Changes in the expression and function of the PDE5 pathway in the obstructed urinary bladder

Abstract Our study aims to explore changes in bladder contractility and the phosphodiesterase type 5 (PDE5) signalling pathway in response to partial bladder outlet obstruction (PBOO). A surgically induced male rat PBOO model and human obstructed bladder tissues were used. Histological changes were examined by H&E and Masson's trichrome staining. Bladder strip contractility was measured via organ bath. The expressions of nitric oxide synthase (NOS) isoforms, PDE5, muscarinic cholinergic receptor (CHRM) isoforms and PDE4 isoforms in bladder were detected by RT‐PCR and Western blotting. The immunolocalization of the PDE5 protein and its functional activity were also determined. PBOO bladder tissue exhibited significant SM hypertrophy and elevated responsiveness to KCl depolarization and the muscarinic receptor agonist carbachol. NOS isoforms, PDE5, CHRM2, CHRM3 and PDE4A were up‐regulated in obstructed bladder tissue, whereas no change in PDE4B and PDE4D isoform expression was observed. With regard to PDE5, it was expressed in the SM bundles of bladder. Interestingly, obstructed bladder exhibited less relaxation responsiveness to sodium nitroprusside (SNP), but an exaggerated PDE5 inhibition effect. The up‐regulation of PDE5 could contribute to the lack of effect on Qmax for benign prostatic hyperplasia/lower urinary tract symptom (BPH/LUTS) patients treated with PDE5 inhibitors. Moreover, PDE5 (with presence of NO) and PDE4 may serve as new therapeutic targets for bladder diseases such as BPH‐induced LUTS and overactive bladder (OAB).


| INTRODUC TI ON
Bladder outlet obstruction (BOO) resulting from benign prostate hyperplasia (BPH) is a common problem among elderly males, with a high incidence of lower urinary tract symptoms (LUTS), such as incomplete bladder emptying, weak urine stream, urinary frequency, urgency, urge incontinence and nocturia. 1 However, the mechanisms of LUTS induced by BOO remain controversial. LUTS can be caused not only by prostatic obstruction, but also by bladder dysfunction, 2 such as detrusor overactivity (DO) and overactive bladder (OAB).
DO is a urodynamic observation characterized by involuntary detrusor contractions during the filling phase which may be spontaneous or provoked. 3 OAB syndrome is characterized by urinary urgency, daytime frequency and nocturia with or without urgency urinary incontinence. 4 Currently, oral drug treatment for male BPH/LUTS recommended by guidelines 5,6 includes α1-adrenoceptor antagonists (α1-blockers), 5α-reductase inhibitors (5-ARIs), muscarinic receptor antagonists (MRAs) and a 'newly emerging treatment' that utilizes phosphodiesterase type 5 inhibitors (PDE5is). PDE5is are the first-line treatment for erectile dysfunction (ED) with sildenafil, vardenafil and tadalafil commonly used. Since the first clinical trial conducted by Sariam 7 in 2002 employing sildenafil for treating BPH/LUTS/ED patients, the effect of PDE5is on BPH/LUTS has been extensively explored.
However, only tadalafil (5 mg once daily), a long-acting PDE5is, has been licensed for moderate-to-severe LUTS with or without ED. 5,6 The majority of randomized controlled trials (RCTs) have demonstrated that PDE5is could reduce International Prostate Symptom Score (IPSS), relieve storage and voiding LUTS and improve the quality of life (QoL) without significantly altering the maximal urine flow rate (Q max ). 6 Consistently, our previous network meta-analysis study 8 showed that PDE5is could reduce the total IPSS score, storage IPSS and voiding IPSS, and rank higher for the improvement of LUTS/BPH when compared with other therapies. 9 Again, no significant effect on Q max was found for PDE5is. 8 Gacci 10 postulated that PDE5is not only relaxed the prostate SM and bladder neck to reduce the IPSS score, but also relaxed detrusor SM, which may counteract the relaxation effect on the bladder outlet and lead to no improvement on Q max . Different from α1-blockers treating BPH/LUTS through lessening the resistance from the urethra muscle, the potential mechanism of PDE5is in treating BPH/LUTS may be multifaceted. Besides the expression and functional activity of PDE5 in bladder, prostate and urethra, 11,12 blood vessels that supply the prostate and bladder also express high level of active PDE5. 13 In general, PDE5is could relax SM via acting on the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signalling pathway, which was believed to play a central role in the pathophysiology of LUTS. Specifically, NO produced by nitric oxide synthase (NOS) enters SM cells then via guanylate cyclase increases the production of cGMP, a kind of second messenger which can relax SM cells. PDE5 can degrade cGMP and stop the relaxation of SM. On the other hand, its inhibitors could block PDE5 activity which results in SM relaxation. In addition, the plausible mechanism of PDE5is in treating BPH/LUTS may be summarized as follows: (a) relief of SM tone in prostate and bladder 14,15 ; (b) relaxation of blood vessels so as to increase LUT perfusion and oxygenation 16 ; (c) decreasing the activity of afferent nerves in the LUT to modulate the micturition reflex [17][18][19] ; (d) anti-proliferation effect on bladder 20 and prostate 12 ; and (e) blunting of intraprostatic inflammation 21 and an anti-fibrotic effect on bladder. 22 However, bladder PDE5 has been less studied. The aim of current study was to explore whether expression and functional activity of PDE5 in the bladder wall were altered in response to partial bladder outlet obstruction (PBOO) in rats and humans. Also, some other molecules related to bladder SM contraction and relaxation pathways were evaluated.

