Extracellular vesicles derived from human amniotic fluid stem cells improve bladder dysfunction in rat model of diabetic atherosclerosis

Abstract

BACKGROUND

The incidence of diabetic atherosclerosis (DMA) is increasing worldwide, but its pathogenesis remains incompletely understood. In addition to cardiovascular complications, bladder dysfunction is one of the common comorbidities associated with DMA but is often refractory to current treatments.

AIM

To investigate the therapeutic effect of human amniotic fluid stem cell-derived extracellular vesicles (hAFSC-EVs) on the recovery of bladder dysfunction in DMA rats.

METHODS

Eighty rats were divided into normal control, streptozotocin-induced diabetic rats, diabetic rats subjected to arterial balloon endothelial injury of common iliac artery (DMA), and DMA rats treated with hAFSC-EVs (DMA + hAFSC-EVs). At 4 weeks and 12 weeks after DMA induction, levels of blood glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, homeostasis model assessment (HOMA)-insulin resistance, and HOMA-β were measured. Cystometry, common iliac artery wall thickness, and bladder tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β1, Smad3, connective tissue growth factor (CTGF) and fibronectin were also evaluated.

RESULTS

Bladder weight and blood glucose, triglyceride, HOMA-insulin resistance, common iliac artery intima thickness, voided volume, intercontraction interval, bladder capacity, and mRNA expression of TNF-α, IL-6, TGF-β1, Smad3, CTGF and fibronectin were significantly increased at 4 weeks and 12 weeks after induction, while the HOMA-β level decreased at 4 weeks and 12 weeks, and the high-density lipoprotein cholesterol level decreased at 12 weeks. hAFSC-EVs treatment in DMA rats significantly reduced bladder weight and blood glucose, thickness of common iliac arterial intima, voided volume, intercontraction interval and bladder capacity at 4 weeks. The mRNA expression of TNF-α, TGF-β1, and CTGF in DMA rats treated with hAFSC-EVs were significantly decreased at 4 weeks, while the mRNA expressions of IL-6 and Smad3 were significantly decreased 12 weeks.

CONCLUSION

hAFSC-EVs treatment can help restore DMA-induced bladder dysfunction, which is associated with lowered blood glucose levels, reduced arterial wall thickness, and decreased TNF-α, IL-6, TGF-β1, Smad3, and CTGF expression.

Key Words: Atherosclerosis; Bladder; Diabetes; Exosome; Extracellular vesicles; Stem cell

Core Tip: In addition to cardiovascular complications, bladder dysfunction is one of the common comorbidities associated with diabetes, but is often refractory to current treatments. Human amniotic fluid stem cells (hAFSCs) demonstrated the efficacy in preclinical studies of diabetic bladder dysfunction and arterial atherosclerosis-induced bladder dysfunction. Here, we demonstrate that extracellular vesicles derived from hAFSCs could help restore diabetic atherosclerosis-induced bladder dysfunction, which is associated with lowered blood glucose levels, reduced arterial wall thickness, and decreased tumor necrosis factor-α, interleukin-6, transforming growth factor-β1, Smad3, and connective tissue growth factor expression. Our study highlights the potential of extracellular vesicles derived from hAFSCs in cell-free regenerative therapy of diabetic atherosclerosis.

INTRODUCTION

Diabetes mellitus (DM) is a serious and growing global health burden[1]. DM can cause serious cardiovascular diseases, such as stroke, myocardial ischemia and renal disease, all of which are related to atherosclerosis[2]. The incidence of diabetic atherosclerosis (DMA) is increasing worldwide; however, its pathogenesis remains incompletely elucidated. Although stem cells have demonstrated efficacy in clinical studies targeting glycemic control[3] and in preclinical studies on diabetic bladder dysfunction[4,5], effective therapies to halt the progression of DM and DMA in humans remain unavailable.

In addition to cardiovascular complications, bladder dysfunction is one of the most common comorbidities associated with DM. Approximately 80% of patients develop diabetic cystopathy[6], including detrusor overactivity, poor emptying, overflow incontinence, urgency and urgency incontinence. These symptoms are often refractory to current treatments and significantly impair quality of life. Our recent studies have shown that human amniotic fluid stem cells (hAFSCs) can improve bladder dysfunction in DM rats[5], and also improve ischemic bladder dysfunction induced by iliac arterial atherosclerosis[7]. Nevertheless, stem cell therapies remain limited by several factors, including potential risks of tumor formation, microvascular obstruction, and low engraftment rates of transplanted cells into ischemic tissues[8].

