Changes in transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 in patients with lower urinary tract dysfunction.

PURPOSE: We investigated the association between transient receptor potential vanilloid (TRPV) expression in human urothelium tissue and lower urinary tract dysfunction (LUTD). MATERIALS AND METHODS: We prospectively enrolled men who planned to undergo surgical treatment for benign prostatic obstruction to analyze TRPV1 and TRPV4 expression in the urothelium using enzyme-linked immunosorbent assay and immunofluorescence staining. Patients were divided into two groups based on urodynamics: the detrusor underactivity (DU) group and the non-DU group. Levels of TRPV1 and TRPV4 were compared between the two groups. We also divided patients into two groups according to degree of subjective urinary urgency symptoms using a 5-point urinary sensation scale and compared the differences in TRPV1 and TRPV4 levels between the two groups. The correlations between urodynamic parameters with TRPV1 or TRPV 4 in all patients were also analyzed. RESULTS: The levels of TRPV1 and TRPV 4 were not significantly different between the DU group (n=10) and the non-DU group (n=11). When we divided the patients according to degree of subjective urgency, the level of TRPV1 was not significantly different between the urgency group (n=10) and the non-urgency group (n =11), but the level of TRPV4 was significantly increased in the urgency group (p=0.029). There was no significant correlation between the level of TRPV1 or TRPV4 and urodynamic parameters in any patients. CONCLUSIONS: TRPV4 could be a useful diagnostic biomarker for patients with LUTD.

Correlation Between Nitric Oxide and Urodynamics in Men With Bladder Outlet Obstruction.

PURPOSE: To investigate the correlation between nitric oxide (NO) and urodynamics in men with bladder outlet obstruction (BOO) by analyzing nitric oxide synthase (NOS) in the urothelium. METHODS: We prospectively enrolled 25 men who planned to undergo surgical treatment for benign prostatic obstruction and identified as BOO in the preoperative urodynamics. Bladder tissue was taken during surgical prostate resection. Expressions of endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in the urothelium were analyzed, and their correlation with urodynamic parameters was also assessed in all patients. We also compared the expressions of eNOS, iNOS, and nNOS between BOO with detrusor underactivity (DU) group and BOO without DU group. RESULTS: In all patients, the level of eNOS positively correlated with maximal flow rate and with maximum cystometric capacity (MCC). The level of iNOS positively correlated with MCC. nNOS levels were positively correlated with detrusor pressure at maximal flow and with bladder contractility index in all patients. The level of eNOS, iNOS, and nNOS did not significantly differ between BOO without DU group and BOO with DU group. CONCLUSION: This study suggests that NO was correlated with bladder dysfunction in men with BOO. Particularly, nNOS may reflect the change in detrusor function.

Evaluation of Collagen Gel-Associated Human Nasal Septum-Derived Chondrocytes As a Clinically Applicable Injectable Therapeutic Agent for Cartilage Repair.

BACKGROUND: Articular cartilage injury has a poor repair ability and limited regeneration capacity with therapy based on articular chondrocytes (ACs) implantation. Here, we validated the hypothesis that human nasal septum-derived chondrocytes (hNCs) are potent therapeutic agents for clinical use in cartilage tissue engineering using an injectable hydrogel, type I collagen (COL1). METHODS: We manufactured hNCs incorporated in clinical-grade soluble COL1 and investigated their clinical potential as agents in an articular defect model. RESULTS: The hNCs encapsulated in COL1 (hNC-collagen) were uniformly distributed throughout the collagen and showed much greater growth rate than hACs encapsulated in collagen for the 14 days of culture. Fluorescent staining of hNC-collagen showed high expression levels of chondrocyte-specific proteins under clinical conditions. Moreover, a negative mycoplasma screening result were obtained in culture of hNC-collagen. Notably, implantation of hNC-collagen increased the repair of osteochondral defects in rats compared with implantation of collagen only. Many human cells were detected within the cartilage defects. CONCLUSION: These results provide reliable evidences supporting for clinical applications of hNC-collagen in regenerative medicine for cartilage repair.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3-RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin ß3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3-RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1.

