Magnetic chitosan nanoparticle-exosome hydrogel enhances bladder function in diabetic bladder dysfunction via activating the FAK-p38 MAPK-GATA4 axis in adipose-derived mesenchymal stromal cells
Junhao Zheng, Daofeng Zhang, Haorui Li, Rongyang Jin, Hao Chen, Xiaoliang Sun, Haiyang Zhang
TL;DR
A magnetic hydrogel containing exosomes from treated cells improves bladder function in diabetic rats by promoting blood vessel and nerve growth.
Contribution
A novel magnetic chitosan nanoparticle-exosome hydrogel is developed to treat diabetic bladder dysfunction by activating specific cellular pathways.
Findings
DLSW activates the FAK-p38 MAPK-GATA4 axis in ADSCs, increasing VEGF and NGF secretion.
The magnetic CSNP-Exo hydrogel effectively targets and improves bladder function in diabetic rats.
Exosome-mediated GATA4 upregulation enhances vascular and neural repair in bladder tissue.
Abstract
Previous attempts to combine adipose-derived mesenchymal stromal cells (ADSCs) with defocused low-energy shock wave (DLSW) have shown effectiveness in treating diabetic bladder dysfunction (DBD). However, the specific mechanisms underlying their therapeutic effects and strategies to enhance the colonization of ADSCs at the disease site remain challenging. Hereby, our investigation revealed that DLSW activated the FAK-p38 MAPK-GATA4 axis in ADSCs, resulting in enhanced secretion of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Moreover, tube formation assay and major pelvic ganglia culture showed that the effects of VEGF and NGF on angiogenesis and nerve fiber growth were hindered by adding GATA4 inhibitors. We developed a thermosensitive hydrogel using chitosan nanoparticles (CSNP) incorporated with β-glycerophosphate and Fe3O4, and loaded it with exosomes…
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Figure 7| Cystometric pamameters | Normal group | DBD group | CSNP-Exo group |
|---|---|---|---|
| ( | ( | ( | |
| MI (s) | 230.1 ± 37.2 | 1538.1 ± 450.1 | 422.1 ± 142.8 |
| UV (mL) | 0.4 ± 0.2 | 2.8 ± 1.7 | 1.2 ± 0.5 |
| Pmax (cmH2O) | 30.6 ± 7.8 | 58.9 ± 14.4 | 40.5 ± 12.3 |
| RV (mL) | 0.06 ± 0.02 | 0.5 ± 0.19 | 0.4 ± 0.08 |
- —Shandong Provincial Natural Science Foundation10.13039/501100007129
- —National Natural Science Foundation of China10.13039/501100001809
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Taxonomy
TopicsUrinary Bladder and Prostate Research · Tissue Engineering and Regenerative Medicine · Pelvic floor disorders treatments
Introduction
Diabetic bladder dysfunction (DBD) encompasses voiding abnormalities in diabetes mellitus patients, characterized by increased urinary frequency, urgency, incontinence and retention [1]. DBD is often attributed to a range of physiological insults, including neuropathy, vasculopathy and detrusor atrophy, posing significant challenges for urologists due to its poor response to conventional therapies.
In 2012, adipose-derived mesenchymal stromal cells (ADSCs) were first introduced for the treatment of DBD [2]. As mesenchymal stromal cells (MSCs), ADSCs share similar surface markers, gene profiles, and differentiation potentials with bone marrow-derived MSCs [3, 4]. Their regenerative effects are attributed to the production of growth factors such as fibroblast growth factor 2, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), hepatocyte growth factor and insulin-like growth factor 1 [2, 5–9]. However, only 40% of DBD animals in the treatment group responded to ADSCs implantation [2].
Defocused low-energy shock wave (DLSW) was applied in 2017 to accelerate the regeneration of injured bladder tissues [9]. DLSW has been utilized to treat peripheral nerve injuries [10], chronic wounds [11] and erectile dysfunction [12], although its precise mechanisms remain unknown. Revascularization and innervation are commonly considered to be the main pathophysiological changes of wound tissues triggered by shock waves [9, 13–15]. We previously reported that DLSW activates ADSCs in vitro via the MAPK pathway, promoting angiogenesis and nerve regeneration [5].
However, several challenges persist, such as enhancing ADSCs engraftment in the bladder. Intravenous administration leads to minimal ADSCs migration to the bladder muscle and submucosa, insufficient for therapeutic effects [2]. Local injection into the bladder faces difficulties due to urine flushing, causing ADSCs apoptosis within days of transplantation.
ADSC-derived exosomes (Exo) are vital for tissue repair, inflammation inhibition and immune response regulation [16, 17]. They offer several advantages over stem cell therapies, such as immune privilege, stability, targeted delivery to damaged sites, ease of transportation and storage and potential for genetic modification [18, 19]. However, low targeting efficiency and short residence time in the bladder due to urine flushing have hindered the application of Exo in the treatment of bladder diseases [16]. Therefore, improving the stability and retention of Exo in vivo is crucial for exerting its reparative effects.
To tackle the challenges associated with Exo-based therapy, a magnetic thermosensitive hydrogel using chitosan (CS) as the primary component for encapsulating Exo was developed in previous studies. Thermosensitive CS hydrogels are biocompatible materials widely used as cell and drug carriers in therapeutic applications [20–22]. Cross-linked CS hydrogels prolong the residence time of cells or drugs in damaged tissues and promote cell adhesion, migration and proliferation by mimicking the natural extracellular matrix microenvironment [23]. Our previous work demonstrated the feasibility of Fe_3_O_4_-based magnetic thermosensitive chitosan/β-glycerophosphate (CS/GP) hydrogel as a drug delivery matrix, enabling continuous intravesical drug release for over 48 h [24]. Additionally, CS has been utilized as a carrier to deliver MSCs-derived Exo to the corpus cavernosum of the penis for the treatment of erectile dysfunction [25]. However, the large molecular size and low solubility of CS constrained its drug-loading capacity and stability in vivo, prompting nanosizing to enhance drug adsorption, sustained release and stability [22, 26].