Extracellular vesicles (EVs) derived from stem cells have been shown to exert regenerative and functional effects comparable to those of their parent cells[9,10]. EVs are nanosized, membrane-bound particles, including exosomes, microvesicles, apoptotic bodies and growth factors[11,12]. They play a crucial role in intercellular communication. Since EVs can recapitulate the same beneficial responses of stem cell transplantation by delivering bioactive cargos without the need for cell transplantation, they offer significant advantages over conventional cell therapies as immunologically unresponsive agents[13]. In addition to lower immunogenicity, EVs exhibit reduced tumorigenic potential compared to traditional cell-based therapies, making them as attractive candidates for therapeutic applications in DM and related complications[14-16]. Recent studies have demonstrated that the beneficial effects observed after stem cell transplantation in several preclinical models of experimental ischemic disease may be mediated by EVs derived from stem cells[9]. These include the activation of anti-apoptotic and pro-survival pathways, induction of angiogenic, anti-inflammatory and anti-fibrotic responses, and stimulation of resident endogenous progenitor cells, leading to an overall enhancement of organ function[17]. Many studies have reported the potential regenerative effects of EVs from adult mesenchymal stem cells in providing cardioprotection against acute myocardial infarction[18], promoting wound healing[19], reducing renal injury[20] and stimulating neuroplasticity after stroke[21].

Almost all cell types can release EVs[22], but amniotic fluid appears to be a better source of EVs for clinical applications compared to adult stem cells such as bone marrow stem cells[23]. Although bone marrow mesenchymal stem cell-derived EVs have been reported to ameliorate diabetic-exacerbated atherosclerosis[24], the role of hAFSC-derived EVs (hAFSC-EVs), which are derived from amniotic fluid (a non-invasive method) and remain genetically stable even after long-term culture[25], in DMA-associated bladder dysfunction has not been reported. Several studies have explored the regenerative potential of hAFSC-EVs in models of chemotherapy-induced ovarian damage[26], skeletal muscle atrophy[9], and renal fibrosis in Alport syndrome[27], their effects on diabetes-induced bladder dysfunction remain unexplored. Therefore, this study aimed to investigate whether hAFSC-EVs can promote bladder function recovery in rats of DMA.

MATERIALS AND METHODS

Animal model and ethical approval

All protocols were approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of Chang Gung Memorial Hospital (No. 2019121712) and the Institutional Review Board of Linkou Chang Gung Memorial Hospital (No. 201902019A3). Experimental protocols also adhered to the ARRIVE guidelines to ensure quality and reproducibility. Female Sprague-Dawley rats (10-12 weeks old) were maintained under controlled conditions at a room temperature of 21-23 °C and 47% humidity, with a 12-hour light/dark cycle. They had ad libitum access to standard laboratory chow and tap water.

Rats in the study groups underwent bilateral ovariectomy via bilateral incisions. Following surgery, the control group was provided with a regular diet, whereas the study groups received a high-fat diet (60% kcal fat, D12492, Research Diets, New Brunswick, NJ, United States). After 4 weeks of dietary manipulation, 80 rats were divided into 4 groups (n = 20 per group): Normal control, streptozotocin (STZ)-induced diabetic rats (DM), DM rats subjected to arterial balloon endothelial injury (AEI) of the common iliac artery (DMA), and DMA rats treated with hAFSC-EVs (DMA + hAFSC-EVs). Immediately after DMA induction, the hAFSC-EVs transplanted group received a single tail vein injection of hAFSC-EVs [100 μg total protein in 0.5 mL phosphate buffered saline (PBS), approximately 1 × 10⁹ particles]. The treatment dose of hAFSCs was determined according to the previous study that used bone marrow-derived stem cells to treat diabetic kidney disease of STZ-diabetic rats[28].

