Continuous administration of mirabegron has advantages in inhibition of central sensitization compared with short-term treatment cessation in a mouse model of overactive bladder.

AIMS: There is no clear pathophysiologic evidence determining how long overactive bladder (OAB) medication should be continued. We, therefore, investigated the effect of mirabegron using cessation (CES) or continuation (CON) treatment in an OAB animal model. METHODS: Female C57BL/6 mice were divided into four groups (N = 8 each): Sham, OAB, CES, and CON groups. The OAB-like condition was induced by three times weekly intravesical instillations of KCl mixture with hyaluronidase. After the last intravesical instillation for inducing OAB, mirabegron (2 mg/kg/day) was administered in CES and CON groups for 10 and 20 days, respectively. Final experiments were carried out on 20 days from the last intravesical instillation in all groups. After cystometry, mRNA levels of bladder muscarinic, ß-adrenergic, and P2X purinergic receptors were measured to investigate bladder efferent and afferent activity. In addition, mRNA levels of CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG) were measured to assess afferent sensitization. Immunofluorescent staining of CX3CR1, GFAP, and CCR2 in the L6 spinal cord was also conducted to investigate glial activation and central sensitization. RESULTS: OAB mice showed bladder overactivity evidenced by decreased intercontraction interval (3.56 ± 0.51 vs. 5.76 ± 0.95 min in sham mice), increased non-voiding contractions (0.39 ± 0.11 vs. 0.13 ± 0.07/min in sham mice), and inefficient voiding (72.1 ± 8.6% vs. 87.1 ± 9.5% in sham mice). Increased M2, M3, ß2, ß3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG indicate bladder efferent and afferent hyperexcitability. In addition, CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in OAB mice. However, the CON group exhibited reduced ß2, ß3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder, reduced CCL2 and CCR2 in DRG, which are markers of afferent hyperexcitability, and reduced immunoreactivities of CX3CR1, GFAP, and CCR2 in the L6 spinal cord, which are markers of the central sensitization. Moreover, the CON group showed better improvements in nonvoiding contractions (0.16 ± 0.09 vs. 0.44 ± 0.17/min) and voiding efficiency (93.9 ± 7.4% vs. 76.5 ± 13.1%) and reductions in bladder ß3 receptors and CCL2 of L6-S1 DRG, and immunoreactivities of CX3CR1 and GFAP in the L6 spinal cord compared to the CES group. CONCLUSIONS: Continuous mirabegron treatment seems to prevent central sensitization and, thus, might be desirable for long-term disease control of OAB.

Antifibrosis treatment by inhibition of VEGF, FGF, and PDGF receptors improves bladder wall remodeling and detrusor overactivity in association with modulation of C-fiber afferent activity in mice with spinal cord injury.

AIMS: Spinal cord injury (SCI) above the sacral level causes bladder dysfunction and remodeling with fibrosis. This study examined the antifibrotic effects using nintedanib, an inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors, on detrusor overactivity (DO) and bladder fibrosis, as well as the modulation mechanisms of C-fiber afferent pathways. METHODS: Thirty female C57BL/6 mice were divided into group A (spinal intact), group B (SCI with vehicle), and group C (SCI with nintedanib). At 2 weeks after SCI, vehicle or 50 mg/kg nintedanib was administered subcutaneously for 2 weeks. Then, cystometry was conducted, followed by RT-PCR measurements of fibrosis-related molecules, muscarinic, ß-adrenergic, TRP and purinergic receptors in the bladder or L6-S1 dorsal root ganglia (DRG). Trichrome stain and Western blot analysis of transforming growth factor-beta and fibronectin were performed in the bladder. TRPV1 expression in L6 DRG was measured by immunohistochemistry. RESULTS: In cystometry, intercontraction intervals, nonvoiding contractions, voided volume, and voiding efficiency were significantly improved in group C versus group B. RT-PCR, Western blotting, and trichrome staining revealed the fibrotic changes in the bladder of group B, which was improved in group C. Increased messenger RNA levels of TRPV1, TRPA1, P2X2 , and P2X3 in DRG of group B were significantly decreased in group C. TRPV1 immunoreactivity in DRG was increased in group B, but decreased in group C. CONCLUSIONS: Nintedanib improves storage and voiding dysfunctions and bladder fibrosis in SCI mice. Also, nintedanib-induced improvement of DO is associated with reduced expression of C-fiber afferent markers, suggesting the modulation of bladder C-fiber afferent activity.