BACKGROUND: Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice. METHODS: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1ß, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting. RESULTS: A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1ß. The serum levels of TNF-α, IL-1ß, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups. CONCLUSION: The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

MicroRNA expression profiling and Notch1 and Notch2 expression in minimal deviation adenocarcinoma of uterine cervix.

BACKGROUND: MicroRNA (miRNA) expression is known to be deregulated in cervical carcinomas. However, no data is available about the miRNA expression pattern for the minimal deviation adenocarcinoma (MDA) of uterine cervix. We sought to detect deregulated miRNAs in MDA in an attempt to find the most dependable miRNA or their combinations to understand their tumorigenesis pathway and to identify diagnostic or prognostic biomarkers. We also investigated the association between those miRNAs and their target genes, especially Notch1 and Notch2. METHODS: We evaluated miRNA expression profiles via miRNA microarray and validated them using.real-time PCR assays with 24 formalin-fixed, paraffin-embedded tissue blocks of MDA and 11 normal proliferative endocervical tissues as control. Expression for Notch1 and 2 was assessed by immunohistochemistry. RESULTS: MiRNA-135a-3p, 192-5p, 194-5p, and 494 were up-regulated, whereas miR-34b-5p, 204-5p, 299-5p, 424-5p, and 136-3p were down-regulated in MDA compared with normal proliferative endocervical tissues (all P<0.05). Considering the second-order Akaike Information Criterion consisting of likelihood ratio and number of parameters, miR-34b-5p showed the best discrimination power among the nine candidate miRNAs. A combined panel of miR-34b-5p and 194-5p was the best fit model to discriminate between MDA and control, revealing 100% sensitivity and specificity. Notch1 and Notch2, respective target genes of miR-34b-5p and miR-204-5p, were more frequently expressed in MDA than in control (63% vs. 18%; 52% vs. 18%, respectively, P<0.05). MiR-34b-5p expression level was higher in Notch1-negative samples compared with Notch1-positive ones (P<0.05). Down-regulated miR-494 was associated with poor patient survival (P=0.036). CONCLUSIONS: MDA showed distinctive expression profiles of miRNAs, Notch1, and Notch2 from normal proliferative endocervical tissues. In particular, miR-34b-5p and 194-5p might be used as diagnostic biomarkers and miR-494 as a prognostic predictor for MDA. The miR-34b-5p/Notch1 pathway as well as Notch2 might be important oncogenic contributors to MDA.

Additive Effect of preservative-free sodium hyaluronate 0.1% in treatment of dry eye syndrome with diquafosol 3% eye drops.

PURPOSE: The aim of this study was to evaluate the treatment effect of diquafosol 3% with preservative-free sodium hyaluronate 0.1% eye drops in dry eye syndrome. METHODS: In total, 150 patients with dry eye syndrome were divided randomly into 3 groups. Group 1 (50 patients) was treated 4 times daily with preserved sodium hyaluronate 0.1%, group 2 (50 patients) was treated 4 times daily with diquafosol 3%, and group 3 (50 patients) was treated 4 times daily with diquafosol 3% and preservative-free sodium hyaluronate 0.1% eye drops for 3 months. Ocular surface disease index (OSDI) score, tear film break-up time, Schirmer I test, corneal fluorescein staining, and impression cytology were evaluated. RESULTS: There were significant improvements in the OSDI score, tear film break-up time, Schirmer I score, fluorescein and Rose Bengal staining, goblet cell density, and impression cytological findings in groups 2 and 3 compared with those for group 1 in patients with dry eye syndrome at 1, 2, and 3 months (P < 0.05). There were statistically significant improvements in the OSDI score (-8.48 ± 0.97, -5.69 ± 0.78; P = 0.02), fluorescein (-1.43 ± 0.21, -1.02 ± 0.18; P = 0.03), and Rose Bengal staining (-1.12 ± 0.26, -0.75 ± 0.12; P = 0.03), goblet cell density (89.65 ± 14.39, 70.36 ± 16.75; P = 0.03), and impression cytological findings (-0.53 ± 0.12, -0.34 ± 0.90; P = 0.01) in group 3 compared with those in group 2 at 3 months. CONCLUSIONS: Treatment with diquafosol 3% with preservative-free sodium hyaluronate 0.1% was more effective than diquafosol 3% monotherapy or treatment with preserved sodium hyaluronate 0.1% in dry eye syndrome. Preservative-free sodium hyaluronate 0.1% eye drops can increase the effect of diquafosol 3% in dry eye syndrome.

