Microenergy Acoustic Pulse Therapy Restores Urethral Wall Integrity and Continence in a Rat Model of Female Stress Incontinence

Abstract

OBJECTIVE:

To determine the outcomes and mechanisms of microenergy acoustic pulse (MAP) therapy in an irreversible rat model of female stress urinary incontinence.

MATERIALS AND METHODS:

Twenty-four female Sprague-Dawley rats were randomly assigned into 4 groups: sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO+β-aminopropionitrile (BAPN), and VBDO+β-aminopropionitrile treated with MAP (MAP). MAP therapy was administered twice per week for 4 weeks. After a 1-week washout period, all 24 rats were evaluated with functional and histological studies. The urethral vascular plexus was examined by immunofluorescence staining with antibodies against collagen IV and von Willebrand factor (vWF). The urethral smooth muscle stem/progenitor cells (uSMPCs) were isolated and functionally studied in vivo and in vitro.

RESULTS:

Functional study with leak point pressure (LPP) measurement showed that the MAP group had significantly higher LPPs compared to VBDO and BAPN groups. MAP ameliorated the decline in urethral wall thickness and increased the amount of extracellular matrix within the urethral wall, especially in the urethral and vaginal elastic fibers. MAP also improved the disruption of the urethral vascular plexus in the treated animals. In addition, MAP enhanced the regeneration of urethral and vaginal smooth muscle, and uSMPCs could be induced by MAP to differentiate into smooth muscle and neuron-like cells in vitro.

CONCLUSION:

MAP appears to restore urethral wall integrity by increasing muscle content in the urethra and the vagina and by improving the urethral vascular plexus and the extracellular matrix.

INTRODUCTION

Stress urinary incontinence (SUI) is a prevalent disease that commonly affects women and is reported in as many as 45.9% of the adult female population (1). Microenergy Acoustic Pulses (MAP) has shown potential as a novel, non-invasive technology for the treatment of SUI related to both birth trauma and obesity (16) (29). The MAP consists of a single predominantly positive-pressure pulse followed by a relatively larger stretched-wave component. Compared to low-intensity extracorporeal shock wave therapy (Li-ESWT), the MAP has a lower peak pressure (up to 21.8 mpa), slower pressure rises (5 milliseconds), and longer duration (~15 milliseconds). Most importantly, the beam of the MAP is defocused: this more evenly distributes energy to the target tissue and eliminates the potential for tissue damage at the focal point if a focused device is used. In 2019 (16) (29)and 2020 (37), we reported that MAP could successively improve urethral leakage pressure (LPP) in an animal model, activate tissue-resident stem cells in the urethra and pelvic floor muscles (PFM), and repair urethral sphincter and pelvic floor muscles. Since that time, additional clinical reports have further confirmed the therapeutic effects of Li-ESWT in patients with female SUI (21). However, the specific biological mechanisms of such therapies have not been thoroughly studied.

The closure pressure of the female urethra mainly comes from three anatomic features: the urethral muscle, the extracellular matrix, and the vascular plexus (36). The urethral smooth muscle, including the longitudinally distributed smooth muscle near the urethral lumen and the outer circular urethral smooth muscle, courses through the entire urethra (36). A study from 2019 found that MAP therapy can activate stem/progenitor cells within urethral striated muscle tissue in situ, significantly improving the regeneration of urethral striated muscle and thereby increasing the urethral closure pressure (16) (30). Recent studies have also shown that MAP significantly increases the number of EdU-label retaining cell (LRC) in the urethra as well as the number of cells positive for stem cell markers as assayed through fluorescence activated cell sorting (FACS) (16). In that study, however, before isolating urethral cells for flow cytometry, the urethral smooth muscles were not completely separated from the striated muscles. Therefore, in these activated cells, there is theoretically a mixture of the smooth and striated muscle stem/progenitor cells (SMPCs) from the urethra.

