Mesenchymal Stem Cells Overexpressing Hepatocyte Growth Factor (HGF) Inhibit Collagen Deposit and Improve Bladder Function in Rat Model of Bladder Outlet Obstruction

Abstract

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly male. This study was performed to examine the capability of human mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF) to inhibit collagen deposition in rat model of bladder outlet obstruction (BOO). HGF is known for its antifibrotic effect and the most promising agent for treating bladder fibrosis. BM3.B10 stable immortalized human MSC line (B10) was transduced to encode human HGF with a retroviral vector was prepared (B10.HGF). Two weeks after the onset of BOO, B10, and B10.HGF cells were injected into the rat's bladder wall. After 4 weeks, bladder tissues were harvested and Masson's trichrome staining was performed. Transgene expression in HGF-expressing B10 cells was demonstrated by reverse transcriptase polymerase chain reaction and immunohistochemical staining, and the high levels of HGF secreted by B10. HGF cells was confirmed by ELISA. The mean bladder weight in BOO rats was 5.8 times of the normal controls, while in animals grafted with B10.HGF cells, the weight was down to four times of the control [90.2 ± 1.6 (control), 89.9 ± 2.8 (sham), 527.9 ± 150.9 (BOO), 447.7 ± 41.0 (BOO + B10), and 362.7 ± 113.2 (BOO + B10. HGF)]. The mean percentage of collagen area increased in BOO rats, while in the animals transplanted with B10.HGF cells, the collagen area decreased to the normal control level [12.2 ± 1.3, (control), 12.8 ± 1.1 (sham), 26.6 ± 2.7 (BOO), 19.9 ± 6.0 (BOO + B10), and 13.3 ± 2.1 (BOO + B10.HGF)]. The expression of collagen and TGF-β protein increased after BOO, while the expression of HGF and c-met protein increased in the group with B10.HGF transplantation after BOO. Intercontraction interval decreased after BOO, but it recovered after B10.HGF transplantation. Maximal voiding pressure (MVP) increased after BOO, and it recovered to levels of the normal control after transplantation of B10.HGF cells. Residual urine volume (RU) increased after BOO, but the RU increase was not reversed by transplantation of B10.HGF cells. Human MSCs overexpressing HGF inhibited collagen deposition and improved cystometric parameters in bladder outlet obstruction of rats. The present study indicates that transplantation of MSCs modified to overexpress HGF could serve as a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.

Keywords

Bladder outlet obstruction Bladder fibrosis Collagen deposit Human mesenchymal stem cells Cell transplantation Hepatocyte growth factor

Introduction

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly male. The collagen deposition in bladder occurs frequently during development of various pathological processes and eventually cumulates in bladder fibrosis. This bladder fibrosis adversely affects the smooth muscle function and the capacity of bladder (3). Although numerous treatments have previously been developed, the improvement in voiding dysfunction is not achieved. Reducing the collagen deposit could improve voiding dysfunction in the bladder outlet obstruction caused by fibrosis.

Mesenchymal stem cells (MSCs) are self-renewing cells with pluripotent capacity to differentiate into various cell types including osteoblasts, chondrocytes, myocytes, adipocytes, and neurons (7,18,23,24,26). Previous studies have reported that reduction in collagen deposition in fibrosis in liver, lung, and heart following transplantation of bone marrow-derived MSCs (1,4,20,21). Recently, we have reported that transplantation of primary human MSCs labeled with nanoparticles containing superparamagnetic iron oxide into the bladder wall of a rat BOO model inhibited bladder fibrosis and induced improvement of bladder dysfunction (11). MSCs could secrete growth factors such as hepatic growth factor (HGF) and contribute to reducing fibrosis through paracrine mechanisms rather than by cell incorporation (1,10,16). HGF, initially identified as a potent mitogen for hepatocytes, is secreted by mesenchymal stem cells, plays an essential part in the angiogenesis and regeneration of the tissue, and acts as a potent antifibrotic agent (19,25). Thus, HGF could be a promising agent for treating bladder fibrosis.