Therefore, CS nanoparticles (CSNP) were used in the present study. The results suggest that magnetic CSNP, owing to their favorable biophysical and chemical properties, can enhance the in vivo retention of Exo, thereby improving therapeutic efficacy and promoting DBD regeneration. The molecular mechanisms underlying DLSW-mediated regulation of Exo secretion from ADSCs and the therapeutic effects of magnetic CSNP-encapsulated Exo (CSNP-Exo) for treating DBD were also explored.
Materials and methods
Part I: to determine the role of FAK-p38 MAPK-GATA4 pathway in vitro in activating ADSCs by DLSW
Isolation and culture of ADSCs
All animal care, treatments and procedures were conducted according to the principles expressed in the Declaration of Helsinki and approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (No. SD 2023-0006). Male Sprague–Dawley (SD) rats (8 weeks old) were obtained from the Model Animal Research Centre of Shandong University. Rat ADSCs were isolated from the visceral fat pads of the rats using a standardized protocol [2]. Briefly, a midline abdominal incision was made to expose the perigonadal fat pad. A paratesticular fat specimen was harvested and placed in ice-cold phosphate-buffered saline (PBS). The adipose tissue was rinsed with PBS, minced into small pieces and then incubated in a solution containing 0.075% collagenase type IA for 1 h at 37°C with vigorous shaking for 15 s in 20-min intervals. The top lipid layer was removed, and the remaining liquid portion was centrifuged at 1000 g for 10 min. The pellet was treated with 160 mM NH_4_Cl for 10 min to lyse red blood cells. The remaining cells were suspended in 10 mL Dulbecco’s Modified Eagle Medium (DMEM, obtained from the Cell Culture Facility at our institution) supplemented with streptomycin, fungizone, penicillin and 10% fetal bovine serum (FBS). The suspension was filtered through a 70-µm cell strainer, plated at a density of 1 × 10^6^ cells in a 10-cm dish and cultured at 37°C in 5% CO_2_ for 3–5 days to allow ADSCs colonies to form. Cells were subcultured upon reaching approximately 90% confluence. Cells at passage 4 (P4) were utilized consistently in this study.
Grouping and preprocessing of ADSCs
The shock wave treatment on cultured ADSCs was performed using an extracorporeal shock wave machine (derma-PACE device, SANUWAVE, USA). The shock wave probe remained in contact with the culture flask housing adherent ADSCs via a water-filled cushion coated with standard ultrasound gel (Supplementary Figure S1). The cells were subjected to 800 impulses of DLSW at an energy flux density of 0.1 mJ/mm^2^. Shock wave treatment was administered prior to each passage until P4. These shocked cells were labeled as the DLSW group. Unshocked ADSCs were cultured simultaneously and served as the control group.
ADSCs were cultured in the presence of the focal adhesion kinase (FAK)-specific inhibitor BI 853520 (1 nM, Bio-Genesis Technologies, China), the p38 MAPK inhibitor SB203580 (50 μM, APExBIO Technology, USA) and the GATA4 inhibitor SGC-CBP30 (2 μM, Selleck, China) and exposed to DLSW shock prior to each passage, referred to as the DLSW+FAK(−) group, DLSW+p38(−) group, and DLSW+GATA4(−) group.
The ADSCs of P4 in each group were collected and subjected to the following procedures.
Cell morphology test
To visualize the actin cytoskeleton, ADSCs from both the Control and DLSW groups were treated with FITC-conjugated phalloidin (Sigma-Aldrich, USA). The cells were fixed for 5 min in 3.7% formaldehyde solution in PBS and then stained with a 50 µg/mL fluorescent phalloidin solution for 40 min at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich, USA.
Flow cytometry
The ADSCs in the control group and DLSW group were resuspended and analyzed by flow cytometry for cell surface antigen expression. The cells were incubated with antibodies including CD29-PE (Elabscience, China), CD44-FITC (Santa Cruz Biotechnology, USA), CD90-PE (Elabscience, China), CD34-PE (AbDSerotec, UK) and CD45-FITC (Abcam, China) at room temperature for 1 h. After incubation, the cells were then rinsed twice with wash buffer, fixed with 1% paraformaldehyde in PBS, and analyzed by FACSCalibur flow cytometer (Beckman Coulter, USA). The raw data were further analyzed using FlowJo software (Tree Star Inc, USA). Three independent measurements were performed for each marker.
Identification of genes affected by DLSW
ADSCs from the control group and DLSW group were harvested and thoroughly washed twice with pre-cooled PBS buffer. Total RNA was extracted using the RNeasy Isolation Kit (Qiagen, USA). ABioanalyzer 2100 system (Agilent Technologies, USA) was used to examine RNA integrity. The purified total RNA was forwarded to Shanghai Bohao Biotech for RNA-seq analysis employing an Illumina HiSeq system post-transcription (Illumina, USA). The DESeq2 software was utilized to identify differentially expressed genes (DEGs), followed by conducting biological functional enrichment analysis on these genes. Genes exhibiting a log2 (fold change) >1 and P < 0.05 were classified as DEGs. Gene cluster analyses and enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted based on the DEGs.
Immunofluorescence staining
The ADSCs in the control group and DLSW group were incubated with the primary antibody of phosphorylated-FAK (p-FAK) (Santa Cruz Biotechnology, USA) overnight at 4°C and were then exposed to secondary antibody for 30 min. Nuclei were counterstained with DAPI.
Immunofluorescence staining of phosphorylated-p38 (p-p38) MAPK in the control group, DLSW group and DLSW+FAK(−) group, and GATA4 in the control group, DLSW group and DLSW+p38(−) group, was performed utilizing the same methodology as described above with primary antibodies of p-p38 MAPK (Cell Signaling Technology, USA) and GATA4 (Abcam, USA).
