the suppression of inflammation by decreasing PGE2 production. It was recently reported that NSAID usage increased ED incidence in men [13]. Therefore, PGI2 may be a good candidate for vascular protection and ED treatment. However, prostacyclin is quickly broken down into 6-keto-PGF1 due to its short half-life, which makes it a much weaker vasodilator and limits its clinical application [14]. Finding a way to specifically increase the production of the vascular protector, PGI2, will be an intriguing project. Recently, Ruan et al. reported an innovative engineering of a recombinant protein COX-2-10aaPGIS with triple catalytic activities directly converting AA into PGI2 [6]. The advantages of the newly engineered COX-2-10aa-PGIS are the following: (i) multiple catalytic enzyme activities can be configured within a single protein molecule if an approximate protein configuration is adopted; (ii) the engineered protein with tricatalytic functions not only accumulates the individual enzymes’ activities but also has a faster turnover rate when compared with the mixture of the parent enzymes [6]. This study was designed to apply Ad-COX210aa-PGIS (the replication deficient recombinant adenoviruses carry the COX-2-10aa-PGIS gene) to the bilateral cavernous nerve crush (BCNC) rat model, which mimics ED after RP, to explore the potential benefit of COX-2-10aa-PGIS
Figure 1 Prostanoids synthesis
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1478 gene therapy and to understand the underlying mechanisms. Materials and Methods
Prepare Ad-COX-2-10aa-PGIS Amplification, Purification, and Identification of the Ad-COX-2-10aa-PGIS The primary Ad-COX2-10aa-PGIS viral stock was established as previously described [15]. The primary Ad-COX2-10aa-PGIS was amplified and purified following the standard protocol of Puresyn’s Adenopure TM kit. Briefly, the HEK-293 cells were subcultured in 150-cm2 tissue culture plates with 20 mL of Dulbecco’s Modified Eagle Medium (DMEM) growth medium for 48 hours. Transfection of Ad-COX2-10aa-PGIS was performed until cells were approximately 75–85% confluent. Fifty microliter of Primary Ad-COX210aa-PGIS (1.3 ¥ 1012 viral particle [VP]/mL) in fresh 20 mL DMEM growth medium was immediately applied to the plate of cells and swirled gently to distribute the suspension evenly in the medium. After incubating for 64–72 hours at 37°C, the cells with DMEM growth medium were scraped into the tube and subjected to four rounds of freeze/thaw by alternation of the tube between the dry ice-ethanol bath and the 37°C water bath. The cellular debris was collected by centrifugation at 10,000 ¥ g for 10 minutes and the supernatant (Ad-COX2-10aa-PGIS virus) was stored at -80°C. The supernatant was then concentrated and purified using the Puresyn’s Adenopure TM kit. Virus particle concentration was calculated from the absorbance at 260 nm (A260). The infectious titer was determined by plaque assay. Finally, 3.8 ¥ 1011 VP/mL (1.25 ¥ 1010 plaque forming units [PFU]/mL) Ad-COX2-10aa-PGIS virus in elution buffer was acquired and stored at -20°C for use in the next step. Verification of the Purified Ad-COX2-10aa-PGIS Special Gene with Polymerase Chain Reaction (PCR) PCR primer was designed using deoxyribonucleic acid (DNA) tools (OligoPerfectTM Designer) from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA): forward (5′-TGGTGGAGAAGTG GGTTTTC-3′); reverse (5′-GTCCAGGAGAA CGGTGACAT-3′). Five micron of each sample (Ad-COX2-10aa-PGIS, null control recombinant adenovirus [NCRA, Cell Biolabs, Inc., San Diego, CA, USA] and positive control group [Baculovirus-COX2-10aa-PGIS]) was added into J Sex Med 2013;10:1476–1487
Lin et al. 100 mL digest buffer (1¥ PCR buffer, 0.45% NP40, 0.45% Tween 20, 10 mL Proteinase K [10 mg/mL]) at 60°C for 1 hour. The sample was further denatured by boiling for 15 minutes and then cooled on ice for 5 minutes. Two microliter of the isolated genetic DNA was subjected to PCR analysis using PCR Reaction Buffer (1¥ PCR buffer, 2.5 mM MgCl2, 200 mM deoxynucleotide triphosphates (dNTPs), 1 mM each primer, and 1 unit Taq Polymerase with conditions). After overlay with 30–40 mL light mineral oil, the DNA was put into the cycler and the following program was run: hold at 94°C for 4.5 minutes, 30¥ step cycles of 94°C for 30 seconds and 54°C for 20 seconds, and 72°C for 1 minute and hold at 4°C until ready to analyze. The PCR product was analyzed by agarose gel (Figure 2A).
Ad-COX2-10aa-PGIS Enzyme Activity Determination Using High-Performance Liquid Chromatography HEK293 cells were respectively transfected with baculovirus-COX2-10aa-PGIS (positive control), pcDNA3.1(+) (Vector), and purified Ad-COX210aa-PGIS. To determine the activities of the synthases that converted AA to PGI2 through the tricatalytic functions, [14C]-AA (3 mM) was added to each group in a total volume of 30 mL PBS. After 0.5 minute of incubation, the reaction was stopped by adding 50 mL of the solvent containing 0.1% acetic acid and 30% acetonitrile (solvent A). After centrifugation (12,000 rpm for 5 minutes), the supernatant was injected into a C18 column (Varian Microsorb-MV 100-5, 4.6 ¥ 250 mm) using the solvent A with a gradient from 35% to 100% of acetonitrile for 45 minutes at a flow-rate of 1.0 mL/minute. The [14C]-labeled AA metabolites, including 6-keto-PGF1R (degraded PGI2), were monitored directly by a flow scintillation analyzer (Packard 150TR) [6] (Figure 2B). Determination of COX-2-10aa-PGIS Protein Expression with Western Blot The purified Ad-COX2-10aa-PGIS, baculovirusCOX2-10aa-PGIS (positive control), and NCRA were applied to HEK293 cells. The HEK 293 cells were incubated in a 10-cm plate for 48 hours after infection. The plate was washed with PBS to remove residual media. A 400 mL of 1¥ radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, MO, USA) was added to the plate. The plate was incubated on ice for
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Figure 2 (A) PCR confirmed the amplified and purified Ad-COX2-10aa-PGIS contain COX2-10aa-PGIS gene. (B) HPLC show COX-2-10aa-PGIS enzyme activity. Ad-COX-2-10aa-PGIS group produce 6-keto-PGF1 (product of PGI2) by consuming substrate AA. No 6-keto-PGF1 was produced in vector group. (C) Western Blot verified the special hybrid enzyme expression.
5 minutes. Then, the cells were scraped, sonicated briefly, and spun for 10 minutes at 14,000 ¥ g in a cold microcentrifuge. Supernatant was removed for use. Protein concentration was determined by bicinchoninic acid (Pierce Chemical Company, Rockford, IL, USA) method. Equal amounts of supernatant protein (20 mg) were electrophoresed according to the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). The membrane was incubated with PGIS (1:1,000, Cayman Chemical Company, Ann Arbor, MI, USA) and COX-2 (1:1,000, Cayman Chemical Company) antibodies at 4°C overnight. Detection of the reactive protein on the membrane was performed with horseradish peroxidase-conjugated secondary antibody (1:5,000, Cayman Chemical Company) for 4 hours at room temperature (RT), followed by exposure to X-ray films. The resulting images were analyzed with Image J 1.44 (US National
Institutes of Health, Bethesda, MD, USA) to determine the integrated density value of each protein band [16] (Figure 2C).