Previous studies have shown that the urethral vascular plexus contributes 30% of the urethral closure pressure (2). In 2018, we first reported that Li-ESWT significantly increased vasculature within the urethral wall (35). Therefore, we questioned whether MAP therapy might also improve the vascular plexus within the urethral wall and what its mechano-biological mechanisms might be.

The extracellular matrix, principally the elastic fibers, of the urethral wall also play an important role in maintaining urethral closure pressure. In 2011, we reported on an animal model of irreversible stress urinary incontinence created by inhibition of LOX1 with β-aminopropionitrile (BPAN), which results in impaired repair of urethral damage (31). In 2020 (37), we reported that Li-ESWT can increase the urethral LPP in this animal model by promoting the regeneration of urethral muscle. Exploring the effect of MAP on the extracellular matrix will further clarify the biological mechanisms of MAP in treating SUI.

In 2008, we also described the paravascular stem cells (18) within adipose tissues. Stem cell antigen 1 (Sca-1) is a member of the Ly6 family and is an 18 kD glycosylphosphatidylinositol-anchored cell surface protein (GPI-AP) (10). Although the biological significance of Sca-1 is still unknown, it is believed to be involved in balancing stem cell self-renewal and differentiation (8), stem cell homing (4), and cell-cell adhesion (25). In 2004, Hu et al reported that Sca-1 is the cell maker of vascular smooth muscle cell (VSMC) progenitor cells residing in the adventitia (11). The Sca-1 is expressed by many different stem cell types, including muscle-derived stem cells (15), mesoangioblasts (5), side population cells (17), and muscular stem cells (34). In addition to being a VSMC progenitor cell marker, Sca1 has also been used as a stem cells marker to localize cardiomuscular stem progenitor cells (28), mammary gland stem progenitor cells (33), and ovary stem progenitor cells (9)

In the current study, we administered MAP therapy to a parturition-induced, irreversible SUI rat animal model in order to define the therapeutic effects of MAP and to explore the mechanisms of action of MAP for the treatment of SUI.

MATERIALS AND METHODS

Experimental Animals and Design

All experiments were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco. Twenty-four female Sprague-Dawley rats (12 weeks old) purchased from Charles River Laboratories (Wilmington, MA, USA) were randomly divided into 4 groups including the control (sham), vaginal balloon dilation plus ovariectomy (VBDO), vaginal balloon dilation plus ovariectomy and β-aminopropionitrile (BAPN), and VBDO plus BANP treated with MAP twice a week for 4 weeks (MAP) groups. The VBDO procedure was performed as previously reported (31). Briefly, under proper anesthesia (ketamine/xylazine, 90mg/kg 10mg/kg respectively, intraperitoneally), the balloon of an 18Fr latex Foley catheter was placed into the rat’s vagina and filled with 4 ml of water. A 130-g weight was placed on the suspended end of the catheter, providing a constant pull to direct the force to the pelvic floor. The balloon was left in place for 4 hours. Seven days later, the rats were anesthetized, and bilateral ovaries were removed. From the second week after initial VBDO, all animals in the BAPN and MAP groups received intraperitoneal injections of 300 mg/kg of BAPN twice a week for 4 weeks. The rats in the MAP group were then treated with MAP. After a 1 week wash-out period after the final MAP treatment, all rats underwent LPP measurement. The rats were then sacrificed, and the urethras harvested for histological study (Figure 1). Three additional female SD rats were used for isolation of smooth muscle stem/progenitor cells.

Figure 1. Experimental design of the MAP therapy experiment.

Experimental protocol from day 0 to week 12.

Microenergy Acoustic Pulses (MAP) Treatment

Application of MAP was performed using a compact electromagnetic unit with an unfocused acoustic pulse source (LiteMed Inc., Taipei, Taiwan). The shockwave probe was applied directly to the shaved pelvis of the rat, overlying the area of the urethra, and was coupled to the skin using ultrasound gel (Aquasonic, Parker Laboratories Inc, Fairfield, NJ, USA). MAP (with an energy flux density of 0.033 mJ/mm² and 500 pulses) was delivered at 3 Hz. Rats in the MAP group were treated twice a week for 4 weeks. After a 1-week washout period, all rats underwent LPP measurement followed by sacrifice and tissue harvest for histological studies.

