Primary hBSMCs (Cat. No. 4310, ScienCell, San Diego, CA, USA) were cultured with special smooth muscle cell medium (SMCM, ScienCell), supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL) in a humidified atmosphere with 5% CO₂. Mycoplasma testing has been done for the hBSMCs in cell culture, and hBSMCs were identified by immunofluorescence. Two to six passages of primary hBSMCs were used for all experiments. Selective ADRB1 agonist, dobutamine hydrochloride (D0676), ADRB2 agonist, formoterol fumarate dihydrate (F9552), ADRB3 agonist, BRL 37344 (B169), ADRB3 antagonist SR 59230A (S8688), selective inhibitor of PKA-H89 dihydrochloride hydrate (B1427), specific inhibitor of EPAC ESI-09 (SML0814) were purchased from Sigma (St. Louis, MO, USA). Selective ADRB1 antagonist Metoprolol (ab120711) and ADRB2 antagonist ICI 118551 (ab120808) were obtained from Abcam (Pudong, Shanghai, China). Selective SMAD2 inhibitor LY2109761 (HY-12075) and SMAD3 inhibitor SIS3 (HY-13013) were purchased from MCE (Shanghai, China). The solvent of all the above drugs was Dimethyl sulfoxide (DMSO), so the control group was also treated with DMSO accordingly. ADRB2 agonist Procaterol was obtained from Otsuka (Guangdong, China), and ADRB3 agonist Mirabegron was purchased from Astellas (Tokyo, Japan). hBSMCs were preincubated for 60 min with ADRB agonist before the application of pressure or preincubated for 60 min with the selective ADRB antagonist, protein kinase A (PKA) or exchange protein directly activated (EPAC) inhibitor before the treatment of ADRB agonist.

hBSMCs cultured in 6-well plates at 80–90% confluence were exposed to hydrostatic pressure using a custom-designed motorized pressure apparatus, which was placed in a CO₂ incubator with the adapted condition for cell growth [21]. hBSMCs in the CO₂ incubator can be exposed to the hydrostatic pressure of 0–100 cm H₂O. After pilot experiments, 100 cm H₂O is the optimal condition for the treatment of hydrostatic pressure. hBSMCs in the control group were exposed to a static condition of 0 cm H₂O. The pressure sensor in the chamber conducted the pressure data to the computer every hour and constant pressure can be obtained by regulating the inlet and outlet valves of the chamber. Moreover, the parameters of culture medium, such as PH, PCO₂ and PO₂ can be monitored by the Abbott i-stat 300 hand-held Blood Gas Analyzer (Abbott, Princeton, NJ, USA).

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was eluted in 40 µL nuclease-free water and stored at –80 °C. RNA concentration was determined by the Spectrophotometer (IMPLEN Nanophotometer, München, Germany). Then, cDNA was synthesized using Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time quantitative PCR was performed by using Bio-Rad CFX Manager^TM software version 3.1 (CFX96, Bio-Rad, Hercules, CA, USA) and the specific primers were listed in Table 1. The housekeeping gene GAPDH was used as an internal control. The polymerase chain reaction (PCR) reactions were performed using IQ^TM SYBR Green Supermix reagent (Bio-Rad) under the following conditions: 95 °C for 3min and 40 cycles of 95 °C for 10 s, and 55 °C for 30 s. The real-time data were analyzed by the 2^{-Δ⁢Δ⁢Ct} method. Experiments were performed in triplicate.