| Rat partial bladder outlet obstruction model
Thirty specific-pathogen-free (SPF) grade male Sprague Dawley (SD) rats bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were used and randomly divided into a PBOO group and sham group. All animals were kept on the normal chow with a 12 hour day/night light cycle for more than one week to adapt to the new environment. As previously reported, 23 rats from the PBOO group were anaesthetized with pentobarbital (35 mg/kg) via an intraperitoneal (ip) injection. A 2-cm midline vertical incision was made from the penoscrotal junction to the midscrotum to gain access to the bulbous urethra. The urethra was then isolated from the cavernosum bodies. A sterile metal bar (19-gauge needle) with a 1.06 mm diameter was placed on the bulbous urethral surface, and a 3-0 polypropylene suture was used to place a tie around both the bulbous urethra and the bar. As soon as the suture was secured, the bar was removed, leaving the bulbous urethra partial obstructed. A 4-0 silk suture was used to reapproximate the muscle layer, and a 4-0 nylon suture was used to close the skin. Sham surgery was performed the same as described above except that the urethral ties were not placed. All animals were kept for 2 weeks on normal chow with a 12 hour day/night light cycle. At the end of 2 weeks, rats were anaesthetized by isoflurane (in 100% oxygen, 5% for induction and 1.5% for maintenance) at a flow rate of 1 L/min and killed by cervical dislocation. Their bladder, prostate and seminal vesicles were quickly obtained. Animal experiments were conducted at the Animal Center of Zhongnan Hospital of Wuhan University, and all animal protocols were approved by the Medical Ethics Committee for Experimental animals of Zhongnan Hospital of Wuhan University. The pain and suffering of experimental rats were kept to a minimum.

| Human bladder tissue acquisition
Human obstructed bladder tissue was obtained from ten male patients that needed to undergo radical cystectomy and whose IPSS score was above 19 points and pathological results demonstrated BPH. All samples were identified by two separate pathologists and showed no tumour infiltration. Normal bladder tissue was obtained from ten male brain-dead men undergoing donation in the transplant centre of Zhongnan Hospital, who were less than 40 years old, and pathological examination showed no hyperplasia (human data are listed in Table 1). All human samples were obtained after the approval of the Hospital Committee for Investigation in Humans and after receiving written informed consent from all patients or their relatives. All human studies were conducted in accordance with the principles of the Declaration of Helsinki.

| H&E Staining and Masson's Trichrome Staining
Rat and human bladder tissues fixed in 10% neutral buffered formalin for 48 hour were processed routinely for paraffin embedding.
The paraffin-embedded tissue sections (4 μm) were stained with haematoxylin and eosin using standard techniques. The paraffin sections were deparaffinized in xylene, followed by graded alcohols.
Masson's composite staining solution (Fuzhou Maxim Biotech Co., Ltd., Fuzhou, China) was added dropwise for 10 minutes. The sections were subsequently washed with distilled water, differentiated in phosphomolybdic-phosphotungstic acid solution for 10 minutes and incubated with blue staining solution for 5-10 minutes. Next, the sections were rinsed briefly in distilled water and differentiated in 1% acetic solution for 2 minutes. After being dehydrated quickly through 95% alcohol and absolute alcohol, the sections were cemented using neutral gum for observation. Using this procedure, bladder SM cells were stained red and collagen fibres were stained blue. In each sample, we analysed three areas under magnification (×100). The choice of three fields was randomized without specific areas of a demarcated slide. The area percentage of SM and collagen fibres were quantitated with Image-Pro Plus 6.0, respectively.
Moreover, in each sample, three random areas under magnification (×25) were chosen and the diameter and number of detrusor SM cells in these micrographs were measured.