Measurement of LPP

LPP was assessed as previously described (32). Briefly, under anesthesia with urethane (i.P), a polyethylene-90 tube was inserted into the bladder dome and secured with a purse string suture. The bladder was slowly filled with warmed phosphate buffered saline (PBS), and the volume was recorded. Bladder capacity was recorded as the point at which leakage of urine was observed. This procedure was repeated 3 times, and the average bladder capacity was obtained. The bladder was subsequently emptied via aspiration and manual pressure. Intravesical pressure changes were recorded by a computer with LabView 6.0 software (National Instruments, Austin, TX, USA) at a sample rate of 10 samples/sec. The bladder was then filled to 40% of capacity and increasing manual extravesical pressure was applied until leakage was noted. This procedure was repeated six times, and the LPPs were recorded. The rats were subsequently sacrificed, and tissues harvested.

Elastin, Masson’s Trichrome, and Phalloidin Staining

For elastin staining tissue sections were rinsed in water and dipped in oxalic acid until clear. After washing in water for 5 min, the sections were incubated in rescoring-fuchsin work solution overnight at room temperature. All tissue sections were stained simultaneously to eliminate variations of staining intensity. The sections were counterstained with Van Geisen solution for 1 min; thereafter, they were dehydrated through a graded series of alcohols, cleared in Histo-Clear, and cover-slipped using Histomount as a sealant.

For the Masson’s trichrome staining, the urethral tissue sections were immersed in warm (58°C) Bouin solution for 20 minutes, rinsed, stained with Weigert Hematoxylin for 10 minutes, and then rinsed until only the nuclei remained stained. Next, the sections were stained with Biebrich Scarlet-Acid Fuchsin for 3 minutes, rinsed, and immersed in phosphomolybdic acid for 45 minutes. Thereafter, the sections were stained with Aniline Blue for 3 minutes, rinsed in distilled water for 2 minutes, immersed in 1% acetic acid for 2 minutes, and rinsed in distilled water for 2 minutes twice. Finally, the sections were dehydrated through increasing concentrations of ethanol then air dried and mounted.

For phalloidin staining, the tissue sections were incubated with Alexa-488/594-conjugated phalloidin (1:500; Invitrogen, Carlsbad, CA, USA), which was diluted 1:200 in 1% bovine serum albumin, for 20 min at room temperature. After rinse with PBS, the tissues were stained with 4’,6-diamidino-2-phenylindole (DAPI, for nuclear staining, 1 μg/ml, Sigma-Aldrich, St. Louis, MO, USA).

Immunohistochemical and Immunofluorescence Staining

Tissue samples were fixed in cold 2% formaldehyde and 0.002% saturated 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 (Sakura Finetic USA, Torrance, CA) and stored at −70 °C until use. Fixed frozen tissue specimens were cut at 10 microns, mounted onto SuperFrost-Plus charged slides (Fisher Scientific, Pittsburgh, PA), and air dried for 5 min. The slides were then placed in 0.3% H₂O₂/methanol for 10 min, washed twice 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 overnight at 4°C with primary antibodies for Sca-1, MHC, SMA, Col-IV, and vWF (Table 1). Control tissue sections were similarly prepared, but no primary antibody was added. Staining of the tissue was performed with the Elite ABC kit (Vector Labs, Burlingame, CA), followed by hematoxylin counterstain. For image analysis, five randomly selected fields per tissue were photographed and recorded using a Retiga Q Image digital still camera and ACT-1 software (Nikon Instruments Inc., Melville, NY). For immunofluorescence staining, the tissue was incubated with primary antibody (Table 1) followed by secondary antibody conjugated with FITC or Texas Red (Vector Labs, Burlingame, CA). Nuclear staining was performed with 4',6-diamidino-2-phenylindole (DAPI). Stained tissues were examined by fluorescence microscopy and confocal microscopy. Subsequent image analysis was done as described above.

