Benign prostatic hyperplasia nodules in patients treated with celecoxib and/or finasteride have reduced levels of NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, a mitochondrial protein essential for efficient function of the electron transport chain.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a condition generally associated with advanced age in men that can be accompanied by bothersome lower urinary tract symptoms (LUTS) including intermittency, weak stream, straining, urgency, frequency, and incomplete bladder voiding. Pharmacotherapies for LUTS/BPH include alpha-blockers, which relax prostatic and urethral smooth muscle and 5ɑ-reductase inhibitors such as finasteride, which can block conversion of testosterone to dihydrotestosterone thereby reducing prostate volume. Celecoxib is a cyclooxygenase-2 inhibitor that reduces inflammation and has shown some promise in reducing prostatic inflammation and alleviating LUTS for some men with histological BPH. However, finasteride and celecoxib can reduce mitochondrial function in some contexts, potentially impacting their efficacy for alleviating BPH-associated LUTS. METHODS: To determine the impact of these pharmacotherapies on mitochondrial function in prostate tissues, we performed immunostaining of mitochondrial Complex I (CI) protein NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3) and inflammatory cells on BPH specimens from patients naïve to treatment, or who were treated with celecoxib and/or finasteride for 28 days, as well as prostate tissues from male mice treated with celecoxib or vehicle control for 28 days. Quantification and statistical correlation analyses of immunostaining were performed. RESULTS: NDUFS3 immunostaining was decreased in BPH compared to normal adjacent prostate. Patients treated with celecoxib and/or finasteride had significantly decreased NDUFS3 in both BPH and normal tissues, and no change in inflammatory cell infiltration compared to untreated patients. Mice treated with celecoxib also displayed a significant decrease in NDUFS3 immunostaining and no change in inflammatory cell infiltration. CONCLUSIONS: These findings suggest that celecoxib and/or finasteride are associated with an overall decrease in NDUFS3 levels in prostate tissues but do not impact the presence of inflammatory cells, suggesting a decline in mitochondrial CI function in the absence of enhanced inflammation. Given that BPH has recently been associated with increased prostatic mitochondrial dysfunction, celecoxib and/or finasteride may exacerbate existing mitochondrial dysfunction in some BPH patients thereby potentially limiting their overall efficacy in providing metabolic stability and symptom relief.

Age-Dependent Effects of Voluntary Wheel Running Exercise on Voiding Behavior and Potential Age-Related Molecular Mechanisms in Mice.

BACKGROUND: Older men frequently develop lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). Risk factors for LUTS/BPH include sedentary lifestyle, anxiety/depression, obesity, and frailty, which all increase with age. Although physical exercise may reduce the progression and/or severity of LUTS/BPH, the age-related mechanisms responsible remain unknown. METHODS: Voiding symptoms, body mass, and frailty were assessed after 4-weeks of voluntary wheel running in 2-month (n = 10) and 24-month (n = 8) old C57Bl/6J male mice. In addition, various social and individual behaviors were examined in these cohorts. Finally, cellular and molecular markers of inflammation and mitochondrial protein expression were assessed in prostate tissue and systemically. RESULTS: Despite running less (aged vs young X¯ = 12.3 vs 30.6 km/week; p = .04), aged mice had reduced voiding symptoms (X¯ = 67.3 vs 23.7; p < .0001) after 1 week of exercise, which was sustained through week 4 (X¯ = 67.3 vs 21.5; p < .0001). Exercise did not affect voiding symptoms in young mice. Exercise also increased mobility and decreased anxiety in both young and aged mice (p < .05). Exercise decreased expression of a key mitochondrial protein (PINK1; p < .05) and inflammation within the prostate (CD68; p < .05 and plasminogen activator inhibitor-1; p < .05) and in the serum (p < .05). However, a frailty index (X¯ = 0.17 vs 0.15; p = .46) and grip strength (X¯ = 1.10 vs 1.19; p = .24) were unchanged after 4 weeks of exercise in aged mice. CONCLUSIONS: Voluntary aerobic exercise improves voiding behavior and mobility, and decreases prostatic mitochondrial protein expression and inflammation in aged mice. This promising model could be used to evaluate molecular mechanisms of aerobic exercise as a novel lifestyle intervention for older men with LUTS/BPH.