At 4 weeks and 12 weeks following DM or DMA induction, bladder function was assessed using conscious cystometry (n = 10 per group at each time point). Serum glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, homeostasis model assessment insulin resistance (HOMA-IR) index and homeostasis model assessment-β (HOMA-β) index were also measured. The animals were then sacrificed, and the common iliac arteries and bladders were dissected. The common iliac artery wall thickness and the expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β1, Smad3, connective tissue growth factor (CTGF) and fibronectin in bladders were evaluated using real-time polymerase chain reaction (PCR). The experimental procedure is shown in Figure 1.

Figure 1 Flowchart of the experimental procedures. IV: Intravenous injection; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles; STZ: Streptozotocin; AEI: Arterial balloon endothelial injury; DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; HFD: High-fat diet; B/S: Blood sugar; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; HOMA-IR: Homeostasis model assessment insulin resistance; HOMA-β: Homeostasis model assessment-β; PCR: Polymerase chain reaction.

Induction of diabetes

Hyperglycemia was induced in experimental rats via intraperitoneal injection of STZ at a dose of 35 mg/kg[29,30], which resembles the condition of human type 2 DM. Rats in the control group were administered citrate buffer alone. Successful induction of DM was confirmed by measuring serum glucose levels at 3 days after STZ administration. Rats continued feeding on their respective diets until euthanasia, and blood glucose levels were measured with the ACCU-CHEK advantage blood glucose monitoring system (Roche Diagnostics, Indianapolis, IN, United States). Rats exhibiting fasting blood glucose levels of 300 mg/dL or higher (fasted for 12 hours before measurement) were considered diabetic and suitable for study.

Common iliac AEI

Following induction of inhalation anesthesia with isoflurane, the femoral artery was isolated, and a 2 Fr Fogarty balloon catheter (Edwards Lifesciences LLC, Irvine, CA, United States) was passed through the femoral artery into the common iliac arteries[31,32]. In order to induce endothelial injury, the balloon was inflated with air and subsequently retracted from the common iliac artery back the femoral artery. This procedure was repeated 10 times on each side.

hAFSCs isolation and characterization

The hAFSCs were obtained from amniotic fluid via routine amniocentesis from healthy pregnant donors. The cells were cultured in StemPro^® mesenchymal stem cell serum free medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, United States) and incubated at 37 °C with 5% carbon dioxide. The specific surface antigens of hAFSCs were characterized by flow cytometry, as previously described in our earlier work[5]. The cultured cells were trypsinized and stained with phycoerythrin-conjugated antibodies against CD44, CD73, CD90, CD105, CD117, and CD45 (BD PharMingen, CA, United States). The stained cells were subsequently analyzed using the Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany), and the results of flow cytometric analysis were shown in Supplementary Figure 1. hAFSCs from passages 4 to 8 were harvested and diluted in PBS to a final concentration of 3 × 10⁶ cells/0.3 mL.

Isolation and characterization of hAFSC-EVs

EVs were harvested from hAFSC-conditioned medium collected after 48 hours of serum-free culture. The conditioned medium was centrifuged at 1200 × g for 10 minutes at 4 °C. Next, the supernatant was processed by high-speed centrifugation in a SORVALL^® RC-5C PLUS Superspeed Centrifuge (US) 2 times at 10000 × g for 30 minutes at 4 °C[33]. The resulting supernatant was then passed through a 0.22-μm filter. The filtered liquid was ultracentrifuged in an Optima XE (Beckmann Coulter, CA, United States) at 100000 × g for 120 minutes at 4 °C using SW28 rotor. The precipitate was then next washed with PBS and ultracentrifuged at 100000 × g for 120 minutes at 4 °C. The EVs-enriched fraction was next reconstituted in 0.1-μm filtered PBS for further studies. To characterize hAFSC-EVs, isolate them from conditioned medium, and then use a combination of techniques: Transmission electron microscopy (TEM, Figure 2A) for morphology, western blot (Figure 2B) analysis for protein markers and nanoparticle tracking analysis (NTA, Figure 2C) for size and concentration.

Figure 2 Characterization of human amniotic fluid stem cell-derived extracellular vesicles. Transmission electron microscopy shows that human amniotic fluid stem cell-derived extracellular vesicles exhibit a bilayered spherical morphology with diameters ranging from 50 nm to 150 nm. Bars indicate 100 nm. Western blot analysis confirms the expression of exosomal surface markers CD63, CD81, and CD9. Nanoparticle tracking analysis reveals that the majority of human amniotic fluid stem cell-derived extracellular vesicles fall within the 50-150 nm size range. A: Transmission electron microscopy; B: Western blot analysis; C: Nanoparticle tracking analysis. hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles; hAFSCs: Human amniotic fluid stem cells.

