Inhibition of Fibroblast Differentiation of Muscle-Derived Stem Cells in Cell Implantation Treatment of Stress Urinary Incontinence

Abstract

The aim of this study was to investigate the effects of transforming growth factor-β1 (TGF-β1) stimulation and the blocking of the TGF-β1/Smad3 signaling pathway by vector-mediated Smad3 shRNA on muscle-derived stem cells (MDSCs) in cell implantation treatment of stress urinary incontinence (SUI) of the rat. MDSCs were infected with the GC-shSmad3 lentivirus vector. Five days after infection, the cells were treated with TGF-β1. The expression levels of desmin (a marker of muscle differentiation) and vimentin (a marker of fibroblast differentiation) were tested by real-time PCR and Western blot. GC-shSmad3 lentivirus-infected MDSCs were injected into the bladder neck and proximal urethra of SUI rats. Urodynamic test was used to measure leak point pressure (LPP) at 2 weeks and 4 weeks after MDSC transplantation. Upregulated expression of vimentin and downregulated expression of desmin were found in MDSCs after culture with TGF-β1 in vitro. GC-shSmad3 lentivirus infection inhibited fibroblast differentiation of MDSCs but allowed muscle differentiation with desmin expression. In vivo experiments showed that GC-shSmad3 lentivirus infection could improve MDSC-mediated repairing of urethra sphincter function. In conclusion, blocking Smad3 expression inhibits the fibroblast differentiation of MDSCs induced by TGF-β1 in vitro and improves the repairing of urethral sphincter function by inhibiting the fibroblast differentiation of MDSCs in a rat model of SUI in vivo.

Introduction

Intrinsic sphincter deficiency (ISD), which is characterized by malfunction of the urethral closure is an important cause of stress urinary incontinence (SUI), especially in older women. There is still no long-term effective treatment for SUI attributed to ISD. Because patients with ISD have myogenic damage of the urethral sphincter, stem cell transplantation may be a promising way for repairing injured muscle and correcting the defects in structure and function of urethral sphincter muscle thoroughly.

Muscle derived-stem cell (MDSC), a highly undifferentiated multipotential cell, is characterized by rapid proliferation, slow fusing and multipotential differentiation. Its survival rate is significantly higher than that of satellite cells after transplantation, so it may be an ideal cell for SUI treatment. Previous study (Cannon et al., 2003) showed that MDSCs injected into bladder smooth muscle layer and urethra survived and formed myofibrils.

Kwon et al. (2006), however, revealed no significant differences among MDSCs, fibroblasts, and a combination of both regarding their potential for restoring urethral function after injection with equal cell dosage.

The reason why there is no difference between fibroblasts and MDSCs regarding their potential for restoring urethra function remains unknown.

Muscle recovery involves the competition between fibrosis and muscle regeneration. Early stage recovery of traumatic muscle, therefore, needs to be emphasized because longer fibrosis leads to less possibility of muscle recovery (Li et al., 2007; Nozaki et al., 2008; Sato et al., 2003). In our previous study we found that MDSCs could differentiate into fibrotic cells upon stimulation in the case of muscle injury (Yan-zhou, et al., 2009). When the MDSCs were injected into the bladder neck and proximal urethra, the needle might cause the injury of urethral sphincter muscle, so we assumed that the treatment effect of MDSCs may be hindered by fibroblast transdifferentiation of MDSCs.

Muscle injury is associated with such factors as fibroblast growth factor, insulin-like growth factor, and transforming growth factor-β (TGF-β) (Ono et al., 2009; Zhao et al., 2002). TGF-β plays a key role in tissue repair and fibrosis, partly due to its capacity to induce myofibroblast differentiation. Smad3 is central in TGF-β1 signaling pathway during myogenesis (Liu et al., 2001, 2004) and mediates TGF-β1 signaling-related lung fibrosis and bronchial smooth muscle fibrosis (Ono et al., 2009). An in vivo study showed that loss of Smad3 greatly attenuated the morphologic evidence of fibrosis in bleomycin-treated mice, thus implicating Smad3 in the pathogenesis of fibrosis (Zhao et al., 2002). However, the Smad3 pathway and its possible role in mediating TGF-β1-induced fibroblast differentiation of MDSCs have not been determined. We characterized the MDSCs isolated from rat gastrocnemius muscle and examined whether blocking TGF-β1/Smad3 signaling pathway would inhibit the fibroblast differentiation of MDSCs in the SUI rat.

