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Departments of Urology,3
Clinical Research Center,4
Pathology,5
Emergency Medicine,6 Inha University Collegeof Medicine, Incheon 400-103, South Korea
Laboratory of Cell Regulation and Carcinogenesis,7 National Cancer Institute, Bethesda, Maryland 20892-5055
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male sexual function, penis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An understanding of the factors that alter the functional smooth muscle-connective tissue balance is of paramount importance in the continuing development of therapeutic agents for the treatment of vasculogenic ED. Recently, much attention has been focused on the role of transforming growth factor (TGF) ß1 as a fibrogenic cytokine to induce fibrosis in a variety of organs. This protein has been shown to increase collagen synthesis in human corpus cavernosal smooth muscle cell by 2.5- to 4.5-fold [3]. Additionally, TGFß1 is overexpressed in the tunica albuginea of men suffering from veno-occlusive dysfunction [1] and in plaques obtained from those with Peyronie disease [4].
To establish an animal model of cavernous fibrosis, Nehra et al. [5] have shown that the intracavernosal injection of recombinant human (rh) TGFß1-impregnated alginate microspheres into the rabbit corpus cavernosum resulted in a dose-dependent decrease in the percentage of corporal smooth muscle, but this change lasted only for a short period. This can be explained, in part, by the short-term effects of the rhTGFß1 protein, which result from the vascular nature of the corpus cavernosum and the short half-life of the free TGFß1 in the bloodstream. Therefore, a new method for long-term, cost-effective delivery of such cytokines is essential for the establishment of a long-term cavernous fibrosis model that is useful in investigating and understanding TGFß1 signaling. Recently, Lee et al. [6] reported that fibroblasts containing an exogenous TGFß1 gene (NIH 3T3-TGFß1 cells) successfully induced cartilage regeneration when injected into the knee joint of rabbits with artificial cartilage defects and were able to express the transgene for at least 4 wk after injection in vivo.
Therefore, we hypothesized that the injection of NIH 3T3 fibroblasts containing a transgene, TGFß1, into the corpus cavernosum could achieve a long-term, sustained production of TGFß1, which could cause cavernous fibrosis with resultant ED.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NIH 3T3-TGFß1 cell line was donated by the National Cancer Institute in National Institutes of Health (Bethesda, MD). The technique to establish this cell line has been described previously [6]. Briefly, pMTMLVß1 was constructed by cloning a 1.2-kilobase porcine TGFß1 cDNA into the polylinker site of the replication-defective retroviral vector pMTMLV. The pMTMLV vector was derived from the retroviral vector MFG by deleting entire gag and env sequences as well as some of the 
package sequence. All the U3 of 5' long terminal repeat (LTR) except for 36 base pairs at the 5' end was repeated with the metallothionein (MT) promoter [7]. The pMTMLVß1 and pVSVG were cotransfected into GP293 cells by the calcium phosphate method. After 48 h of culture, the supernatant was filtered (pore size, 0.45 µm), and half of it was saved at –70°C for later use. The NIH 3T3 cells were seeded in 60-mm culture dishes, and 18 h later, they were infected with the filtrate plus 8 µg/ml of Polybrene (Sigma Chemical Co., St. Louis, MO). After 4 h of incubation, the medium was replaced with fresh medium. Infection was repeated 24 h later with the saved viral supernatant. Transduced cells were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum, and selection with neomycin (10 µM/ml) started 48 h after transfection. The secretion rate of TGFß1 protein of NIH 3T3-TGFß1 or NIH 3T3-neo cells was measured by ELISA.