TEM and NTA

The morphology of hAFSC-EVs was confirmed by TEM; while particle size and concentration were analyzed using NTA (NanoSight NS300, Malvern, United Kingdom). NTA measurements were performed in filtered PBS using NS Xplorer software for automated analysis. The data of hAFSC-EVs size distribution and concentration were collected using a NanoSight NS300 (Malvern Panalytical, United Kingdom). For TEM, EVs were fixed in 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH = 7.4), applied to copper-mesh grids, and negatively stained with 4% uranyl acetate. Samples were visualized on a HT7800 transmission electron microscope (Hitachi, Tokyo, Japan). Protein content was determined using a Micro BCA Protein Assay Kit (Thermo Fisher, MA, United States).

Western blot

Surface markers of hAFSC-EVs were analyzed using western blot method. Proteins were denatured in electrophoresis sample buffer and boiled at 95 °C for 5 minutes prior to running sodium-dodecyl sulfate gel electrophoresis. EVs and cell lysates (10-20 μg protein) were separated on 13% sodium-dodecyl sulfate gel electrophoresis gels, transferred to polyvinylidene fluoride membranes, and blocked before incubation with primary antibodies against CD9, CD63, CD81 (1:500, Santa Cruz Biotechnology, CA, United States), and calnexin (1:1000, ABclonal, China) overnight at 4 °C. After washing with Tween-Tris buffered saline, polyvinylidene fluoride membranes were incubated with HRP-conjugated secondary antibodies in Tween-Tris buffered saline at room temperature for 1 hour and visualized using a UVP BioSpectrum system (UVP, LLC, CA, United States).

Conscious cystometry

At 4 weeks and 12 weeks following DM induction, conscious rats were placed in metabolic cages (Med Associates, VT, United States) for cystometric evaluation according to previously established protocols[5]. Parameters assessed over five micturition cycles included peak voiding pressure, peak voided volume, intercontraction interval, bladder capacity, and residual volume. Data were processed using Cystometry Analysis Version 1.05 (Catamount Research, VT, United States).

Collection of blood samples for detecting the levels of serum component

The blood samples collected from all rats were centrifuged to separate the serum, and stored at -80 °C for according to previously the levels of relevant serum components[34]. Meanwhile, the visceral adipose tissue and other organs were harvested, weighed, rinsed in saline, immersed in liquid nitrogen, and immediately stored at -80 °C until further use. Automatic biochemical analyzer (HITACHI 7600-020, Hitachi Ltd., Tokyo, Japan) was used to measure the levels of low-density lipoprotein cholesterol, HDL-C, triglyceride, and total cholesterol. Additionally, fasting insulin levels were determined using a radioimmunoassay kit (Atom Hi-Tech Co., Ltd., China). HOMA-IR index and HOMA-β index were used for estimating insulin resistance and the function of β-cells, respectively.

Histological examination of blood vessels

Animals were sacrificed immediately after cystometry, and the common iliac arteries and bladders were harvested. Cross-sections of the common iliac arteries were fixed in freshly prepared 4% paraformaldehyde for 2.5 hours at 4 °C. The specimens were then transferred to 25% sucrose in phosphate buffer at 4 °C until fully equilibrated. The specimens were then fixed in optimal cutting temperature compound and sectioned into 10-μm slices for staining with hematoxylin and eosin staining. The thickness of common iliac arterial wall was determined by averaging measurements from 4 distinct locations per sample[32].