Western blot analysis
The expressions of p-FAK and FAK in the ADSCs in the control group and DLSW group; p-p38 MAPK and p38 MAPK in the ADSCs in the control group, DLSW group and DLSW+FAK(−) group; and GATA4 in the control group, DLSW group and DLSW+p38(−) group were analyzed by western blot analysis. The antibodies used were p-FAK (Santa Cruz Biotechnology, USA), FAK (Santa Cruz Biotechnology, USA), p-p38 MAPK (Cell Signaling Technology, USA), p38 MAPK (Santa Cruz Biotechnology, USA) and GATA4 (Abcam, USA). All membranes were then reprobed with anti-glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, USA) as an internal control. The resulting images were analyzed using Image-Pro Plus 6.0 software to determine the integrated density value of each protein band. Each experiment was performed in triplicate.
Enzyme linked immunosorbent assay
The levels of VEGF and NGF secreted by the cells in the control group, DLSW group, and DLSW+GATA4(−) group were quantified using enzyme-linked immunosorbent assay (ELISA). The culture media were replaced with 2 mL of FBS-free DMEM, and after 24 h, the conditioned media were collected and centrifuged at 1000 g for 10 min. The resulting supernatants were analyzed using VEGF and NGF commercial ELISA kits (ABclonal Technology Co., Ltd, China). To generate statistically significant data, three independent ELISA experiments were performed for each sample, with each experiment including triplicate wells per sample.
Matrigel-based capillary-like tube formation assay
Human umbilical vein endothelial cells (HUVECs) were purchased from Allcells (China) and maintained in EGM2 medium (Lonza, Switzerland). HUVECs were trypsinized, and 1.5 × 10^5^ cells were seeded into 12-well culture plates that were pre-coated with 300 μL of growth factor reduced matrigel (BD Biosciences, USA). The cells were maintained in three different conditioned media of ADSCs (P4) in the control group, DLSW group and DLSW+GATA4(−) group. Then all the HUVECs were incubated in a 37°C incubator with 5% CO_2_ for 16 h to allow the formation of tubes. Each experiment included triplicate wells per sample. For each medium, the number of endotubes was quantified by counting three random fields under the microscope (×100).
Rat major pelvic ganglia culture
The dissection of major pelvic ganglia (MPG) from five 8-week-old male SD rats and culture were performed as previously described [27–29]. In brief, bilateral MPG were completely isolated and excised from each rat. Following a PBS rinse, each isolated MPG was further dissected to isolate the dorsocaudal region (DCR, the origin of the cavernous nerve) of the MPG. Each DCR was then divided into three equally sized pieces, resulting in a total of 30 specimens. Each specimen was embedded in a 40 μL drop of growth factor-reduced Matrigel (BD Biosciences, USA) and treated with 3 mL of one of the three distinct conditioned media derived from ADSCs (P4) as described earlier. The cultures were maintained at 37°C in a humidified atmosphere with 5% CO_2_ for 48 h. Neurite growth from the MPG fragments was captured using a Nikon DXM1200 digital camera attached to a Zeiss Axiovert microscope, using ACT-1 software (Nikon Instruments Inc., USA). The neurite growth areas in the digital images were quantified using Image-Pro Plus 6.0 software.
Part II: to detect the physical and chemical properties of the magnetic CSNP-Exo hydrogel
Isolation and grouping of Exo from ADSCs
Exo from ADSCs in the control group and DLSW group were isolated as previously reported [17], referred to as the Exo group and DLSW-Exo group, respectively. In brief, the cells were incubated for 24 h in serum-free medium. The supernatant was filtered through a 0.22-μm filter and then centrifuged at 10 000 g for 50 min. The resulting supernatant was then transferred to a new ultracentrifugation tube and centrifuged at 100 000 g for 90 min. The resulting pellet was dissolved in PBS.
Examinations of characterizations of Exo from ADSCs
The obtained Exo were analyzed using Nanoparticle Tracking Analysis (NTA) (Zeta View PMX 110, ParticleMetrix, Germany) to measure their diameters. The sample pool was rinsed with deionized water, and the Zeta View PMX110 was calibrated with 110 nm polystyrene microspheres. The sample cells were diluted with PBS buffer and subjected to detection. The Exo were examined under a transmission electron microscope (TEM) for morphological identification.
Western blot analysis was performed to detect the levels of CD63 and CD81 in the Exo group, and GATA4 in the Exo group and DLSW-Exo group, using the same methodology as described above. The antibodies used were GATA4 (Abcam, USA), CD63 (Santa Cruz Biotechnology, USA) and CD81 (Santa Cruz Biotechnology, USA).
Total RNA was isolated from the Exo group and the DLSW-Exo group using the RNeasy Isolation Kit (Qiagen, USA). The isolated RNA was reverse transcribed into complementary DNA libraries. Real-time polymerase chain reaction (PCR) was performed using reagents and primers for rat GATA4 and GAPDH purchased from Applied Biosystems, USA. The primer sequences were as follows: GATA4 (Forward: 5′-CTGTCATCTCACTATGGGCA-3′; Reverse: 5′-CCAAGTCCGAGCAGGAATTT-3′); GAPDH (Forward: 5′-ATGATTCTACCCACGGCAAG-3′; Reverse: 5′-CTGGAAGATGGTGATGGGTT-3′). The reactions were run on the Applied Biosystems’ PRISM 7300HT sequence detection system using a 96-well plate format. The cycling conditions included an initial phase at 95°C for 3 min, 40 cycles at 95°C for 15 s and 55°C for 1 min, followed by a melting curve analysis. The real-time PCR results were analyzed using Applied Biosystems’ SDS 7000 software to determine the expression levels of GATA4 relative to GAPDH.