Maternal Characteristics, Short Mid-Trimester Cervical Length, and Preterm Delivery.

We aimed to determine the maternal characteristics (demographics, an obstetric history, and prior cervical excisional procedure) associated with a short mid-trimester cervical length (CL, defined as a CL of ≤ 25 mm) and whether having a short cervix explains the association between these maternal characteristics and spontaneous preterm delivery (SPTD, defined as a delivery before 34 weeks). This is a single-center retrospective cohort study of 3,296 consecutive women with a singleton pregnancy who underwent routine CL measurement between 20 and 24 weeks. Data were collected on maternal age, weight, height, parity, obstetric history (nulliparity; a history of at least 1 SPTD; and at least 1 term birth and no preterm birth [low-risk history group]), and prior cervical excisional procedure. In the multivariate regression analysis, an obstetric history, prior cervical excisional procedure, and gestational age at measurement were the variables significantly associated with short CL. In contrast, maternal weight, height, age, and parity were not significantly associated with short CL. By using the likelihood of SPTD as an outcome variable, logistic regression indicated that short CL and obstetric history, but not prior cervical excisional procedure, were significantly associated with SPTD after adjustment for potential confounders. A history of SPTD and prior cervical excisional procedure were associated with an increased risk of a short mid-trimester CL. A history of SPTD, but not prior cervical excisional procedure, is associated with an increased risk of SPTD, independent of a short CL.

Idiopathic pulmonary arterial hypertension associated with a novel frameshift mutation in the bone morphogenetic protein receptor II gene and enhanced bone morphogenetic protein signaling: A case report.

RATIONALE: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by intense remodeling of small pulmonary arteries. Loss-of-function mutation of bone morphogenetic protein receptor II (BMPR2) gene and exaggerated activation of transforming growth factor (TGF)-ß signaling play a critical role in this process. PATIENT CONCERNS AND DIAGNOSIS: We report a novel frameshift mutation (c.117InsT, p.Y40fsX48) of the BMPR2 gene identified in a 19-year-old IPAH patient with syncope. Despite BMPR2 mutation, the phosphorylation of Smad2/3 and Samd1/5/8 was increased in the patient's peripheral blood mononuclear cells, and this event was accompanied by the upregulation of bone morphogenetic protein (BMP) signaling target genes, but not TGF-ß signaling target genes. Moreover, we observed an increased expression of other BMPRs, that is, anti-Mullerian hormone type-2 receptor and the activin receptor-like kinases (ALK) 1, ALK3, and ALK6. INTERVENTIONS AND OUTCOMES: The patient was prescribed a combination of macitentan, sildenafil, and nifedipine, which successfully controlled her symptom of syncope and normalized N-terminal pro-brain natriuretic peptide level after 3 months of medication. LESSONS: In light of these results, we propose a new pathogenetic mechanism for IPAH, based on enhanced BMP signaling via the functional replacement of mutated BMPR2 by other BMP receptors.

Enhanced Activation of Rac1/Cdc42 and MITF Leads to Augmented Osteoclastogenesis in Autosomal Dominant Osteopetrosis Type II.

The autosomal dominant osteopetrosis type II (ADOII) caused by the mutation of chloride channel 7 (ClC-7) gene is the most common form of adult-onset osteopetrosis. Despite dysfunctional bone resorption, an augmented osteoclast differentiation was reported recently in ADOII patients. DNA sequencing analysis of the ADOII patient's ClC-7 gene identified a known heterozygous mutation, c.643G>A in exon 7, encoding p.Gly215Arg. In vitro osteoclast differentiation from the ADOII patient's peripheral blood mononuclear cells (PBMCs) increased compared with control despite their dysfunctional bone resorbing capacity. Osteoclasts from the ADOII patient's PBMCs and ClC-7 knockdown bone marrow monocytes (BMMs) showed an enhanced Ser-71 phosphorylation of Rac1/Cdc42 and increase of the microphthalmia-associated transcription factor (MITF) and receptor activator of NF-κB (RANK) that can be responsible for the enhanced osteoclast differentiation. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Degrading products of chondroitin sulfate can induce hypertrophy-like changes and MMP-13/ADAMTS5 production in chondrocytes.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.