Materials and Methods

The study was carried out from September 2006 to March 2009 at the Department of Obstetrics and Gynecology, First Affiliated Hospital, Third Military Medical University, Chongqing, China. The experimental protocol was approved by the Ethical Committee for Animal Studies of the Third Military Medical University.

Experimental Animals

The experiments were performed on female Sprague-Dawley (SD) rats (3–4 weeks old) purchased from the Experimental Animal Center, Research Institute of the Third Military Medical University.

Isolation, culture, and purification of MDSCs

The hind limbs (gastrocnemius muscles) were resected from the SD rats, and the skin and bones were removed. The muscle mass was minced and chopped with razor blades, and the cells were dissociated using two enzymes (collagenase XI and dispase) for 1 h at 37°C. The muscle cell extract was preplated in culture flasks as described by Qu-Petersen et al. (2002). The phenotypic characteristics of MDSCs were detected by immunostaining of Sca-1, CD34, and desmin.

Immunocytochemical staining for MDSCs

Cells were plated in a six-well culture dish and fixed with cold methanol (−20°C) for 1 min. After rinsing with phosphate-buffered saline (PBS), the cells were blocked with 5% horse serum at room temperature for 30 min. The primary antibodies against desmin (1:200; Sigma, St. Louis, MO), goat Sca-1 (1:200; R&D Systems, Minneapolis, MN), and vimentin (1:200; Sigma) were applied overnight at room temperature. Fluorescent group-labeled secondary antibodies (FITC-labeled secondary antibodies or TRITC-labeled secondary antibodies) were applied at 37°C for 60 min. FITC-labeled anti-CD34 antibody was used to stain the MDSCs, and the nuclei were stained with DAPI (Beyotime, China). The cells were then rinsed with PBS and examined by fluoroscopy.

Generation of lentiviral vector for silencing rat Smad3 expression

From the Smad3 cDNA coding sequence, four pairs of cDNA oligonucleotides targeting rat Smad3 mRNA at different locations were chosen (Table 1). These primers were annealed and inserted into pGCSIL-GFP. One negative control shRNA containing a scrambled sequence (TTCTCCGAACGTGTCACGT) was also constructed. Different shRNAs (Genechem, Inc., Shanghai, China) were screened by cotransfection with a rat Smad3 cDNA plasmid into 293T cells with Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA). To examine the efficiency of these shRNAs in silencing Smad3 expression, Smad3 expression levels were measured using real-time PCR and Western blot.

Table 1.

Selected Sequences of Rat Smad3 siRNAs

Marker	Gene	Species	Target Seq	GC%
Target-1	Smad3	Rat	GGATGAAGTGTGTGTAAAT	36.84%
Target-2	Smad3	Rat	CACAGATTCTCTCAACTAA	36.84%
Target-3	Smad3	Rat	GCTGTGTCTGCTGCATATA	47.36%
Target-4	Smad3	Rat	CCACATTGAATGAATACAT	31.57%

The optimal sequence of shRNA (target-1 GGATGAAGTGTGTGTAAAT) against rat Smad3 was then cloned into plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP cassette flanked by loxP sites. The resulting lentiviral vector containing rat Smad3 shRNA was called GC-shSmad3, and its sequence was confirmed by PCR and sequencing (Zhang et al., 2008). Lentiviruses were packaged with Lipofectamine 2000 (Invitrogen) in the presence of lentiviral vectors using 293 cells. The resultant viral supernatant was harvested and concentrated to about 3×10⁸ TU/mL. Lentivirus preparations were produced by Shanghai GeneChem Co. Ltd., China. MDSCs were infected at a multiplicity of infection (MOI) of approximately 100. Five days after infection, MDSCs were serum-starved for 24 h and then treated with TGF-β1.