Preparation of magnetic CSNP-Exo
The magnetic CSNP solution was prepared following a previously established method [24]. In brief, CSNP (>75% deacetylation, Mw 310–375 kDa, viscosity 800-2000 cP Shandong Quanhang Marine Biological Technology Co., Ltd, China) were dissolved in 0.1 M hydrochloric acid and stirred magnetically at room temperature for 2 h. GP powders (Sigma-Aldrich, USA) were dissolved in distilled water and both solutions were cooled in an ice bath for 10 min. The GP and CS solutions were then mixed under magnetic agitation at 4°C to produce a clear and uniform solution. Fe_3_O_4_ magnetic nanoparticles (>99.5% purity, 50 nm, Changsha Jingkang Co., Ltd, China) were subsequently added into the CS/GP mixture under stirring and then dispersed by ultrasound to produce magnetic CSNP. The magnetic CSNP-Exo hydrogel was prepared by mixing DLSW-Exo with CSNP at a ratio of 100 μg DLSW-Exo per 100 μL CSNP solution.
Examinations of physicochemical properties of magnetic CSNP-Exo
The magnetic targeting capability of CSNP-Exo was assessed by placing a clot of CSNP-Exo gel at the bottom of a beaker filled with PBS, followed by observing its movement in response to a magnet placed outside the beaker.
To characterize the functional groups and bonds of CS and CSNP, Fourier transform-infrared (FTIR) spectroscopy analysis was conducted using a Nicolet 6700 spectrometer (Thermo, USA). FTIR spectra were recorded over the wavenumber range of 4000–400 cm^−1^ at a resolution of 4 cm^−1^.
The cured CSNP and CSNP-Exo hydrogels were rapidly frozen in liquid nitrogen, fractured and then subjected to lyophilization. Subsequently, the samples were analyzed using scanning electron microscopy (SEM).
Rheological characterization was conducted using an Anton Paar MCR 301 rotational rheometer configured with a PP 25 plate-plate system. CSNP solution was loaded between the plates, and mineral oil was applied to the solution’s edges to reduce water evaporation during the measurements. Dynamic oscillatory tests were performed at 5% strain, a condition that ensures G′ and G″ remain unaffected by strain amplitude. A dynamic temperature ramp from 10 to 45°C at 2.5°C/min was implemented, with the frequency maintained at 10 rad/s throughout.
The swelling ratio of CSNP was analyzed using gravimetric analysis. Magnetic CSNP and CSNP-Exo gels were freeze-dried and weighed to obtain their initial dry weight (W0). These xerogels were then placed in 10 ml PBS at pH 6.0 and incubated in a water bath at 37°C. At time intervals of 0, 2, 4, 6, 8, 10, 12, 18 and 24 h, the gels were removed, excess surface water was gently wiped off and the wet weights (Wt) were recorded. The swelling rate was calculated by the formula of swelling ratio = (Wt − W0)/W0 × 100%.
CSNP-Exo release was assessed using the Transwell system. PKH26-labeled Exo and CSNP-Exo were placed in the upper chamber of a 24-well Transwell plate. The semi-permeable membrane allowed Exo to pass through, and 1.2 mL of PBS containing 10% FBS was added to the lower chamber. The system was then placed in a 37°C constant temperature shaker at 60 r/min. At time points of 0, 1, 2, 4, 6, 8 and 10 days, 400 µl of the lower chamber solution was collected, and the fluorescence intensity of the samples was measured to determine the cumulative release rate.
Part III: to evaluate the efficacy of magnetic CSNP-Exo hydrogel in treating DBD in vivo
Preparation of animals, ADSCs harvest and processing, and preparation of magnetic CSNP-Exo
A rat model of DBD was established following a previously described protocol [2, 6]. In brief, all rats were fed a high-fat diet (obtained from the Model Animal Research Center of Shandong University) for 1 month, followed by two intraperitoneal injections of streptozotocin (STZ, Sigma-Aldrich, USA) at a dose of 30 mg/kg, with a 1-week interval between injections. Their blood glucose levels were monitored weekly by sampling the tail vein blood. Rats with fasting blood glucose levels ≥200 mg/dl were identified as diabetic and selected for further testing. A total of 25 such rats were assigned to a DBD group (n = 5) and a CSNP-Exo group (n = 20). Rats fed a regular diet during the same period were used as the Normal group (n = 5).
Subsequent isolation and culture of ADSCs from the rats*,* shock wave treatment of isolated ADSCs, isolation of Exo, PKH26 labeling and preparation of magnetic CSNP-Exo were performed following the above protocols. To avoid data bias, the rats in all groups underwent the same surgical procedures simultaneously.
Intravesical instillation of CSNP-Exo and conscious cystometry
The rats in the Normal group and DBD group were administered 1 mL of PBS via insertion of a self-made 3F catheter into the bladder, while the CSNP-Exo group rats received magnetic CSNP-Exo instillation containing 100 μg DLSW-Exo. The rats were then placed in a cage surrounded by a magnetic field of 0.4 T.
One month later, all rats underwent awake cystometry following established procedures [6]. A polyethylene-90 catheter was surgically implanted into the bladder under halothane anesthesia 24 h prior to conscious cystometry. Additionally, a second polyethylene-90 tube connected to a latex balloon was inserted into the intra-abdominal space to measure abdominal pressure. During cystometry, the bladder was slowly filled with PBS at a rate of 0.1 mL/min via the bladder catheter, while simultaneous bladder and abdominal pressures were recorded using a pressure transducer and Laboratory View 6.0 software (National Instruments, USA). Rats were given approximately 20 min to stabilize their voiding pattern, after which micturition was recorded for 1 h. Cystometric parameters assessed included micturition interval, urine volume per void, maximum voiding pressure and residual volume.
Histological analysis
On the first, third and fifth days post-instillation, three rats from the CSNP-Exo group were selected, euthanized and their bladders were harvested. Following tissue fixation, PKH26 staining was performed using the aforementioned method to observe the retention of DLSW-Exo in the bladder.
After conscious cystometry, all rats were humanely euthanized. Their bladders were harvested and divided into three parts.
One-third of the samples were subjected to immunofluorescence staining according to previous protocols [2, 6]. Primary antibodies included rabbit anti-Collagen IV (Col IV, Abcam Inc., USA) and mouse anti-S100 (Santa Cruz Biotechnology, USA). Secondary antibodies were conjugated with Texas Red.
