Bone marrow mesenchymal stem cells reduce ureteral stricture formation in a rat model via the paracrine effect of extracellular vesicles

Abstract With no effective therapy to prevent or treat ureteral stricture (US), a multifactorial fibrotic disease after iatrogenic injury of the ureter, the need for new therapies is urgent. Mesenchymal stem cells (MSCs) have been widely studied for treating tissue defects and excessive fibrosis, and recent studies established that one of the main therapeutic vectors of MSCs is comprised in their secretome and represented by extracellular vesicles (EVs). Thus, we have determined to explore the specific role of MSCs‐derived EVs (MSC‐EVs) treatment in a pre‐clinical model of US. The results firstly showed that either a bolus dose of MSCs or a bolus dose of MSC‐EVs (administration via renal‐arterial) significantly ameliorated ureteral fibrosis and recuperated ureter morphological development in a US rat model. We confirmed our observations through MSCs or MSC‐EVs treatment alleviated hydronephrosis, less renal dysfunction and blunted transforming growth factor‐β1 induced fibration. Due to MSC‐EVs are the equivalent dose of MSCs, and similar curative effects of transplantation of MSCs and MSC‐EVs were observed, we speculated the curative effect of MSCs in treating US might on account of the release of EVs through paracrine mechanisms. Our study demonstrated an innovative strategy to counteract ureteral stricture formation in a rat model of US.

represent valid and successful treatments for complicated US. However, the major drawback of these techniques is the recurrent US because of the formation of hyperplastic muscle or scar tissue in anastomotic stoma. 2 Anti-fibrotic drugs, such as glucocorticoids, 5 captopril 6 and halofuginone 7 have been tested to limit re-stricturing after US surgery. Regrettably, none of these drugs exhibit validation of therapeutic benefit thus have not been applied in the clinic.
Mesenchymal stem cells (MSCs) have been widely studied for treating tissue defects and excessive fibrosis, due to their ability to differentiate into cells needed for tissue repair and secrete a broad range of bioactive molecules (eg, growth factors, cytokines and chemokines). 8,9 However, with the recognition that only limited MSCs are recruited in the injury site after MSCs transplanted, 10 many investigators have suggested its therapeutic effects on tissue repair might be rather by stimulating the activity of tissue-resident recipient cells via paracrine mechanisms. 11,12 Extracellular vesicles (EVs) are small membrane vesicles originating from multivesicular bodies, which have been identified as a new kind of major paracrine factor naturally secreted by cells. 12,13 EVs can be categorized into exosomes (40-200 nm), microvesicles (50-1000 nm) and apoptotic bodies (50-5000 nm), etc. 14 Recently, the therapeutic capacity of MSCs-derived EVs (MSC-EVs) to treat fibrosis has been studied extensively. These cell-free agents have shown promise in decreasing fibrosis in pre-clinical models of lung fibrosis, liver fibrosis, cardiac fibrosis, kidney fibrosis and skin fibrosis. [15][16][17][18][19] Based on above information, we hypothesized that MSC-EVs such as bone marrow MSCs-derived EVs could reduce US formation.
With this scope, we try to develop a rodent model mimicking US after iatrogenic ureteral injury and to investigate the efficacy of administration of MSC-EVs to prevent fibrosis.

| MSCs and MSC-EVs preparation
Sprague Dawley (SD) rat bone marrow MSCs line (Cat. No SCSP-402) was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). MSCs were cultured in a-Minimal Essential Medium (Gibco/Thermo Fisher Scientific) with 1% L-glutamine supplemented with 10% Exosome-depleted foetal bovine serum (System Biosciences, NO. 082615). Once at 80% confluence, MSCs were starved (replaced with serum-free medium) overnight and then the conditioned medium was collected. Then, MSCs-EVs from the supernatant were isolated by the ExoQuick TM (System Biosciences) reagent. EVs were further detected by a transmission electron microscopy analysis (Hitachi, Japan), nanoparticle tracking analysis (Malvern NanoSight, UK) and EVs characteristic surface marker proteins (CD9, CD63 and CD81). For subsequent animal experiment, a dose of 3.5 9 10 6 MSCs from a 100 mm cell culture dish (80% of cell confluent) was administered for one animal; and an EVs dosing (25 lg protein, quantitated by the Micro Bicinchoninic Acid Protein Assay Kit (Pierce), diluted with 100 lL PBS) was determined by corresponding to the amount produced by 3.5 9 10 6 MSCs equivalents from cell culture supernatants.

| Study design
We established an original US animal model using microscopic vascular clamp placed on the left proximal ureter and subsequently removed 6 hours later. Rats were randomly divided into four groups. Sham rats had their left ureters exposed but not clamped We preferred to do the injection using a 29-G insulin syringe (Becton, Dickinson and Company, USA) filled with 100 lL PBS/MSCs/ MSC-EVs.

| Magnetic resonance imaging assessment
All rats were scanned by Magnetic resonance imaging (MRI) at 2 weeks and 4 weeks following the treatment for assessing the presence of hydronephrosis. Based on the MRI images (sagittal, coronal and transverse section), the left renal pelvis volume (RPV) of each rat was quantified in accordance with a previously described method, 20 following the calculation formula: "maximum anteroposterior diameter" 9 "maximum length diameter" 9 "maximum transverse diameter" 9 0.523.