Animal Groups and Study Animal Model, Grouping, and Tissue Harvesting The BCNC animal model was previously described [17]. Briefly, rats were anesthetized, shaved, and disinfected as required. They were placed in a supine position. A midline suprapubic incision was made to expose the bladder and prostate. The major pelvic ganglion (MPG) lying on the dorsal aspect of the ventral prostate was found after carefully separating any overlying adipose tissues. Cavernous nerves were isolated and identified. At the point of 5 mm distal to the MPG, the bilateral cavernous nerves were crushed using an ultrafine hemostat with full tip closure for 30 seconds, removed for 30 seconds, and then reapplied for another 30 seconds. The incision was J Sex Med 2013;10:1476–1487
1480 closed with 3-0 Vicryl sutures. Analgesia and antibiotics were applied before and after the operation as required. Thirty-two Sprague-Dawley rats (male, 200– 224 g, Harlan Laboratories, Indianapolis, IN, USA) were randomized into four groups: group 1 (sham, N = 8), sham surgery, no cavernous nerve crush; group 2 (BCNC, N = 8), surgery for BCNC, without any therapy; group 3 (BCNC + NCRA, N = 8), surgery for BCNC, with NCRA penile injection; and group 4 (BCNC + Ad-COX-2-10aa-PGIS, N = 8), surgery for BCNC, with Ad-COX2-10aa-PGIS penile injection. The intracavernosal injection of NCRA or Ad-COX2-10aa-PGIS was given at day 1 and day 14. The transfection time, determined at the pilot study, which revealed the highest expression of the new protein, was found at day 7 and weakened after the first week (supplementary file). At day 28, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the blood pressure was monitored. At the end of the measurement, the penes were harvested and processed for the following: (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (a-SMA), and transforming growth factor beta-1 (TGF-b1); (ii) Masson’s trichrome stain for smooth muscle/ collagen ratios; (iii) Western blot of eNOS, a-SMA, TGF-b1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. All animal procedures were approved by the Animal Welfare Committee.
Intracavernous Injection of Ad-COX2-10aa-PGIS and NCRA The rats were transferred into the biohazard room prior to administration of adenovirus. The animals were anesthetized with inhalant isoflurane at the flow-rate 3–4 level. Either 50 mL Ad-COX2-10aaPGIS solution containing 1.25 ¥ 1010 PFU/mL or 50 mL NCRA was injected into the corpora cavernosum using Hamilton Microliter with a 25-gauge needle. The transfection dosage that revealed the best expression of COX2-10aa-PGIS was determined at the pilot study and was identified in the 1.25 ¥ 1010 PFU/mL group (supplementary file). An elastic band was placed at the base of the penis prior to injection and was removed 5 minutes after the injection. The rats stayed in the biohazard room for next 72 hours before being transferred back to regular housing. This procedure was J Sex Med 2013;10:1476–1487
Lin et al. repeated in 14 days to maintain the high expression of PGI2.
Erectile Function Evaluation ICP under electric stimulation of the cavernous nerve was measured by the ICP/mean arterial pressure (MAP) set (Grass S48 stimulate, Powerlab/4SP, AD instruments QUAD bridge, NL108A pressure amplifier, data acquisition module: DI-194RS and Windaq/lite recording software) before the rats were sacrificed. After being anesthetized intraperitoneally, with 40 mg/kg pentobarbital sodium, the MPG, and cavernous nerve were exposed on either side of the prostate. A 25-gauge needle connected to a PE-50 tube and filled with 100 U/mL heparin sodium solution was inserted into the right crura for measurement of ICP. The left carotid artery was exposed and cannulated with a PE-50 tube to record the MAP. Stimulations were performed at a parameter of 16 Hz, 5 milliseconds duration, and 7.5 V for 60 seconds with a period of 5 minutes between subsequent stimulations. The maximal increase of ICP during nerve electrostimulation was selected for statistical analysis in each animal. The ICP/MAP ratios were analyzed to evaluate the erectile function [18] (Figure 3). Histology Penile tissues were fixed in 4% paraformaldehyde overnight, after which the tissues were taken for routine processing and paraffin embedding. Tissue samples were cut into 5-mm sections from the midshaft of the penis. Then the tissue slides, showing the cross section of the corpora cavernosa, were deparaffinized for the following studies. Masson’s Trichrome Staining Slides were rehydrated and refixed in Bouin’s solution overnight to improve staining quality prior to the following standard protocol [19]. Smooth muscle/collagen ratios were analyzed using Image J v.1.44 (National Institutes of Health, Bethesda, MD, USA). Five sections per rat were quantified at 200¥ by Image J. The entire section was used for analysis. Sections from four rats per group were analyzed. Thus, in each group (sham, BCNC, BCNC + NCRA, and BCNC + Ad-COX2-10aaPGIS), a total of 20 sections were reviewed for histomorphometry [20]. Immunohistochemistry-Paraffin Staining Tissue sections were rehydrated according to standard protocols. Antigen retrieval was performed
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Figure 3 (A) Representative ICP response to cavernous nerve stimulation. (B) Ad-COX2-10aa-PGIS gene therapy group had a significant increase of ICP/MAP ratios compared with BCNC and BCNC + NCRA group (P < 0.001).
using 10 mM citrate acid buffer (pH 3.0) at 37°C for 30 minutes blocked by superblock (either 10% goat serum or horse serum, depending on the primary antibody) for 10 minutes at RT. Penile sections were then incubated with primary antibodies (a-SMA [1:200, Abcam, Cambridge, MA, USA], eNOS [1:250, BD Transduction Laboratories, Lexington, KY, USA], TGF-b1 [1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA]) overnight at 4°C, followed by biotinylated secondary antibody working solution (1:200) for 30 minutes at RT. Then, the stain section was incubated in diluted ABC Reagent (VectorLabs, Burlingame, CA, USA) at RT for 30 minutes, followed by diaminobenzidine (DAB)/H2O2 development solution (VectorLabs). Finally, the stained sections were counterstained with hematoxylin, dehydrated, and mounted with Clearium mounting medium [21]. Five sections per rat were quantified at 400¥ magnification by Image J 1.44. Five rats per group were quantified.
Apoptosis Assessment In Situ Cell Death Detection Kit (fluorescein, Roche Applied Science, Indianapolis, IN, USA) was used for TUNEL assay and was performed following the manufacturer’s instructions to assess apoptosis. Apoptotic cells were traced with fluo-
rescein and the nucleus was marked with 4′, 6′ diamidino-2-phenylindole (DAPI, VectorLabs). One section from each rat was quantified by counting the number of apoptotic cells over total cells stained with DAPI in five regions chosen randomly per section. Four rats per group were quantified by this methodology. The ratio of the percentage of fluorescent apoptotic cells to total number of cells was recorded as the apoptotic index (AI) [20].
Western Blot The nerve bundle and urethra were stripped from the corpora prior to homogenization. A 1¥ RIPA lysis buffer with PMSF was added to the corpora cavernosa that was cut into tiny pieces by scissors. The tissue samples were vortexed for 60 seconds, incubated on ice for 45 minutes, homogenized with a polytron (3 ¥ 20 seconds), and centrifuged at 14,000 ¥ g for 10 minutes at 4°C. Supernatant was removed for use. First antibodies used in Western blot were a-SMA (1:1,500), eNOS (1:1,000), TGF-b1 (1:1,000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000, Santa Cruz Biotechnology Inc.), and PGIS. The detailed protocol was described in the above Western blot section. Quantification of a-SMA, J Sex Med 2013;10:1476–1487
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Figure 4 Ad-COX2-10aa-PGIS gene therapy preserved smooth muscle/collagen ratios. (A) Representative figures of Masson’s trichrome staining in each group (original magnification ¥100). (B) Masson’s trichrome stain was used for muscle/collagen ratios. Compared with BCNC and BCNC + NCRA group, Ad-COX2-10aa-PGIS group was found to have higher smooth muscle/collagen ratios (P < 0.001).
eNOS, and TGF-b1 were performed relative to GAPDH in five rats per group using Image J 1.44.