Real-time PCR of bladder

Total RNAs were extracted using a Trizol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. Reverse transcription mixture was performed at 25 °C for 5 minutes, 50 °C for 1 hour, 70 °C for 15 minutes, and then cooled to 4 °C for 5 minutes. Gene expression of TNF-α, IL-6, TGF-β1, Smad3, CTGF and fibronectin in bladder tissues were analyzed by real-time PCR using inventoried TaqMan assays from Applied Biosystems (Life Technologies, Grand Island, NY, United States). The assay codes of TNF-α, IL-6, TGF-β1, Smad3, CTGF, and fibronectin were Rn01525859_g1, Rn01410330_m1, Rn00572010_m1, Rn00565331_m1, Rn01537279_g1 and Rn00569575_m1, respectively (Applied Biosystems, Oster City, CA, United States). The GAPDH assay code (Rn99999916_s1) was used as an endogenous control to allow for semi-quantification of relative gene expression. TaqMan Universal PCR Master Mix Kit and ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, United States) were used for measurement. Relative gene expression levels were calculated using the 2^-ΔΔCt method. Data were expressed as mean ± SD and compared between normal control group and each experimental time point.

Statistical analysis

Data were analyzed using Prism 9 (GraphPad Software, CA, United States) and presented as mean ± SD. One-way ANOVA was applied to assess differences among groups, with post hoc comparisons conducted using the Tukey-Kramer method. A P value < 0.05 was considered statistically significant.

RESULTS

hAFSC-EVs improve bladder weight and blood glucose level in DMA rats

Compared with the normal control group, the bladder weight, blood glucose, triglyceride, and HOMA-IR of the DM and DMA rats were significantly increased at 4 weeks and 12 weeks after induction, while the HOMA-β level decreased at 4 weeks and 12 weeks after induction, and the HDL-C level decreased at 12 weeks. Compared with untreated rats, DMA rats treated with hAFSC-EVs showed significant reductions in bladder weight and blood glucose at 4 weeks after induction, but no significant decreases after 12 weeks, which may be because the effects of a single injection of EVs did not persist for 12 weeks (Table 1).

Table 1 General characteristics and fasting serum parameters, mean ± SD.

Parameter	4 weeks				12 weeks
	Normal	DM	DMA	DMA + hAFSC-EVs	Normal	DM	DMA	DMA + hAFSC-EVs
Body weight (initial; gm)	255.30 ± 28.04	248.40 ± 7.38	256.79 ± 23.59	235.52 ± 7.95	256.73 ± 20.53	256.99 ± 10.66	257.90 ± 15.08	246.40 ± 9.52
Body weight (final; gm)	360.98 ± 20.36	335.24 ± 46.20	400.47 ± 49.36	338.70 ± 8.06	383.88 ± 37.12	361.81 ± 61.66	384.80 ± 9.62	350.90 ± 11.03
Bladder weight (mg)	128.75 ± 13.15	176.25 ± 14.93a	185.80 ± 30.01a	131.25 ± 25.29c	112.50 ± 22.17	183.13 ± 28.78a	214.38 ± 41.18a	193.60 ± 29.41a
Glucose (initial; mg/dL)	94.33 ± 7.97	93.38 ± 9.35	98.38 ± 15.30	115.00 ± 15.48	94.14 ± 8.43	93.20 ± 19.76	98.62 ± 13.62	102.40 ± 9.50
Glucose (final; mg/dL)	147.40 ± 22.63	328.71 ± 72.18a	391.67 ± 50.87a	223.80 ± 63.06b,c	135.33 ± 9.03	282.75 ± 46.25a	281.08 ± 64.84a	298.50 ± 80.98a
Total cholesterol (mg/dL)	88.25 ± 7.27	91.40 ± 7.73	99.00 ± 13.00	78.60 ± 19.11	78.00 ± 7.91	97.33 ± 10.41	91.09 ± 16.45	73.40 ± 14.55
Triglyceride (mg/dL)	64.00 ± 14.14	157.50 ± 15.00a	161.50 ± 16.26a	132.00 ± 12.29a	59.00 ± 12.41	162.50 ± 27.00a	157.25 ± 4.03a	145.60 ± 22.35a
HDL-C (mg/dL)	34.33 ± 2.07	24.83 ± 5.64a	24.83 ± 4.67a	26.83 ± 2.48a	30.00 ± 3.83	27.86 ± 3.85	27.10 ± 3.60	25.20 ± 3.70
LDL-C (mg/dL)	5.33 ± 0.58	4.60 ± 0.89	5.67 ± 1.21	5.00 ± 1.15	4.20 ± 0.45	4.33 ± 0.52	5.11 ± 1.17	3.75 ± 1.26
Insulin (μU/mL)	7.33 ± 0.22	7.49 ± 0.76	7.65 ± 0.88	7.19 ± 0.27	7.09 ± 0.18	7.38 ± 0.93	7.79 ± 0.97	7.83 ± 0.51
HOMA-IR	2.51 ± 0.20	5.84 ± 0.77a	7.09 ± 1.25a	4.92 ± 1.79a	2.11 ± 0.49	5.73 ± 0.82a	6.32 ± 0.60a	6.20 ± 1.80a
HOMA-β	35.94 ± 7.96	10.90 ± 2.52a	9.02 ± 1.86a	16.18 ± 7.18a	35.89 ± 2.07	12.38 ± 2.58a	12.17 ± 4.83a	12.39 ± 5.48a