| Renal function test
Blood samples were collected from the inferior vena cava for detection of serum creatinine (Cr) and blood urea nitrogen (BUN) at 4 weeks after treatment. Cr and BUN were measured using Automated Chemistry Analyzers (Beckman Coulter Chemistry Analyzer AU5800, CA, USA) according to the manufacturer's instructions.

| Histology
After rats were killed, the stenotic tissue of the ureter segment (proximal and distal to the injury site, about 1.5 cm) and the homolateral kidney were harvested, fixed and further processed for histology.
Haematoxylin and eosin (HE) and immunohistochemical staining procedures were performed according to a standard protocol. The degree of US was evaluated by the quantification of the ureteral lumen diameter. We assessed the degree of tissue damage in the renal cortex and outer medulla (ie, tubular necrosis and tubular enlargements.) on a scale of 0 to 2 (none to marked) according to Toyohara et al's study. 21 The scoring of immunoreactivity was obtained by adding the score of positive cell percentages and staining intensities.

| qRT-PCR and Western blot
The mRNA and protein expression levels of collagen I (Col I), collagen III (Col III), fibronectin (Fib), transforming growth factor-b1 (TGF-b1) and Smad3 (phosphorylation of Smad3 (p-Smad3) was used in Western blot (WB)) in experimental ureter tissue samples were examined.
b-actin was employed as the internal normalization. The sequences of the PCR primers are as follows are listed in Table S1. All the relevant antibodies were purchased from Abcam Biotechnology.

| Statistical analysis
The results were analysed using SSPS 16.0 software (SPSS, Inc., Chicago, IL) and expressed as mean AE standard deviation of the mean.
One-way analysis of variance followed by Bonferroni's multiple comparison tests was used to evaluate whether differences between groups were significant. Statistical significance was set at P < .05.

| Characterization of MSC-EVs
Transmission electron microscopy revealed that EVs were grapelike clusters of vesicles with double layer membrane structure and diameters about 120 nm (

| Animals
One rat of the MSCs group died due to perioperative infection after microscopic vascular clamp implantation. The body weight of rats at initial and 2 weeks after treatment did not differ significantly among the 4 groups (P > .05, for all). At 4 weeks after treatment, the body weight of the Sham group was significantly greater than that of the other three groups (P < .05, for all). The body weight of MSCs and MSC-EVs groups increased a slight but not statistically as compared with the US group (P = 1.000 and 1.000, respectively; Table S2).

| Mri
As listed in Figure 3, the MSCs group and MSC-EVs treated group showed significantly less hydronephrosis in comparison with the US group at 2 weeks and 4 weeks after treatment. As expected, the Sham group had the lowest values of RPV, whereas the US group had the highest values of these parameters (Table 1). Notably, the RPV values were statistically alleviated in the MSCs as well as MSC-EVs treatment groups compared to the US group (P < .01 for all).
Furthermore, the MSC-EVs group had the better alleviation of hydronephrosis when compared to MSCs groups at 2 weeks and 4 weeks, but the difference was not statistically significant (P = 1.000, P = 1.000).

| Renal function
As shown in Figure S1A, MSCs and MSC-EVs rats exhibited lower but not statistically Cr than those in the US group at 4 weeks after injection (P = 1.000, P = .928). The BUN was significantly lower in

A B
F I G U R E 1 Ureteral stricture rat model. A, The upper ureter was exposed and positioned with a microscopic vascular clamp. B, After 3 d, the clamp was removed from the ureter via the initial incision LUO ET AL.   Figure 5D. Changes of renal structure in US rats were characterized by tubular dilation, tubular destroy, apoptosis and glomerulus atrophy, whereas these disorganizations were significantly improved in MSCs and MSC-EVs treated rats ( Figure 5C).
Corresponding histological scoring of the areas with tubular necrosis and tubular enlargements in each group were listed in Figure 5E and F.
TGF-b1 is a recognized fibrosis factor. Col I, one of the extracellular matrix molecules, is a key biomarker during fibrosis process.

| Gene and protein expression
Activation of TGF-b1/Smad3 pathways is a key mediator not only of epithelial-mesenchymal transition induction, but also the synthesis of extracellular matrix molecules such as Col I, Fib and Col III, leading to tissue fibrosis. As shown in Figure

| DISCUSSION
In the present study, we found that transplantation of a bolus dose of MSCs by intra-arterial route counteracts US formation through the anti-fibrotic action in a rat model. Importantly, we provided evidence that EVs derived from MSCs might be involved in this func-   Smads (ie, Smad3), that is the classic TGF-b1/Smad signalling leading to tissue fibrosis. 37 In the present study, we found that the stenotic ureter of MSCs and MSC-EVs groups had a greater than 36% and 45% reduction in the level of TGF-b1 protein expression compared with the US group as judged by WB. In addition, the protein expression of p-Smad3 had a trend similar to that of TGF-b1 protein expression when comparison was made in these groups. These data