One-third of the samples were fixed in 4% paraformaldehyde (Yayu Bio Medical Co., Ltd, China) for at least 24 h and embedded in paraffin. Immunohistochemical staining was performed using the avidin-biotin-peroxidase method (Abcam, USA) with anti-endothelial cell antigen-1 (RECA-1) (AbD Serotec, UK).
The remaining one-third of the bladder samples were frozen and treated with ice-cold Tissue Extraction Reagent (Invitrogen Corp., USA) containing protease inhibitors. After a 30-min incubation on ice, the samples were centrifuged at 10 000 g for 5 min. The resulting supernatants were collected and analyzed using ELISA kits for VEGF, NGF (ABclonal Technology Co., Ltd, China) and GATA4 (LSBio, USA). Three tissue samples were selected from each group, and three independent ELISA experiments were performed for each sample.
Image and statistical analysis
Images of immunofluorescence staining were captured by fluorescence microscope (Nikon Ti-S, Nikon Inc., Japan), and digital histomorphometric analysis was performed with Image-Pro Plus 6.0 software. Each sample was examined in triplicate, with three high-power fields counted at ×200 magnification for each cellular section and ×100 magnification for each bladder tissue section. To determine the expression of S100 in bladder tissue, the number of positive pixels over the total number of pixels of each slide was calculated by Image-Pro Plus 6.0 software.
The data were analyzed using GraphPad Prism 8.0.2 and expressed as mean ± standard deviation. Statistical significance between groups was determined using the Student’s t-test. The chi-square test with Fisher’s exact test was employed to assess bladder function measured by conscious cystometry among groups. A significance level of P < 0.05 was set for statistical analysis.
Results
The effects of DLSW on ADSCs
The ADSCs exhibited elongated, spindle-shaped cell bodies with 2–4 processes. No significant morphological changes were observed between the groups before and after DLSW treatment (Figure 1A). Fluorescent staining of the F-actin cytoskeleton with phalloidin showed a slightly increased alignment of F-actin fibers in the DLSW group (Figure 1B). However, there were no significant differences observed between the two groups.
*(A) Microscopic observation of cultured ADSCs before and after DLSW treatment, with an enlarged view of the circled area (×1000 magnification). (B) Phalloidin staining to visualize the F-actin cytoskeleton. (C) Flow cytometry analysis of CD markers in the ADSCs of the control and DLSW groups. Expression ratios are presented in (D). P > 0.05.
The expressions of ADSCs surface antigens (CD29, CD34, CD44, CD45, CD90) were analyzed by flow cytometry (Figure 1C). DLSW did not cause statistically significant changes in the expression levels of surface antigens in ADSCs (P > 0.05) (Figure 1D).
Identification of genes affected by DLSW
A volcano plot was utilized to identify genes affected by DLSW (Figure 2A). A total of 69 DEGs were identified in ADSCs following DLSW treatment, with 46 genes showing upregulation and 23 genes showing downregulation. The top three significantly upregulated genes were Mapk14, Socs1 and Stmn4. KEGG analysis revealed the top 16 pathways modulated by DLSW, with the most common pathways being the MAPK pathway, NF-kappa B signaling pathway, Gap junction and DNA replication (Figure 2B). The violin plot of FPKM values indicated a significantly higher expression level of MAPK mRNA in the DLSW group than in the control group (P < 0.01) (Figure 2C).
*(A) Volcano plot displaying gene expression levels in ADSCs treated by DLSW, identifying 69 DEGs, including 46 upregulated and 23 downregulated genes. The red dots represent upregulated genes, the blue dots represent downregulated genes, and the black dots represent genes with stable expression levels. (B) Pathway enrichment results. Spot areas indicate numbers of genes; different colors indicate q values. (C) Violin plot of MAPK mRNA expression levels in the control and DLSW groups. *P < 0.01.
Establishing the role of the FAK-p38 MAPK-GATA4 axis in the activation of ADSCs by DLSW
Immunofluorescence staining revealed significantly higher expression of p-FAK in the ADSCs of the DLSW group than in the control group (P < 0.001) (Figure 3A). This finding was corroborated by western blot analysis, which showed higher p-FAK/FAK density in the DLSW group than in the control group (P < 0.001) (Figure 3B).
*Establishing the role of the FAK-p38 MAPK-GATA4 axis in the activation of ADSCs by DLSW. (A and B) The levels of p-FAK in the ADSCs of the control group and the DLSW group were investigated by immunofluorescence staining and Western blot. (C and D) The expressions of p-p38 MAPK in the ADSCs of the control group, the DLSW group and the DLSW+FAK(−) group were examined by immunofluorescence staining and Western blot. (E and F) GATA4 expressions in the ADSCs of the control group, the DLSW group and the DLSW+p38(−) group were assessed by immunofluorescence staining and Western blot. (G) Quantifications of VEGF and NGF levels secreted by the cells using ELISA. (H) Representative photographs of endothelial-like tube formation in the control group, DLSW group and DLSW+GATA4(−) group (left panel). Differences in the number of endotubes are presented as fold change. Rat MPG were cultured in the respective culture medium (right panel). The longest neurite in each group was identified (indicated by a white arrow). To enhance clarity, the neurite outgrowth was delineated by dashed lines. Quantification was conducted by measuring the neurite growth areas in each treatment group. (I) Proposed mechanism of the involvement of the FAK-p38 MAPK-GATA4 axis in the activation of ADSCs by DLSW. *P < 0.05, **P < 0.01, **P < 0.001.
The expression of p-p38 MAPK in the DLSW group was significantly upregulated compared with the control group (P < 0.001). Inhibition of FAK resulted in downregulation of p-p38 MAPK expression (DLSW+FAK(−) group vs. DLSW group, P < 0.001), as confirmed by both immunofluorescence staining and western blot analysis (Figure 3C and D).