^aP < 0.05 vs normal control (4 weeks and 12 weeks).

^bP < 0.05 vs diabetes mellitus (4 weeks and 12 weeks).

^cP < 0.05 vs diabetic atherosclerosis (4 weeks and 12 weeks).

DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; HOMA-IR: Homeostasis model assessment insulin resistance; HOMA-β: Homeostasis model assessment-β.

hAFSC-EVs improve bladder dysfunction subsequent to DMA

At 4 weeks and 12 weeks after induction, the peak voided volume, intercontraction interval and bladder capacity were significantly increased in both DM and DMA groups compared with the control group, while the residual volume was elevated only in the DM group (Figure 3A-E). Furthermore, compared with the DM group, DMA rats had significantly reduced residual volume at 4 weeks and 12 weeks after induction and the peak voided volume was decreased at 12 weeks. In DMA rats treated with hAFSC-EVs, the peak voided volume, intercontraction interval and bladder capacity were significantly reduced at 4 weeks after induction. Figure 3F shows representative cystometry tracings of the normal control, DM, DMA, and DMA + hAFSC-EVs groups at 4 weeks and 12 weeks.

Figure 3 Cystometric evaluation of bladder function. A-F: At 4 weeks and 12 weeks after induction, peak voided volume, intercontraction interval and bladder capacity were significantly increased in both diabetes mellitus (DM) and diabetic atherosclerosis (DMA) groups compared with the control group, whereas residual volume was elevated only in the DM group. Furthermore, compared with the DM group, DMA rats had significantly reduced residual volume at 4 weeks and 12 weeks after induction, and peak voided volume was decreased at 12 weeks. In DMA rats treated with human amniotic fluid stem cell-derived extracellular vesicles, peak voided volume, intercontraction interval and bladder capacity were significantly reduced at 4 weeks after induction. P values are shown in each comparison. Cystometric variables include: Peak voiding pressure (A); peak voided volume (B); intercontraction interval (C); bladder capacity (D); residual volume (E); representative cystometry tracings for normal, DM, DMA, and DMA + human amniotic fluid stem cell-derived extracellular vesicles groups at 4 weeks and 12 weeks after induction (F). ^aP < 0.05 vs normal control, ^bP < 0.05 vs diabetes mellitus, ^cP < 0.05 vs diabetic atherosclerosis. DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles.

hAFSC-EVs improve arterial wall thickness in DMA rats

Hematoxylin and eosin staining of the common iliac arteries shows that the intima thickness of the DMA group was significantly increased compared with both the control and DM groups. However, the intima thickness of DMA rats treated with hAFSC-EVs was significantly reduced at 4 weeks after induction (Figure 4).

Figure 4 Histological findings of the common iliac arteries. The intima thickness of the common iliac arteries in the diabetic atherosclerosis group significantly increased compared with the control and diabetes mellitus groups. However, the intima thickness of diabetic atherosclerosis rats treated with human amniotic fluid stem cell-derived extracellular vesicles was significantly reduced at 4 weeks after induction. Bars indicate 400 μm. A: Hematoxylin and eosin staining of the common iliac arteries; B: Common iliac artery wall thickness. ^aP < 0.05 vs normal control, ^bP < 0.05 vs diabetes mellitus, ^cP < 0.05 vs diabetic atherosclerosis. DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles.

hAFSC-EVs attenuate the expression of TNF-α, IL-6, TGF-β1, Smad3, CTGF and fibronectin in DMA rats

Compared with the control group, DM and DMA bladders showed significant increases in the mRNA expression of TNF-α, IL-6, TGF-β1, Smad3, CTGF and fibronectin at 4 weeks and 12 weeks after induction. Compared with the DM and DMA groups, the mRNA expression of TNF-α, TGF-β1, and CTGF in DMA rats treated with hAFSC-EVs were significantly decreased at 4 weeks after induction, while the mRNA expression of IL-6 and Smad3 were significantly decreased 12 weeks (Figure 5).