Considering evidence of reduction in fibrosis in liver, lung, and heart in animals following transplantation of MSCs (4,11,20,21), we wished to investigate whether the human MSCs overexpressing HGF, by pairing clonal human MSCs with HGF gene, can lead to the reduction of collagen deposit and functional improvement in rat BOO model.

Materials and Methods

Stable Human MSC Line

An amphotropic replication-incompetent retroviral vector encoding v-myc oncogene [transcribed from mouse leukemia virus long terminal repeat (LTR) plus neomycin-resistant gene transcribed from an SV40 early promotor] was used to infect human fetal bone marrow MSCs inducing propagation of stable immortalized human bone marrow MSC lines. Individual clones were designated as HM3 human MSC lines, and one of these clones, B10, was subjected to further study (18).

Transfection of Human HGF Gene Into MSCs

PT67 mouse packaging cell line was transfected with pLPCX-HGF vector (Fig. 1A) using LipofectAMINE (Invitrogen, Carlsbad, CA), and stable PT67 cell line was selected using 3 μg/ml puromycin for 3 days. Replication incompetent retroviral vector collected from PT67.HGF cells were collected and used for transfection of B10 MSCs. Puromycin-resistant clones were isolated, screened, expanded, and used for the transplantation. Expression of HGF in B10.HGF cell line was analyzed by RT-PCR, ELISA, and immunohistochemistry.

Figure 1.

Hepatocyte growth factor (HGF) transfection of B10 human mesenchymal stem cell (MSC) line. (A) HGF was transfected with recombinant retrovirus from the pLPCX.HGF. (B) Transgene expression of HGF in B10 and B10.HGF cells as demonstrated by RT-PCR. Note the higher expression of HGF message in B10.HGF cells. (C, D) Immunocytochemical staining of HGF in B10 and B10.HGF cells. Scale bar: 100 μm. (E) Levels of HGF expression in B10 and B10.HGF cells by ELISA (*B10 vs. B10.HGF, p < 0.05).

Transplantation of MSCs Into Rat Bladder

All procedures were approved by the Institutional Animal Care and Use Committee of Soonchunhang University Hospital. Fifty 6-week-old female Sprague–Dawley rats weighing 200 g were used in this study (group 1: control, n = 10; group 2: sham operation, n = 10; group 3: bladder outlet obstruction BOO, n = 10; group 4: B10 transplantation 2 weeks after BOO, n = 10; and group 5: B10.HGF transplantation 2 weeks after BOO, n = 10). Rats were anesthetized intraperitoneally with 1% ketamine (30 mg/kg) and xylazine hydrochloride (4 mg/kg), and BOO operation was conducted. The lower abdominal skin incision was made, and urethra was dissected and 4–0 silk sutures were placed around the urethra, including a length of metal rod with an outer diameter of 1 mm, which was positioned extraluminally. After the suture was tied, the rod was removed. The abdominal wall was then closed after the removal of the rod. At 2 weeks after the onset of BOO, approximately 1.0 × 10⁶ B10 cells were injected into the bladder wall in group 4, approximately 1.0 × 10⁶ B10.HGF cells were injected into the bladder wall in group 5 using a 500-μl syringe with a 26-G needle. Flomoxef (Cephalosporin, Ildong, Seoul, South Korea) at 10 mg/kg was injected daily to prevent infection.