Table 1.

List of primary antibodies information.

Antibody	Supplier	Dilution
Sca-1	EMD Millipore	1:500
α-SMA	SIGMA	1:1000
CXCR-4	Abcam	1:500
PDGF-R2	Abcam	1:500
PCNA	Abcam	1:500
Id-1	Abcam	1:500
OCT-4	Abcam	1:500
Nanog	Abcam	1:500
Calponin	Abcam	1:500
LN-28	SANTA CRUZ Bio	1:500
S100	DAKO	1:500
NF	Abcam	1:500
SSEA-1	Abcam	1:500
Laminin	Abcam	1:500
RECA	SANTA CRUZ Bio	1:500
CD34	SANTA CRUZ Bio	1:500
Shh	SANTA CRUZ Bio	1:500

Urethral Smooth Muscle Sca-1⁺ Cell Isolation

The procedure was similar to that we have described previously (6) (30). The female urethral tissues were removed from 12-week-old rats. Under a dissection microscope, the urethral tissues were carefully harvested and prepared by removing surrounding connective tissues. The urethral striated muscle (the proximal 2/3 of the urethra) and pelvic floor striated muscle were dissected, and the residual urethral wall was minced into small pieces followed by 5ml Collagenase II (0.2% collagenase II in DMEM contain 10% FBS) incubation at 37°C for 60 minutes with shaking. Dispase (final concentration: 0.04U/ml) was added to the collagenase solution then incubated at 37°C for another 30 minutes with shaking. The cell suspension was then filtered with a 40 μm nylon cell strainer (Falcon) and centrifuged (2000rpm, 4°C, 5mins) to obtain the cell layer, which was then re-suspended with 200 μl DMEM with 2% FBS. The cells were detached using trypsin and washed with PBS containing 0.5% BSA before incubation with anti–Sca-1 and anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cell suspension was applied over a column equipped with a magnetic cell sorting system (MACS). After washing, Sca-1⁺ CD34⁺ cells were collected. For higher purity of Sca-1⁺ CD34⁺ cells, the same process was repeated using a second column. Purity of isolated Sca-1⁺ CD34⁺ cells was evaluated by immunostaining. The cells were then characterized with different cellular markers (Table 1) and utilized for in vitro studies.

Multiple Differentiation of Sca-1⁺ CD34⁺ In Vitro

For smooth muscle cell differentiation, the Sca-1⁺ CD34⁺ were cultured in six-well plates with 15% FBS DMEM. After 24 hours, the medium was changed to induction medium composed of α-MEM, 10% FBS, and 5 ng/ml TGF-β1. For neuron-like cell differentiation, the Sca-1⁺ CD34⁺ cells were induced with DMEM supplemented with 10μM RA for 12 days. The expression of specific cellular markers (SMA, calponin, S100, and Neurofilament (NF)) was assayed with IF as previously reported (19).

Statistical Analysis

Results were analyzed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard error of the mean. Statistical analyses were performed using one-way analysis of variance followed by the Tukey’s post hoc test for multiple comparisons. Differences among groups were considered significant at P < 0.05.

RESULTS

1. MAP improved urethral leakage point pressure.

Overall, the urethral injury recovered quickly in the VBDO only group. The urethral LPP of VBDO alone (34.6±1.8 cmH₂O) recovered more than that of the BAPN group (28.2±4.2 cmH₂O), with both LPPs significantly lower than that of the sham control (57.3±8 cmH₂O) (n=6, P<0.05). After BAPN treatment, the urethral injury remained extensive even at the 10th week after injury. The urethral LPP was significantly lower in the BAPN group as compared to sham and VBDO only groups. Excitingly, MAP therapy significantly increased the LPP of the VBDO animals treated with BAPN to 52.8±8.4 cmH₂O (Figure 2).