Figure 5 Relative mRNA expressions of tumor necrosis factor-α, interleukin-6, transforming growth factor-β1, Smad3, connective tissue growth factor, and fibronectin in the bladder of control and diabetic groups. A-F: Compared with the control group, diabetes mellitus and diabetic atherosclerosis (DMA) bladders showed significant increases in the mRNA expression of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1), Smad3, connective tissue growth factor (CTGF), and fibronectin at 4 weeks and 12 weeks after induction. Compared with the diabetes mellitus and DMA groups, the mRNA expression of TNF-α, TGF-β1, and CTGF in DMA rats treated with human amniotic fluid stem cell-derived extracellular vesicles were significantly decreased at 4 weeks after induction, while the mRNA expression of IL-6 and Smad3 were significantly decreased 12 weeks. ^aP < 0.05 vs normal control (4 weeks and 12 weeks), ^bP < 0.05 vs diabetes mellitus (4 weeks and 12 weeks), ^cP < 0.05 vs diabetic atherosclerosis (4 weeks and 12 weeks). TNF: Tumor necrosis factor; IL: Interleukin; TGF: Transforming growth factor; CTGF: Connective tissue growth factor; DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles.

DISCUSSION

Our results demonstrated that bladder weight and residual urine volume were significantly higher in DM rats compared with the control group at 4 weeks and 12 weeks after induction. Increased bladder weight and residual urine volume in hyposensitive DM rats indicate incomplete bladder emptying, whereas residual urine volume in DMA rats was also increased, but the increase was not significant. Experimental studies have demonstrated that the effects of STZ-induced DM on bladder function and bladder mass may appear shortly after DM onset, peak within 7 days to 14 days, and then remain relatively stable for up to 8 weeks[35,36]. In DM rats, peak voiding pressure was decreased and residual urine volume was increased between 12 weeks to 20 weeks following STZ treatment[37]. Our results revealed that the peak voided volume, intercontraction interval and bladder capacity were significantly increased in both DM and DMA rats compared with normal controls. Previous studies have also reported similar results, namely that the intercontraction interval increases between 4 weeks and 12 weeks following STZ administration[38]. However, Sasaki et al[39] demonstrated that the intercontraction interval in the DM rats was significantly shorter than that in the controls at 4 weeks and 12 weeks after STZ administration. These findings regarding intercontraction interval in DM rats highlight the need for additional research.

In this study, chronic bladder ischemia was established by inducing luminal atherosclerosis through balloon injury to the common iliac arteries combined with a high-fat diet over an 8-week period. This animal model demonstrated that DM and DMA rats exhibited thickening of the common iliac artery intima, along with increased voided volume, intercontraction interval and bladder capacity. However, DMA rats exhibited significantly lower residual urine volume and voided volume at 4 weeks and/or 12 weeks after induction compared with the DM group. A previous study demonstrated that cystometric evaluations in AEI rats without DM revealed decreased peak voiding pressure, voided volume and intercontraction interval, along with increased residual urine amount[8].

Several studies have reported the beneficial effects of stem cells in improving diabetic bladder dysfunction[4,5]. Our previous study demonstrated that diabetic bladder dysfunction in STZ-induced DM rats can be improved by hAFSCs treatment, which may be related to the recovery of bladder nerve growth factor and muscarinic receptors[5]. Zhang et al[4] observed that adipose-derived stem cells improve diabetic bladder dysfunction in STZ-induced DM rats through paracrine effects and some cellular differentiation following stem cell transplantation. In our previous study, neointimal hyperplasia of the common iliac arteries in AEI rats was significantly reduced after hAFSCs transplantation for 7 days, suggesting that hAFSCs treatment may have the potential to attenuate atherosclerosis progression[7]. Although hAFSCs have demonstrated remarkable regenerative potential in various preclinical disease models[5,40,41], only a few studies have reported hAFSC-EVs to date[9,26,27]. Stem cell-derived EVs represent a novel cell-free therapy for the corresponding cells. Compared to their parental cells, EVs have fewer membrane proteins in number, smaller size, and lower risk of immunogenicity. This study demonstrated that DMA rats treated with hAFSC-EVs exhibited significant reductions in common iliac artery wall thickness, bladder weight, and blood glucose levels at 4 weeks after induction compared with untreated DMA rats. In addition, in DMA rats treated with hAFSC-EVs showed significant decreases in peak voided volume, intercontraction interval and bladder capacity were significantly decreased.

