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It has been previously demonstrated that adipose tissue-derived stem cell (ADSC) can differentiate into muscle and neuron-like cells in vitro. In this study, we investigate the utility of ADSC in the treatment of overactive bladder (OAB) in obese hyperlipidemic rats (OHR).
Hyperlipidemia was induced in healthy rats by administration of a high fat diet. The resulting OHR were then treated with bladder injection of saline or ADSC or tail vein injection of ADSC. Bladder function was assessed by 24-h voiding behavior study and conscious cystometry. Bladder histology was assessed using immunostaining and trichrome staining followed by image analysis.
Serum total cholesterol and low-density lipoprotein levels were significantly higher in OHR than in normal rats (p < 0.01). Micturition intervals were shorter in the saline-treated OHR relative to normal rats, OHR that received ADSC via tail vein, and OHR that received ADSC by bladder injection (143 ± 28.7 vs 407 ± 77.9 vs 281 ± 43.9 vs 368 ± 66.7 seconds respectively, p = 0.0084). Smooth muscle content of the bladder wall was significantly lower in OHR than in normal animals (p = 0.0061) while there was no significant difference between OHR groups. Nerve content and blood vessel density were lower in control than in ADSC-treated OHR.
Hyperlipidemia is associated with increased urinary frequency and diminished bladder blood vessel and nerve density in rats. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential new therapy for OAB.
Lower urinary tract symptoms (LUTS) consist of storage, voiding and postmicturition symptoms. Storage symptoms include increased micturition frequency, nocturia, urinary urgency, and urinary incontinence. Voiding symptoms include weak stream, hesitancy, and intermittemcy. Postmicturition symptoms include the sensation of incomplete emptying and postmicturition dribble.1
It is estimated that moderate to severe LUTS (AUA Symptom Score 8) are present in 36% of men aged 50–59 years, 50% of men aged 60–69 years and 60% of men aged 70–79 years.2 LUTS in men are frequently coincident with obesity and benign prostatic hyperplasia (BPH);3, 4 however, LUTS may also arise from other disorders, such as detrusor dysfunction. Overactive bladder (OAB), a subset of storage LUTS often associated with detrusor overactivity (DO), is defined as urgency and is often associated with frequency, nocturia, and urinary incontinence.1
Many treatment options are available for patients with LUTS. Mild LUTS may not be bothersome enough to necessitate treatment; if therapy is desired, oral medication is typically the initial treatment choice. Antimuscarinic drugs are currently the first-line pharmacotherapy of choice for patients with bothersome OAB not thought to be related to BPH. However, antimuscarinics do not produce adequate relief in all patients and they do not resolve the underlying functional disorder.5 In addition, these medications have potential adverse effects including dry mouth, constipation, headache and blurred vision.6
Surgical interventions such as transurethral resection of the prostate (TURP) are appropriate for men with moderate/severe LUTS associated with BPH who (i) do not improve after medical therapy, (ii) do not want medical therapy, or (iii) present with a BPH-related complications developed.7 TURP is considered the gold standard therapy for BPH-associated LUTS. Symptomatic improvement has been reported in up to 88% of patients and the magnitude of reduction in obstructive symptoms may be as great as 85%.8 However, it has been reported that TURP may often (20–40% of cases) fail to alleviate storage symptoms especially nocturia.9–11 In addition, long-term follow up reveals that OAB symptoms recur in over 60% of the patients who have undergone TURP. Recurrence of symptoms may be secondary to prostate regrowth but may also be attributable to comorbid OAB.12 Furthermore, TURP is not a therapeutic option for LUTS in women.
Bladder ischemia is a potential factor in the pathogenesis of LUTS.13 A clinical model of DO has been reported in rats fed a high-fat diet (obese hyperlipidemic rats).14 It has been demonstrated that diminution in blood flow may injure the intrinsic nerves of detrusor muscles and induce denervation supersensitivity.15 Furthermore, in a rabbit model, high-fat diet was demonstrated to induce endothelial denudation and disruption of vascular supply to the bladder.16
Adipose-derived stem cells (ADSC) are multipotent progenitor cells isolated from the stromal vascular fraction of adipose tissue.17 Unlike bone marrow stem cells, ADSC can be obtained in large qualities at low risk.18 In addition to being more abundant and easily accessible, adipose tissue yields far more stem cells than bone marrow on a per-gram basis (5,000 vs 100–1,000).19 In previous studies, we have demonstrated that ADSC can differentiate into endothelial and neuron-like cells in vitro.20 This capacity makes ADSC an attractive potential therapy for conditions known to be associated with disruption of endothelial and neuronal tissues.
In this study, we investigate the efficacy of ADSC in the treatment of DO and bladder histological changes associated with high-fat diet in the rat model animal.