RNA isolation and real-time PCR

Total RNA from MDSCs was extracted using Trizol reagent, and first-strand cDNA was generated using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI). Real-time PCR was performed using the primers of Smad3 (5′-ACACAATAACTTGGACCTACAG-3′; 5′-GTGAAGCGTGGAATGTCTC-3′), vimentin (5′-TCCCTGAACCTGAGAGAAAC-3′; 5′-ATCGTGGTGCTG-AGAAGTC-3′), and desmin (5′-CCTACACCTGCGAGATTGATG-3′; 5′-GCGATGTTGTCCTGATAGCC-3′). Amplifications were performed in 45 cycles using an Opticon continuous fluorescence detection system (MJ Research, Incline Village, NV) with SYBR green. Each cycle consisted of 5 s at 95°C, 30 s at 60°C, 30 s at 72°C. All samples were quantified using the comparative CT method for relative quantification of gene expression, normalized to GAPDH.

Western blot

Two hundred thousand MDSCs or MDSCs infected with GC-shSmad3 were seeded into 25-cm² flasks in Dulbecco's modified Eagle's medium containing 10 ng/mL of TGF-β1 for 0, 3, 6, 12, and 24 h. Then the cells were lysed in buffer and the lysates were separated by 10% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). After incubation with mouse antivimentin monoclonal antibody (1:1000 dilution; Sigma), mouse antidesmin monoclonal antibody (1:1000 dilution; Sigma) or rabbit anti-Smad3 antibody (1:200 dilution; Cell Signaling, Beverly, MA), the membrane was washed with PBS and incubated with goat antimouse IgG conjugated with horseradish peroxidase (HRP; Zhongshan Biotech, China). Immunoreactive complexes were visualized by ECL and exposed to an X-ray film. The density of vimentin and desmin protein bands was assayed by gel documentation system and Quantity One software.

Establishing rat model of SUI

SD rats were given pentobarbital sodium anesthesia. Surgery was performed through a dorsal incision in the skin and an incision in the muscle over the ischiorectal fossa. The pudendal nerve was exposed on each side and a 2-cm segment was removed just distal to the origin of the pudendal nerve from the sciatic nerve (Lee et al., 2004).

MDSCs injection

Female SD rats (250–300 g) were divided into four groups (n=10 in each group): control SD rats (CON), denervated SD rats (SUI), denervated SD rats + MDSCs infected with a lentiviral vector as negative control (MOCK), and denervated SD rats + MDSCs infected with GC-shSmad3 (KD). The rats were given pentobarbital sodium anesthesia, and a low midline incision was made to expose the bladder and urethra. Fifty microliters of cell suspension were injected with a microsyringe at about 1×10⁶ MSDCs per rat.

Urodynamic test

At 2 and 4 weeks after injection, urodynamic tests were performed. Five rats were taken from each group. The rats were mounted on a table and placed in the dorsal position. A transvesical epidural catheter was inserted into the dome of the bladder, and the other end of the epidural catheter was connected with a urodynamic detection and microinfusion pump through a three-way stopcock. The bladder was emptied, and then saline solution was injected at a rate of 0.3 mL/min. The pressure at the first drop leaked out from the urethral orifice was taken as leak point pressure (LPP).

Statistical analysis

Values are shown as means±SD. One-way ANOVA was used to compare the means from two or more experimental groups, followed by t-tests. Statistical differences between groups were considered to be significant at p<0.05.

Results

Isolation and identification of MDSCs

We isolated MDSCs, desmin(+), CD34(+), Sca-1(+), and CD45(−) as shown in immunofluorescence staining (Fig. 1), from rat gastrocnemius muscles.

FIG. 1.

Immunostaining of muscle-derived stem cells. The muscle-derived stem cells are Desmin positive CD34 positive, Sca-1 positive, and CD45 negative. The nucleus is blue (DAPI staining). Arrows: positive stain (A) Desmin. (B) CD34(+). (C) Sca-1 (+). (D) CD45(−). (Original magnification;×100.)