Improvement in Voiding Function After MSC Transplantation

Voiding response was assessed at 4 weeks after transplantation. Female Sprague–Dawley rats (n = 10, each group) were anesthetized with isoflurane (2% oxygen) for surgical insertion of bladder catheter. The bladder was exposed via a midline abdominal incision. A catheter (PE-50), the bladder end of which was heated to create a collar, was inserted through a small incision in the bladder dome, and a suture was tightened around the collar. The other end of the catheter was passed through subcutaneous tissue and exited through the skin. After closing, the abdominal incision by suturing the muscle and skin, rats that were to be studied without anesthesia were placed in a restraining cage for 5–6 h (including 2 h of recovery from isoflurane anesthesia) that was large enough to permit them to adopt a normal crouching posture but narrow enough to prevent them from turning around. The rats were subsequently allowed to recover from isoflurane anesthesia. The bladder catheter was connected via a T-stopcock to a pump for continuous infusion of physiological saline and to a pressure transducer. Physiological saline was infused at room temperature into the bladder at a constant rate of 0.04 ml/min to elicit repeated voiding responses. The intercontraction interval (ICI; the interval between voids or reflex bladder contractions), maximal voiding pressure (MVP), pressure threshold (PT), and residual urine volume (RU) were measured.

Histology and Immunohistochemistry

At the end of the cystometry, half of the animals (n = 5, each group) were sacrificed and the bladder bodies were extracted. After any adjacent tissue was removed, the extracted bladders were weighed. The bladders were then immediately frozen and stored in liquid nitrogen until use in further biochemical and molecular biological analyses. At the end of the cystometry, half of the animals (n = 5, each group) were perfused through the heart with 100 ml cold saline and 100 ml of 4% paraformaldehyde in PBS. After 24 h of fixation in 4% paraformaldehyde, the bladder was cryoprotected in 30% sucrose for 24 h, cut into 20-μm sections in a cryostat (Leica CM 1900), and stained with H&E and Masson's trichrome. Each slide was inspected microscopically, and 10 randomly chosen representative areas from light microscope images were captured. Captured video images were then displayed on a color monitor and simultaneously digitized and analyzed using an IBM computer. To evaluate the results of Masson's trichrome staining, representative portions of each slide were calculated in blinded fashion with a square micrometer and the mean area was expressed as the relative percent. The mean percent collagen area was defined according to the formula: (collagen)/(collagen + muscle). This technique was predicated on the area calculation of the smooth muscle, which stained red, and connective tissue, which stained blue. Quantitative image analysis was done using OPTIMAS (Media-Cybernetic, Bethesda, MD).

For immunofluorescence examination, adjacent serial sections were processed for fluorescent staining with human nuclear matrix antigen (hNuMA, 1:100, mouse monoclonal, Abcam, Cambridge, MA) to identify the transplanted human MSCs.

Antibodies specific for smooth muscle α-actin (SMA, 1:1,000, rabbit, Sigma, St. Louis, MO) and desmin (1:1,000, rabbit, Chemicon, Temecula, CA). Bladder sections were incubated in mixed solution of primary antibodies overnight at 4°C as free floating sections, followed by mixed secondary antibodies of Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000, Molecular Probe, Eugene, OR) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:1,000, Molecular Probe) for 1 h at room temperature (RT). Negative control sections from each animal were prepared in an identical manner, except the primary antibodies were omitted. Stained sections were then examined under an Olympus laser confocal fluorescence microscope.

Western Blot Analysis

Bladder tissues were homogenized in radio-immunoprecipitation assay lysis buffer on ice. After 20 min of 10,000 rpm centrifugation at 4°C, the supernatants were collected. Tissue homogenates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis with 15% polyacrylamide for transforming growth factor (TGF)-β and 8% for all other proteins, followed by transfer to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% nonfat dry milk and PBS containing 0.05% Tween-20 and then incubated overnight with primary antibody at 4°C. The primary antibodies used were anti-rat TGF-β (1:500, rabbit, SC-146, Santa Cruz Biotech, Santa Cruz, CA), anti-rat HGF (1:500, goat, SC-1357, Santa Cruz Biotech), and anti-rat c-met (1:250, rabbit, SC-162, Santa Cruz Biotech). The blots were washed in PBS and incubated for 1 h in HRP-conjugated secondary anti-rabbit or anti-goat antibodies at RT, after which detection was done by enhanced chemiluminescence (Amersham Bioscience, Piscatawy, NJ). Quantitative analysis of several animals was provided by scanning of the blots and calculating the relative intensities of collagen I, HGF, c-met, and TGF-in relation to the corresponding actin signal.