Rebamipide suppresses collagen-induced arthritis through reciprocal regulation of th17/treg cell differentiation and heme oxygenase 1 induction.

OBJECTIVE: Rebamipide, a gastroprotective agent, has the ability to scavenge reactive oxygen radicals. Increased oxidative stress is implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to investigate the impact of rebamipide on the development of arthritis and the pathophysiologic mechanisms by which rebamipide attenuates arthritis severity in a murine model of RA. METHODS: Collagen-induced arthritis (CIA) was induced in DBA/1J mice. Anti-type II collagen antibody titers and interleukin-17 (IL-17) levels were determined using enzyme-linked immunosorbent assay. The expression of transcription factors was analyzed by immunostaining and Western blotting. Frequencies of IL-17-producing CD4+ T cells (Th17 cells) and CD4+CD25+FoxP3+ Treg cells were analyzed by flow cytometry. RESULTS: Rebamipide reduced the clinical arthritis score and severity of histologic inflammation and cartilage destruction in a dose-dependent manner. The joints isolated from rebamipide-treated mice with CIA showed decreased expression of nitrotyrosine, an oxidative stress marker. Rebamipide-treated mice showed lower circulating levels of type II collagen-specific IgG, IgG1, and IgG2a. Whereas the number of Th17 cells in spleens was decreased in rebamipide-treated mice with CIA, a significant increase in the number of Treg cells in spleens was observed. In vitro, rebamipide inhibited Th17 cell differentiation through STAT-3/retinoic acid receptor-related orphan nuclear receptor γt and reciprocally induced Treg cell differentiation through FoxP3. Rebamipide increased Nrf2 nuclear activities in murine CD4+ T cells and LBRM-33 murine T lymphoma cells. Heme oxygenase 1 (HO-1) expression in the spleens was markedly increased in rebamipide-treated mice. CONCLUSION: The inhibitory effects of rebamipide on joint inflammation are associated with recovery from an imbalance between Th17 cells and Treg cells and with activation of an Nrf2/HO-1 antioxidant pathway.

Expression of miRNAs and ZEB1 and ZEB2 correlates with histopathological grade in papillary urothelial tumors of the urinary bladder.

Histopathological grading of papillary urothelial tumors (PUTs) of the urinary bladder is subjective and poorly reproducible. We investigated the relationship between the expression of frequently deregulated microRNAs (miRNAs) as well as their target genes (ZEB1/ZEB2) and bladder cancer histopathological grade in an attempt to find a miRNA that might allow more reliable grading of PUTs. We measured the expression levels of four miRNAs (miR-145, miR-205, miR-125b, and miR-200c) in 120 formalin-fixed, paraffin-embedded bladder tumor tissue samples using real-time PCR assays. ZEB1 and ZEB2 expression was assessed in the same bladder tissues by immunohistochemistry. MiR-205 distinguished low-grade papillary urothelial carcinoma (LG) from high-grade papillary urothelial carcinoma (HG), and miR-145 distinguished HG from infiltrating carcinoma (CA) with an area under the receiver operator characteristic curve (AUC) of 0.992 and 0.997, respectively (sensitivity/specificity of 95.8/96.7 % and 100/91.7 %, respectively; p < 0.05). The expression level of miR-125b was significantly lower in LG than in PUNLMP, with an AUC value of 0.870 (93.3 % sensitivity and 84.2 % specificity; p < 0.05). ZEB1 immunoreactivity was more frequently detected in HG than in LG (57 % vs 13 %, p < 0.01) and in HG than in CA (57 % vs 17 %, p < 0.01). ZEB2 immunoreactivity was more frequent in CA than in HG (83 % vs 54 %, p < 0.05). ZEB1/ZEB2 and miRNAs expression seems to reliably distinguish between different grades of PUTs of the urinary bladder. They might well serve as useful complementary diagnostic biomarkers for grading of papillary urothelial tumors.