Membrane proteins were extracted as per the protocol of a special membrane protein kit from Thermo Scientific (Men-PER^TM Plus, Waltham, MA, USA, 89842). The treated cells were scraped off the petri dish and further cleaned with cell wash solution. Next, the cells were incubated with permeabilization buffer for 10 min at 4 °C and centrifuged for 15 min at 16,000 g. Furthermore, the cells were incubated with solubilization buffer for 30 min at 4 °C and centrifuged for 15 min at 16,000 g. Finally, the solubilized membrane and membrane-associated proteins were extracted in the supernatant. The proteins were detected using primary antibodies against three subtypes of ADRBs. Total proteins were extracted using the RIPA buffer (Biosharp, Hefei, China) containing protease inhibitors and phosphatase inhibitors. Proteins (20 µg) were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bio-Rad) and transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% skimmed milk for 1 h and incubated with the primary antibodies at 4 °C overnight followed by secondary anti-rabbit IgG or anti-mouse IgG (MBL, Nagoya, Japan) for 1 h at RT. The primary antibodies used for western blotting were as follows: ADRB1 (ab3442, Abcam, Cambridge, UK; 1:1000), ADRB2 (ab13989; Abcam; 1:1000), ADRB3 (ab76249; Abcam; 1:1000), SMAD2 (ab40855; Abcam; 1:2000), SMAD3 (ab40854; Abcam; 1:1000), p-SMAD2 (phospho S467; ab53100; Abcam; 1:500); p-SMAD3 (phospho S423+S425; ab52903; Abcam; 1:2000); Collagen I (ab34710; Abcam; 1:1000); Collagen III (ab7778; Abcam; 1:5000); Fibronectin (ab2413; Abcam; 1:1000); anti-$\alpha$-tubulin (ab7291; Abcam; 1:5000); Na⁺/K⁺-ATPase (252101 ZenBio, Chengdu, China 1:1000). Then the membranes were exposed and the grey intensities of the bands was quantified using Gel-pro software version 6.3 (Media Cybernetics, Rockville, MD, USA).

A total of 24 eight weeks old female Sprague-Dawley rats (weight 200–250 g, purchased from Dasuo Experimental Animals Limited Company, Chengdu, China) were divided into four groups randomly: sham-operated group, pBOO group, pBOO+Procaterol group, pBOO+Mirabegron group. The steps of pBOO rat model are as flows: Rats were anesthetized with isoflurane (5% in oxygen). A urethral catheter (1 mm diameter) was placed through the urethra of anesthetized female rats, and then 3-0 silk was used to ligate around the proximal urethra to simulate pBOO, the catheter was carefully removed finally. The sham group underwent the same procedure except for urethral ligation. All rat groups were treated one day after the operation. The rats of the sham-operated group and pBOO group were intragastrically administered with normal saline, and rats of the BOO+Procaterol group or BOO+Mirabegron group were intragastrically administered with Procaterol at 0.01 mg/kg/d or Mirabegron at 5.0 mg/kg/d respectively (the dosage was determined based on the equivalent dose of clinical application). Then the bladder tissue was harvested after rats were sacrificed with CO₂ 3 weeks later.

The bladder tissue of rats was fixed with 4% paraformaldehyde, bedded in paraffin, then cut into 4 µm sections. The sections were stained using Masson’s trichromatic staining kit (Solarbio, Beijing, China). The images of collagen accumulation of fibrotic lesions were obtained at 100 $\times$ magnification in 5 different fields and analyzed the proportion of collagen area.

The paraffin sections of bladder tissue were immersed in the distilled water, washed with PBS–T, and then blocked with 3% peroxide-methanol at room temperature (RT) for endogenous peroxidase ablation. Sections were incubated in primary antibody COL1 (ab34710; Abcam, Cambridge, UK; 1:100), FN (ab2413; Abcam; 1:50) respectively overnight at 4 °C. Then the sections were washed with PBS and incubated in rabbit IgG antibody-horseradish peroxidase complex (ab6721; Abcam; 1:10,000) for 30 min at RT, after which the sections were reacted with diaminobenzidine and were preserved for additional analysis.

The results were presented as means $\pm$ SD. The data were analyzed by Student t-test or a one-way analysis of variance (ANOVA) by using GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA). p 0.05 was considered as the statistical difference.