Forty (40) 3-month old male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA, USA). All animal care, treatments and procedures were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco (AN080146-01).
Ten rats fed a diet of standard rat chow served as negative controls. The remaining 30 rats (obese hyperlipidemic rats, OHR) were fed a high-fat diet consisting of 2% cholesterol and 10% lard (Zeigler Brothers, Gardner, PA, USA). At five months, all rats in the negative control group and ten rats randomly selected from the OHR were placed in a metabolic cage for 24 hours, to perform voiding behavior studies.
After metabolic cage study, all rats underwent para-testicular fat harvest for procurement of ADSC. After culture and purification of ADSC the 30 OHR were divided into three groups: 1) injection of phosphate buffered saline (PBS) into the detrusor through a laparotomy (OHR+PBS), 2) Injection of ADSC via tail vein (OHR+TV), or 3) Injection of ADSC into the detrusor through a laparotomy (OHR+B). Negative control animals underwent laparotomy and PBS injection into the detrusor (NR+PBS).
One month after these treatments all rats underwent conscious cystometry. Animals were then euthanized and serum samples obtained for assessment of total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglyceride and glucose. Bladder tissues were harvested for immunohistochemical examination for Masson’s trichrome, 5-ethynyl-2′-deoxyuridine (EdU), α-smooth muscle actin (α-SMA), rat endothelial cell antigen-1 (RECA-1), and rat choline acetyltransferase (ChAT) staining.
All rats undergoing 24 hour voiding behavior studies were placed in a metabolic cage for 24 hours on a 12/12 hour dark/light photocycle.21 Each monitoring period started at 16:00. Micturition time and volumes were recorded via a fluid collector connected to a pressure transducer (Utah Medical Products, Midvale, UT, USA). The pressure transducer was connected to a Dell Pentium 4 computer with Laboratory View 6.0 software (National Instruments, Austin, TX, USA) to monitor the cumulative weight of the collected urine. All rats received water and food ad-libitum throughout the duration in the cage. Voiding frequency and volume were recorded for 24 hours.
Animals were anesthetized with isoflurane. A midline abdominal incision was made to expose the perigonadal fat pad. A specimen of para-testicular fat was harvested and placed in ice cold PBS. The animal was then closed in two layers with absorbable suture and anesthesia was weaned.
Fat tissue was rinsed with PBS in a 50 ml conical tube. Adipose tissue was removed from the upper layer to a fresh tube, and digested in 0.075% collagenase I (Sigma-Aldrich Cp., St Louis, MO, USA) for 1 hr at 37° C with shaking. The top lipid layer was removed and the remaining liquid portion was centrifuged at 220 × g for 10 min. The pellet was then treated with 160 mM NH4Cl for 10 min to lyse red blood cells. The remaining cells were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and plated at a density of 1 × 106 cells in a 10-cm dish. The culture dish was placed in a 5% CO2 incubator for 3–5 days to allow the formation of ADSC colonies. For the purpose of cell tracking, ADSC were treated with 10 μM EdU (Invitrogen, Carlsbad, CA, USA) overnight. A total of 3 × 106 EdU-labeled ADSC were collected into a 2-ml conical tube containing 1 ml PBS; these ADSC were subsequently utilized for injection.
All animals were anesthetized with isoflurane and underwent midline laparotomy to expose the bladder. Animals underwent injection of 1 ml PBS (NR+PBS and OHR+PBS group), or 3 million of autologous ADSC in 1 mL PBS (OHR+B) into the bladder. Animals in the OHR+TV group received 3 million of autologous ADSC transplantation via the tail vein. After treatment the laparotomy was closed in two layers.
Bladder catheters were implanted under isoflurane anesthesia 24 hours prior to conscious cystometry. The abdomen was opened through a midline incision and a polyethylene catheter (PE-90, Clay-Adams, Becton Dickinson, Parsippany, NJ, USA) was implanted into the bladder through the dome. The catheter was tunneled subcutaneously and brought out through a skin incision on the rat’s back.
Conscious cystometry was performed by placing an awake rat in a custom-made tunnel within a metabolic cage (Braintree Scientific, Braintree, MA, USA). Conscious cystometry enables the recording of micturition time and volumes via a fluid collector connected to a pressure transducer connected to surgically implanted bladder catheters (Utah Medical Products, Midvale, UT, USA). The bladder catheter was connected via a T-tube to both a pressure transducer (Utah Medical Products, Midvale, UT, USA) and an infusion pump (Harvard Model 22, KD Scientific, Holliston, MA, USA). After calibration of the pressure transducer to zero, the bladder was filled with room temperature normal saline at a rate of 0.1 mL/min, while recording simultaneous pressure on a sensor input module (model SCXI 1121, National Instruments, Austin, TX, USA) connected to a Dell Pentium 4 computer with Laboratory View 6.0 software (National Instruments). All rats were given around 10 min for the voiding patterns to stabilize. Thereafter micturition were recorded for 30 min .