Statistical Analysis All measurements are expressed as mean ⫾ standard deviation. Data were analyzed using Student’s t-test or anova with Origin 6.1 for multiple comparisons between groups. A P value < 0.05 was considered statistically significant. Results
Erectile Function Assessment The typical ICP tracings of sham, BCNC, BCNC + NCRA, and BCNC + Cox-2-10aa-PGIS were shown in Figure 3. Each group had six rats for the ICP/MAP ratio analysis. The ICP/MAP ratio in the sham group was 0.84 ⫾ 0.06, which was significantly higher compared with BCNC group (0.34 ⫾ 0.08, P < 0.001), BCNC ⫾ NCRA group (0.29 ⫾ 0.07, P < 0.001), and BCNC + AdCOX2-10aa-PGIS group (0.62 ⫾ 0.09, P < 0.01). Ad-Cox2-10aa-PGIS therapy significantly improved ICP/MAP when compared with BCNC by 82% (P < 0.001) and 114% with the BCNC + NCRA groups (P < 0.001). J Sex Med 2013;10:1476–1487
Masson’s Trichrome Staining Analysis Smooth muscle/collagen ratios were evaluated by Masson’s trichrome method. The sham group revealed smooth muscle/collagen ratios of 0.46 ⫾ 0.13, which had the highest ratio among all study groups (P < 0.05). The smooth muscle/ collagen ratio for the BCNC Ad-COX2-10aaPGIS group was 0.38 ⫾ 0.08, which was significantly greater than those from the BCNC group (0.21 ⫾ 0.04, P < 0.001) and BCNC + NCRA group (0.23 ⫾ 0.04, P < 0.001) (Figure 4). Ad-Cox2-10aa-PGIS therapy significantly improved smooth muscle/collagen ratios when compared with BCNC by 81% (P < 0.001) and 65% with the BCNC + NCRA groups (P < 0.001). Apoptosis Analysis At 4 weeks after BCNC, the BCNC ⫾ COX-210aa-PGIS group demonstrated a significant reduction in apoptosis within the corporal tissue with a mean AI of 0.23 ⫾ 0.08, compared with BCNC group (AI: 0.64 ⫾ 0.15, P < 0.001) and BCNC + NCRA group (AI: 0.6 ⫾ 0.17, P < 0.001). The AI value in the sham group was 0.11 ⫾ 0.10, which was notably lower compared with all other groups (P < 0.001)
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Figure 5 (Left) was the representative figure of apoptotic cell stained by fluorescein (green), nucleus stained by DAPI (Blue) and the merged figure from each group. A, B, C and D respectively represent Sham, BCNC, BCNC + NCRA and BCNC + AdCOX2-10aa-PGIS groups. On the right figure, the ratio of apoptotic cell/Nucleus was calculated and analyzed as the apoptotic index. The Ad-COX2-10aa-PGIS therapy significantly decreased the apoptosis compared with BCNC and BCNC + NCRA groups (P < 0.001).
(Figure 5). Ad-Cox2-10aa-PGIS therapy significantly decreased AI when compared with BCNC by 64% (P < 0.001) and 62% with the BCNC + NCRA groups (P < 0.001).
Immunohistochemistry Analysis Immunohistochemical staining showed that Ad-COX2-10aa-PGIS therapy group had significant increases of eNOS (128% and 97%) and a-SMA (67% and 97%) in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). Ad-COX2-10aa-PGIS therapy group also had decreased TGF-b1 (54% and 49%) expression level compared with BCNC and BCNC + NCRA groups (P < 0.05) (Figure 6). Western Blot Analysis Western blot results denoted that the Ad-COX210aa-PGIS therapy group significantly increased the expression of eNOS (121% and 95%) and a-SMA (118% and 146%) in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). Ad-COX2-10aa-PGIS therapy group also decreased TGF-b1 expression level
(45% and 46%) compared with BCNC and BCNC + NCRA groups (P < 0.05). In addition, Western blot confirmed that Ad-COX2-10aaPGIS therapy group was expressing COX2-10aaPGIS protein at the end of the experiment (Figure 7). Discussion
ED following RP is due to damage to the cavernous nerve, known as neuropraxia. Neuropraxia can be caused by mechanically induced nerve stretching that may occur during prostate retraction, thermal damage to the nerve caused by electrocauterization, ischemia of the nerve secondary to disruption of blood supply while attempting to control surgical bleeding, and local inflammatory effects associated with surgical trauma [22]. Even in the most meticulous nerve-sparing dissection, some degree of neuropraxia is unavoidable because of the close proximity of the nerve to the prostate gland. These nerves tend to recover slowly; it may take as long as 18–24 months for them to reach a new baseline functional status [23]. Apoptosis J Sex Med 2013;10:1476–1487
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Figure 6 Immunohistochemical staining of TGF-b1, eNOS, and a-SMA. (Upper) Representative photos of TGF-b1, eNOS, and a-SMA at ¥400 magnification. (Lower) Positive area/total area (%) was used to denote the expression of TGF-b1, eNOS, and a-SMA. Ad-COX2-10aa-PGIS therapy increase eNOS and a-SMA in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). TGF-b1 was also significant decreased in BCNC + Ad-COX2-10aa-PGIS group compared with BCNC and BCNC + NCRA groups (P < 0.05).
of penile smooth muscle caused by cavernous nerve injury leads to decreased erectile function [24,25]. Lack of erections can cause poor oxygenation of the corporal bodies and fibrosis resulting in venous leak [26]. In an experimental model, significant J Sex Med 2013;10:1476–1487
overexpression of hypoxia-related substances, such as hypoxia-inducible factor (HIF-1-a) and TGF-b1 and collagen I and III, was found in rats that had undergone bilateral excision of the cavernosal nerves compared with controls [27]. When human penile smooth muscle cell is exposed to
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Figure 7 The protein expression in rat penis was detected by Western blot. (a–d) respectively represent Sham (a), BCNC (b), BCNC + NCRA (c) and BCNC + Ad-COX2-10aa-PGIS (d) groups. (A) represents typical bands of Western Blot. Bands from up to down represent a-SMA, TGF-b1, eNOS, COX2-10aa-PGIS, and GAPDH. (B–E) respectively represent column figures of a-SMA, TGF-b1, eNOS, and COX2-10aa-PGIS. Each figure denotes means ⫾ standard errors of each protein expression determined by densitometry relative to GAPDH. Compare with BCNC group (N = 5) and BCNC + NCRA group (N = 5), BCNC + COX2-10aa-PGIS group (N = 5) significantly increased the expression of eNOS and a-SMA (P < 0.05), decreased the expression of TGF-b1 (P < 0.05). COX2-10aa-PGIS protein was expressed in BCNC + COX2-10aa-PGIS treatment group at the end of experiment.
a prolonged hypoxic environment, TGF-b1dependent endothelin-1 (ET-1) synthesis is increased. ET-1 is a potent constrictor of penile smooth muscle and a profibrotic peptide [28]. Studies also show that low oxygen tension in human cavernosal tissue inhibits production of prostaglandin-E1 (PGE1). PGE1 inhibits collagen formation by inhibiting TGF-b1, which induces collagen synthesis. With the inhibition of PGE1, TGF-B1 is thought to induce connective tissue synthesis [26]. Collagen deposition is increased, which leads to the loss of veno-occlusive mechanism [29,30]. A reduction in arterial inflow was also reported by several authors. This is associated with the ligation of accessory internal pudendal arteries during prostatectomy [31,32]. Combination of nerve damage with decreased arterial inflow may inten-
sify hypoxia and ultimately lead to apoptosis, which is an underlying cause of post-RP-induced ED. User et al. performed bilateral neurotomy of the rat penis and found that there was significant apoptosis in the subalbugineal smooth muscle cells. With apoptosis in the region of the subtunical venular plexus, a defect in the veno-occlusive mechanism of the corpus cavernosum occurs [33]. McVary et al. recently confirmed that both intrinsic and extrinsic apoptotic pathways were activated in rats whose cavernous nerves were disrupted [34]. In our study, intracavernous administration of Ad-COX2-10aa-PGIS obviously improved erectile function in the BCNC rat model. Our data also showed that COX-2-10aa-PGIS gene therapy upgraded a-SMA and eNOS levels, downgraded TGF-b1 levels, increased smooth muscle/collagen ratios, and decreased apoptosis in corpus cavernoJ Sex Med 2013;10:1476–1487
1486 sum. The detailed molecular mechanism of these findings was not studied. However, a potential mechanism could be postulated as follows. It is believed that endogenous AA is directly converted into PGI2 by the high efficiency hybrid enzyme COX-2-10aa-PGIS without affecting other prostanoids, especially TXA2. PGI2 binds to endothelial PGI receptor (IP) and Gs protein-coupled receptor (prostacyclin receptor) is then activated. This activation, in turn, signals adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP). The cAMP activates protein kinase A (PKA) that will eventually induce a cascade of phosphorylation and inhibition of myosin lightchain kinase, which leads to smooth muscle relaxation and vasodilation [35]. The relaxation of the smooth muscle will then increase arterial inflow and is followed by increased tissue oxygenation. This will then inhibit TGF-b1 generation and suppress fibrosis and cell apoptosis as shown in our study. The preservation of smooth muscle cells in corporeal cavernosum will maintain the venoocclusive integrity and ED can be prevented after BCNC. Ad-COX2-10aa-PGIS gene therapy appears to be a reasonable candidate in early penile rehabilitation after RP. (i) Ad-COX2-10aa-PGIS gene therapy may directly dilate the artery and sinusoid trabeculae and improve the cavernous nerve injury barrier. (ii) PGE1 injection has the same effect on vasodilation and theoretically inhibits collagen synthesis [26]. However, frequent injections are painful and cumbersome, with a significant challenge for compliance, particularly for young patients [36]. A two-time injection within a month may increase the compliance. This study also has some limitations. The possible PGI2-IP-cAMP-PKA signal pathway needs to be further verified. Although, from our data, the COX-2-10aa-PGIS increases the eNOS level, the relationship between the PGI2 signal pathway and NO signal pathway is not clear. In addition, the direct evidence that Ad-COX2-10aa-PGIS gene therapy may increase the arterial blood flow and tissue oxygenation is not studied. The use of virus for gene transfer may be limited in future human studies and we are currently planning to use stem cells as the vector for the COX-2-10aa-PGIS gene transfer in the future studies. Conclusion
Intracavernous injection of Ad-COX2-10aa-PGIS has a beneficial effect on improving erectile funcJ Sex Med 2013;10:1476–1487
Lin et al. tion on BCNC rats. This improvement appears to be related to its antifibrotic and anti-apoptotic mechanisms. Acknowledgments
This study was supported by NIH Grants HL56712 and HL79389 (Ke-He Ruan). The authors would like to thank Dorothy Stradinger for her editorial assistance, and Ms. Shui-Ping So and Ms. Vanessa Cervantes for their technical support. Corresponding Author: Run Wang, MD, FACS, Department of Urology, University of Texas Medical School at Houston and MD Anderson Cancer Center, Houston, TX 77030, USA. Tel: (713) 500-7337; Fax: (713) 500-0546; E-mail: [email protected]; Yutian Dai, MD, PhD, Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing 210008, China. Tel: 86-25-83105665; Fax: 86-25-83317016; E-mail: ytdai@ hotmail.com Conflict of Interest: The authors have no conflicts of interest to report. Statement of Authorship
Category 1 (a) Conception and Design Haocheng Lin; Jiuhong Yuan; Ke-He Ruan; Yutian Dai; Run Wang (b) Acquisition of Data Haocheng Lin; Jiuhong Yuan; Wenli Yang; Junlan Zhang (c) Analysis and Interpretation of Data Haocheng Lin; Jiuhong Yuan; Wenli Yang; Junlan Zhang
Category 2 (a) Drafting the Article Haocheng Lin (b) Revising It for Intellectual Content Haocheng Lin; Ke-He Ruan; Yutian Dai; Run Wang
Category 3 (a) Final Approval of the Completed Article Haocheng Lin; Ke-He Ruan; Yutian Dai; Run Wang References 1 Jemal ASR, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. 2 Walsh PC, Marschke P, Ricker D, Burnett AL. Patientreported urinary continence and sexual function after anatomic radical prostatectomy. Urology 2000;55:58–61.