To investigate whether the pathological hydrostatic pressure in vitro stimulated the ECM expression of hBSMCs, cells were cultured in a hydrostatic apparatus with sustained hydrostatic pressure of 40 cm H₂O. The expression of ECM genes including COL1, COL2, COL3, COL4, FN was detected. Firstly, COL2 and COL4 expression was relatively low in hBSMCs, indicating these two proteins may not the predominant components of hBSMC ECM. Secondly, the pressure of 40 cm H₂O cannot upregulate the expression of hBSMC ECM (p $>$ 0.05) (Fig. 1A). Expression of COL2 and COL4 in hBSMCs qPCR detection showed that the number of PCR amplification cycles of the two genes COL2 and COL4 was more than 30 cycles (Cq value $>$ 33), indicating low expression levels in hBSMCs. Therefore, in subsequent studies, Changes in COL2 and COL4 expression were no longer detected (Supplementary Fig. 1). These results were consistent with the previous report [22]. Hence, sustained hydrostatic pressure of 100 cm H₂O was used to study the expression of hBSMC ECM. The mRNA levels of COL1, COL3, FN did not change significantly at 2 h, and significantly increased at 6 h and 12 h under 100 cm H₂O (100 cm H₂O 6 h/12 h vs. Ctrl, p 0.01) (Fig. 1B). To keep the cells under good condition, we treated hBSMCs with 100 cm pressure at 6 h, and the results of Western blot (WB) were consistent with the previous experiments (100 cm H₂O 6 h vs. Ctrl, p 0.01) (Fig. 1C). Therefore, the hBSMCs were treated with the hydrostatic pressure of 100 cm H₂O for 6 h for the following experiments.

Fig. 1.

100 cm H₂O hydrostatic pressure increases COL1, COL3 and FN expression in hBSMCs. (A) Hydrostatic pressure of 40 cm H₂O cannot upregulate the expression of hBSMC ECM compared to the control (0 cm H₂O). (B) The mRNA expression of COL1, COL3 and FN increased in hBSMCs under 100 cm H₂O hydrostatic pressure for 6 h and 12 h. (C) The protein levels of COL1, COL3 and FN in hBSMCs were determined by western blotting and the grey intensities of the brands were shown. Ctrl, control group; controlHP, hydrostatic pressure; FN, fibronectin; hBSMCs, human bladder smooth muscle cells. Three independent experiments have been performed. **, p 0.01.

To explore the relationship between the expression of ADRBs and hydrostatic pressure in hBSMCs, the mRNA levels of three subtypes of ADRBs were detected in hBSMCs exposed to the hydrostatic pressure of 100 cm H₂O for 6 h. The results showed that the mRNA expression of ADRB1 did not change significantly, and the expression of ADRB2 and ADRB3 significantly decreased at 6 h under hydrostatic pressure when compared to the control group (Fig. 2A, p 0.05). ADRB is a membrane protein receptor, and Na⁺/K⁺-ATPase is used as an internal control of membrane protein [23]. The protein levels of ADRBs in hBSMCs exposed to hydrostatic pressure for 6 h were consistent with its mRNA levels (Fig. 2B, p 0.05). Thus, hydrostatic pressure could suppress the expression of ADRB2 and ADRB3 in hBSMCs at 6 h. Importantly, this result is also consistent with the microarray analysis of our previous study using a 3-week pBOO rat model [24]. So the expression of ADRB2 and ADRB3 in hBSMCs was markedly suppressed by the hydrostatic pressure.

Fig. 2.

Hydrostatic pressure suppresses the expression of ADRB2 and ADRB3. (A) The mRNA expression of ADRB2 and ADRB3 was decreased at 6 h in hBSMCs under hydrostatic pressure when compared to the control. (B) The protein levels of ADRBs were identified by western blotting, and Na⁺/K⁺-ATP was used for normalization. HP, hydrostatic pressure. Three independent experiments have been performed. *, p 0.05.