Figure 2. Effect of MAP on urethral leak point pressure (LPP).

(a). Representative graphs of LPP (cm H₂O) measurement among the 4 groups: sham, VBDO, BAPN, and MAP. (b). Average LPP of the 4 groups: sham, VBDO, BAPN, and MAP (n=6 * p < 0.05, compared with the sham group ** 0.01 < p < 0.05, compared with the VBDO and BAPN group.)

2. MAP promoted angiogenesis within the urethral wall and regeneration of the urethral vascular plexus.

In 2000, Augsburger and Muller reported that the female canine urethral submucosa had a vascular plexus structure with small arteries open directly into sinusoids (2). In this study, we found a similar structure in the urethra of female rats. In our research, through the detection of basement membrane specific antigen Col IV, we found that such vascular plexus structures are also present between the smooth muscle bundles and in the urethral submucosa. Our results clearly demonstrate a layer of endothelial cells covering the cavity of these vascular plexus structures and expressing the specific antigen of endothelial cells, vWF (Figure 3a&3b).

Figure 3. Urethral vascular plexus structure affected by MAP.

(a). Cross sections of mid-urethra depict urethral vascular plexus outlined by collagen IV (Col IV, green) and urethral smooth muscle (phalloidin, red). (b). The urethral vascular plexus was enveloped by endothelium (vWF, green). (c). Representative staining of urethral vascular plexus in sham, VBDO, BAPN, and MAP. (d). Breaking cicle: individual plexus. Arrows: normal vascular plexus. Arrow heads: damaged vascular plexus. Data were presented as the number of vascular plexi per high power field (PHF) N=6, * p < 0.05 compared with the sham, VBDO, and BAPN.

In the normal rat urethra, these vascular plexus structures are arranged in an orderly fashion. In the animals treated with VBDO and BAPN, however, the urethral vascular plexus structures became disorganized, with variable sizes. There was also extensive muscle atrophy and damage to the endothelial layer of the sinusoids. Strikingly, MAP therapy significantly increased the number, organization, and integrity of the urethral vascular plexus structures (Figure 3c&3d).

We agree with Augsburger and Muller(2) that the urethral vascular plexus is an integral part of the urethral continence mechanism, and we believe that improvement of these structures after MAP therapy may contribute to continence recovery.

3. MAP increased the thickness of the urethral wall.

Previous studies have demonstrated that the urethral muscles, extracellular matrix, and vascular plexus are the main components of the urethral wall and are also the key factors in maintaining urethral closure pressure. In this study, measuring with the method of radar polar chart, the urethral wall thickness of the mid-urethra was quantitatively analyzed, and the regeneration of urethral tissue in sham, VBDO, BAPN, and MAP groups was examined. The results reveal that the urethral wall was thinner after VBDO, especially at the 3, 6, and 9 o'clock positions though the difference of average urethral wall thickness in the polar graph did not reach statistical differences. Interestingly, after BAPN treatment, the entire structure of the urethra was completely disrupted and the tension of the urethral ring disappeared, resulting in urethral collapse. MAP treatment significantly increased the thickness of the urethral wall and restored ring tension and the integrity of the urethral structure (Figure 4).

Figure 4. MAP ameliorated the decline of urethral wall thickness.

(a) Middle section of urethra presents entire urethral wall by staining of urethral striated muscle (MHC, green) and urethral smooth muscle (a-SMA, red). Arrow: disrupted urethral ring. (b). Polar graphs were used to overlap the entire urethral wall to demonstrate the urethral wall morphology and thickness of each component. (c) Data were presented as average thickness (um) of urethral wall, urethral striated muscle, and urethral smooth muscle at four directions of the polar graph. N=6, * p < 0.05 compared with the VBDO and BAPN group.

4. Changes of Vaginal Submucosal Tissues after MAP

Through trichrome staining, we found that that the vaginal submucosal tissues became thinner in all three groups treated with VBDO. MAP therapy did improve vaginal submucosal tissue thickness but without statistical significance (Figure 5a).