Statistics

Two-way ANOVA and the post hoc Tukey test were used for analysis of stem cell transplantation. Data are presented as mean ± SE. A value of p < 0.05 was considered statistically significant.

Results

Stable Human MSC Line Overexpressing Human HGF

B10 human MSC line was infected with a retroviral vector encoding human HGF gene; clones resistant to puromycin were selected and then cloned again by limited dilution. The morphology of B10.HGF human MSCs does not differ from the parental B10 MSCs with bipolar or multipolar morphology (Fig. 1). Transgene expression in HGF expressing B10 was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining. B10.HGF cells showed higher density of the RT-PCR band and immunohistochemical staining for HGF than B10.

Levels of HGF in the supernatant of cultured B10 and B10.HGF cells were determined with ELISA kit specific for human HGF. The amount of HGF in the medium of B10.HGF was 2,389 ± 270 pg/10⁶ cells/day, which was 10 times higher than that from B10 (276 ± 10 pg/10⁶ cells/day, p < 0.05) (Fig. 1).

Transplanted MSCs Differentiated Into Smooth Muscle

The presence of B10 cells was confirmed 4 weeks after transplantation into the bladder with anti-human nuclear antigen antibody staining. In bladder sections transplanted with B10 cells, B10 cells expressing immunoreaction against human HGF, smooth muscle actin, or desmin were found indicating that the B10 cells secreted HGF and differentiated into smooth muscle cells (Fig. 2).

Figure 2.

The presence of B10.HGF human MSCs (human-specific anti-nuclear antigen-positive; hNuMA) was confirmed 4 weeks after transplantation into the bladder of animals with bladder outlet obstruction (BOO). In bladder sections transplanted with B10 and B10.HGF cells (A, B, C). Positive immunoreaction against desmin (D, E, F) and smooth muscle α-actin (SMA) (G, H, I) in B10.HGF cells is indicative of differentiation of B10.HGF cells into smooth muscle like cells. Scale bar: 100 μm.

Weight of Whole Body and Bladder

The mean body weight of animals was not significantly different in groups 1–5.

The mean body weights (g) of animals were 240 ± 1 (control), 229 ± 1 (sham), 224 ± 2 (BOO), 245 ± 4 (BOO + B10), 240 ± 5 (BOO + B10.HGF) in groups 1–5, respectively.

The group with BOO showed increased bladder weight than the groups of control and sham-operated animals (p < 0.05). The BOO + B10 (group 4) showed decreased bladder weight as compared with the BOO group without MSC transplantation (group 3). The BOO + B10.HGF (group 5) showed decreased bladder weight as compared with the BOO (group 3) or BOO+ B10 (group 4) (Fig. 3). The mean bladder weights (mg) were 90.2 ± 1.6 (control), 89.9 ± 2.8 (sham), 527.9 ± 150.9 (BOO), 447.7 ± 41.0 (BOO + B10), and 362.7 ± 113.2 (BOO + B10.HGF) in groups 1–5, respectively.

Figure 3.

Changes in weight of whole body and bladder after transplantation of B10 and B10.HGF human MSCs after bladder outlet obstruction (BOO). There was no significant difference of body weight between groups. The BOO animals showed increased bladder weight over the normal controls and sham operation group (p < 0.05) but returned to the control level following transplantation of B10 or B10.HGF cells. The animals transplanted with B10.HGF cells showed lower bladder weight than those transplanted with nonmodified B10 cells (*BOO vs. BOO + B10, p < 0.05; **BOO + B10 cells vs. BOO + B10.HGF, p < 0.05).