To investigate the relationship among ADRBs, hBSMCs ECM and hydrostatic pressure, the agonists or antagonists of ADRBs were used to study their effects on hBSMCs ECM under hydrostatic pressure, and the optimal concentrations of these reagents were determined as per their half maximal inhibitory concentration (IC₅₀) or concentration for 50% of maximal effect (EC₅₀). According to our previous study [24, 25], the optimal concentration of ADRB1 antagonist Metoprolol 200 nM (IC₅₀ for ADRB1 120 nM), ADRB2 antagonist ICI 118551 10 nM (IC₅₀ for ADRB2 1.2 nM), ADRB3 antagonist SR 59230A 100 nM (IC₅₀ for ADRB3 40 nM), ADRB1 agonist Dobutamine 500 nM [26], ADRB2 agonist Formoterol 500 nM [27], ADRB3 agonist BRL 37344 100 nM [28] were selected for the next experiments.

Firstly, hBSMCs were pretreated with 200 nM of Metoprolol, 10 nM of ICI 118551, or 100 nM of SR 59230A for 1 h, and then exposed to 100 cm H₂O hydrostatic pressure for 6 h. In Fig. 3A, the expressions of COL1,COL3 and FN in hBSMCs treated with hydrostatic pressure after $\beta$-blocker intervention were significantly higher than those in the control group. However, compared with hBSMCs treated with hydrostatic pressure alone, there was no significant difference in the expression of COL1,COL3 and FN in hBSMCs treated with hydrostatic pressure after the intervention of $\beta$-blockers (p $>$ 0.05) . In addition, hBSMCs were also preincubated with 500 nM of Dobutamine, 500 nM of Formoterol, or 100 nM of BRL 37344 for 1 h. After 6 h of 100 cm H₂O hydrostatic pressure, the mRNA expression of COL1 and FN decreased in hBSMCs treated with Formoterol and BRL 37344, and no statistical difference of COL3 mRNA expression was observed (HP+For vs. HP, p 0.05; HP+BRL vs. HP, p 0.05) (Fig. 3B). Furthermore, the COL1, COL3 and FN expression at the protein level was consistent with its mRNA level (HP+For vs. HP, p 0.05; HP+BRL vs. HP, p 0.05) (Fig. 3C).

Fig. 3.

Formoterol or BRL 37344 significantly suppresses the expression of COL1 and FN in hBSMCs under hydrostatic pressure. (A) The antagonists of different ADRBs had no effect on the COL1, COL3 and FN expression at the mRNA level of hBSMCs under hydrostatic pressure for 6 h. (B) The agonists of ADRBs, Formoterol and BRL 37344, remarkably decreased the COL1 and FN mRNA expression of hBSMCs under hydrostatic pressure for 6 h when compared to the hydrostatic group. (C) COL1 and FN protein levels were determined by western blotting. HP, hydrostatic pressure; Met, Metoprolol; ICI, ICI 118551; SR, SR 59230A; Dob, Dobutamine; For, Formoterol; BRL, BRL 37344. Three independent experiments have been performed. *, p 0.05, **, p 0.01.

To further identify the effects of Formoterol and BRL 37344 on hBSMCs ECM, hBSMCs were pretreated with the antagonists ICI 118551 or SR 59230A for 1 h, and then incubated with Formoterol or BRL 37344 for another one hour followed by hydrostatic pressure. ICI 118551 and SR 59230A could respectively inhibit the effects of Formoterol and BRL 37344 on hBSMCs ECM, namely the suppressive effects of Formoterol and BRL 37344 on hBSMCs ECM under hydrostatic pressure can be reversed by their corresponding antagonists (p 0.05) (Fig. 4A). The protein levels of COL1 and FN in hBSMCs under the same condition were consistent with its mRNA levels (p 0.05) (Fig. 4B). Hence, the ADRB2 and ADRB3 were indeed associated with the hBSMCs ECM under hydrostatic pressure.

Fig. 4.