Genetic alterations in CREBRF influence prostate cancer survival and impact prostate tissue homeostasis in mice.

BACKGROUND: Risk factors for prostate cancer include age, environment, race and ethnicity. Genetic variants in cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) gene are frequently observed in Pacific Islanders, a population with elevated prostate cancer incidence. CREBRF has been shown to play a role in other cancers, however its function in prostate homeostasis and tumorigenesis has not been previously explored. We determined the incidence of CREBRF alterations in publicly available databases and examined the impact of CREBRF deletion on the murine prostate in order to determine whether CREBRF impacts prostate physiology or pathophysiology. METHODS: Alterations in CREBRF were identified in prostate cancer patients via in silico analysis of several publicly available datasets through cBioPortal. Male Crebrf knockout and wild-type littermate mice were generated and examined for prostate defects at 4 months of age. Immunohistochemical staining of murine prostate sections was used to determine the impact of Crebrf knockout on proliferation, apoptosis, inflammation and blood vessel density in the prostate. Serum adipokine levels were measured using a Luminex Multiplex Assay. RESULTS: CREBRF alterations were identified in up to 4.05% of prostate tumors and the mutations identified were categorized as likely damaging. Median survival of prostate cancer patients with genetic alterations in CREBRF was 41.23 months, compared to 131 months for patients without these changes. In the murine model, the prostates of Crebrf knockout mice had reduced epithelial proliferation and increased TUNEL+ apoptotic cells. Circulating adipokines PAI-1 and MCP-1 were also altered in Crebrf knockout mice compared to age-matched controls. CONCLUSIONS: Prostate cancer patients with genetic alterations in CREBRF had a significantly decreased overall survival suggesting that wild type CREBRF may play a role in limiting prostate tumorigenesis and progression. The murine knockout model demonstrated that CREBRF could modulate proliferation and apoptosis and macrophage density in the prostate. Serum levels of adipokines PAI-1 and MCP-1 were also altered and may contribute to the phenotypic changes observed in the prostates of Crebrf knockout mice. Future studies focused on populations susceptible to CREBRF mutations and mechanistic studies will be required to fully elucidate the potential role of CREBRF in prostate tumorigenesis.

The SARS-CoV-2 E protein induces Toll-like receptor 2-mediated neonatal lung injury in a model of COVID-19 viremia that is rescued by the glucocorticoid ciclesonide.

SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.

E-cadherin deficiency promotes prostate macrophage inflammation and bladder overactivity in aged male mice.

Decreased E-cadherin immunostaining is frequently observed in benign prostatic hyperplasia (BPH) and was recently correlated with increased inflammation in aging prostate. Homozygous E-cadherin deletion in the murine prostate results in prostate inflammation and bladder overactivity at 6 months of age. However, this model is limited in that while E-cadherin is significantly reduced in BPH, it is not completely lost; BPH is also strongly associated with advanced age and is infrequent in young men. Here, we examined the functional consequences of aging in male mice with prostate luminal epithelial cell-specific E-cadherin heterozygosity. In control mice, aging alone resulted in an increase in prostate inflammation and changes in bladder voiding function indicative of bladder underactivity. At 24 months of age, mice with prostate-specific Cre-mediated heterozygous deletion of E-cadherin induced at 7 weeks of age developed additional prostatic defects, particularly increased macrophage inflammation and stromal proliferation, and bladder overactivity compared to age-matched control mice, which are similar to BPH/LUTS in that the phenotype is slow-progressing and age-dependent. These findings suggest that decreased E-cadherin may promote macrophage inflammation and fibrosis in the prostate and subsequent bladder overactivity in aging men, promoting the development and progression of BPH/LUTS.

Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum.

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1-5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.

Claudin-1 down-regulation in the prostate is associated with aging and increased infiltration of inflammatory cells in BPH.

INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) is an age-related disease that is frequently associated with chronic prostatic inflammation. In previous studies, we detected the presence of PSA protein in the stroma of BPH nodules and down-regulation of junction proteins E-cadherin and claudin-1. Transmission electron microscopy (TEM) imaging showed a decrease in tight junctions suggesting the luminal epithelial barrier in BPH tissues may be compromised. Recent in vitro studies showed that stimulation of benign prostate epithelial cell lines with TGF-ß1 induced a decrease in claudin-1 expression suggesting that inflammation might be associated with alterations in the prostate epithelial barrier. This study explored the potential associations between aging and loss of junction proteins and the presence of inflammatory cells in prostate tissue specimens from young healthy donors and aged BPH patients. METHODS: Immunostaining of serial prostate sections from 13 BPH patients and five healthy young donors was performed for claudin-1, CD4, CD8, CD20 and CD68. H-Scores and the number of inflammatory cells were calculated for the same area in donor, normal adjacent prostate (NAP) to and BPH specimens. Quantification and statistical correlation analyses were performed. RESULTS: Claudin-1 immunostaining was inversely associated with increasing age, and inflammation in prostate specimens. B-cell infiltration increased with age and BPH was associated with an increased infiltration of T-cells and macrophages compared to NAP. CONCLUSIONS: These findings suggest that aging is associated with down-regulation of claudin-1 and claudin-1 is further decreased in BPH. Claudin-1 down-regulation was associated with increased infiltration of inflammatory cells in both NAP and BPH tissues. Claudin-1 down-regulation in the aging prostate could contribute to increased prostatic inflammation, subsequently contributing to BPH pathogenesis.

Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings.

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Prostate-Specific Deletion of Cdh1 Induces Murine Prostatic Inflammation and Bladder Overactivity.

Benign prostatic hyperplasia (BPH) is an age-related debilitating prostatic disease that is frequently associated with prostatic inflammation and bothersome lower urinary tract symptoms (LUTS). Animal models have shown that formalin- and bacterial-induced prostatic inflammation can induce bladder dysfunction; however, the underlying mechanisms contributing to prostatic inflammation in BPH and bladder dysfunction are not clear. We previously reported that E-cadherin expression in BPH is downregulated in hyperplastic nodules compared with expression in adjacent normal tissues. Here, we explored the potential consequences of prostatic E-cadherin downregulation on the prostate and bladder in vivo using an inducible murine model of prostate luminal epithelial-specific deletion of Cdh1. The prostate-specific antigen (PSA)-CreERT2 transgenic mouse strain expressing tamoxifen-inducible CreERT2 recombinase driven by a 6-kb human PSA promoter/enhancer was crossed with the B6.129-Cdh1tm2Kem/J mouse to generate bigenic PSA-CreERT2/Cdh1-/- mice. Deletion of E-cadherin was induced by transient administration of tamoxifen when mice reached sexual maturity (7 weeks of age). At 21 to 23 weeks of age, the prostate, bladder, and prostatic urethra were examined histologically, and bladder function was assessed using void spot assays and cystometry. Mice with Cdh1 deletion had increased prostatic inflammation, prostatic epithelial hyperplasia, and stromal changes at 21 to 23 weeks of age, as well as changes in bladder voiding function compared with age-matched controls. Thus, loss of E-cadherin in the murine prostate could result in prostatic defects that are characteristic of BPH and LUTS, suggesting that E-cadherin downregulation could be a driving force in human BPH development and progression.

Effects of dutasteride in a rat model of chemically induced prostatic inflammation-Potential role of estrogen receptor ß.