Figure 5. Effect of MAP on the extracellular matrix around the urethral wall.

(a) Representative sections of the mid-urethra stained by trichrome staining in four groups: sham, VBDO, BAPN, and MAP; (b) Representative sections showing elastic fibers within vaginal submucosa; (c) Data represent the maximal length (um) of elastic fibers in four groups. N=6, * p < 0.05 compared with the sham group, ** p < 0.05 compared with the VBDO and BAPN group.

We also studied the elastic fibers under the vaginal mucosa. In normal rats the elastic fibers in the vaginal submucosal tissue are abundant and resemble tree roots distributed under the mucosa. In the VBDO treated groups, the elastic fibers were distorted and irregularly distributed, especially in the submucosa. After BAPN treatment, the elastic fibers were even more chaotic. After MAP treatment, the elastic fibers in the vaginal submucosal tissues recovered to a certain extent, but they did not return to the pre-treatment, normal state (Figure 5b&c). The elastic fiber within urethral submucosa, smooth muscle layer and striated muscle layer have the similar outcomes (sFig 1).

5. MAP promoted urethral smooth muscle regeneration.

The urethral smooth muscle is mainly composed of two parts, the inner longitudinal smooth muscle and the outer circular smooth muscle. Of these, the inner longitudinal smooth muscle comprises the majority of the urethral smooth muscle. This study demonstrates that vaginal balloon dilation and BAPN treatment trigger muscle atrophy and disarray of muscle fiber arrangement, increasing the space between the muscular bundles significantly. MAP therapy significantly increased the urethral smooth content and restored the arrangement and organization of smooth muscle bundles.

Both urethral and vaginal wall smooth muscle decreased significantly in the VBDO and BAPN groups, while MAP therapy increased the content of smooth muscle in the urethra and vaginal walls (Figure 4&6). Interestingly, the smooth muscle bundles became thinner and weaker than those in the control.

Figure 6. MAP enhanced the regeneration of urethral and vaginal smooth muscle.

(a). Schematic representation of urethral wall and anterior vaginal wall. (b). Urethral smooth muscle (SMA, red); (c). Vaginal muscle (Pha, green). (d). Vaginal mucosa and submucosa. (e) Data represent the percentage of urethral smooth muscle to total area per field (PF); (f) Data represent the percentage of vaginal wall smooth muscle to total area per field (PF); Arrow: muscle atrophy and disarray of muscle fiber arrangement. Arrowhead: increased space between the muscular bundles. * p < 0.05 compared with the sham group, ** p < 0.05 compared with the VBDO and BAPN group.

6. Identification of urethral tissue resident stem/progenitor cells.

Historically, smooth muscle stem/progenitor cells, especially urethral smooth muscle stem/progenitor cells (uSMPCs), have been challenging to identify. Stem cell marker, SCA-1, has been used to identify and localize smooth muscle stem cells in multiple different organs. Therefore, in this study, we used immunofluorescence staining to detect the location of these cells and to characterize the SCA-1 positive cells in the urethra.

Consistent with previous publications, there are two layers of urethral smooth muscle - inner longitudinal and outer circular fibers - within the female urethral wall (36). Within the smooth muscle bundles, there are several cell types which are not smooth muscle cells (non-SMC). To further study these cells, we used two markers to localize the basal membrane of smooth muscle bundle, collagen IV, and laminin. The results indicate that non-SMC cells are SMA−, Lam−, Col IV− (Figure 7a, 7b, and 7c).

Figure 7. Urethral smooth muscle stem/progenitor cells.