Suppressing rat Smad3 expression by GC-shSmad3

ShRNA plasmids showed variable efficacy, and the most effective shRNA was shRNA1 (Fig. 2). The optimal sequence of shRNA against rat Smad3 (5′-GGATGAAGTGTGTGTAAAT-3′) was then cloned into plasmid pGCL-GFP to obtain lentivirus vector GC-shSmad3. MDSCs were infected with GC-shSmad3. The transfection efficiency was more than 80% as shown by fluorescent microscopy analysis. The expression of Smad3 was monitored after the MDSCs were cultured with TGF-β1 (10 ng/mL) for 24 h. The effective silencing of endogenous rat Smad3 by GC-shSmad3 was confirmed by Western blot. The Smad3 silencing rate was 80% as proven by real-time PCR (Fig. 3).

FIG. 2.

Screen optimal sequence of siRNA against rat Smad3 (C, control; PC, rat Smad3 cDNA plasmid + pcDNA3.1; NC, rat Smad3 cDNA plasmid + negative control (shRNA;1#,2#,3#,4#): rat Smad3 cDNA plasmid + shRNA target-1, −2, −3, −4.

FIG. 3.

Smad3-specific lentivirus-mediated RNA interference inhibited TGF-β-induced Smad3 expression. (A) MDSCs were infected with GC-shSmad3 lentiviral vector (KD)or negative control lentiviral vector(MOCK) at an MOI of 100 for 5 days, and then subjected to fluorescence assays. (B) MDSCs were infected with GC-shSmad3 for 5 days, then stimulated with TGF-β1 (10 ng/mL) for 24 h. The expression of Smad3 were monitored by real-time PCR. Smad3 silencing rate was 80%. (C) The effective silencing of endogenous rat Smad3 by GC-shSmad3 was confirmed by Western blot analysis.

TGF-β1/Smad3 signaling in the fibroblast differentiation of MDSCs

Stimulating MDSCs with TGF-β1 induced the expression of fibroblastic marker vimentin in a time-dependent manner, but suppressed the expression of muscle differentiation marker desmin (Fig. 4). Inhibition of Smad3 with vector-mediated Smad3 shRNA significantly suppressed TGF-β1-induced fibroblast differentiation (Fig. 5).

FIG. 4.

TGF-β1 stimulated fibroblast differentiation of muscle-derived stem cell. Western blot indicated an induction of vimentin expression in MDSCs cells stimulated with TGF-β in a time-dependent manner. Lane 1 represents nonstimulated MDSCs cells. Lanes 2 to 5 represent MDSCs cells stimulated with TGF-β at 3, 6, 12, and 24 h, respectively.

FIG. 5.

Smad3-specific lentivirus-mediated RNA interference inhibited TGF-β1-induced Vimentin expression and TGF-β1-induced Desmin downregulation. (A) MDSCs were infected with GC-shSmad3 lentiviral vector (KD) or negative control lentiviral vector (MOCK) at an MOI of 100 for 5 days, and then stimulated with TGF-β for 0, 3, 6, 12, and 24 h, respectively, and then subjected to Western blot assays using antibody to Vimentin and antibody to Desmin. (B) mRNA levels of Vimentin and Desmin were assessed using Q-RT-PCR. : vs. MOCK p<0.05.

Urodynamic test after cell transplantation

Urodynamic test for SUI rat model showed that LPP decreased significantly. The phenomenon of SUI became weakened or disappeared after cell injection. LPP in the MOCK group was significantly lower at 4 weeks than at 2 weeks. There was no such difference in the CON and KD groups. LPP in the KD group was significantly higher than that in the MOCK group at 4 weeks after cell injection (Fig. 6).

FIG. 6.

Leak point pressure at 2 and 4 weeks. Leak point pressure in the SUI group were significantly lower at 2 and 4 weeks than Leak point pressure in the CON, MOCK, and KD groups. No such differences were found between the CON, MOCK, and KD groups at 2 weeks. Leak point pressure in the CON group were significantly higher than in the MOCK group at 4 weeks, Leak point pressure in the MOCK groups were significantly lower at 4 week than at 2 weeks. There were no such differences between 2 and 4 weeks in the CON and KD groups. Leak point pressure in the KD group at 4 weeks were significantly higher than MOCK in the group at 4 week. Compared with CON group (p<0.05); ^§significant difference between KD and MOCK groups (p<0.05); ^#significant difference between 2 and 4 weeks (p<0.05).