BACKGROUND: Dutasteride administration reportedly improves lower urinary tract symptoms in patient with chronic, histologically-identified prostatic inflammation, potentially through estrogen receptor ß (ERß), activation of which has anti-inflammatory effects in the prostate tissue. Therefore, we investigated the effect of dutasteride on intraprostatic inflammatory responses and bladder activity using a rat model of chemically induced prostatic inflammation. METHODS: Male Sprague-Dawley rats at 10 weeks old were used. Prostatic inflammation was induced by 5% formalin injection into ventral lobes of the prostate and saline was injected in the control group (control, n = 5). Rats with prostatic inflammation were divided into dutasteride therapy (dutasteride, n = 5) and placebo groups (placebo, n = 5). Dutasteride was administrated at a dose of 0.5 mg/kg daily from 2 days before induction of prostatic inflammation whereas placebo rats received vehicle only. Twenty-eight days later, cystometry was performed in a conscious condition to measure non-voiding contractions (NVCs), intercontraction intervals (ICI) and postvoid residual volume (RV). After cystometry, the prostate was excised for analysis of messenger RNA (mRNA) expression levels of ERα, ERß, interleukin-1ß (IL-1ß), and IL-18 by quantitative polymerase chain reaction. RESULTS: The mean number of NVCs was significantly greater in placebo group than that of control group without prostatic inflammation (p < .05), and ICI were significantly decreased in placebo group compared with control group (p < .05). On the contrary, there was no significant change in NVCs or ICI between control and dutasteride groups. RV was not significantly different among three groups. Gene expression levels of ERα, IL-1ß, and IL-18 was significantly increased in placebo rats compared with control rats (p < .05), but not significantly different between control and dutasteride rats. On the other hand, the mRNA expression level of ERß was significantly decreased in placebo rats (p < .05), but not in dutasteride rats, compared with control rats. CONCLUSION: Dutasteride treatment improved not only prostatic inflammation evident as increased gene expression levels in IL-1ß and IL-18, but also bladder overactivity shown by increased NVCs during bladder filling. These therapeutic effects were associated with the restored expression of anti-inflammatory ERß. Therefore, dutasteride might be effective via ERß modulation for the treatment of prostatic inflammation in addition to its previously known, anti-androgenic effects on benign prostatic hyperplasia.

Differential impact of paired patient-derived BPH and normal adjacent stromal cells on benign prostatic epithelial cell growth in 3D culture.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related disease characterized by nonmalignant abnormal growth of the prostate, which is also frequently associated with lower urinary tract symptoms. The prostate with BPH exhibits enhanced growth not only in the epithelium but also in the stroma, and stromal-epithelial interactions are thought to play an important role in BPH pathogenesis. However, our understanding of the mechanisms of stromal-epithelial interactions in the development and progression of BPH is very limited. METHODS: Matched pairs of glandular BPH and normal adjacent prostate specimens were obtained from BPH patients undergoing simple prostatectomy for symptomatic BPH. Tissues were divided further into fresh specimens for culture of primary prostatic stromal cells, and specimens were embedded in paraffin for immunohistochemical analyses. Proliferation assays, immunohistochemistry, and immunoblotting were used to characterize the primary prostate stromal cells and tissue sections. Coculture of the primary stromal cells with benign human prostate epithelial cell lines BHPrE1 or BPH-1 was performed in three-dimensional (3D) Matrigel to determine the impact of primary stromal cells derived from BPH on epithelial proliferation. The effect of stromal-conditioned medium (CM) on BHPrE1 and BPH-1 cell growth was tested in 3D Matrigel as well. RESULTS: BPH stromal cells expressed less smooth muscle actin and calponin and increased vimentin, exhibiting a more fibroblast and myofibroblast phenotype compared with normal adjacent stromal cells both in culture and in corresponding paraffin sections. Epithelial spheroids formed in 3D cocultures with primary BPH stromal cells were larger than those formed in coculture with primary normal stromal cells. Furthermore, CM from BPH stromal cells stimulated epithelial cell growth while CM from normal primary stromal cells did not in 3D culture. CONCLUSIONS: These findings suggest that the stromal cells in BPH tissues are different from normal adjacent stromal cells and could promote epithelial cell proliferation, potentially contributing to the development and progression of BPH.

Tight junction protein claudin-1 is downregulated by TGF-ß1 via MEK signaling in benign prostatic epithelial cells.

BACKGROUND: Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-ß1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-ß1 in prostatic epithelial cells are not clear. METHODS: The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-ß1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-ß1 treatment using Western blot analysis. RESULTS: Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-ß1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-ß1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. CONCLUSION: Our findings suggest that downregulation of claudin-1 by TGF-ß1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-ß1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.