After euthanasia, tissue samples were fixed in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, PH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (American Master Tech Scientific, Inc, Lodi, CA, USA) and stored at 70°C until use. Sections were cut at 5 μm, mounted into charged slides and air dried for 5 min. The slides were stained with Masson’s trichrome for connective tissue and smooth muscle histology.
EdU staining with immunostaining for α-SMA was performed by placing slides in 0.3% H2O2/methanol for 10 min. Slides were then twice-washed in PBS for 5 min and incubated with 3% horse serum in PBS/0.3% Triton X-100 for 30 min at room temperature. After draining this solution from the tissue section, the slides were incubated at room temperature with anti-α-SMA antibody (Abcam Inc., Cambridge, MA, USA) overnight. After rinsing with PBS, sections were incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After three rinses with PBS, the slides were incubated with freshly made Click-iTTM reaction cocktail (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature without light followed by Dilute Hoechst (Invitrogen, Carlsbad, CA, USA) for nuclear staining.
We used RECA-1 as a marker for endothelial cells to study the changes in vascular profile and ChAT in cholinergic nerves profile. The tissue sections were similarly prepared as described above. After draining excess fluid, the sections was incubated overnight at room temperature with the following antibodies: mouse anti-RECA-1 (Abcam Inc, Cambridge, MA, USA) and goat anti-ChAT (Chemicon, Temecula, CA, USA). Immunostaining of the tissue was performed with the avidin-biotin-peroxidase method (Elite ABC, Vector Labs, Burlingame, CA, USA), with 3,3-diaminobenzidine as chromagen, followed by hematoxylin counterstain.
For image analysis, five randomly selected fields per slide were photographed and recorded at 200x magnification for Masson’s trichrome staining and at 400x magnification for RECA-1 and ChAT staining using a digital still camera (Nikon DXM1200) and ACT-1 software (Nikon Instruments Inc., Melville, NY, USA). Image-Pro Plus imaging software was used for quantification of differential staining (Media Cybernetics, Silver Spring, MD, USA). The percentage of smooth muscle was calculated as the sum of the smooth muscle (red stained) areas divided by the sum of all smooth muscle and connective tissue (blue stained) areas.15
Data was analyzed with Prism 4 (GraphPad Software, Inc., San Diego, CA, USA) and expressed as mean ± standard error of the mean for continuous variables. The continuous data was compared the groups using Student’s t-test and one-way analysis of variance. The Bonferroni test was used for post-hoc comparisons. Statistical significance was set at p < 0.05.
Micturition frequency of animals fed the high-fat diet was significantly higher than that of controls at five months (14.7 ± 1.51 vs 9.9 ± 0.78 time/day, p = 0.0114). Mean volume per void was significantly lower in OHR relative to controls (0.57 ± 0.059 mL vs 1.01 ± 0.154 mL, p = 0.0115).
Total body, bladder, and prostate weights were significantly higher in OHR than in rats fed a normal diet for 6 months (p < 0.05). Serum total cholesterol and LDL levels were significantly higher in OHR than in rats fed a normal diet for six months (p < 0.05). There were no significant differences in glucose, triglyceride or HDL levels between the four groups (table 1).
Representative bladder pressure and voiding measurements from conscious cystometry in NR+PBS and OHR+PBS rats are presented in figure 1. Micturition interval was significantly shorter in the OHR+PBS group (143 ± 28.7 sec) compared to the NR+PBS (407 ± 77.9 sec) and OHR+B (368 ± 66.7 sec) groups (table 2). The OHR+TV group (281 ± 43.9 sec) had longer micturition intervals than the OHR+PBS group but the difference did not reach statistical significance.
The mean volume per void in the OHR+PBS group (0.23 ± 0.048 ml) was significantly smaller than the NR+PBS (0.665 ± 0.131 ml) and OHR+B (0.628 ± 0.122 ml) groups. The OHR+TV group (0.472 ± 0.054 ml) had larger voided volume than the OHR+PBS group but the difference did not reach statistical significance. There was no statistically significant difference in post-void residual urine volume and maximal voiding pressure among the four groups.
Autologous EdU-labeled ADSC were transplanted via tail vein (OHR+TV group) or bladder (OHR+B group) and identified by Alexa-594 stain. The histological data indicated the presence of EdU-labeled cells in the submucosal connective tissue and muscular tissue of the bladder. Many more EdU-labeled cells were identified in the OHR+B group relative to the OHR+TV group. A few EdU-positive nuclei appeared to co-localize with α-SMA staining, suggesting the possibility of stem cell differentiation into smooth muscle cells (fig. 2).