Vitamin D-binding protein in cervicovaginal fluid as a non-invasive predictor of intra-amniotic infection and impending preterm delivery in women with preterm labor or preterm premature rupture of membranes.

OBJECTIVE: To determine whether vitamin D-binding protein (VDBP) in cervicovaginal fluid (CVF) is independently predictive of intra-amniotic infection and imminent spontaneous preterm delivery (SPTD, delivery within 48 hours) in women with preterm labor with intact membranes (PTL) or preterm premature rupture of membranes (PPROM). METHOD: This was a single-center retrospective cohort study. CVF samples for VDBP assays were obtained along with serum C-reactive protein (CRP) levels immediately after amniocentesis in consecutive women with PTL (n = 148) or PPROM (n = 103) between 23.0 and 34.0 weeks of gestation. VDBP levels in CVF were determined by enzyme-linked immunosorbent assay kits. The primary outcome measures were intra-amniotic infection [defined as positive amniotic fluid (AF) culture] and SPTD within 48 hours after sampling. RESULTS: In the multivariable analysis, elevated VDBP levels in CVF samples of PTL women were significantly associated with intra-amniotic infection and imminent preterm delivery, even after adjusting for potential confounders (e.g., gestational age at sampling, parity, and serum CRP). However, these relationships were not found in women with PPROM. In women with PTL, the areas under receiver operating characteristic curves of CVF VDBP level for predicting intra-amniotic infection and imminent preterm delivery were 0.66 and 0.71, with cut-off values of 1.76 µg/mL (sensitivity of 64.3% and specificity of 78.4%) and 1.37 µg/mL (sensitivity of 65.4% and specificity of 72.6%), respectively. The CVF VDBP levels were significantly higher in women with PPROM than in those with PTL. CONCLUSIONS: VDBP in the CVF independently predicts intra-amniotic infection and imminent preterm delivery in women with PTL, whereas in women with PPROM, an elevated VDBP level in CVF is not associated with increased risks of these two outcome variables.

Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis.

An activation of osteoclasts and subchondral bone remodeling is a major histologic feature of early-stage osteoarthritis (OA), which can be accompanied by an increase of calcium (Ca) and phosphate (Pi) level in the subchondral milieu. Considering articular cartilage gets most of nutrition from subchondral bone by diffusion, these micro-environmental changes in subchondral bone can affect the physiology of articular chondrocytes. Here, we have shown that Ca is increased and co-localized with Pi in articular cartilage of early-stage OA. The Ca-Pi complex increased the production of MMP-3 and MMP-13 in the hypertrophic chondrocytes, which was dependent on nuclear factor-kappa B (NF-kB), p38 and extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein (MAP) kinase and Signal transducer and activator of transcription 3 (STAT3) signaling. The Ca-Pi complexes increased the expression of endocytosis markers, and the inhibition of the formation of the Ca-Pi complex ameliorated the Ca-Pi complex-mediated increases of MMPs expression in hypertrophic chondrocytes. Our data provide insight regarding the Ca-Pi complex as a potential catabolic mediator in the subchondral milieu and support the pathogenic role of subchondral bone in the early stages of cartilage degeneration.

Frequency and clinical significance of short cervix in patients with preterm premature rupture of membranes.