Immunofluorescence staining demonstrated significantly higher expression of GATA4 in the ADSCs of the DLSW group than in the control group (P < 0.001). Inhibition of p38 MAPK led to downregulation of GATA4 expression (DLSW+p38(−) group vs. DLSW group, P < 0.001), a result consistent with western blot analysis (Figure 3E and F).
The ELISA results indicated that the ADSCs in the DLSW group secreted elevated levels of NGF and VEGF compared with the control group, which were reversed by adding GATA4 inhibitor (DLSW+GATA4(−) group vs. DLSW group, NGF, P < 0.05; VEGF, P < 0.01) (Figure 3G).
The angiogenic potentials of conditioned medium of ADSCs in three groups were assessed using tube formation assay. Results depicted in Figure 3H demonstrated a significant enhancement in the ability of HUVECs to form endothelial-like tubes in the DLSW group compared with the control group (P < 0.05). This ability was inhibited by adding GATA4 inhibitor (DLSW+GATA4(−) group vs. DLSW group, P < 0.05). Examinations of neurite growth from the MPG fragments revealed the same pattern (Figure 3H).
Figure 3I summarizes the role of the FAK-p38 MAPK-GATA4 pathway in the activation process of ADSCs triggered by DLSW.
Characterizations of Exo from ADSCs
Exo derived from ADSCs were characterized using TEM, revealing a spherical morphology with diameters ranging from 30 to 200 nm and featuring discernible lipid bilayer structures (Figure 4A). NTA indicated that the majority of Exo were approximately 120 nm in size (Figure 4B). Western blot analysis confirmed the presence of Exo markers CD63 and CD81 (Figure 4C), verifying their identity as Exo.
*Characterization of exo from ADSCs. (A) Morphology observed under TEM. (B) NTA analysis of exo size distribution. (C) Western blot was used to detect exosomes’ surface markers. (D) Real-time PCR was performed to analyze the expression of GATA4 mRNA in the exo group and the DLSW-Exo group. (E) Western blot was performed to assess the expression of GATA4 in the exo group and the DLSW-Exo group. *P < 0.05, *P < 0.01.
Real-time PCR was employed to assess GATA4 mRNA expression in the Exo and DLSW-Exo groups. Results demonstrated a significant upregulation of GATA4 mRNA expression in the DLSW-Exo group compared with the Exo group (P < 0.05) (Figure 4D). This finding was further corroborated by western blot analysis (P < 0.01) (Figure 4E).
Characterizations of magnetic CSNP-Exo
The magnetic CSNP-Exo developed in this study demonstrated rapid transformation from liquid to gel within 3 min, a crucial feature for bladder perfusion. Additionally, it exhibited effective magnetic targeting, enabling immediate adherence to the target (Figure 5A).
*Characterization of magnetic CSNP-Exo. (A) Demonstration of the magnetic property of CSNP-Exo. (B) FTIR spectra of CS and CSNP. (C) SEM images of CSNP. (D) SEM images of CSNP-Exo. (E) Rheological properties of CSNP analyzed with temperature and time changes. (F) Both CSNP gel and CSNP-Exo gel exhibited favorable swelling characteristics. (G) The cumulative release rate curve illustrates the sustained release properties of CSNP gel for exo. *P > 0.05, **P < 0.05, **P < 0.01.
To understand the binding mechanism, the FTIR spectra of CS and magnetic CSNP were analyzed (Figure 5B), revealing a peak around 3480 cm^−1^ associated with the O–H group. The superimposed O–H and N–H stretching vibration absorption peaks suggested the formation of hydrogen bonds between the N–H on the CSNP molecular chain and the O–H of GP [30].
SEM analysis revealed that the CSNP gel had a porous structure (Figure 5C), while the outer surface of the CSNP-Exo gel appeared rough and granular, probably due to Exo being embedded in the gel matrix (Figure 5D). As depicted in Figure 5E, the thermo-responsive hydrogel exhibited a gelation temperature of 28.57°C, and the gelation process took 4 min.
Swelling data (Figure 5F) indicated that both CSNP and CSNP-Exo gels swelled rapidly, reaching equilibrium after 2 h of incubation in PBS. The swelling ratios of the two gels were 198.5% ± 13.4% and 189.4% ± 9.3% (P > 0.05) at 2 h, and then plateaued. The differences at all time intervals were not significant (P > 0.05).
Transwell system was utilized to investigate the release of Exo. As shown in Figure 5G, the Exo group exhibited rapid release, with a cumulative release of 89.1% ± 7.3% at 1 day, indicating limited local retention of Exo. In contrast, the CSNP-Exo group displayed a slow release pattern, with a cumulative release of 54.1% ± 3.5% at 10 days. At each observation time point, there was a statistically significant difference in the cumulative release of Exo between the two groups (P < 0.01 at 1, 2, 4 and 6 days; P < 0.05 at 8 and 10 days). This suggested that the CSNP gel exhibited excellent local retention of Exo, and as the CSNP gel biodegraded, Exo was released from the gel system, exerting therapeutic effects.
CSNP-Exo restored damaged bladder function
To assess the therapeutic impact of CSNP-Exo on DBD, a DBD animal model was established, and bladder instillation of CSNP-Exo gel was performed. The study design flowchart is depicted in Figure 6A.
*(A) Flow chart of animal study design. (B) Conscious cystometry demonstrated representative normal and abnormal voiding patterns. Rats exhibiting a normal voiding pattern were significantly more prevalent in the CSNP-Exo group compared to the DBD group. **P < 0.01.
Representative cystometric graphs of rats exhibiting normal and abnormal voiding patterns are presented in Figure 6B. In the normal group, all rats displayed a normal voiding pattern, voiding 8–9 times per 30 min during cystometry. Conversely, rats in the DBD group exhibited an abnormal voiding pattern, voiding 1–2 times per 30 min, with unstable bladder activity before urination and higher maximum voiding pressure. A substantial proportion of rats in the CSNP-Exo group (64.3%, P < 0.01 vs. DBD group) showed a normal voiding pattern, with improved cystometric parameters compared to the DBD group, indicating maintained bladder contractile function (Table 1).