| In vitro organ bath studies
As previously described, 23 bladder strips containing urothelium with identical length were mounted longitudinally in a 10 mL organ bath The maximum force of KCl depolarization was taken as 100%, and the force generated by CC was normalized to a percentage of this value. After the contraction experiment completed, the strips were washed several times using buffer and the tension was reduced to the baseline. Next, strips were pre-contracted with 1 μmol/L carbachol (a dose that induces about 50% maximal contraction) and allow to reach stable tension and then the relaxant effects of increasing doses of sodium nitroprusside (SNP) with or without 100 nmol/L vardenafil were evaluated. The concentration of 100 nmol/L was chosen because it reflects the C max of vardenafil reached in clinical studies with 10 mg dosing to humans. 24 The effects of SNP were also compared with vehicles (saline). Moreover, the relaxant effects of increasing doses of vardenafil were also evaluated. Finally, for the concentration-effect curve, EC50s and a maximal effect value were determined using the four parameter logistic model by GraphPad Prism 7.0.

| Total RNA extraction and real-time RT-PCR
Total RNA was isolated from the tissues using TaKaRa MiniBEST Universal RNA Extraction Kit (Takara Bio. Inc, Otsu, Shiga, Japan) according to the manufacturer's protocol. 100 ng of RNA was added in the one-step real-time RT-PCR reaction system (Takara Bio. Inc).

| SDS-PAGE and Western blotting analysis
As previously described, proteins were extracted from tissues using RIPA (radioimmunoprecipitation assay) lysis buffer (Sigma-Aldrich,

| Immunofluorescence microscopy
Human and rat urinary bladders were embedded in Tissue-Tec OCT compound (Sakura Finetek Japan, Tokyo, Japan) and snap-frozen.
Then, the tissue was sectioned in 10-μm-thick slices, thawed and then mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar,

TA B L E 3 Primer sequences used to amplify target genes in human by real-time RT-PCR
Target gene Primer sequence

| Statistical analysis
Results are expressed as mean ± SEM for n experiments. Statistical analysis used either the Student's t test with Excel software (twosample treatments compared) or ANOVA and Bonferroni's posttests with GraphPad Prism 7.0 (multiple means compared). P < 0.05 was considered significant.

| Rat bladder weight is increased in PBOO model
In rats, enlarged bladder mass was observed in the PBOO group ( Figure 1) with mean bladder weight significantly increased from 128.0 ± 29.7 mg to 263.0 ± 89.3 mg (P < 0.01) representing a 2.05fold increase. Since the bodyweight did not increase as much in the PBOO rats, the bladder weight-to-bodyweight ratio actually increased by 2.43-fold (Table 5).

| Histological and pathological changes in PBOO rat and human bladder
H&E staining showed that PBOO rat and human bladder exhibited SM hypertrophy, arrangement disorder and fibrous tissue invaded the SM bundles ( Figure 2). Note that the rat bladder wall was observed obviously thickened in the PBOO group ( Figure 2C,D).

| In vitro contractility of bladder strips in response to KCl and carbachol are altered in obstructed bladder
As shown in Figure 4, in response to KCl depolarization and carbachol (CC), human and rat bladder strips produced significant force, and in a dose-dependent manner for CC. The left panels of Figure 4A

| In vitro relaxation effect of SNP is attenuated and restored by vardenafil in bladder strips from PBOO human and rat
As shown in Figure 5, CC pre-contracted strips (in Figure S2A

| Co-immunolocalization of α-SMA and PDE5 in human and rat bladder
We further carried out a confocal microscopy study ( Figure 6A,B).
Both for human and rat, the PDE5 exhibited almost entirely in the SM bundles, and this co-immunolocalized with α-SMA. Both positive and negative controls reacted as expected.