Transforming growth factor beta 1 impairs benign prostatic luminal epithelial cell monolayer barrier function.

Our recent studies identifying the presence of luminal secretory protein PSA in the stroma, decreased E-cadherin expression, and reduced number of tight junction kiss points in benign prostatic hyperplasia (BPH) tissues suggest that epithelial barrier permeability is increased in BPH. However, the cause of increased epithelial permeability in BPH is unclear. Transforming growth factor beta 1 (TGF-ß1) has been reported to be up-regulated in clinical BPH specimens and TGF-ß1 overexpression induced fibrosis and inflammation in a murine model. TGF-ß1 was reported to repress the expression of E-cadherin in benign prostatic cells. However, whether and how TGF-ß1 up-regulation affects epithelial barrier permeability is unknown. Here, in vitro benign prostatic epithelial cell lines BHPrE1 and BPH-1 were utilized to determine the impact of TGF-ß1 treatment on epithelial barrier, tight junctions, and expression of E-cadherin and claudin 1 by transepithelial electrical resistance (TEER) measurement, FITC-dextran trans-well diffusion assays, qPCR, as well as transmission electron microscopy (TEM) observation. Laser capture micro-dissection (LCM) combined with reverse transcription-polymerase chain reaction (qPCR) were utilized to determine the expression of E-cadherin and claudin 1 in BPH patient specimens. TGF-ß1 treatment decreased TEER, increased FITC-dextran diffusion, and reduced the mRNA expression of junction protein claudin 1 in cultured cell monolayers. Claudin 1 mRNA but not E-cadherin mRNA was down-regulated in the luminal epithelial cells in BPH nodules compared to normal prostate tissues. Our studies suggest that TGF-ß1 could increase the permeability through decreasing the expression of claudin 1 and inhibiting the formation of tight junctions in BHPrE1 and BPH-1 monolayers. These results suggest that TGF-ß1 might play an important role in BPH pathogenesis through increasing the permeability of luminal epithelial barrier in the prostate.

E-cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer.

BACKGROUND: We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. METHODS: The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. RESULTS: The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. CONCLUSIONS: Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

The role of prostaglandin and E series prostaglandin receptor type 4 receptors in the development of bladder overactivity in a rat model of chemically induced prostatic inflammation.

OBJECTIVES: To evaluate, using a rat model of non-bacterial prostatic inflammation, the prostaglandin production and expression profiles of E-series prostaglandin (EP) receptor subtypes, which are reportedly implicated in the development of overactive bladder, in the bladder mucosa, and to investigate the effect of EP receptor type 4 (EP4) blockade on bladder overactivity after prostatic inflammation. METHODS: Male Sprague-Dawley rats were used. Prostatic inflammation was induced by formalin injection (5%; 50 µL per lobe) into the bilateral ventral lobes of the prostate. At 10 days after induction of prostatic inflammation or vehicle injection, bladder tissues from the deeply anaesthetized rats were harvested and separated into mucosal and detrusor layers. Then, prostaglandin E2 (PGE2) concentrations and protein levels of PGE2 receptors (EP1-4) in the bladder mucosa and detrusor were measured by ELISA and Western blotting, respectively. In separate groups of control and formalin-treated rats, awake cystometry was performed to evaluate the changes in bladder activity after prostatic inflammation. In addition, the effect of intravesical administration of a selective EP4 antagonist (ONO-AE3-208; 30 µm) on bladder activity was evaluated in control rats and rats with prostatic inflammation. RESULTS: PGE2 concentration and protein levels of EP4, but not other EP receptor subtypes, in the bladder mucosa and detrusor layers were significantly increased in formalin-injected rats vs vehicle-injected control rats. In cystometry, rats with prostatic inflammation exhibited a significant decrease in intercontraction intervals (ICIs) compared with control rats. Intravesical application of ONO-AE3-208 (30 µm), but not vehicle application, significantly increased ICIs in rats with prostatic inflammation, whereas ONO-AE3-208 at this concentration did not significantly affect any cystometric values in control rats. CONCLUSIONS: Because intravesical administration of an EP4 antagonist effectively improved bladder overactivity after prostatic inflammation, EP4 activation, along with increased PGE2 production in the bladder mucosa, seems to be an important contributing factor to bladder overactivity induced by prostatic inflammation. Thus, blockade of EP4 in the bladder could be a therapeutic approach to male lower urinary tract symptoms attributable to benign prostatic hyperplasia with prostatic inflammation.