Intracavernosal Injection of NIH 3T3-TGFß1 Cells
Adult male Sprague-Dawley rats (weight, 200–250 g) were used. Before the experiments, the rats were maintained in standard cages under clean conditions in separate quarters under a 12L:12D photoperiod with free access to water and pellets. The present experiments were approved by the Institutional Animal Care and Use Subcommittee of our university. Rats were randomly assigned to the control group or to treatment groups (Table 1). Treatment animals received 1 x 105, 1 x 106, or 3 x 106 cells in 0.1 ml of NIH 3T3-TGFß1 cells, whereas control animals received 3 x 106 cells in 0.1 ml of NIH 3T3-neo cells. Based on these initial results, three separate groups of rats were subjected to the next animal experiment: In group 1, culture medium (0.1 ml) was injected into the corpora cavernosa of the rats as a control (n = 12). In group 2, rhTGFß1 (200 ng in 0.1 ml) was injected into the cavernosa (n = 12). In group 3, NIH 3T3-TGFß1 cells (3 x 106 cells in 0.1 ml) were injected into the cavernosa (n = 36). Using sterile technique, penile skin was incised, and tunica albuginea was exposed. Culture medium, rhTGFß1 protein, or cells were injected into the cavernosum under magnification (10x to 16x) via a 26-gauge needle. Injection was made into the midportion of the left side of corpus cavernosum of each rat. The incision was closed with 6-0 vicryl sutures. The rats in groups 1 and 2 were killed at 20 days and those in the group 3 at 20, 40, and 60 days after electrical stimulation of cavernous nerve.
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Measurement of Erectile Function
The rats from each group were anesthetized with chloral hydrate (20 mg/kg) administered i.p. and were placed on a thermoregulated surgical table. Supplemental doses of chloral hydrate were administered as needed to maintain a uniform level of anesthesia. A carotid artery was cannulated (PE-50 tubing) for the measurement of systemic arterial pressure. The animal was placed in a supine position, and the bladder and prostate were exposed through a midline abdominal incision. With the aid of a Zeiss dissecting microscope, the major pelvic ganglion and cavernous nerve were identified posterolaterally to the prostate on one side, and bipolar platinum-wire electrodes were placed around the cavernosal nerve for electrical stimulation. The penis was denuded of skin, and a 26-gauge needle filled with 250 U/ml of heparin was inserted into one side of the corpus cavernosum for monitoring of the intracavernous pressure (ICP). Systemic arterial and intracavernosal blood pressures were measured with a Statham P23 pressure transducer connected to a computerized system for data acquisition (Biopac Systems, Goleta, CA), which was interfaced to a personal computer for recording and data analysis. Each rat underwent electrical field stimulation at a frequency of 12 Hz, a pulse width of 1 msec, and a duration of 1 min. The application of 5 V was used in the current protocol to achieve a significant and consistent erectile response. During tumescence, maximal ICP and latency to maximal erection were recorded.
Tissue Harvest
After the functional study was completed, a midportion of penile segment was harvested for histologic staining. The cavernous tissue specimens were immediately fixed in 10% formalin phosphate buffer solution before paraffin embedding. The specimens were stained with Masson trichrome or hematoxylin-eosin.
Immunohistochemical Staining for Mouse Major Histocompatibility Class I, 
-Smooth Muscle Actin, TGFß1, and Its Type II Receptor
Sections, prepared as described above, were deparaffinized and hydrated by sequential incubations in xylene and ethanol. After washing in 1x PBS for 2 min, the sections were blocked with 3% H2O2 for 10 min. The primary antibody against mouse major histocompatibility class I (Accurate Chemical, Westbury, NY), -smooth muscle actin (-SMA; DAKO, Trappes, France), TGFß1 (Sigma), or TGFß1 receptor type II (TGFßRII; Santa Cruz Biotechnology Inc., Santa Cruz, CA) was applied to the sections and incubated for 1 h. Control sections were incubated without the primary antibody at this step. The sections were washed and blocked with 5% milk in 1x PBS for 20 min before incubation with the horseradish peroxidase-conjugated secondary antibody. The chromogen reaction was performed with 0.05% diaminobenzidine in 1x PBS for 5 min. The sections were subsequently stained with hematoxylin and then mounted.