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COX-2-10aa-PGIS Gene Therapy for Penile Rehabilitation 3 Wang R. Penile rehabilitation after radical prostatectomy: Where do we stand and where are we going? J Sex Med 2007;4:1085–97. 4 Barry MJ, Gallagher PM, Skinner JS, Fowler FJ Jr. Adverse effects of robotic-assisted laparoscopic versus open retropubic radical prostatectomy among a nationwide random sample of medicare-age men. J Clin Oncol 2012;30:513–8. 5 Yuan J, Westney OL, Ruan KH, Wang R. A new strategy, SuperEnzyme gene therapy in penile rehabilitation. J Sex Med 2009;6(suppl 3):328–33. 6 Ruan KH, Deng H, So SP. Engineering of a protein with cyclooxygenase and prostacyclin synthase activities that converts arachidonic acid to prostacyclin. Biochemistry 2006;45:14003–11. 7 Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowith JB. Arachidonic acid metabolism. Annu Rev Biochem 1986;55:69–102. 8 Bunting S, Gryglewski R, Moncada S, Vane JR. Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac ateries and inhibits platelet aggregation. Prostaglandins 1976;12:897–913. 9 Moncada S, Herman AG, Higgs EA, Vane JR. Differential formation of prostacyclin (PGX or PGI2) by layers of the arterial wall. An explanation for the anti-thrombotic properties of vascular endothelium. Thromb Res 1977;11:323–44. 10 Weksler BB, Ley CW, Jaffe EA. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J Clin Invest 1978;62:923–30. 11 Ingerman-Wojenski C, Silver MJ, Smith JB, Macarak E. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J Clin Invest 1981;67:1292–6. 12 Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B. Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci U S A 1999;96: 7220–5. 13 Shiri R, Koskimaki J, Hakkinen J, Tammela TL, Auvinen A, Hakama M. Effect of nonsteroidal anti-inflammatory drug use on the incidence of erectile dysfunction. J Urol 2006;175: 1812–5; discussion 1815–6. 14 Cawello W, Schweer H, Muller R, Bonn R, Seyberth HW. Metabolism and pharmacokinetics of prostaglandin E1 administered by intravenous infusion in human subjects. Eur J Clin Pharmacol 1994;46:275–7. 15 Ruan K-H. Hybrid protein that converts arachidonic acid into prostacyclin. Patent application number: 20100015120. 2010. 16 Gassmann M, Grenacher B, Rohde B, Vogel J. Quantifying Western blots: Pitfalls of densitometry. Electrophoresis 2009;30:1845–55. 17 Mullerad M, Donohue JF, Li PS, Scardino PT, Mulhall JP. Functional sequelae of cavernous nerve injury in the rat: Is there model dependency. J Sex Med 2006;3:77–83. 18 Cellek S, Bivalacqua TJ, Burnett AL, Chitaley K, Lin CS. Common pitfalls in some of the experimental studies in erectile function and dysfunction: A consensus article. J Sex Med 2012;9:2770–84. 19 Goldner J. A modification of the masson trichrome technique for routine laboratory purposes. Am J Pathol 1938;14:237–43. 20 Muller A, Akin-Olugbade Y, Deveci S, Donohue JF, Tal R, Kobylarz KA, Palese M, Mulhall JP. The impact of shock wave therapy at varied energy and dose levels on functional and structural changes in erectile tissue. Eur Urol 2008;53:635–42. 21 Shi SR, Key ME, Kalra KL. Antigen retrieval in formalinfixed, paraffin-embedded tissues: An enhancement method
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for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39: 741–8. Burnett AL. Rationale for cavernous nerve restorative therapy to preserve erectile function after radical prostatectomy. Urology 2003;61:491–7. Dean RC, Lue TF. Neuroregenerative strategies after radical prostatectomy. Rev Urol 2005;7(suppl 2):S26–32. Fraiman MC, Lepor H, McCullough AR. Changes in penile morphometrics in men with erectile dysfunction after nervesparing radical retropubic prostatectomy. Mol Urol 1999;3: 109–15. Walsh PC. Patient-reported urinary continence and sexual function after anatomic radical prostatectomy. J Urol 2000;55:58–61. Moreland RB, Traish A, 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. Leungwattanakij S, Bivalacqua TJ, Usta MF, Yang DY, Hyun JS, Champion HC, Abdel-Mageed AB, Hellstrom WJ. Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum. J Androl 2003;24:239–45. Granchi S, Vannelli GB, Vignozzi L, Crescioli C, Ferruzzi P, Mancina R, Vinci MC, Forti G, Filippi S, Luconi M, Ledda F, Maggi M. Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells. Mol Hum Reprod 2002;8:1053–64. Moreland RB, Gupta S, Goldstein I, Traish A. Cyclic AMP modulates TGF-beta 1-induced fibrillar collagen synthesis in cultured human corpus cavernosum smooth muscle cells. Int J Impot Res 1998;10:159–63. Gontero P, Kirby R. Proerectile pharmacological prophylaxis following nerve-sparing radical prostatectomy (NSRP). Prostate Cancer Prostatic Dis 2004;7:223–6. Mulhall JP, Graydon RJ. The hemodynamics of erectile dysfunction following nerve-sparing radical retropubic prostatectomy. Int J Impot Res 1996;8:91–4. Mulhall JP, Slovick R, Hotaling J, Aviv N, Valenzuela R, Waters WB, Flanigan RC. Erectile dysfunction after radical prostatectomy: Hemodynamic profiles and their correlation with the recovery of erectile function. J Urol 2002;167: 1371–5. User HM, Hairston JH, Zelner DJ, McKenna KE, McVary KT. Penile weight and cell subtype specific changes in a postradical prostatectomy model of erectile dysfunction. J Urol 2003;169:1175–9. McVary KT, Podlasek CA, Wood D, McKenna KE. Apoptotic pathways are employed in neuropathic and diabetic models of erectile dysfunction. J Urol 2006;175:387 (Abstract 1203). Evans DH, Gunderson MP. A prostaglandin, not NO, mediates endothelium-dependent dilation in ventral aorta of shark (Squalus acanthias). Am J Physiol 1998;274:R1050–7. Goldstein I. Early penile rehabilitation following radical prostatectomy: Overview and rationale. Contemp Urol 2006; 18(1 suppl):3–6.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Appendix S1 Pilot study.