RECA-1 staining for endothelial tissue showed intense positivity in the submucosal layer of bladders from the NR+PBS, OHR+TV and OHR+B groups; there was minimal RECA-1 positivity in the OHR+PBS group (fig. 3). There was no significant difference between the NR+PBS, OHR+TV and OHR+B groups (table 3), suggesting that ADSC transplantation maintained vascular endothelial integrity in the bladder of ADSC-treated OHR.
The OHR+PBS group had significantly lower cholinergic nerve content relative to NR+PBS, suggesting that cholesterol may adversely affect nerve content (fig. 4). Cholinergic nerve content in the OHR+B was significantly greater than what was observed in the OHR+PBS group; there was no significant difference between OHR+TV and OHR+PBS groups (table 3).
All OHR groups had significantly thicker individual muscle bundles relative to the NR+PBS group (fig. 5). Tissue sections from the NR+PBS group had higher smooth muscle percentage than the OHR+PBS, OHR+TV and OHR+B groups (table 3). However, there was no significant difference between animals fed a high-fat diet.
OAB is a complex syndrome attributed to numerous factors. In addition to neurologic disease, bladder outlet obstruction, congenital malformations, dysfunctional behavioral, tissue senescence, myogenic disease, and inflammation, ischemia is a potential cause of OAB.22 The mechanism of ischemia related bladder dysfunction is complex but may be related to ischemic denervation of the detrusor; this may produce supersensitivity of the muscarinic receptors to acetylcholine and OAB.15 In addition to neurological injury, ischemia may lead to smooth muscle fibrosis, and desquamation of urothelium.13 Azadzoi et al. found that hyperlipidemia-induced chronic ischemia increases transforming growth factor-β1 (TGF-β1) expression in the bladder leading to fibrosis and non-compliance. Moreover, increased serum low density lipoprotein (LDL) deposition has been shown to enhance TGF-β1 production and fibronectin synthesis in various other organs.23 Frisbee et al. demonstrated that obesity led to progressive microvascular rarefaction (a reduction in microvascular density) within multiple tissues/organs with the likely end result of progressive tissue ischemia.24
In the current study, it was determined that micturition frequency over a 24 hours period is higher, and voided volume significantly lower, in rats fed a high-fat diet for five months relative to those fed a normal diet. Four weeks after ADSC transplantation, these functional parameters improved toward normalcy in the OHR+TV and OHR+B groups. It is therefore implied that 1) hyperlipidemia and obesity produces a phenotype similar to OAB in rats, and 2) ADSC may ameliorate obesity related OAB. In addition, application of ADSC by direct injection appears to be more efficacious than application by tail vein injection.
The precise mechanism(s) by which ADSC enhance bladder function remains to be elucidated. ADSC have the capability to differentiate into a variety of cells types such as adipocytes, cardiomyocytes, chondrocytes, endothelial cells, smooth muscle cells, neuronal-like cells, and osteoblasts in-vitro.20, 25, 26 Furthermore, ADSC secrete cytokines and growth factors such as vascular endothelial growth factor and hepatocyte growth factor that stimulate angiogenic recovery in a paracrine manner.27–29 The angiogenic and vascular restorative properties of ADSC by either intravenous or local administration have been demonstrated in multiple in vivo studies.28–30 In addition to angiogenic mechanisms, ADSC may provide antioxidant chemicals, free radical scavengers and heat shock proteins in ischemic tissues.27 Histological data from our study suggests that ADSC treatment enhances microvessel and neuronal content of the detrusor muscle. However, while it is possible that ADSC may have differentiated into various somatic cells within our study animals, the scant staining for EdU in the bladder tissues suggests that the therapeutic effect of ADSC was likely mediated primarily by the paracrine release of cytokines and growth factors. Further study of the specific cytokines and growth factors secreted by ADSC in vivo is required.
Specific limitations of our study include the lack of pre-obesity conscious cystometry and the unknown durability of ADSC treatment effect. While it would be more convincing to demonstrate that DO is a direct result of high fat diet and obesity by performing pre-obesity conscious cystometry, this would necessitate multiple surgeries and catheter placements, thus increasing the risk of iatrogenic damages. For future studies, it would be useful to know the durability of ADSC effects and whether coupling ADSC therapy with dietary modification could enhance the therapeutic effects. Above all, the greatest need will be the assessment of the long term safety of this form of stem cell therapy before clinical trials in humans can be considered.
To date, no treatment has been proven capable of repairing the tissue alterations resulting from bladder ischemia. The present study suggests that ADSC administered by local injection enhance angiogenesis and cholinergic nerve innervations after induction of ischemic injury by hyperlipidemic diet. The mechanism of action is likely via cytokine secretion although stem cell differentiation may also play a role. ADSC may hold promise in the treatment of overactive bladder induced by obesity; further studies are warranted.