Smooth Muscle Quantification Using Automated Computer Morphometric Analysis System
For better identification of smooth muscle cells lining the cavernosal spaces, we used sections immunolabeled with the anti--SMA antibody, which is specific for a single -actin isoform. Quantitative analysis of smooth muscle and collagen fibers in cavernous tissue was done with an image analyzer system combined with a light microscope equipped with a video camera at a final magnification of 40x. The images were discriminated interactively, and the measurements were performed on the resulting binary images. We divided the total area of the corpus cavernosum into four fields, and then the relative area of smooth muscle cells was determined in each field as described previously [2]. We analyzed five slides containing two penile sections each per animal.
Reverse Transcriptase-Polymerase Chain Reaction Analysis
The corpus cavernosal tissues were carefully dissected free from the surrounding tunica albuginea at 3, 5, 7, 10, 15, 20, and 30 days after injection of NIH 3T3-TGFß1 or control NIH 3T3-neo cells, and mRNA was extracted from the cavernosal tissue each time. First-strand cDNA was synthesized using reverse transcriptase (RT) with oligo(dT) primers, and then the polymerase chain reaction (PCR) was performed with an LTR-specific primer (forward, 5'-TCG TCC GGG ATC GGG AGA-3') and TGFß1-specific primer (reverse, 5'-TCG CGG GRA CTG TTG TAA AGA GC-3') for 35 cycles. Each cycle consists of a denaturation step at 94°C for 1 min, an annealing step at 56°C for 2 min, and an extension step at 72°C for 3 min. ß-Actin was used as an internal control. The set of primers for ß-actin included the following: forward primer, 5'-TCT ACA ATG AGC TGC GTG TG-3'; reverse primer, 5'-AAT GTC ACG CAC GAT TTC CC-3'.
Statistical Analysis
Results are expressed as the mean ± SD. The means of more than three groups were compared by ANOVA. The statistical significance of differences between two groups was assessed by the Scheffe test. Probability values of less than 5% were considered to be significant. All statistical analyses were performed using the SPSS-Win 10.0 package (SPSS Inc., Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore the possibility of establishing a new cavernous fibrosis model using fibroblasts expressing TGFß1, NIH 3T3 murine fibroblast cells were infected with recombinant TGFß1 retroviruses in which the expression of active TGFß1 was driven by the MT promoter. Several stable cell lines (NIH 3T3-TGFß1) were generated by G418 selection. The average rates of secretion of TGFß1 as measured by ELISA from a stable cell line, which was used in the present experiment, was approximately 30 ng per 105 cells per 24 h (Fig. 1).
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Expression of Exogenous TGFß1 In Vivo after Injection
To investigate the expression of exogenous TGFß1 transcript after injection of the TGFß1-producing fibroblasts, RT-PCR was performed with mRNA extracted from the cavernosal tissues up to 30 days postinjection. To detect exogenous TGFß1 only, a set of primers was derived from the retroviral LTR sequence and the 5'-end of the TGFß1 gene of pMTMLVß1. We found that the expression of exogenous TGFß1 transcripts peaked at 5 days postinjection and continued to 10 days after injection (Fig. 2).
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Cavernous Histologic Change after Injection of NIH 3T3-TGFß1 Cells
To determine whether TGFß1-producing fibroblasts can induce cavernous fibrosis, three different cell numbers of NIH 3T3-TGFß1 cells (1 x 105, 1 x 106, or 3 x 106 in 0.1 ml of PBS) were injected into the cavernosa. Then, the degree of cavernous fibrosis was evaluated by Masson trichrome staining of specimens at 5, 10, 15 and 20 days after injection. Specimen injected with NIH 3T3-neo cells (3 x 106 in 0.1 ml) was used as the control. The results showed that the NIH 3T3-TGFß1 cells sufficiently induced cavernous fibrosis in a dose-dependent manner, whereas the NIH 3T3-neo cells did not show any significant changes in a histological examination (Fig. 3). At 5 days postinjection, multiple inflammatory nodules were found in the sinusoidal space, which consisted mainly of injected mouse fibroblasts as well as some neutrophils and myofibroblasts. At 10 or 15 days after injection, these inflammatory nodules started to produce collagen fibrils and then formed fibrous scars, which consisted mainly of lymphocytes, plasma cells, and fibroblasts as well as some myofibroblasts (Fig. 4). In the group injected with the high-dose NIH 3T3-TGFß1 cells (3 x 106 in 0.1 ml), these fibrous scars lasted up to 20 days postinjection and reduced gradually thereafter until 40 days (data not shown).