(a) Representative longitudinal section shows Sca-1 + (red, Arrow) cells at the edges of urethral smooth muscle (SMA, green); cross section depicts CD34 + (red, Arrow) cells at the edges of urethral smooth muscle (SMA, green). cross section shows that Sca-1 + (red, Arrow) cells are Lam −. (b) Schematic diagraph of the localization of a urethral smooth muscle stem/progenitor cell and its cellular markers. (c) Sca-1 + (red) uSMPCs in sham, VBDO, BAPN and MAP. Arrow: Sca-1 + (red) uSMPC. (d). Data represent the uMPC numbers in four groups. N=6, * p < 0.05 BAPN compared with other groups.

It has been reported that vascular smooth muscle progenitor cells have a genotype of Sca-1+ CD117+ CD34+. In this study, all three of the specific stem cell markers were used to isolate the SMPCs, while alpha-SMA and calponin were used to identify the mature smooth muscle cells. The non-SMCs were not stained by alpha-SMA and Calponin. The non-SMCs were detected underneath the basal membrane of the smooth muscle bundle and are Sca-1+ CD34. These cells were mainly distributed around the smooth muscular bundles and existed under the sarcolemma on the surface of the smooth muscle cell. The cell phenotype is Sca-1+, CD34+, SMA - (Figure 7d). Therefore, we classified this cell type as the urethral smooth muscle stem/progenitor cell (uSMPCs). Most of the Sca-1+ cells localized near the blood vessels.

7. Isolation and in vitro differentiation of urethral tissue resident stem/progenitor cells.

In order to further confirm the existence of uSMPCs, we isolated these cells through the MACS method and induced cell differentiation in vitro (Figure 8a). These cells are Sca-1+, CXCR4+, PDGFR2 +, CD34+, PCNA+, ID-1 +/−, OCT4−, Nanog−, SHH−, RECA−, SSEA1−, and LN28−. These results indicate that these uSMPCs cells can differentiate into smooth cells (α-SMA+, Calponin+) and also have the potential to differentiate into nerve-like cells (S-100+, NF+). (Figure 8b & 8c).

Figure 8. Isolation, characterization, and differentiation of urethral smooth muscle stem/progenitor cells.

(a). Schematic illustration of the procedure for isolating uSMPCs with MACS. (b). IF staining shows that uSMPCs are Sca-1+, CXCR4+, PDGFR2 +, CD34+, PCNA+, ID-1 +/−, OCT4−, Nanog−, SHH−, RECA−, SSEA1−, and LN28−. (c). α-SMA+ (red) and Calponin+ (green) in induced smooth cells from uSMPCs (Ind); S100 + (red) and NF + (green) in neuron-like cells induced from uSMPCs (Ind) (X200).

DISCUSSION

The maintenance of urethral closure pressure is a complex physiological process involving multiple anatomic components, including urethral muscle, the vascular system, and the extracellular matrix. Previous studies have focused primarily on one of these three factors and seldom conducted in-depth studies on the integrity of the three as a whole. Only the integrity of these three components functioning in unison can maintain the stability of the urethral wall and the intrinsic tension of the urethral tubular structure. Two kinds of urethral muscle contribute the urethral closure pressure, striated muscle and smooth muscle (36). The urethral smooth muscles are distributed within the entire urethral wall, consisting of two layer of muscle fibers, the inner longitudinal smooth muscle fibers and the outer circular smooth muscle fibers. Smooth muscle can be divided into two types: single unit smooth muscle and multi-unit smooth muscle. In single unit smooth muscle, cells are electrically connected through gap junctions and contract as a coordinated unit. In multi-unit smooth muscle, cells have no electrical connection, and each muscle cell operates independently. Both longitudinal and circular urethral smooth muscles are single-unit type of smooth muscle, so they contract at same time when the neuronal signals arrive (12). Therefore, any injury to a portion of the muscular urethral wall, such as during birth trauma, may impair the coordinated function of the entire smooth muscle unit.

In the current study, we studied the female urethra as a whole rather than studying each component separately. We found that the combination of urethral muscular atrophy, injury to the vascular plexus structures, and damage to the extracellular matrix, especially the elastic fibers of the vaginal submucosa, resulted in irreversible SUI in a female rat model (31) (37). This study revealed that MAP therapy improves all of these consequences of urethral trauma. The prospect of identifying a non-invasive treatment modality, such as MAP, that has potential to repair damage to these three kinds of tissues at the same time is appealing.