Percent of Collagen Area

The group with BOO (group 3) showed increased ratio of collagen/collagen + muscle (%) than the group with sham operation (group 2) and controls (group 1) (p < 0.05). The BOO + B10 transplantation group (group 4) showed decreased ratio than the group with BOO group (group 3). The BOO + B10.HGF transplantation group (group 5) showed decreased ratio than the group with the BOO+ B10 (group 4). The ratio of collagen/collagen + muscle (%) was 12.2 ± 1.3 (control), 12.8 ± 1.1 (sham), 26.6 ± 2.7 (BOO), 19.9 ± 6.0 (BOO + B10), and 13.3 ± 2.1 (BOO + B10.HGF) in groups 1–5, respectively. The collagen deposition increased in the group with BOO and decreased after transplantation of MSCs as shown by Masson's trichrome staining (Figs. 4 and 5).

Western Blot Analysis of HGF, TGF-β and c-met

The group with BOO showed increased expression of collagen and TGF-β than the sham-operated group. The group with B10 transplantation after BOO showed decreased amount of collagen and TGF-β than the BOO group without cell transplantation. The BOO + B10. HGF transplantation group (group 5) showed decreased amount of collagen and TGF-β than the group with the BOO + B10 (group 4). The group with BOO + B10 transplantation (group 5) showed increased levels of HGF and c-met than the groups with sham (group 2) or BOO alone (group 3). The BOO + B10.HGF transplantation group (group 5) showed increased levels of HGF and c-met than the group with the BOO + B10 (group 4) (Fig. 6).

Quantitative analysis of collagen, HGF, c-met, and TGF-β in membrane fraction from the bladders of sham, BOO, BOO + B10, and BOO + B10.HGF indicated that levels of HGF and c-met were significantly increased and those of collagen and TGF-β were significantly decreased in BOO + B10.HGF animals (p < 0.05: BOO vs. BOO + B10.HGF) (Fig. 6).

Improvement in Voiding Function After MSC Transplantation

Intercontraction interval (ICI) in BOO animals decreased and recovered after transplantation of B10 or B10.HGF cells (p <0.05). ICI (second) was 342 ± 15 in controls, 361 ± 35 in sham, 280 ± 25 in BOO, 338 ± 55 in BOO + B10, and 368 ± 35 in BOO + B10.HGF group, respectively (Fig. 7). Maximal voiding pressure (MVP) increased after BOO and recovered after transplantation of B10.HGF cells (p <0.05). MVP (cm H₂O) was 48.2 ± 6.2 (control), 44.5 ± 1.4 (sham), 64.8 ± 11.9 (BOO), 61.1 ± 6.6 (BOO + B10), and 45.5 ± 9.5 (BOO + B10.HGF) in groups 1–5, respectively (Fig. 7). Pressure threshold (PT) had no change among groups. PT (cm H₂O) was 8.5 ± 0.7 (control), 8.6 ± 0.5 (sham), 10.1 ± 1.3 (BOO), 10.5 ± 1.0 (BOO + B10), and 8.3 ± 0.8 (BOO + B10.HGF) in groups 1–5, respectively (Fig. 7). Residual urine volume (RU) increased after BOO and was not reversed after transplantation of B10. HGF cells. RU (ml) was 0.04 ± 0.03 (control), 0.08 ± 0.02 (sham), 0.39 ± 0.19 (BOO), 0.35 ± 0.11(BOO + B10), and 0.32 ± 0.14 (BOO + B10.HGF) in groups 1–5, respectively (Fig. 7).

Figure 4.

Histological change of collagen deposition after MSC transplantation. The collagen deposition increased in the group with bladder outlet obstruction (BOO) and recovered after transplantation of B10.HGF cells. (A, B) Control. (C, D) Sham. (E, F) BOO. (G, H) BOO + B10 cells. (I, J) BOO + B10.HGF cells. Masson's trichrome staining. Sham, sham operation; BOO, bladder outlet obstruction; BOO + B10, B10 MSC transplantation at 2 weeks after BOO; BOO + B10.HGF, B10.HGF MSC transplantation at 2 weeks after BOO. Scale bar: 100 μm.

Figure 5.