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Based on these preliminary results, we compared the effects of three different groups (rhTGFß1 protein, NIH 3T3-TGFß1 cells, and culture medium as the control) on cavernous fibrosis at 20 days postinjection. Examination of serial sections stained with Masson trichrome in the group receiving the NIH 3T3-TGFß1 cells revealed multiple fibrous scars on the corpus cavernosum, which not only were distributed evenly around the injection site but also were identified bilaterally. However, no histological changes were found in either the rhTGFß1 protein or the control group (Fig. 5). Immunohistochemical staining of the cavernosal tissues performed with the TGFß1 and TGFßRII antibody 20 days after postinjection of NIH 3T3-TGFß1 cells showed higher levels of TGFß1 and TGFßRII protein expression, especially in the area of fibrosis, compared to the rhTGFß1 protein or the medium control group (Fig. 6).
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Computer-assisted histomorphometric analysis was used to determine the mean trabecular smooth muscle content for each treatment and the control group. Interestingly, the content of trabecular smooth muscle was increased significantly in the NIH 3T3-TGFß1 group (15.2% ± 3.7%), especially around the fibrous scars, compared to the culture medium (8.9% ± 3.2%) and the rhTGFß1 protein group (8.8% ± 1.2%; P <0.001) (Fig. 7).
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Penile Erection after Injection of NIH 3T3-TGFß1 Cells
Electrical stimulation of the cavernous nerve in the group injected with the NIH 3T3-TGFß1 cells elicited a significantly lower maximal ICP and a more prolonged latency to maximal ICP at 20 days postinjection compared to those in the control group. However, a significant recovery of erectile function was noted at 40 and 60 days after injection of the NIH 3T3-TGFß1 cells. The group injected with the rhTGFß1 protein did not show any significant difference in erectile function compared to the control group (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RT-PCR analysis of the cavernosal tissues showed that the fibroblasts containing an exogenous TGFß1 gene were able to express the transgene for at least 10 days after injection in vivo. This finding suggests that active TGFß1 proteins secreted from the injected cells can be available for more than 10 days to induce cavernous fibrosis. This sustained expression of the active TGFß1 protein is believed to be a principal cause of the cavernous fibrosis shown in the present study. To explore whether the fibroblast itself may influence cavernous fibrosis, we also performed a cavernosal injection with NIH 3T3-neo cells that did not contain an exogenous TGFß1. The results showed that the NIH 3T3-neo cells alone did not induce cavernous fibrosis, suggesting that fibroblasts itself may not be a determining factor for cavernous fibrosis. In the present study, a single dose of rhTGFß1 protein did not induce cavernous fibrosis or subsequent deterioration of erectile function, which is similar to the results of another rat study (T.F. Lue, personal communication). However, other studies have demonstrated that injection of TGFß1 protein into the tunica albuginea of the rat induced long-term histological changes to the tunica albuginea similar to those found in human Peyronie disease [8, 9]. Lack of an effect in the present study can be explained by rapid turnover or clearance of TGFß1 protein because of the vascular nature of the corpus cavernosum. In a rabbit model to establish corporal fibrosis, an intracavernous injection of TGFß1-impregnated sodium alginate microspheres elicited corporal fibrosis and a significant decrease in the trabecular smooth muscle content for 3–5 days postinjection [5].