Long-lasting bladder overactivity and bladder afferent hyperexcitability in rats with chemically-induced prostatic inflammation.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the major causes of lower urinary tract symptoms (LUTS), including storage LUTS such as urinary frequency and urgency. Recently, a growing number of clinical studies indicate that prostatic inflammation could be an important pathophysiological mechanism inducing storage LUTS in patients with BPH. Here we aimed to investigate whether nonbacterial prostatic inflammation in a rat model induced by intraprostatic formalin injection can lead to long-lasting bladder overactivity and changes in bladder afferent neuron excitability. METHODS: Male Sprague-Dawley rats were divided into four groups (n = 12 each): normal control group, 1-week prostatic inflammation group, 4-week inflammation group, and 8-week inflammation group. Prostatic inflammation was induced by formalin (10%; 50 µL per lobe) injection into bilateral ventral lobes of the prostate. Voiding behavior was evaluated in metabolic cages for each group. Ventral lobes of the prostate and the bladder were then removed for hematoxylin and eosin (HE) staining to evaluate inflammation levels. Continuous cystometrograms (CMG) were recorded to measure intercontraction intervals (ICI) and voided volume per micturition. Whole-cell patch clamp recordings were performed on dissociated bladder afferent neurons labeled by fluorogold injected into the bladder wall, to examine the electrophysiological properties. RESULTS: Results of metabolic cage measurements showed that formalin-treated rats exhibited significantly (P < 0.05) increases in micturition episodes/12 hours and decrease in voided volume per micturition at every time point post injection. Continuous CMG illustrated the significant ( P < 0.05) higher number of nonvoiding contractions per void and shorter ICI in formalin-treated rats compared with control rats. HE staining showed significant prostatic inflammation, which declined gradually, in prostate tissues of formalin-induced rats. In patch clamp recordings, capsaicin-sensitive bladder afferent neurons from rats with prostatic inflammation had significantly ( P < 0.05) lower thresholds for spike activation and a "multiple" firing pattern compared with control rats at every time point post injection. CONCLUSIONS: Formalin-induced prostatic inflammation can lead to long-lasting bladder overactivity in association with bladder afferent neuron hyperexcitability. This long-lasting model could be a useful tool for the study of inflammation-related aspects of male LUTS pathophysiology.

Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation.

KEY POINTS: There is clinical evidence showing that prostatic inflammation contributes to overactive bladder symptoms in male patients; however, little is known about the underlying mechanisms In this study, we investigated the mechanism that prostatic inflammation causes detrusor overactivity by using a rat model of chemically induced prostatic inflammation. We observed a significant number of dorsal root ganglion neurons with dichotomized afferents innervating both prostate and bladder. We also found that prostatic inflammation induces bladder overactivity and urothelial NGF overexpression in the bladder, both dependent on activation of the pelvic nerve, as well as changes in ion channel expression and hyperexcitability of bladder afferent neurons. These results indicate that the prostate-to-bladder cross-sensitization through primary afferent pathways in the pelvic nerve, which contain dichotomized afferents, could be an important mechanism contributing to bladder overactivity and afferent hyperexcitability induced by prostatic inflammation. ABSTRACT: Prostatic inflammation is reportedly an important factor inducing lower urinary tract symptoms (LUTS) including urinary frequency, urgency and incontinence in patients with benign prostatic hyperplasia (BPH). However, the underlying mechanisms inducing bladder dysfunction after prostatic inflammation are not well clarified. We therefore investigated the effects of prostatic inflammation on bladder activity and afferent function using a rat model of non-bacterial prostatic inflammation. We demonstrated that bladder overactivity, evident as decreased voided volume and shorter intercontraction intervals in cystometry, was observed in rats with prostatic inflammation versus controls. Tissue inflammation, evident as increased myeloperoxidase activity, and IL-1α, IL-1ß, and IL-6 levels inside the prostate, but not in the bladder, following intraprostatic formalin injection induced an increase in NGF expression in the bladder urothelium, which depended on activation of the pelvic nerve. A significant proportion (18-19%) of dorsal root ganglion neurons were double labelled by dye tracers injected into either bladder or prostate. In rats with prostatic inflammation, TRPV1, TRPA1 and P2X2 increased, and Kv1.4, a potassium channel α-subunit that can form A-type potassium (KA ) channels, decreased at mRNA levels in bladder afferent and double-labelled neurons vs. non-labelled neurons, and slow KA current density decreased in association with hyperexcitability of these neurons. Collectively, non-bacterial inflammation localized in the prostate induces bladder overactivity and enhances bladder afferent function. Thus, prostate-to-bladder afferent cross-sensitization through primary afferents in the pelvic nerve, which contain dichotomized afferents, could underlie storage LUTS in symptomatic BPH with prostatic inflammation.