OBJECTIVE: Cervical length measurement has been uggested as a useful tool for predicting intra-amniotic infection/inflammation in preterm labor, but little information is available in the setting of preterm premature rupture of membranes (pPROM). We aimed to determine whether a short cervical length is independently associated with an increased risk of intra-amniotic infection or inflammation and impending preterm delivery in women with pPROM. METHODS: This was a retrospective cohort study involving 171 consecutive singleton pregnant women with pPROM (21+0-33+6 weeks' gestation), who underwent amniocentesis. Amniotic fluid (AF) was cultured, and assayed for interleukin (IL)-6 and IL-8. Cervical length was measured at the time of amniocentesis by transvaginal ultrasonography with an aseptic technique. Short cervical length was defined as a cervical length of ≤15 mm. Intra-amniotic infection was defined as a positive AF culture for microorganisms and intra-amniotic inflammation was defined as elevated AF concentrations of IL-6 or IL-8 (IL-6 ≥1.5 ng/mL and/or IL-8 ≥1.3 ng/mL). RESULTS: Fifty (29.2%) women had a sonographic cervical length of ≤15mm. On univariate analysis, short cervical length was associated with an increased risk for intra-amniotic infection and/or inflammation; no other parameters studied showed a significant association. Multivariable analyses indicated that short cervical length was significantly associated with a higher risk of impending preterm delivery (within 2 days of measurement, within 7 days of measurement, and before 34 weeks), and remained significant after adjustment for potential confounders. CONCLUSION: In women with pPROM, short cervical length is associated with an increased risk for intra-amniotic infection/inflammation and associated with impending preterm delivery, independent of the presence of intra-amniotic infection/inflammation.

Short cervical lengths initially detected in mid-trimester and early in the third trimester in asymptomatic twin gestations: Association with histologic chorioamnionitis and preterm birth.

OBJECTIVE: To determine whether short cervical lengths (≤20 mm) that were initially detected in mid-trimester and early in the third trimester are independently associated with increased risks of subsequent histologic chorioamnionitis and spontaneous preterm birth (SPTB, defined as a delivery before 34 weeks) in asymptomatic women with twin pregnancies. MATERIAL AND METHODS: This is a prospective study including 292 consecutive asymptomatic women with twin gestations. Cervical length measurements were carried out at 20 to 24 weeks' gestation and at 28 to 32 weeks' gestation. Both placentas of each twin pair were examined histologically after delivery. The generalized estimation equations models and logistic regression analysis were used for statistical analyses. RESULTS: Multivariable generalized estimation equations analysis revealed that short cervical length at mid-trimester was independently associated with an increased risk for subsequent histologic chorioamnionitis, whereas short cervical length initially detected early in the third trimester was not. By using the likelihood of SPTB as an outcome variable, multivariable logistic regression analysis indicated that short mid-trimester cervical length and histologic chorioamnionitis were independently associated with a greater risk for SPTB. Similarly, based on the multivariable analysis, a short third trimester cervical length was independently and significantly associated with a greater risk for SPTB. CONCLUSIONS: In asymptomatic women with twin pregnancies, a short mid-trimester cervical length is independently associated with an increased risk of both subsequent histologic chorioamnionitis and SPTB, whereas a short cervical length initially detected early in the third trimester is independently associated with preterm delivery, but not subsequent histologic chorioamnionitis.

Intracellular delivery of desulfated heparin with bile acid conjugation alleviates T cell-mediated inflammatory arthritis via inhibition of RhoA-dependent transcellular diapedesis.

Heparin has a potential regulatory role in inflammatory diseases. However, the anticoagulant activity and poor oral bioavailability of heparin limit its use as an anti-inflammatory agent. Conjugation of bis-deoxycholic acid to 6-O-desulfated low molecular weight heparin (6DSHbD) was efficiently internalized by activated endothelial cells via a 2-step model, in which heparin attaches to adhesion molecules that facilitate accessibility of the bile acid conjugate to membrane transporters. The critical role of P-selectin during endothelial cell uptake of 6DSHbD by arthritic tissue was confirmed in p-selectin(-/-) arthritic mice. Intracellular 6DSHbD inhibited transcellular diapedesis of T cells through activated endothelial cells and impaired both the formation of ICAM-1-rich docking structures at the T cell contact surface and subsequent cytoskeletal rearrangement. Furthermore, 6DSHbD blocked activation of RhoA-GTPase and phosphorylation of ezrin/radixin/moesin induced by ICAM-1 cross-linking on activated endothelial cells, thereby impairing lymphocyte transcellular transmigration. After oral administration 6DSHbD was preferentially delivered to inflamed joint tissue, particularly in and around post-capillary venular endothelium and inhibited effector T cell homing to arthritic joints. Aggravation of collagen-induced arthritis conferred by the transfer of effector T cells was suppressed by oral 6DSHbD. Thus, intracellular heparin exerts anti-inflammatory effects through the inhibition of RhoA-dependent transendothelial recruitment of T cells and may have applications in the treatment of chronic inflammatory arthritis.