ICI118551 and SR 59230A rescue the inhibitory effects of Formoterol and BRL 37344 on FN and COL1 in hBSMCs, respectively. (A) The suppressive effects of Formoterol and BRL 37344 on the mRNA expression of FN and COL1 of hBSMCs under hydrostatic pressure were rescued by ICI118551 and SR 59230A. (B) The protein levels of COL1 and FN were determined by western blotting and the grey intensities of bands were shown. HP, hydrostatic pressure; ICI, ICI 118551; SR, SR 59230A; For, Formoterol; BRL, BRL 37344. Three independent experiments have been performed. *, p 0.05.

To unravel the regulatory mechanism of ADRBs, inhibitors of PKA and EPAC, H89 250 nM (IC₅₀ = 140 nM) and ESI09 5 µM (EPAC1 IC₅₀ = 3.2 µM, EPAC2 IC₅₀ = 1.4 µM) were used to treat hBSMCs for 1 h before the treatment with Formoterol or BRL 37344 for another 1 h. Furthermore, the cells were exposed to 100 cm H₂O cm hydrostatic pressure for 6 h. The mRNA expression of FN and COL1 increased in hBSMCs pretreated with ESI09 when compared to the groups of HP+For or HP+BRL, however, the mRNA expression of FN and COL1 did not change significantly in hBSMCs pretreated with H89 (Fig. 5A, p 0.05). The protein levels of FN and COL1 were consistent with the mRNA levels (Fig. 5B, p 0.05).

Fig. 5.

EPAC regulates the expression of COL1 and FN of hBSMCs under hydrostatic pressure. (A) The mRNA levels of COL1 and FN in hBSMCs pretreated with ESI09 significantly increased when compared to that of hBSMCs treated with Formoterol or BRL37344. (B) The protein levels of COL1 and FN in hBSMCs were determined by western blotting. HP, hydrostatic pressure; For, Formoterol; BRL, BRL 37344; ESI, ESI09. Three independent experiments have been performed. *, p 0.05, **, p 0.01.

To further study the factors regulating COL1 and FN expression, the roles of well-known regulators of collagen and fibronectin, SMAD2 and SMAD3 were investigated. The protein levels of p-SMAD2 and p-SMAD3 were increased in hBSMCs under hydrostatic pressure when compared to the control group (Fig. 6A, p 0.05). The LY2109761 [29], a selective SMAD2 inhibitor, rather than SIS3 [30], a selective SMAD3 inhibitor, significantly decreased FN expression in hBSMCs under hydrostatic pressure (Fig. 6B,C, p 0.05). Interestingly, the expression of COL1 in hBSMCs was decreased by treating with SIS3 rather than LY2109761 (Fig. 6B,C, p 0.05).

Fig. 6.

SMAD2 and SMAD3 respectively regulate FN and COL1 expression in hBSMCs under hydrostatic pressure. (A) The protein level of p-SMAD2 and p-SMAD3 increased in hBSMCs under hydrostatic pressure when compared to the control group. (B) 1 µM of LY2109761 inhibited the mRNA expression of FN and 3 µM of SIS3 inhibited the mRNA expression of COL1 in hBSMCs under hydrostatic pressure. (C) The protein levels of COL1 and FN in hBSMCs were determined by western blotting. HP, hydrostatic pressure; LY, LY2109761. Three independent experiments have been performed. *, p 0.05.