Effects of Estrogen Receptor ß Stimulation in a Rat Model of Non-Bacterial Prostatic Inflammation.

BACKGROUND: There is increasing evidence showing that chronic non-bacterial prostatic inflammation is involved in the pathogenesis of benign prostatic hyperplasia (BPH) and male lower urinary tract symptoms (LUTS). It has also been reported that estrogen receptor ß (ERß) could have an immunoprotective role in prostatic tissue. Therefore, we investigated the effect of ERß-activation on not only prostatic inflammation, but also bladder overactive conditions in a rat model with nonbacterial prostatic inflammation. METHODS: Male Sprague-Dawley rats (8 weeks, n = 15) were divided into three groups: sham-saline group (n = 5), formalin-vehicle group (n = 5), and formalin-treatment group (n = 5). The sham-saline group had sham operation and 50 µl normal saline injected into each ventral lobe of the prostate. The formalin-vehicle group had 50 µl 5% formalin injection into bilateral ventral lobes of the prostate. The formalin-treatment group was treated with 3α-Adiol (a selective ERß agonist precursor) at a dose of 3 mg/kg daily from 2 days before induction of prostatic inflammation, whereas formalin-vehicle rats received vehicle (olive oil). In each group, conscious cystometry was performed on day 28 after intraprostatic formalin injection or sham treatment. After cystometry, the bladder and prostate were harvested for evaluation of mRNA expression and histological analysis. RESULTS: In cystometric investigation, the mean number of non-voiding contractions was significantly greater and voiding intervals were significantly shorter in formalin-vehicle rats than those in sham-saline rats (P < 0.05). In RT-qPCR analysis, mRNA expression of NGF, P2X2, and TRPA1 receptors was significantly increased in the bladder mucosa, and mRNA expression of TNF-α, iNOS and COX2 in the ventral lobes of prostate was significantly increased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). In addition, relative mRNA expression ratio of ERß to ERα (ERß/ERα) in the ventral lobes of prostate was significantly decreased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). These changes were ameliorated by 3α-Adiol administration in formalin-treatment rats. CONCLUSIONS: These results indicate that ERß activation by 3α-Adiol administration, which normalized the ERß/ERα expression ratio in the prostate, can improve not only prostatic inflammation, but also bladder overactivity. Therefore, ERß agonists might be useful for treating irritative bladder symptoms in patients with symptomatic BPH associated with prostatic inflammation. Prostate 77:803-811, 2017. © 2017 Wiley Periodicals, Inc.

Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor ß (ERß) Response to 5α-Reductase Inhibition in Prostate Epithelial Cells.

Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor-driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent androgen receptor ligand dihydrotestosterone. Because dihydrotestosterone is the precursor for estrogen receptor ß (ERß) ligands, 5AR inhibitors could potentially limit ERß activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell adhesion protein E-cadherin by the 5AR inhibitor dutasteride requires both ERß and TGFß. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative feedback loop in TGFß and ERß signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERß action through its effect on the expression of a number of steroidogenic enzymes in the ERß ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue-protective action of ERß.