Expression of miRNAs and PTEN in endometrial specimens ranging from histologically normal to hyperplasia and endometrial adenocarcinoma.

We investigated the relationship between frequently deregulated microRNAs (miRNAs) and enodometrial pathology in an attempt to find the most dependable miRNA or combination of miRNAs to identify normal, hyperplastic and malignant endometrial tissues. We also investigated the association between those miRNAs and PTEN status. We measured the expression of six miRNAs (miR-21, 182, 183, 200a, 200c and 205) in 75 formalin-fixed, paraffin-embedded normal, hyperplastic, and malignant endometrial tissue blocks using Taqman-based real-time PCR assays. PTEN loss of expression was assessed in the same endometrial tissues by immunohistochemistry. Expression of five miRNAs (miR-182, 183, 200a, 200c and 205) was significantly higher in endometrial carcinoma (CA) when compared with complex atypical hyperplasia (CAH), simple hyperplasia (SH) and normal endometrial tissue (P<0.05, respectively). Considering the likelihood ratio and number of parameters, the composite panel of six miRNAs was the best marker, revealing a sensitivity of 91% and a specificity of 94% in differentiating endometrial CA from endometrial hyperplasia or normal endometrium while the individual miRNAs exhibited 64-77% sensitivity and 66-91% specificity. Interestingly, in distinguishing endometrial CA from CAH, the composite panel of four miRNAs (miR-182, 183, 200a, 200c) was the best marker, producing 95% sensitivity and 91% specificity. The percentage of PTEN loss was significantly higher in endometrial CA compared with SH (68% vs 24%, P<0.05), and it was also higher in CAH compared with SH (71% vs 24%, P<005). Aberrant expression of miRNAs and loss of PTEN expression are common in endometrial hyperplasia and CA. They might serve to increase the diagnostic reproducibility and improve discrimination, especially, between CAH and CA by miRNA expression profiles and between simple and complex hyperplasia through PTEN expression patterns. Those expression profiles of biomarkers also might be used to predict the potential for progression from endometrial hyperplasia to invasive CA.

The inhibitory effects of bevacizumab eye drops on NGF expression and corneal wound healing in rats.

PURPOSE: To investigate the effect of bevacizumab eye drops on corneal epithelial wound healing in rats. METHODS: One hundred twenty Sprague-Dawley male rat corneas were divided into two groups and de-epithelized with a microblade. The bevacizumab group was treated with 5% bevacizumab and antibiotic 0.5% levofloxacin eye drops four times daily and the control group with antibiotic eye drops only. Corneal wound healing was evaluated by fluorescein staining at initial wounding and 24, 48, and 72 hours after epithelial debridement. Nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) proteins were measured in rat corneas by ELISA. Immunofluorescent staining for NGF and VEGF was performed in rat corneas. NGF mRNA and VEGF mRNA was measured in rat corneas by real-time PCR. RESULTS: The corneal wound healing rate was decreased in the bevacizumab group compared with that in the control group. Twenty-four, 48, and 72 hours after epithelial debridement, corneal NGF and VEGF proteins in the bevacizumab group were lower than that in the control group (P<0.05). Immunofluorescent staining showed that NGF and VEGF expression was stronger in the control group than in the bevacizumab group. NGF mRNA and VEGF mRNA levels in the bevacizumab group were also lower than in the control group (P<0.05). CONCLUSIONS: After corneal epithelial damage, VEGF and NGF increased normally in the rat corneas. In contrast, when VEGF was inhibited by bevacizumab eye drops, the wound healing rate was decreased, and NGF was downregulated. Bevacizumab eye drops have an inhibitory effect on corneal wound healing in rats.