Our results showed that the mRNA expression levels of TNF-α, IL-6, TGF-β1, Smad3, CTGF, and fibronectin were significantly increased in DM and DMA rats at 4 weeks and 12 weeks after induction compared with the control group. A previous study demonstrated that the expressions of 8-hydroxy-2’-deoxyguanosine, malondialdehyde and TNF-α were elevated in AEI rats, which improved following hAFSCs treatment[7]. An experimental study demonstrated that hypoxia induced by partial bladder outlet obstruction-induced hypoxia significantly increased hypoxia-inducible factor-1 alpha mRNA expression and promoted other pro-inflammatory cytokines, TGF-β1 and IL-1β[42]. Elevated TGF-β1 may activate TGFβ-Smad signaling pathway[43], which in turn may promote bladder fibrosis secondary to partial bladder outlet obstruction[44]. Activation of the TGFβ-Smad pathway can lead to downstream activation of CTGF, which modulates cell growth and collagen synthesis. Moreover, tissue hypoxia has been shown to induce the expression and secretion of CTGF. In this study, the mRNA expression levels of TGF-β1, Smad3, and CTGF were significantly downregulated in DMA rats treated with hAFSC-EVs at 4 weeks and/or 12 weeks after DMA induction, indicating that hAFSC-EVs may suppress activation of the TGFβ-Smad pathway. However, we must acknowledge that lack of validation at the protein level prevents us from concluding that the improvement of DMA-induced bladder dysfunction is mechanistically achieved through inactivation of the TGFβ-Smad pathway.

While these findings are promising, the study has several limitations. First, as a preclinical study in a rodent model, the results may not be directly translatable to human DMA patients. Second, this study used female rats as subjects to investigate the therapeutic effect of hAFSC-EVs on the recovery of bladder dysfunction in DMA rats. Whether the female rat model is applicable to male rats requires further investigation. Female rats were selected for this study because our previous study[5] explored the therapeutic effect of hAFSCs on bladder dysfunction in type 2 DM rats. To understand the difference in the effects of hAFSC-EVs and hAFSCs in treating bladder dysfunction in type 2 DM rats, we used the same female rats for comparison. Third, lack of a “DMA + vehicle” control group in this study, which cannot rule out that improvements in bladder function/vascular injury are due to PBS injection (e.g., volume effects) rather than hAFSC-EVs. However, our previous study on the improvement of bladder dysfunction by hAFSCs treatment showed that hAFSCs could improve bladder function in rats with partial bladder outlet obstruction (pBOO + hAFSCs), but PBS alone (pBOO + PBS) did not improve bladder function in pBOO rats[45]. Fourth, this study lacked protein-level verification. mRNA levels do not always correlate with protein activity in key TGFβ-Smad pathway proteins (for example, Smad3 requires phosphorylation for activation).

CONCLUSION

This study is the first to investigate the use of hAFSC-EVs as a potential treatment for diabetic bladder dysfunction. Results show that hAFSC-EV treatment can help restore DMA-induced bladder dysfunction, which is associated with lowered blood glucose levels, reduced arterial wall thickness, and decreased expression of TNF-α, IL-6, TGF-β1, Smad3, and CTGF. The mechanism by which hAFSC-EVs transplantation improves bladder dysfunction requires further investigation.

ACKNOWLEDGEMENTS

The authors wish to thank the technical assistance of the Microscopy Core Laboratory of Chang Gung Memorial Hospital, Linkou, Taiwan for their technical assistance. We also thank the Laboratory Animal Center of Chang Gung Memorial Hospital, Linkou, Taiwan, for providing animal husbandry and molecular imaging technical support.