Change of collagen deposition after MSC transplantation. The group with bladder outlet obstruction (BOO) showed increased collagen deposition than the group of sham operation group (p < 0.05). The BOO animals transplanted with B10 cells or B10.HGF cells showed significantly decreased collagen deposition than the BOO animals without cell transplantation. The animals transplanted with B10.HGF cells showed lower collagen deposition than the group with B10 cell transplantation (p < 0.05). Sham, sham operation; BOO, bladder outlet obstruction, BOO + B10 cells, B10 cell transplantation at 2 weeks after BOO; BOO + B10.HGF, B10. HGF transplantation at 2 weeks after BOO. Data are mean ± standard error.

Figure 6.

(A) Protein analyses of HGF, transforming growth factor-β (TGF-β), and c-met in bladder. The levels of collagen and TGF-β increased after bladder outlet obstruction (BOO). The BOO animals transplanted with B10.HGF cells showed increased levels of HGF and c-met and decreased levels of collagen and TGF-β as compared with the group with B10 cell transplantation after BOO. (B) Quantitative analyses of levels of collagen, HGF, c-met, and TGF-β in membrane fractions from the bladder of sham, BOO, BOO + B10, and BOO + B10. HGF animals. Levels of HGF and c-met were significantly increased, and those of collagen and TGF-β were significantly decreased in BOO + B10.HGF animals. *p < 0.05, BOO versus BOO + B10.HGF. Sham, sham operation; BOO, bladder outlet obstruction; BOO + B10 cells, B10 cell transplantation at 2 weeks after BOO; BOO + B10.HGF, B10.HGF cell transplantation at 2 weeks after BOO. Data are mean ± standard error.

Figure 7.

Improvement in cystometric parameters after cell transplantation. (A) The bladder outlet obstruction (BOO) animals showed decreased intercontraction interval (ICI) than sham operation group and reversed after transplantation with B10.HGF cells (p < 0.05). (B) The BOO animals showed increased maximal voiding pressure (MVP) than sham operation group and reversed after transplantation with B10.HGF cells (p < 0.05). (C) There was no difference in pressure threshold (PT) between groups. (D) The BOO animals showed increased residual urine volume (RU) than sham operation group but the RU increase was not reversed after transplantation with B10.HGF cells. Sham, sham operation; BOO, bladder outlet obstruction; BOO + B10, B10 cells transplanted at 2 weeks after BOO; BOO + B10. HGF, B10.HGF cells transplanted at 2 weeks after BOO. Data are mean ± standard error. *BOO versus BOO + B10.HGF.

Discussion

In the present study, transplantation of B10 human MSCs overexpressing HGF in bladder of BOO model rats inhibited collagen deposit and improved bladder function. Histological and immunohistochemical examination of bladder sections indicated that the transplanted B10.HGF cells differentiated into smooth muscle cells.

MSCs are self-renewing adult stem cells with multipotent differentiation potential. MSCs could become many types of tissues either via transdifferentiation or via cell fusion, subsequently allowing the regeneration and function restoration (32) and are an important source for cell replacement (26). They can serve as vehicles for gene transfer, proliferate, and differentiate into bladder smooth muscle cells to repopulate damaged bladder. Recently, we have reported that transplantation of primary human MSCs labeled with nanoparticles containing superparamagnetic iron oxide on the bladder wall in rat BOO model inhibited bladder fibrosis and induced improvement of bladder dysfunction (11).

A previous study has reported that MSCs themselves could not substitute the damaged cells directly but secrete a growth factor and contribute to reducing fibrosis through paracrine mechanisms (25). Our study with primary MSCs agrees with these observations, and there were increased levels of HGF and c-met proteins in bladder tissue in BOO rats transplanted with primary naive MSCs (11). It appears that the inhibition of collagen deposition following transplantation of B10. HGF cells was mainly due to paracrine effect of HGF secreted from the B10.HGF cells rather than SMA- and desmin-positive smooth muscle-like cells differentiated from B10.HGF cells.