A common polymorphism in pre-microRNA-146a is associated with lung cancer risk in a Korean population.

INTRODUCTION: MicroRNAs (miRs) play important roles in the development and progression of human cancers. MiR-146a down-regulates epidermal growth factor receptor and the nuclear factor-κB regulatory kinase interleukin-1 receptor-associated kinase 1 genes that play important roles in lung carcinogenesis. This study was conducted to evaluate the association between rs2910164C>G, a functional polymorphism in the pre-miR-146a, and lung cancer risk. MATERIAL AND METHODS: The rs2910164C>G genotypes were determined in 1094 patients with lung cancer and 1100 healthy controls who were frequency matched for age and gender. RESULTS: The rs2910164 CG or GG genotype was associated with a significantly decreased risk for lung cancer compared to that of the CC genotype (adjusted odds ratio=0.80, 95% confidence interval=0.66-0.96, P=0.02). When subjects were stratified according to smoking exposure (never, light and heavy smokers), the effect of the rs2910164C>G genotype on lung cancer risk was significant only in never smokers (adjusted odds ratio=0.66, 95% confidence interval=0.45-0.96, P=0.03, under a dominant model for the C allele) and decreased as smoking exposure level increased (Ptrend<0.001). In line with this result, the level of miR-146a expression in the tumor tissues was significantly higher in the GG genotype than in the CC or CG genotype only in never-smokers (P=0.02). CONCLUSIONS: These findings suggest that the rs2910164C>G in pre-miR-146a may contribute to genetic susceptibility to lung cancer, and that miR-146a might be involved in lung cancer development.

Association between genetic variants in pre-microRNAs and survival of early-stage NSCLC.

INTRODUCTION: MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. Recent evidence indicates that altered miRNA expression plays an important role in the initiation and progression of lung cancer. Single nucleotide polymorphisms (SNPs) in pre-miRNAs could alter miRNAs processing, or expression, and hence, could influence the prognosis of lung cancer. To test this hypothesis, we evaluated the effects of four SNPs in pre-miRNAs (pre-miR-146a rs2910164, pre-miR-149 rs2292832, pre-miR-196a rs11614913, and pre-miR-499 rs3746444) on the survival outcomes of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: Three hundred sixty-three patients with surgically resected NSCLC were enrolled. The four SNPs were genotyped using a polymerase chain reaction-restriction fragment length polymorphism assay. The genotype associations with overall survival (OS) and disease-free survival (DFS) were analyzed. RESULTS: Of the four SNPs examined, the pre-miR-149 rs2292832T>C and pre-miR-196a rs11614913C>T were found to be significantly associated with OS and DFS. The rs2292832 TC or CC genotype exhibited a significantly better OS and DFS compared with the rs2292832 TT genotype (adjusted hazard ratio [aHR] for OS, 0.66; 95% confidence interval [CI], 0.47-0.92; p = 0.01 and aHR for DFS, 0.64; 95% CI, 0.48-0.87; p = 0.004). For the pre-miR-196a rs11614913C>T, patients with the CT or TT genotype had a significantly better OS and DFS than those with the CC genotype (aHR for OS, 0.70; 95% CI, 0.49-0.99; p = 0.05 and aHR for DFS, 0.66; 95% CI, 0.48-0.90; p = 0.01). When the two SNPs were combined, OS and DFS improved in a dose-dependent manner as the number of good genotypes increased (p = 0.002 and 0.0001, respectively). CONCLUSIONS: These results suggest that miR-149 and miR-196a may be involved in the pathogenesis of NSCLC, and that rs2292832 and rs11614913 can be used as prognostic markers for patients with surgically resected early-stage NSCLC.