Since 2009, Li-ESWT has been extensively used in regenerative medicine (22). It has been reported that Li-ESWT promotes angiogenesis (27), nerve regeneration (14) (24), bone formation (13), and muscular regeneration et al (30). We previously reported that Li-ESWT activates penile stem cells and promotes regeneration of erectile tissue to improve erectile function (20). Recently, we reported on an improved technology, named Microenergy Acoustic Pulses (MAP), to replace Li-ESWT, and we demonstrated that MAP improved urethral function in an animal model of SUI (29). This effect was based on the regeneration of the urethral vascular plexus, muscle structure, and extracellular matrix content. In the current study, we note that MAP enhances urethral smooth muscle regeneration and increases urethral muscle content. More importantly, urethral smooth muscle stem/progenitor cells were increased significantly. We found that MAP therapy repairs tissue damage in the smooth and striated muscle, the vascular plexus, and the ECM, simultaneously promoting the regeneration of urethral muscles, the regeneration of urethral vascular plexus structures, and the repair of the extracellular matrix of the urethra and surrounding tissues.

In this study, we also conducted an in-depth investigation to further elucidate the mechano-biological mechanisms of MAP. Researchers have previously reported that Li-ESWT activates stem cells both in vitro and in vivo (20) (7). In 2013, it was reported that Li-ESWT activates adipose-derived stem cells in vitro and increases the expression of ki-67 and PCNA (26). In 2017, Zissler et al reported that Li-ESWT activates striated muscle satellite cells by increasing H3 phosphorylation (38). In 2017 and 2019, we also reported that penile stem cells are activated by Li-ESWT in a rat model (20; 23). Resident stem/progenitor cells play a critical role in the process of tissue regeneration (3). It is well established that local stem cells contribute to smooth muscle regeneration; this has been extensively studied in vascular smooth muscle. In 2008, we described that vascular stem cells exist in the adventitia and are positive for CD34+, PDGFR2+ (18). More recently, research has demonstrated that vascular SMPCs are Sca-1+, CD34+ and CD117+(15) (17) (28) (9). Though vascular SMPCs have been well studied, uSMPCs have not been as well studied. In the past, we have used Sca-1 and CD34 to isolate the SMPCs with MACS or FACS in several organs (23). In the current study, we also used Sca-1 and CD34 as stem cells markers to localize stem/progenitor cells in the urethral wall in situ. We noted that uSMPCs are located underneath the basal membrane of the smooth muscle bundle and express all smooth muscle markers, Sca-1+ CD34+ SMA−. We identified that the uSMPCs are spindle-shaped cells located on the surface of mature smooth muscle cells.

A major limitation of the current study is that we did not investigate the multiple differentiation potentials of urethral smooth muscle stem/progenitor cells (uSMPCs). In addition, the study did not examine the structural changes and signaling pathways at different time points during the treatment period. Further studies to determine the molecular mechanisms involved in stem cell activation, differentiation, tissue restoration, and functional recovery are warranted.

CONCLUSION

By utilizing an established animal model of irreversible SUI which closely mimics the human condition after birth trauma and menopause, we explored the biological effects of MAP as a potential therapeutic technique to improve female SUI. Through a comprehensive study of the three main urethral tissues (muscle, vascular plexus, and extracellular matrix) that maintain urethral closure pressure, this study illustrates that MAP therapy can promote regeneration in all three of these tissues to improve LPP. Our results suggest that MAP may provide a novel, non-invasive therapeutic approach for female SUI patients in the future.

Supplementary Material

sF1

sFigure 1. Effect of MAP on the extracellular matrix within the urethral wall. Representative sections showing elastic fibers within urethral submucosa, urethral smooth muscle and urethral striated muscle in sham, VBDO, BAPN and MAP group.

DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.