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COX-2-10aa-PGIS Gene Therapy Improves Erectile Function in Rats after Cavernous Nerve Injury Haocheng Lin, MD,*† Jiuhong Yuan, MD,‡ Ke-He Ruan, MD, PhD,§ Wenli Yang, PhD,¶ Junlan Zhang, MD, PhD,¶ Yutian Dai, MD, PhD,* and Run Wang, MD, FACS†** *Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, China; † Division of Urology, University of Texas Medical School at Houston, Houston, TX, USA; ‡Department of Urology, West China Hospital, Sichuan University, Chengdu, China; §Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, Houston, TX, USA; ¶Division of Gastroenterology, Hepatology & Nutrition, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX, USA; **Department of Urology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA DOI: 10.1111/jsm.12147
ABSTRACT
Introduction. Erectile dysfunction (ED) is a very common complication after radical prostatectomy. COX-2-10aaPGIS is a newly engineered protein with COX-2 and prostacyclin synthase activities that converts arachidonic acid directly to prostacyclin (prostaglandin I2 [PGI2]). PGI2 is a potent smooth muscle relaxant. Aim. The purpose of this study was to explore the effect and mechanism of COX-2-10aa-PGIS gene therapy in penile rehabilitation. Methods. Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley rats was used to mimic radical prostatectomy-induced ED. Sprague-Dawley rats were randomly assigned into four groups: 1. sham surgery; 2. BCNC; 3. BCNC + null control recombinant adenovirus intracavernous injection; and 4. BCNC + Ad-COX2-10aaPGIS intracavernous injection. Twenty-eight days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the mean arterial pressure (MAP) was monitored. At the end of the measurement, the penis was harvested and processed for (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (a-SMA), and transforming growth factor beta-1 (TGF-b1); (ii) Masson’s trichrome stain for smooth muscle/collagen ratios; (iii) Western blot of eNOS, a-SMA, TGF-b1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. Main Outcome Measures. Erectile function was evaluated by ICP/MAP. Smooth muscle and endothelium functions in corpora cavernosum were assessed by Masson’s trichrome stain, immunohistochemistry, and Western blot. Apoptosis was identified by TUNEL assay. Results. The results were the following: 1. COX2-10aa-PGIS gene therapy improved erectile function (82%, compared with control) in the BCNC rat model; 2. COX2-10aa-PGIS gene therapy increased eNOS (121%) and a-SMA (118%) expression and decreased TGF-b1 (45%) expression; 3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury (64%); and 4. COX2-10aa-PGIS gene therapy improved smooth muscle/ collagen ratios (81%). Conclusion. Our data demonstrated that COX2-10aa-PGIS improved erectile function after cavernous nerve injury through antifibrotic and anti-apoptotic mechanisms. Lin H, Yuan J, Ruan K-H, Yang W, Zhang J, Dai Y, and Wang R. COX-2-10aa-PGIS gene therapy improves erectile function in rats after cavernous nerve injury. J Sex Med 2013;10:1476–1487. Key Words. Cavernous Nerve Injury; COX2-10aa-PGIS; Erectile Dysfunction; Penile Rehabilitation; Radical Prostatectomy
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© 2013 University of Texas Medical School at Houston Journal of Sexual Medicine © 2013 International Society for Sexual Medicine
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COX-2-10aa-PGIS Gene Therapy for Penile Rehabilitation Introduction
C
ancer is one of the leading causes of death in the United States, with prostate cancer as the most common cancer in men and second as a cause of death [1]. As techniques improve, such as prostate-specific antigen screening, laparoscopic, and robotic surgery, radical prostatectomy (RP) is the primary option for treatment of clinically localized prostate cancer with excellent long-term results [2,3]. However, erectile dysfunction (ED) as a result of RP remains a significant challenge. Recent clinical study showed that the use of robotic-assisted surgery has not improved erectile function outcomes [4]. Currently, post-RP pharmacological sexual rehabilitation is widely practiced. It is commenced early and based primarily on phosphodiesterase type 5 inhibitors, intracavernosal or transurethral administration of vasodilators, or with the vacuum erection device. However, the reported recovery rate is varied and experiences low patient compliance due to cumbersome situation and ineffectiveness [3]. Although animal studies with nerve regeneration regimens showed promising results, their clinical applications were disappointing [3]. Therefore, there is a great need to explore novel rehabilitation modalities after RP. Prostacyclin (prostaglandin I2 [PGI2]) is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase (PGIS). PGH2 is converted from arachidonic acid (AA) by cyclooxygenase (COX) enzymes: COX-1 and/or COX-2. AA released from the membrane phosphoglycerides is converted to the prostaglandin G2 (catalytic step 1) and then to PGH2 (catalytic step 2) by COX-1 or COX-2. PGH2 is further isomerized to the biologically active end products of prostaglandin D2, prostaglandin E2 (PGE2), prostaglandin F2, and PGI2 or thromboxane A2 (TXA2) by individual synthases (catalytic step 3) in tissue-specific processes (Figure 1) [5,6]. Prostanoids act as local hormones in the vicinity of their production site to regulate hemostasis and smooth muscle functions [7]. TXA2 produced from PGH2 by TXA2 synthase has been implicated as a proaggregatory and vasoconstricting mediator in various pathophysiological conditions [8]. PGI2 is the primary AA metabolite in vascular walls, has opposing biological properties to TXA2, and therefore represents the most potent endogenous vascular protector acting as an inhibitor of platelet aggregation [9] and a strong vasodilator on vascular beds [10–12].
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2 activities to reduce the production of all prostanoids, which leads to the thinning of blood by reducing TXA2 production and the suppression of inflammation by decreasing PGE2 production. It was recently reported that NSAID usage increased ED incidence in men [13]. Therefore, PGI2 may be a good candidate for vascular protection and ED treatment. However, prostacyclin is quickly broken down into 6-keto-PGF1 due to its short half-life, which makes it a much weaker vasodilator and limits its clinical application [14]. Finding a way to specifically increase the production of the vascular protector, PGI2, will be an intriguing project. Recently, Ruan et al. reported an innovative engineering of a recombinant protein COX-2-10aaPGIS with triple catalytic activities directly converting AA into PGI2 [6]. The advantages of the newly engineered COX-2-10aa-PGIS are the following: (i) multiple catalytic enzyme activities can be configured within a single protein molecule if an approximate protein configuration is adopted; (ii) the engineered protein with tricatalytic functions not only accumulates the individual enzymes’ activities but also has a faster turnover rate when compared with the mixture of the parent enzymes [6]. This study was designed to apply Ad-COX210aa-PGIS (the replication deficient recombinant adenoviruses carry the COX-2-10aa-PGIS gene) to the bilateral cavernous nerve crush (BCNC) rat model, which mimics ED after RP, to explore the potential benefit of COX-2-10aa-PGIS
Figure 1 Prostanoids synthesis
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1478 gene therapy and to understand the underlying mechanisms. Materials and Methods
Prepare Ad-COX-2-10aa-PGIS Amplification, Purification, and Identification of the Ad-COX-2-10aa-PGIS The primary Ad-COX2-10aa-PGIS viral stock was established as previously described [15]. The primary Ad-COX2-10aa-PGIS was amplified and purified following the standard protocol of Puresyn’s Adenopure TM kit. Briefly, the HEK-293 cells were subcultured in 150-cm2 tissue culture plates with 20 mL of Dulbecco’s Modified Eagle Medium (DMEM) growth medium for 48 hours. Transfection of Ad-COX2-10aa-PGIS was performed until cells were approximately 75–85% confluent. Fifty microliter of Primary Ad-COX210aa-PGIS (1.3 ¥ 1012 viral particle [VP]/mL) in fresh 20 mL DMEM growth medium was immediately applied to the plate of cells and swirled gently to distribute the suspension evenly in the medium. After incubating for 64–72 hours at 37°C, the cells with DMEM growth medium were scraped into the tube and subjected to four rounds of freeze/thaw by alternation of the tube between the dry ice-ethanol bath and the 37°C water bath. The cellular debris was collected by centrifugation at 10,000 ¥ g for 10 minutes and the supernatant (Ad-COX2-10aa-PGIS virus) was stored at -80°C. The supernatant was then concentrated and purified using the Puresyn’s Adenopure TM kit. Virus particle concentration was calculated from the absorbance at 260 nm (A260). The infectious titer was determined by plaque assay. Finally, 3.8 ¥ 1011 VP/mL (1.25 ¥ 1010 plaque forming units [PFU]/mL) Ad-COX2-10aa-PGIS virus in elution buffer was acquired and stored at -20°C for use in the next step. Verification of the Purified Ad-COX2-10aa-PGIS Special Gene with Polymerase Chain Reaction (PCR) PCR primer was designed using deoxyribonucleic acid (DNA) tools (OligoPerfectTM Designer) from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA): forward (5′-TGGTGGAGAAGTG GGTTTTC-3′); reverse (5′-GTCCAGGAGAA CGGTGACAT-3′). Five micron of each sample (Ad-COX2-10aa-PGIS, null control recombinant adenovirus [NCRA, Cell Biolabs, Inc., San Diego, CA, USA] and positive control group [Baculovirus-COX2-10aa-PGIS]) was added into J Sex Med 2013;10:1476–1487
Lin et al. 100 mL digest buffer (1¥ PCR buffer, 0.45% NP40, 0.45% Tween 20, 10 mL Proteinase K [10 mg/mL]) at 60°C for 1 hour. The sample was further denatured by boiling for 15 minutes and then cooled on ice for 5 minutes. Two microliter of the isolated genetic DNA was subjected to PCR analysis using PCR Reaction Buffer (1¥ PCR buffer, 2.5 mM MgCl2, 200 mM deoxynucleotide triphosphates (dNTPs), 1 mM each primer, and 1 unit Taq Polymerase with conditions). After overlay with 30–40 mL light mineral oil, the DNA was put into the cycler and the following program was run: hold at 94°C for 4.5 minutes, 30¥ step cycles of 94°C for 30 seconds and 54°C for 20 seconds, and 72°C for 1 minute and hold at 4°C until ready to analyze. The PCR product was analyzed by agarose gel (Figure 2A).