CSNP-Exo enhanced the regenerations of vascularization and innervation of bladder
ELISA results of bladder tissue demonstrated significantly higher expressions of GATA4 (P < 0.01), NGF (P < 0.001) and VEGF (P < 0.001) in the CSNP-Exo group compared with the DBD group (Figure 7A). Following bladder instillation, PKH26-labeled DLSW-Exo were observed in the bladder lumen, mucosal layer and submucosal layer of rats in the CSNP-Exo group on the first, third and fifth days after instillation (Figure 7B).
*(A) The expressions of GATA4, NGF and VEGF in the bladder tissue of rats after treatments were examined by ELISA. (B) PKH26-labeled DLSW-Exo were observed in the bladder lumen (green arrow), mucosal layer (white arrow), and submucosal layer (yellow arrow) of rats in the CSNP-Exo group on the first, third, and fifth days after instillation. (C) In the normal group, col IV staining revealed a continuous suburothelial capillary network, while in the DBD group, the network appeared fragmented and disappeared. The integrity of the network was enhanced in the CSNP-Exo group. (D and E) RECA-1 staining indicated a higher density of blood vessels in both the normal group and the CSNP-Exo group compared to the DBD group. The number of RECA-1-positive blood vessels was analyzed in all three groups. (F and G) Expressions of S100 in bladder tissues of the three groups were analyzed by staining and quantified by the number of positive pixels over total number of pixels of each slide. ***P < 0.01, ***P < 0.001.
Immunofluorescence staining of bladder tissue revealed a continuous and dense network of Col IV-positive capillaries beneath the urothelium in rats of the normal group (Figure 7C). In the DBD group, the continuity of the Col IV-positive capillary network was disrupted or deficient in certain areas. However, following treatment with CSNP-Exo, there was a significant improvement in the damaged Col IV staining.
RECA-1 staining was intensely positive in the submucosal layer of the normal group and CSNP-Exo group with minimal positivity in the DBD group (Figure 7D). The RECA-1-positive vascular density was significantly higher in the CSNP-Exo group compared with the DBD group (P < 0.001) (Figure 7E).
Results of counting S100-positive pixels showed that bladder instillation of CSNP-Exo reversed the decrease in S100 expression induced by high glucose (CSNP-Exo group vs. DBD group, P < 0.01) (Figure 7F and G).
Discussion
DLSW, acting as a mechanical force, applies significant pressure to cells at the lesion site, while cavitation effects lead to the expansion and bursting of microbubbles, resulting in shear forces on cells. This establishes the theoretical basis at the cellular level for the treatment mechanism of DLSW [31]. The cytoskeleton is recognized for its active role in transmitting extracellular signals to intracellular information. The intensity of mechanical stimulation is a crucial contributing factor, and under high-intensity shock wave application, the structure of ADSCs was found to be altered, leading to a notable reduction in F-actin fibers and cell death [32]. Therefore, appropriate mechanical stimulation intensity provides the necessary energy density to activate ADSCs. In the present study, DLSW did not visibly modify the morphology and F-actin cytoskeleton of ADSCs, nor did it disrupt the cellular phenotype of ADSCs, consistent with previous research [5, 14]. Potential explanations might include the possibility that the low energy and brief duration of DLSW stimulation are insufficient to modify the cytoskeletal structure and shape of stem cells. Alternatively, any subtle modifications might be undetectable through an optical microscope, or the effects could be temporary, dissipating once the external forces are removed, and causing the stem cells to return to their initial state.
Emerging evidence suggests that the effects of ADSCs are primarily mediated by their capacity to produce various bioactive molecules, including VEGF, NGF, CXC ligand 5 and brain-derived neurotrophic factor [6, 9]. In the present study, ADSCs were stimulated by DLSW to secrete VEGF and NGF. Furthermore, the intrinsic molecular pathways activated in ADSCs were also investigated in this study, which will be discussed below.
DLSW has been shown to regulate cellular signal transduction and impact the transcription and modification of intracellular proteins [33]. Specific cellular processes and molecules affected by DLSW include FAK [34], extracellular-signal-regulated kinase (ERK) [35], Wnt [36] and VEGF [37]. ADSCs can be activated by DLSW through the MAPK, PI-3K/AKT and NF-kappaB signaling pathways [5]. The KEGG analysis in the current study supported these findings. Additionally, DLSW treatment did not significantly impact the phosphorylation of ERK and JNK in ADSCs. Hence, it is plausible to suggest that the activation of ADSCs by DLSW occurs through the activation of the p38 MAPK signaling pathway.
FAK, a member of the integrin family and a component of integrin-based focal adhesion complexes, serves as a critical kinase for transducing signals from the extracellular matrix into the cell, thereby regulating cytoskeletal networking and cellular signaling [38]. Numerous studies have highlighted the significant role of FAK in the differentiation and proliferation processes of MSCs induced by DLSW [34, 38, 39]. The p38 MAPK pathway was implicated in mechanotransduction, with DLSW potentially modulating the integrin-p38 MAPK pathway to influence chondrocyte extracellular matrix synthesis [40, 41]. Additionally, the integrin-FAK-p38 MAPK pathway was instrumental in mitigating acute inflammation in human primary tenocytes by DLSW [39]. Our previous work demonstrated the in vitro activation of ADSCs by DLSW through the p38 MAPK pathway [5]. In the present study, we further observed that the activation of p38 MAPK was contingent on the upstream phosphorylation of FAK.