In order to further clarify the regulatory pathways, the relationships between PKA, EPAC pathway and SMAD2/3 were further investigated. The protein levels of p-SMAD2 and p-SMAD3 increased significantly in hBSMCs treated with hydrostatic pressure+Formoterol+ESI09 when compared to the cells treated with hydrostatic pressure+Formoterol and hydrostatic pressure+Formoterol+H89 (Fig. 7A, p 0.05). Meanwhile, the protein levels of p-SMAD2 and p-SMAD3 increased significantly in hBSMCs treated with hydrostatic pressure+BRL 37344+ESI09 when compared to the hBSMCs treated with hydrostatic pressure+BRL 37344 and hydrostatic pressure+BRL 37344+H89 (Fig. 7B, p 0.05). The above results suggested the level of p-SMAD2 and p-SMAD3 was regulated by the EPAC pathway, combined with the previous result that the expression of COL1 and FN was simultaneously regulated by the EPAC pathway, we concluded that ADRB2 agonist Formoterol and ADRB3 agonist BRL 37344, suppressed COL1 and FN expression of hBSMCs under hydrostatic pressure via EPAC/SMAD2/FN and EPAC/SMAD3/COL1 pathways.

Fig. 7.

The protein levels of p-SMAD2 and p-SMAD3 in hBSMCs under hydrostatic pressure were regulated by the EPAC pathway. (A) The protein levels of p-SMAD2 and p-SMAD3 increased significantly in hBSMCs treated with hydrostatic pressure+Formoterol+ESI09 when compared to the cells treated with hydrostatic pressure+Formoterol and hydrostatic pressure+Formoterol+H89. (B) The protein levels of p-SMAD2 and p-SMAD3 increased significantly in hBSMCs treated with hydrostatic pressure+BRL 37344+ESI09 when compared to the cells treated with hydrostatic pressure+BRL 37344 and hydrostatic pressure+BRL 37344+H89. HP, hydrostatic pressure; For, Formoterol; BRL, BRL 37344; ESI, ESI09. Three independent experiments have been performed. *, p 0.05.

In order to further investigate the effect of ADRB2 and ADRB3 on the bladder wall remodeling of pBOO, the pBOO rats were treated with ADRB2 or ADRB3 agonist. Considering the cost and accessibility, we chose the ADRB2 agonist Procaterol and ADRB3 agonist Mirabegron, the commonly two drugs used in the clinic as the intervention drugs. A total of twenty-four SD rats were divided into four groups including the sham-operation group, pBOO group, pBOO+Procaterol group, and pBOO+Mirabegron group. After 3 weeks of treatment, Masson staining of bladder tissue of different groups showed the collagen fibers in the bladder of pBOO group were obviously thickened and the proportion was increased compared to the sham-operation group. The accumulation of collagen in the bladder of the pBOO+Procaterol group or pBOO+Mirabegron group was significantly decreased compared to the pBOO group (pBOO+Procaterol vs. pBOO, p 0.01; pBOO+Mirabegron vs. pBOO, p 0.05) (Fig. 8), but the effect of Procaterol was stronger than that of Mirabegron. Meanwhile, the expression of FN and COL1 in bladder tissue of different groups was detected by immunohistochemistry, the results showed the expression of FN and COL1 was significantly decreased in the bladder of the pBOO+Procaterol group or pBOO+Mirabegron group compared to the pBOO group, which was consistent with previous results of cell experiments in vitro (pBOO+Procaterol vs. pBOO, p 0.01; pBOO+Mirabegron vs. pBOO, p 0.05) (Fig. 9).

Fig. 8.

Procaterol and Mirabegron inhibited collagen deposition in the bladder of pBOO rats. Masson trichrome staining was used to analyze collagen (stained blue) and smooth muscle (stained red). The pictures are the representative images of groups of sham, pBOO, pBOO+Procaterol, and pBOO+Mirabegron. pBOO, partial bladder outlet obstruction. Scale bar represents 50 µm. n = 6. *, p 0.05, **, p 0.01.

Fig. 9.

Procaterol and Mirabegron suppressed the expression of COL1 and FN in the bladder of pBOO rats by immunohistochemistry. The expression of FN and COL1 was significantly decreased in the bladder of the pBOO+Procaterol group or pBOO+Mirabegron group compared to the pBOO group (brown is positive). Red arrows represent the positive expressions of FN or COL1 in tissues. The scale bar represents 50 µm. n = 6. *, p 0.05, **, p 0.01.