Ad-COX2-10aa-PGIS Enzyme Activity Determination Using High-Performance Liquid Chromatography HEK293 cells were respectively transfected with baculovirus-COX2-10aa-PGIS (positive control), pcDNA3.1(+) (Vector), and purified Ad-COX210aa-PGIS. To determine the activities of the synthases that converted AA to PGI2 through the tricatalytic functions, [14C]-AA (3 mM) was added to each group in a total volume of 30 mL PBS. After 0.5 minute of incubation, the reaction was stopped by adding 50 mL of the solvent containing 0.1% acetic acid and 30% acetonitrile (solvent A). After centrifugation (12,000 rpm for 5 minutes), the supernatant was injected into a C18 column (Varian Microsorb-MV 100-5, 4.6 ¥ 250 mm) using the solvent A with a gradient from 35% to 100% of acetonitrile for 45 minutes at a flow-rate of 1.0 mL/minute. The [14C]-labeled AA metabolites, including 6-keto-PGF1R (degraded PGI2), were monitored directly by a flow scintillation analyzer (Packard 150TR) [6] (Figure 2B). Determination of COX-2-10aa-PGIS Protein Expression with Western Blot The purified Ad-COX2-10aa-PGIS, baculovirusCOX2-10aa-PGIS (positive control), and NCRA were applied to HEK293 cells. The HEK 293 cells were incubated in a 10-cm plate for 48 hours after infection. The plate was washed with PBS to remove residual media. A 400 mL of 1¥ radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, MO, USA) was added to the plate. The plate was incubated on ice for
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Figure 2 (A) PCR confirmed the amplified and purified Ad-COX2-10aa-PGIS contain COX2-10aa-PGIS gene. (B) HPLC show COX-2-10aa-PGIS enzyme activity. Ad-COX-2-10aa-PGIS group produce 6-keto-PGF1 (product of PGI2) by consuming substrate AA. No 6-keto-PGF1 was produced in vector group. (C) Western Blot verified the special hybrid enzyme expression.
5 minutes. Then, the cells were scraped, sonicated briefly, and spun for 10 minutes at 14,000 ¥ g in a cold microcentrifuge. Supernatant was removed for use. Protein concentration was determined by bicinchoninic acid (Pierce Chemical Company, Rockford, IL, USA) method. Equal amounts of supernatant protein (20 mg) were electrophoresed according to the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). The membrane was incubated with PGIS (1:1,000, Cayman Chemical Company, Ann Arbor, MI, USA) and COX-2 (1:1,000, Cayman Chemical Company) antibodies at 4°C overnight. Detection of the reactive protein on the membrane was performed with horseradish peroxidase-conjugated secondary antibody (1:5,000, Cayman Chemical Company) for 4 hours at room temperature (RT), followed by exposure to X-ray films. The resulting images were analyzed with Image J 1.44 (US National
Institutes of Health, Bethesda, MD, USA) to determine the integrated density value of each protein band [16] (Figure 2C).
Animal Groups and Study Animal Model, Grouping, and Tissue Harvesting The BCNC animal model was previously described [17]. Briefly, rats were anesthetized, shaved, and disinfected as required. They were placed in a supine position. A midline suprapubic incision was made to expose the bladder and prostate. The major pelvic ganglion (MPG) lying on the dorsal aspect of the ventral prostate was found after carefully separating any overlying adipose tissues. Cavernous nerves were isolated and identified. At the point of 5 mm distal to the MPG, the bilateral cavernous nerves were crushed using an ultrafine hemostat with full tip closure for 30 seconds, removed for 30 seconds, and then reapplied for another 30 seconds. The incision was J Sex Med 2013;10:1476–1487
1480 closed with 3-0 Vicryl sutures. Analgesia and antibiotics were applied before and after the operation as required. Thirty-two Sprague-Dawley rats (male, 200– 224 g, Harlan Laboratories, Indianapolis, IN, USA) were randomized into four groups: group 1 (sham, N = 8), sham surgery, no cavernous nerve crush; group 2 (BCNC, N = 8), surgery for BCNC, without any therapy; group 3 (BCNC + NCRA, N = 8), surgery for BCNC, with NCRA penile injection; and group 4 (BCNC + Ad-COX-2-10aa-PGIS, N = 8), surgery for BCNC, with Ad-COX2-10aa-PGIS penile injection. The intracavernosal injection of NCRA or Ad-COX2-10aa-PGIS was given at day 1 and day 14. The transfection time, determined at the pilot study, which revealed the highest expression of the new protein, was found at day 7 and weakened after the first week (supplementary file). At day 28, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the blood pressure was monitored. At the end of the measurement, the penes were harvested and processed for the following: (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (a-SMA), and transforming growth factor beta-1 (TGF-b1); (ii) Masson’s trichrome stain for smooth muscle/ collagen ratios; (iii) Western blot of eNOS, a-SMA, TGF-b1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. All animal procedures were approved by the Animal Welfare Committee.
Intracavernous Injection of Ad-COX2-10aa-PGIS and NCRA The rats were transferred into the biohazard room prior to administration of adenovirus. The animals were anesthetized with inhalant isoflurane at the flow-rate 3–4 level. Either 50 mL Ad-COX2-10aaPGIS solution containing 1.25 ¥ 1010 PFU/mL or 50 mL NCRA was injected into the corpora cavernosum using Hamilton Microliter with a 25-gauge needle. The transfection dosage that revealed the best expression of COX2-10aa-PGIS was determined at the pilot study and was identified in the 1.25 ¥ 1010 PFU/mL group (supplementary file). An elastic band was placed at the base of the penis prior to injection and was removed 5 minutes after the injection. The rats stayed in the biohazard room for next 72 hours before being transferred back to regular housing. This procedure was J Sex Med 2013;10:1476–1487
Lin et al. repeated in 14 days to maintain the high expression of PGI2.