Discussion

Taken together, we isolated and purified MDSCs by preplate technique. We constructed GC-shSmad3 lentiviral vector for silencing rat Smad3 expression. The upregulated expression of vimentin and downregulated expression of desmin were found in MDSCs after culture with TGF-β1 in vitro, but TGF-β1 was unable to induce fibroblast differentiation of MDSCs in Smad3-deficient MDSCs, which exhibited accelerated myogenesis. In vivo experiments showed that GC-shSmad3 lentivirus infection could improve the MDSC-mediated repairing of urethra sphincter function. Thus, we proved that GC-shSmad3 lentivirus vector inhibited the fibroblast differentiation of MDSCs induced by TGF-β1 in vitro and improved the repairing of urethral sphincter function by inhibiting the fibroblast differentiation of MDSCs in a rat model of SUI in vivo.

Myoblasts are a promising source for cell transplantation because they can easily be harvested from skeletal muscle and proliferated in culture. Previous study has demonstrated a group of skeletal MDSCs, characterized by a variety of stem cell markers (e.g., Sca-1, CD-34, and Bcl2) (Ward and Hilton, 2004). MDSCs are able to circumvent the limitations of myoblasts and could be a superior alternative to myoblasts for the regeneration and repair of skeletal, cardiac, and smooth muscles. Several methods have been used to isolate and purify skeletal MDSCs. The most popular method was established by Qu-Petersen et al. (2002) and Gharaibeh et al. (2008). They used three-step enzyme digestion (collagenase XI, dispase, and trypsin) and the preplate technique to enrich the desired cells. We explored a more convenient way of isolating and purifying skeletal MDSCs by two enzyme (collagenase XI and dispase) digestion and the preplate technique. The MDSCs were then identified with immunocytochemistry.

TGF-β1 has been considered a key factor in the development of fibrosis in various tissues (Choi et al., 2007; Dooley et al., 2000; Kato et al., 2007). It has been suggested that TGF-β1 is capable of inducing the expression of fibroblastic markers in MC13 cells (Li and Huard, 2002). This phenomenon was also demonstrated in our experiment. In addition, we observed that the expression of desmin was dramatically reduced upon TGF-β1 stimulation. These data suggest that TGF-β1 is probably involved in the differentiation of MDSCs toward fibroblasts, which is accompanied with inhibition of myogenesis.

Smad3 is central in TGF-β1 signaling pathway during fibrosis and myogenesis (Liu et al., 2001, 2004). Therefore, we hypothesize that fibroblast differentiation of MDSCs is also mediated by Smad3. We compared the expression levels of vimentin and desmin in the MDSCs infected with GC-shSmad3 lentivirus vector with those in the MDSCs infected with a lentiviral vector as negative control. The results demonstrated that GC-shSmad3 lentivirus vector could attenuate the fibrosis, which was concomitant with increased expression of differentiation-specific myogenic genes such as desmin. The repairing of urethral sphincter function by preventing muscle fibrosis was also observed by transplantation of MDSCs infected with GC-shSmad3 lentivirus vector to a rat model of SUI. But there are still some limitations that need to be acknowledged and addressed regarding the present study: the long-term effect of MDSCs injected into denervated female rat urethral sphincters should be investigated in further empirical evaluation.

Although we cannot exclude other growth factors in the fibroblast transdifferentiation of MDSCs, it seems possible that TGF-β1, a key factor involved in the fibrosis of various tissues, also plays a role in the fibroblast differentiation of MDSCs and contributes to the development of fibrosis. Furthermore, inhibiting Smad3 expression can suppress fibroblast differentiation of MDSCs and improve the repairing of urethral sphincter function in a rat model of SUI in vivo. It may provide a novel strategy for the treatment of SUI and improve the effect of MDSC implantation treatment on female SUI patients.

Footnotes

Acknowledgments

This work was supported by the Natural Science Foundation of China (30500539) to Huicheng Xu.

Author Disclosure Statement

The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

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