GATA4, as a downstream effector molecule of p38 MAPK, is primarily associated with cardiac differentiation [42], the development of the neural crest [43], and the initiation of inflammatory responses [44]. Furthermore, GATA4-modified MSCs have been shown to promote angiogenesis by enhancing the expression of VEGF [45]. In our investigation, DLSW promoted the growth of nerve fibers and the formation of blood vessels. Moreover, the upregulation of VEGF and NGF was reversed by inhibiting GATA4, providing further evidence that VEGF and NGF are the downstream molecules responsible for the therapeutic effects of GATA4. The regulation of VEGF by GATA4 forms a multi-dimensional network. Early evidence supports its direct binding to the promoter. For instance, chromatin immunoprecipitation assays and mutagenesis experiments on the promoter region have clearly demonstrated that GATA4 can directly bind to the VEGF gene promoter [46]. In contrast, recent genome-wide data indicate that regulation via distal enhancers may play a more central role [47]. The realization of this process relies not only on GATA4’s own DNA-binding ability but also requires coordination with numerous coactivators and is subject to the integrated regulation of complex upstream signaling networks. Compared with VEGF, experimental evidence regarding the direct regulation of NGF expression by GATA4 is extremely limited. Furthermore, in our future work, omics approaches including transcriptomics and proteomics will be applied to investigate alterations in the expression profiles of mediators, such as transcription factors, cytokines, lipids, and miRNAs, in Exo before and after DLSW treatment, thereby enabling a more precise characterization of the impact of DLSW on Exo. In summary, the phosphorylation of FAK enables the transmission of DLSW signals from the extracellular matrix into the cell, and subsequently to the nucleus via the p38 MAPK-GATA4 pathway, thereby triggering the activation of ADSCs. This activation leads to therapeutic effects, including the release of VEGF and NGF, among other mechanisms.
Initially, Exo were previously viewed as cellular waste products [48]. However, it is now widely acknowledged that they serve as essential mediators of intracellular communication by facilitating the transfer of proteins and genetic materials, such as mRNA, microRNA and DNA [49]. Recent evidence has indicated the critical role of Exo derived from MSCs in mediating the beneficial effects of MSCs in tissue regeneration [49]. Notably, therapeutic potential has been demonstrated for MSC-derived Exo in erectile dysfunction [25], skin wound healing [50], spinal cord injury [51] and heart repair and regeneration [52]. Exo derived from ADSCs offered benefits over ADSCs, including increased stability, lack of cellular content, slower decay of biological function and reduced immunogenicity [53]. In the present investigation, Exo exhibited a significant capacity to enhance the development of blood vessels in HUVECs. Furthermore, Exo derived from DLSW-preconditioned ADSCs demonstrated an enhanced capability to promote blood vessel formation, potentially attributed to the presence of GATA4 encapsulated within the Exo. However, utilizing Exo for DBD treatment still presents several challenges, including limited targeting specificity and a short bladder residence time due to urine flushing. Thus, an effective drug carrier is required to ensure the continuous delivery of Exo to the damaged tissues.
CS is an abundant natural alkaline polysaccharide known for its non-toxic, non-irritating nature and excellent biocompatibility and biodegradability. Despite its potential, the large molecular weight and poor solubility of CS limit its drug-carrying capacity and make it susceptible to in vivo environmental influences, impacting drug release and stability. Nanosizing CS was employed to improve drug adsorption, sustained release and stability [26, 54]. CSNP hydrogels, with their three-dimensional polymer networks and hydrophilic cavities, are pliable, viscoelastic and tissue-compatible, causing no harm to surrounding tissues when implanted. It was confirmed via immunohistochemistry and Masson’s trichrome staining that CSNP did not induce significant bladder inflammation or fibrosis (Supplementary Figure S2). To enhance the retention of CSNP in the bladder and prevent it from being washed away by urine, Fe_3_O_4_ was incorporated into CSNP in the present study to impart magnetic properties and improve its retention in the bladder. We previously found magnetic CS possessed magnetic targeting and sustained release properties [24].
In contrast to traditional bladder perfusion therapy, the release of Exo from the CSNP gel was gradual, exhibiting a weaker initial burst and sustained release over time as the gel degraded. Additionally, the incorporation of Exo did not compromise the good swelling performance of CSNP. Consequently, magnetic CSNP hydrogels hold significant promise for biomedical applications, including tissue repair and drug delivery.
Based on cystometric analysis, bladder function was partly restored by bladder instillation with CSNP-Exo, indicating it is a promising therapeutic choice. The extended presence of CSNP-Exo in the bladder cavity was crucial for its efficacy, and we observed penetration of some CSNP-Exo into the cells of the bladder mucosal and submucosal layers, ensuring sustained effects. One of the histological improvements was the restoration of the vasculature. The restoration of the “capillary network” in the submucosa of the bladder represented a notable improvement. We first reported the “capillary network” might be essential for the normal physiological properties of urothelium [2]. Exo, serving as carriers for therapeutic agents, facilitated the transportation of cytokines such as GATA4 to the injured tissue. This process led to elevated VEGF and NGF secretion, promoting vascular repair and nerve regeneration at the site of tissue damage. These findings align with those of prior studies [45]. Smooth muscle cell regeneration and fibrotic tissue remodeling are slow processes requiring sustained cellular replacement and paracrine regulation. For instance, histological analyses have shown that even in bladders with partial functional recovery, smooth muscle bundle arrangement, cell density and extracellular matrix ratio may not be fully restored to normal levels [55]. Consequently, in early stem cell therapy, despite histological evidence of incipient vascular and neural recovery, bladder wall stiffness and elasticity remain unaltered. This impairs the efficient conversion of detrusor muscle contractile force into urine evacuation momentum, leading to delayed improvement in residual urine volume.
Conclusion
The study validated our hypothesis that magnetic CSNP exhibited exceptional targeting, swelling, and sustained release properties. Furthermore, the findings indicated that utilizing Exo from ADSCs pretreated by DLSW enhanced bladder function in DBD animals. This improvement was attributed to the transportation of GATA4 by Exo via the FAK-p38 MAPK pathway, promoting vascular and nerve repair. Therefore, magnetic CSNP-Exo demonstrates promising potential in the treatment of DBD.
Supplementary Material
rbag007_Supplementary_Data
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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