Erectile Function Evaluation ICP under electric stimulation of the cavernous nerve was measured by the ICP/mean arterial pressure (MAP) set (Grass S48 stimulate, Powerlab/4SP, AD instruments QUAD bridge, NL108A pressure amplifier, data acquisition module: DI-194RS and Windaq/lite recording software) before the rats were sacrificed. After being anesthetized intraperitoneally, with 40 mg/kg pentobarbital sodium, the MPG, and cavernous nerve were exposed on either side of the prostate. A 25-gauge needle connected to a PE-50 tube and filled with 100 U/mL heparin sodium solution was inserted into the right crura for measurement of ICP. The left carotid artery was exposed and cannulated with a PE-50 tube to record the MAP. Stimulations were performed at a parameter of 16 Hz, 5 milliseconds duration, and 7.5 V for 60 seconds with a period of 5 minutes between subsequent stimulations. The maximal increase of ICP during nerve electrostimulation was selected for statistical analysis in each animal. The ICP/MAP ratios were analyzed to evaluate the erectile function [18] (Figure 3). Histology Penile tissues were fixed in 4% paraformaldehyde overnight, after which the tissues were taken for routine processing and paraffin embedding. Tissue samples were cut into 5-mm sections from the midshaft of the penis. Then the tissue slides, showing the cross section of the corpora cavernosa, were deparaffinized for the following studies. Masson’s Trichrome Staining Slides were rehydrated and refixed in Bouin’s solution overnight to improve staining quality prior to the following standard protocol [19]. Smooth muscle/collagen ratios were analyzed using Image J v.1.44 (National Institutes of Health, Bethesda, MD, USA). Five sections per rat were quantified at 200¥ by Image J. The entire section was used for analysis. Sections from four rats per group were analyzed. Thus, in each group (sham, BCNC, BCNC + NCRA, and BCNC + Ad-COX2-10aaPGIS), a total of 20 sections were reviewed for histomorphometry [20]. Immunohistochemistry-Paraffin Staining Tissue sections were rehydrated according to standard protocols. Antigen retrieval was performed
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Figure 3 (A) Representative ICP response to cavernous nerve stimulation. (B) Ad-COX2-10aa-PGIS gene therapy group had a significant increase of ICP/MAP ratios compared with BCNC and BCNC + NCRA group (P < 0.001).
using 10 mM citrate acid buffer (pH 3.0) at 37°C for 30 minutes blocked by superblock (either 10% goat serum or horse serum, depending on the primary antibody) for 10 minutes at RT. Penile sections were then incubated with primary antibodies (a-SMA [1:200, Abcam, Cambridge, MA, USA], eNOS [1:250, BD Transduction Laboratories, Lexington, KY, USA], TGF-b1 [1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA]) overnight at 4°C, followed by biotinylated secondary antibody working solution (1:200) for 30 minutes at RT. Then, the stain section was incubated in diluted ABC Reagent (VectorLabs, Burlingame, CA, USA) at RT for 30 minutes, followed by diaminobenzidine (DAB)/H2O2 development solution (VectorLabs). Finally, the stained sections were counterstained with hematoxylin, dehydrated, and mounted with Clearium mounting medium [21]. Five sections per rat were quantified at 400¥ magnification by Image J 1.44. Five rats per group were quantified.
Apoptosis Assessment In Situ Cell Death Detection Kit (fluorescein, Roche Applied Science, Indianapolis, IN, USA) was used for TUNEL assay and was performed following the manufacturer’s instructions to assess apoptosis. Apoptotic cells were traced with fluo-
rescein and the nucleus was marked with 4′, 6′ diamidino-2-phenylindole (DAPI, VectorLabs). One section from each rat was quantified by counting the number of apoptotic cells over total cells stained with DAPI in five regions chosen randomly per section. Four rats per group were quantified by this methodology. The ratio of the percentage of fluorescent apoptotic cells to total number of cells was recorded as the apoptotic index (AI) [20].
Western Blot The nerve bundle and urethra were stripped from the corpora prior to homogenization. A 1¥ RIPA lysis buffer with PMSF was added to the corpora cavernosa that was cut into tiny pieces by scissors. The tissue samples were vortexed for 60 seconds, incubated on ice for 45 minutes, homogenized with a polytron (3 ¥ 20 seconds), and centrifuged at 14,000 ¥ g for 10 minutes at 4°C. Supernatant was removed for use. First antibodies used in Western blot were a-SMA (1:1,500), eNOS (1:1,000), TGF-b1 (1:1,000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000, Santa Cruz Biotechnology Inc.), and PGIS. The detailed protocol was described in the above Western blot section. Quantification of a-SMA, J Sex Med 2013;10:1476–1487
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Figure 4 Ad-COX2-10aa-PGIS gene therapy preserved smooth muscle/collagen ratios. (A) Representative figures of Masson’s trichrome staining in each group (original magnification ¥100). (B) Masson’s trichrome stain was used for muscle/collagen ratios. Compared with BCNC and BCNC + NCRA group, Ad-COX2-10aa-PGIS group was found to have higher smooth muscle/collagen ratios (P < 0.001).
eNOS, and TGF-b1 were performed relative to GAPDH in five rats per group using Image J 1.44.
Statistical Analysis All measurements are expressed as mean ⫾ standard deviation. Data were analyzed using Student’s t-test or anova with Origin 6.1 for multiple comparisons between groups. A P value < 0.05 was considered statistically significant. Results
Erectile Function Assessment The typical ICP tracings of sham, BCNC, BCNC + NCRA, and BCNC + Cox-2-10aa-PGIS were shown in Figure 3. Each group had six rats for the ICP/MAP ratio analysis. The ICP/MAP ratio in the sham group was 0.84 ⫾ 0.06, which was significantly higher compared with BCNC group (0.34 ⫾ 0.08, P < 0.001), BCNC ⫾ NCRA group (0.29 ⫾ 0.07, P < 0.001), and BCNC + AdCOX2-10aa-PGIS group (0.62 ⫾ 0.09, P < 0.01). Ad-Cox2-10aa-PGIS therapy significantly improved ICP/MAP when compared with BCNC by 82% (P < 0.001) and 114% with the BCNC + NCRA groups (P < 0.001). J Sex Med 2013;10:1476–1487
Masson’s Trichrome Staining Analysis Smooth muscle/collagen ratios were evaluated by Masson’s trichrome method. The sham group revealed smooth muscle/collagen ratios of 0.46 ⫾ 0.13, which had the highest ratio among all study groups (P < 0.05). The smooth muscle/ collagen ratio for the BCNC Ad-COX2-10aaPGIS group was 0.38 ⫾ 0.08, which was significantly greater than those from the BCNC group (0.21 ⫾ 0.04, P < 0.001) and BCNC + NCRA group (0.23 ⫾ 0.04, P < 0.001) (Figure 4). Ad-Cox2-10aa-PGIS therapy significantly improved smooth muscle/collagen ratios when compared with BCNC by 81% (P < 0.001) and 65% with the BCNC + NCRA groups (P < 0.001). Apoptosis Analysis At 4 weeks after BCNC, the BCNC ⫾ COX-210aa-PGIS group demonstrated a significant reduction in apoptosis within the corporal tissue with a mean AI of 0.23 ⫾ 0.08, compared with BCNC group (AI: 0.64 ⫾ 0.15, P < 0.001) and BCNC + NCRA group (AI: 0.6 ⫾ 0.17, P < 0.001). The AI value in the sham group was 0.11 ⫾ 0.10, which was notably lower compared with all other groups (P < 0.001)
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Figure 5 (Left) was the representative figure of apoptotic cell stained by fluorescein (green), nucleus stained by DAPI (Blue) and the merged figure from each group. A, B, C and D respectively represent Sham, BCNC, BCNC + NCRA and BCNC + AdCOX2-10aa-PGIS groups. On the right figure, the ratio of apoptotic cell/Nucleus was calculated and analyzed as the apoptotic index. The Ad-COX2-10aa-PGIS therapy significantly decreased the apoptosis compared with BCNC and BCNC + NCRA groups (P < 0.001).
(Figure 5). Ad-Cox2-10aa-PGIS therapy significantly decreased AI when compared with BCNC by 64% (P < 0.001) and 62% with the BCNC + NCRA groups (P < 0.001).