In the present study, the inflammatory response was noted up to 5–10 days after injection of the NIH 3T3-TGFß1 cells, and fibrotic processes, as evidenced by Masson trichrome stain, began to appear at 10 days postinjection. These fibrous scars lasted up to 20 days postinjection and decreased gradually thereafter. Furthermore, immunohistochemical staining for TGFß1 and TGFßRII at 20 days after the injection of NIH 3T3-TGFß1 cells showed increased immunoreactivity, especially in the fibrous scar, compared to the group injected with the culture medium or the rhTGFß1 protein. These results imply that all aspects of the fibrotic disease process have been shown to be regulated by TGFß1, including the initial inflammatory phase in which the infiltrating inflammatory cells and macrophages set the stage for the subsequent fibrotic phase in which the mononuclear cells and activated fibroblasts contribute to the pathogenic accumulation of the matrix [10]. The TGFß1 induces penile fibrosis via activation of TGFßRII.
We also performed computer-assisted histomorphometric analysis to determine the mean cavernosal smooth muscle content. Surprisingly, the content of the trabecular smooth muscle was significantly increased, especially around the fibrous scars, in the group injected with the NIH 3T3-TGFß1 cells compared to the other groups. This is quite an unexpected finding, because TGFß1 inhibits smooth muscle proliferation. However, it is unclear whether the increase of the smooth muscle content resulted from the compensatory mechanism during the long-lasting fibrotic process, from the influence of other cytokines involved, or from a problem in the cell-mediated approach to gene delivery. This is a major drawback of the present model, because naturally occurring cavernous fibrosis shows a decrease in the smooth muscle content. Additional studies dealing with the effect of a pure TGFß1 gene on the rat corpus cavernosum or the effect of the NIH 3T3-TGFß1 cells on other animals, such as the rabbit, may offer explanations for this.
Regarding the erectile function study in the rat at 20 days after injection of NIH 3T3-TGFß1 cells, the nerve-induced erection was much more compromised compared to the control group. However it gradually recovered to the normal level at 40 and 60 days after injection. This result was well correlated with histological findings, including cavernous fibrosis.
The TGFß1 has been known to cause fibrotic disease and a resultant functional impairment in a variety of organs, including liver cirrhosis, chronic hepatitis, glomeruolonephritis, and pulmonary fibrosis [11]. Additionally, TGFß1 signaling is responsible for cavernous fibrosis and vasculogenic ED in an animal model of atherosclerosis [12, 13].
The multifaceted effects of TGFß in promoting fibrosis have led to the suggestion that therapies aimed at reducing the expression or activity of TGFß might be efficacious in the treatment or prevention of fibrotic diseases. Several groups have shown a direct correlation between the inhibition of TGFß and the abrogation of fibrosis in the skin and lung. In three notable examples, bleomycin-induced lung fibrosis was prevented in mice by introduction of the inhibitory Smad7 gene via in vivo [14], and the administration of anti-TGFß antibodies prevented cutaneous hyperplasia in a model for chronic graft versus host disease [15]. In addition, disruption of the TGFß1 gene prevented skin fibrosis in tight skin mice [16].
In summary, NIH 3T3 fibroblasts containing a transgene, TGFß1, showed sustained TGFß1 expression and induced cavernous fibrosis that lasted for more than 20 days, whereas a single dose of rhTGFß1 did not result in cavernous fibrosis. We think that a cell-mediated approach to gene delivery using fibroblasts secreting TGFß1 is a useful, cost-effective method for establishing a long-lasting model of cavernous fibrosis. This model may contribute to further investigation of the pathogenesis of penile fibrosis associated with TGFß1 signaling and to the development of new therapeutics targeting this pathway in vasculogenic ED.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jun-Kyu Suh, Department of Urology, Inha University Hospital, 7-206 Third St., Shinheung-Dong, Jung-Gu, Incheon 400-103, South Korea. FAX: 82 32 890 2363; jksuh{at}inha.ac.kr
Received: 10 August 2004.
First decision: 20 September 2004.
Accepted: 16 November 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TGF-ß genes. J Invest Dermatol 2001 116:136-143[CrossRef][Medline]
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