| Expression of molecules related to SM contraction and relaxation pathways are altered in human and rat PBOO bladder
The expression of important molecules in the NO/PDE5 pathway, CHRM isoforms and PDE4 isoforms of human and rat bladder were determined by RT-PCR ( Figure 7A,B) and Western blotting ( Figure 7C,D). For both human and rat, eNOS, nNOS, PDE5, CHRM2, CHRM3 and PDE4A were increased in the PBOO group, whereas PDE4B and PDE4D were not changed, either at mRNA or at protein level.

| D ISCUSS I ON
The current study demonstrates for the first time that pathological conditions of SM area increase resulted from hyperplasia and/or hypertrophy, we found that SM cells from both human and rat obstructed bladder increased in size (diameter) but not in numbers, based upon which we speculated that the pathological changes in obstructed bladder were mainly SM hypertrophy rather than hyperplasia.
Our study also found that the contractility of PBOO bladder strips was heightened to carbachol stimulation both for rats and human. It is well known that M 2 and M 3 receptors predominantly modulated the contraction of bladder SM and their dysregulation may lead to change in bladder SM contractility. In line with a previous study in a PBOO rat model 28,29 and BOO patients, 30  in the PBOO groups, which could be responsible for the increased CC-induced contraction. Also, carbachol EC50 values were different between PBOO and control (or sham) groups (in Figure 4A,B).
There are at least five subtypes of muscarinic receptors that exist in the mammalian bladder (M1-M5), and our current study found the up-regulation of M2 and M3 receptors (in Figure 7)   F I G U R E 4 Contractility of urinary bladder strips and analysis. A, The left panels are force tracings of human urinary bladder SM contractions in response to KCl and carbachol (CC) from control (above) and PBOO patients (below). The x-axis represents time (min), whereas the y-axis represents force (mN). The right panels are summary graphs for contraction force of urinary bladder in response to cumulative doses (10 −8 -10 −4 ) of CC from control and PBOO patients (n = strips obtained from 10 different humans, one strip was used for each human). The maximum force of KCl depolarization was taken as 100% and the force generated by CC was normalized as a percentage of this value. Data are shown as mean ± SEM. *P < 0.05 vs control; **P < 0.01 vs control. B, The left panels are force tracings of rat urinary bladder SM contractions in response to KCl and carbachol (CC) from sham (above) and PBOO rats (below). The x-axis represents time (min), whereas the y-axis represents force (mN). The right panels are summary graphs for contraction force of urinary bladder in response to cumulative doses (10 −8 -10 −4 ) of CC from sham and PBOO group (n = strips obtained from 10 different rats, one strip was used for each rat). The maximum force of KCl depolarization was taken as 100% and the force generated by CC was normalized as a percentage of this value. Data are shown as mean ± SEM. *P < 0.05 vs sham; **P < 0.01 vs sham  Figure S1). Meanwhile, PDE5is may also relax bladder SM through regulating the RhoA/ROCK pathway. 16 It is believed that the relaxant effect of vardenafil was related to PDE5 protein expression, but its accurate mechanism remains undefined and needs further exploration.
Moreover, it should be taken into consideration that full-thickness bladder preparations including urothelium and lamina propria were used in our current study when interpreting the data.
Besides the primary role of the urothelium to form a relatively impermeable barrier to protect underlying tissue, more evidence has demonstrated that ageing seems to induce hypo-function and histological changes of the bladder and urethra in humans. Therefore, it is necessary to perform an age-matched cohort study. However, it is well known that elderly men often suffered BPH/LUTS which increases with age with incidence reaching 90% for men over 85 years old. Thus, it was hard for us to find aged men without bladder outlet obstruction as controls. Actually, a recent study from Silva-Ramos et al 32 demonstrated that ageing did not seem to influence the sensitivity of detrusor SM to cholinergic stimulus in obstructed bladders using correlation analysis. In addition, our age-matched rat study showed consistent results with non-age-controlled human experiments. But it will be intriguing to conduct further study using an age-matched cohort in the future.
In summary, obstructed bladder showed significant changes in histology and physiology, as well as related contractility molecules.
The up-regulation of PDE5 could contribute to the lack of effect on Q max for BPH/LUTS patients treated with PDE5is. However, PDE5 (with presence of NO) and PDE4 may be new therapeutic targets for bladder diseases.

ACK N OWLED G EM ENTS
We thank the staff at Zhongnan Hospital of Wuhan University for their help in completing the study.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.