Immunohistochemistry Analysis Immunohistochemical staining showed that Ad-COX2-10aa-PGIS therapy group had significant increases of eNOS (128% and 97%) and a-SMA (67% and 97%) in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). Ad-COX2-10aa-PGIS therapy group also had decreased TGF-b1 (54% and 49%) expression level compared with BCNC and BCNC + NCRA groups (P < 0.05) (Figure 6). Western Blot Analysis Western blot results denoted that the Ad-COX210aa-PGIS therapy group significantly increased the expression of eNOS (121% and 95%) and a-SMA (118% and 146%) in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). Ad-COX2-10aa-PGIS therapy group also decreased TGF-b1 expression level
(45% and 46%) compared with BCNC and BCNC + NCRA groups (P < 0.05). In addition, Western blot confirmed that Ad-COX2-10aaPGIS therapy group was expressing COX2-10aaPGIS protein at the end of the experiment (Figure 7). Discussion
ED following RP is due to damage to the cavernous nerve, known as neuropraxia. Neuropraxia can be caused by mechanically induced nerve stretching that may occur during prostate retraction, thermal damage to the nerve caused by electrocauterization, ischemia of the nerve secondary to disruption of blood supply while attempting to control surgical bleeding, and local inflammatory effects associated with surgical trauma [22]. Even in the most meticulous nerve-sparing dissection, some degree of neuropraxia is unavoidable because of the close proximity of the nerve to the prostate gland. These nerves tend to recover slowly; it may take as long as 18–24 months for them to reach a new baseline functional status [23]. Apoptosis J Sex Med 2013;10:1476–1487
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Figure 6 Immunohistochemical staining of TGF-b1, eNOS, and a-SMA. (Upper) Representative photos of TGF-b1, eNOS, and a-SMA at ¥400 magnification. (Lower) Positive area/total area (%) was used to denote the expression of TGF-b1, eNOS, and a-SMA. Ad-COX2-10aa-PGIS therapy increase eNOS and a-SMA in corpus cavernosum compared with BCNC and BCNC + NCRA groups (P < 0.05). TGF-b1 was also significant decreased in BCNC + Ad-COX2-10aa-PGIS group compared with BCNC and BCNC + NCRA groups (P < 0.05).
of penile smooth muscle caused by cavernous nerve injury leads to decreased erectile function [24,25]. Lack of erections can cause poor oxygenation of the corporal bodies and fibrosis resulting in venous leak [26]. In an experimental model, significant J Sex Med 2013;10:1476–1487
overexpression of hypoxia-related substances, such as hypoxia-inducible factor (HIF-1-a) and TGF-b1 and collagen I and III, was found in rats that had undergone bilateral excision of the cavernosal nerves compared with controls [27]. When human penile smooth muscle cell is exposed to
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Figure 7 The protein expression in rat penis was detected by Western blot. (a–d) respectively represent Sham (a), BCNC (b), BCNC + NCRA (c) and BCNC + Ad-COX2-10aa-PGIS (d) groups. (A) represents typical bands of Western Blot. Bands from up to down represent a-SMA, TGF-b1, eNOS, COX2-10aa-PGIS, and GAPDH. (B–E) respectively represent column figures of a-SMA, TGF-b1, eNOS, and COX2-10aa-PGIS. Each figure denotes means ⫾ standard errors of each protein expression determined by densitometry relative to GAPDH. Compare with BCNC group (N = 5) and BCNC + NCRA group (N = 5), BCNC + COX2-10aa-PGIS group (N = 5) significantly increased the expression of eNOS and a-SMA (P < 0.05), decreased the expression of TGF-b1 (P < 0.05). COX2-10aa-PGIS protein was expressed in BCNC + COX2-10aa-PGIS treatment group at the end of experiment.
a prolonged hypoxic environment, TGF-b1dependent endothelin-1 (ET-1) synthesis is increased. ET-1 is a potent constrictor of penile smooth muscle and a profibrotic peptide [28]. Studies also show that low oxygen tension in human cavernosal tissue inhibits production of prostaglandin-E1 (PGE1). PGE1 inhibits collagen formation by inhibiting TGF-b1, which induces collagen synthesis. With the inhibition of PGE1, TGF-B1 is thought to induce connective tissue synthesis [26]. Collagen deposition is increased, which leads to the loss of veno-occlusive mechanism [29,30]. A reduction in arterial inflow was also reported by several authors. This is associated with the ligation of accessory internal pudendal arteries during prostatectomy [31,32]. Combination of nerve damage with decreased arterial inflow may inten-
sify hypoxia and ultimately lead to apoptosis, which is an underlying cause of post-RP-induced ED. User et al. performed bilateral neurotomy of the rat penis and found that there was significant apoptosis in the subalbugineal smooth muscle cells. With apoptosis in the region of the subtunical venular plexus, a defect in the veno-occlusive mechanism of the corpus cavernosum occurs [33]. McVary et al. recently confirmed that both intrinsic and extrinsic apoptotic pathways were activated in rats whose cavernous nerves were disrupted [34]. In our study, intracavernous administration of Ad-COX2-10aa-PGIS obviously improved erectile function in the BCNC rat model. Our data also showed that COX-2-10aa-PGIS gene therapy upgraded a-SMA and eNOS levels, downgraded TGF-b1 levels, increased smooth muscle/collagen ratios, and decreased apoptosis in corpus cavernoJ Sex Med 2013;10:1476–1487
1486 sum. The detailed molecular mechanism of these findings was not studied. However, a potential mechanism could be postulated as follows. It is believed that endogenous AA is directly converted into PGI2 by the high efficiency hybrid enzyme COX-2-10aa-PGIS without affecting other prostanoids, especially TXA2. PGI2 binds to endothelial PGI receptor (IP) and Gs protein-coupled receptor (prostacyclin receptor) is then activated. This activation, in turn, signals adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP). The cAMP activates protein kinase A (PKA) that will eventually induce a cascade of phosphorylation and inhibition of myosin lightchain kinase, which leads to smooth muscle relaxation and vasodilation [35]. The relaxation of the smooth muscle will then increase arterial inflow and is followed by increased tissue oxygenation. This will then inhibit TGF-b1 generation and suppress fibrosis and cell apoptosis as shown in our study. The preservation of smooth muscle cells in corporeal cavernosum will maintain the venoocclusive integrity and ED can be prevented after BCNC. Ad-COX2-10aa-PGIS gene therapy appears to be a reasonable candidate in early penile rehabilitation after RP. (i) Ad-COX2-10aa-PGIS gene therapy may directly dilate the artery and sinusoid trabeculae and improve the cavernous nerve injury barrier. (ii) PGE1 injection has the same effect on vasodilation and theoretically inhibits collagen synthesis [26]. However, frequent injections are painful and cumbersome, with a significant challenge for compliance, particularly for young patients [36]. A two-time injection within a month may increase the compliance. This study also has some limitations. The possible PGI2-IP-cAMP-PKA signal pathway needs to be further verified. Although, from our data, the COX-2-10aa-PGIS increases the eNOS level, the relationship between the PGI2 signal pathway and NO signal pathway is not clear. In addition, the direct evidence that Ad-COX2-10aa-PGIS gene therapy may increase the arterial blood flow and tissue oxygenation is not studied. The use of virus for gene transfer may be limited in future human studies and we are currently planning to use stem cells as the vector for the COX-2-10aa-PGIS gene transfer in the future studies. Conclusion
Intracavernous injection of Ad-COX2-10aa-PGIS has a beneficial effect on improving erectile funcJ Sex Med 2013;10:1476–1487
Lin et al. tion on BCNC rats. This improvement appears to be related to its antifibrotic and anti-apoptotic mechanisms. Acknowledgments
This study was supported by NIH Grants HL56712 and HL79389 (Ke-He Ruan). The authors would like to thank Dorothy Stradinger for her editorial assistance, and Ms. Shui-Ping So and Ms. Vanessa Cervantes for their technical support. Corresponding Author: Run Wang, MD, FACS, Department of Urology, University of Texas Medical School at Houston and MD Anderson Cancer Center, Houston, TX 77030, USA. Tel: (713) 500-7337; Fax: (713) 500-0546; E-mail: [email protected]; Yutian Dai, MD, PhD, Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing 210008, China. Tel: 86-25-83105665; Fax: 86-25-83317016; E-mail: ytdai@ hotmail.com Conflict of Interest: The authors have no conflicts of interest to report. Statement of Authorship
Category 1 (a) Conception and Design Haocheng Lin; Jiuhong Yuan; Ke-He Ruan; Yutian Dai; Run Wang (b) Acquisition of Data Haocheng Lin; Jiuhong Yuan; Wenli Yang; Junlan Zhang (c) Analysis and Interpretation of Data Haocheng Lin; Jiuhong Yuan; Wenli Yang; Junlan Zhang
Category 2 (a) Drafting the Article Haocheng Lin (b) Revising It for Intellectual Content Haocheng Lin; Ke-He Ruan; Yutian Dai; Run Wang
Category 3 (a) Final Approval of the Completed Article Haocheng Lin; Ke-He Ruan; Yutian Dai; Run Wang References 1 Jemal ASR, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. 2 Walsh PC, Marschke P, Ricker D, Burnett AL. Patientreported urinary continence and sexual function after anatomic radical prostatectomy. Urology 2000;55:58–61.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Appendix S1 Pilot study.
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