MSCs could secrete many growth factors including HGF, nerve growth factor (NGF), brain-derived growth factor (BDNF), glial-derived growth factor (GDNF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and ciliary neurotrophic growth factor (CNTF) (18) and play an essential part in the antifibrosis effects in injured organ (16,28 –30). It was reported that the transplantation of MSCs in damaged tissues ameliorate fibrosis in aorta, heart, kidney, liver, or lung (4,12 –15,21,22,26). Growth factors have been reported in the bladder development and the remodeling of the bladder wall after outlet obstruction (2). HGF protein released by B10.HGF human MSCs in BOO lesion site provides complementary functions, including suppression of fibrosis and stimulation of tissue regeneration.

For gene therapy to be efficacious, effective gene transfer into stem cells must be achieved without inducing detrimental effects on their biological properties. Although the combination of MSCs and HGF appears to be efficacious for bladder fibrosis therapy, the choice of vector for cell transduction should be carefully considered. The selected vector should have high transduction efficiency and should ensure stable and long-term trans-gene expression from the cell vehicle and be devoid of any damaging effect on cell viability. Retroviral vectors are proven to be capable of transducing cells in multiple mammalian species (9). In this study, modified MSCs encoding HGF gene markedly enhance HGF production after retroviral transduction.

HGF protein levels increased considerably in rats treated with HGF-modified MSCs and stayed at high levels was reported and demonstrated that the therapeutic effects of HGF depended on the duration of HGF elevation (5,6,8). In this study, we demonstrate that MSCs can be transduced by a retroviral vector encoding HGF gene, and the MSCs encoding HGF were capable of secreting high levels of HGF, and HGF suppress the expression of TGF-β (17,31). The expression of TGF-β protein increased after BOO and recovered after transplantation of MSCs. In this study, the group with BOO showed increased expression of collagen and TGF-β protein than the group with sham operation. The group with transplantation of B10 after BOO showed decreased collagen and TGF-β protein than the group with BOO. The group with transplantation of B10 after BOO showed increased HGF and c-met protein than the group with sham and BOO.

MSC transplantation alone showed insufficient effect on restoring liver fibrosis than HGF modified MSCs (6). Modification of MSCs to overexpress HGF has an effective means to maintain or enhance the capacity of MSCs. In this study, the BOO animals transplanted with B10.HGF cells showed increased HGF and c-met protein levels than the group with B10 cells after BOO. The group with transplantation of B10.HGF cells after BOO showed decreased levels of collagen and TGF-β protein as compared with the group transplanted with B10 cells after BOO.

We found that the transplantation of HGF overexpressing MSCs showed significant reduction of the bladder fibrosis in BOO rats than nonmodified MSCs alone. In bladder outlet obstruction, bladder instability was found (27). In this study, ICI was decreased in BOO and was reversed after transplantation of B10. HGF cells. Maximal voiding pressure (MVP) increased in BOO and reversed after transplantation of B10. HGF cells. Residual urine volume (RU) also increased in BOO, but the RU increase was not reversed after transplantation of B10.HGF cells. Bladder overactivity in BOO animals was reversed by transplantation of MSCs overexpressing HGF. It appears that HGF overexpressing MSCs are more effective than the nonmodified naive MSCs in improving bladder function in an experimental BOO model.

In summary, the present study shows that retroviruses that MSCs genetically modified to encode HGF gene not only attenuate bladder fibrosis but also improve bladder function in BOO animal model. This therapeutic approach may provide a new and effective venue for the treatment of bladder fibrosis in patients with bladder outlet obstruction.

Footnotes

Acknowledgments

This research was supported by a grant from the national Research Foundation of Korea (NRF) funded by the Ministry of education, Science and Technology (KRF-2008-313-E00407). The following author contributions are recognized: Conceived and designed the experiments (H.J.L., Y.S.S., S.U.k.), performed the experiments (H.J.L., S.H.D., S.J.L., K.Y.S.), analyzed the data (H.J.L., I.J.L., K.T.C), and wrote the paper (H.J.L., Y.S.S., S.U.k.). The authors declare no conflicts of interest.

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