Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers.

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

BACKGROUND: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. METHODS: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. RESULTS: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. CONCLUSIONS: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Deficiency of Cardiac Natriuretic Peptide Signaling Promotes Peripartum Cardiomyopathy-Like Remodeling in the Mouse Heart.

BACKGROUND: The maternal circulatory system and hormone balance both change dynamically during pregnancy, delivery, and the postpartum period. Although atrial natriuretic peptides and brain natriuretic peptides produced in the heart control circulatory homeostasis through their common receptor, NPR1, the physiologic and pathophysiologic roles of endogenous atrial natriuretic peptide/brain natriuretic peptide in the perinatal period are not fully understood. METHODS: To clarify the physiologic and pathophysiologic roles of the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system during the perinatal period, the phenotype of female wild-type and conventional or tissue-specific Npr1-knockout mice during the perinatal period was examined, especially focusing on maternal heart weight, blood pressure, and cardiac function. RESULTS: In wild-type mice, lactation but not pregnancy induced reversible cardiac hypertrophy accompanied by increases in fetal cardiac gene mRNAs and ERK1/2 (extracellular signaling-regulated kinase) phosphorylation. Npr1-knockout mice exhibited significantly higher plasma aldosterone level than did wild-type mice, severe cardiac hypertrophy accompanied by fibrosis, and left ventricular dysfunction in the lactation period. Npr1-knockout mice showed a high mortality rate over consecutive pregnancy-lactation cycles. In the hearts of Npr1-knockout mice during or after the lactation period, an increase in interleukin-6 mRNA expression, phosphorylation of signal transducer and activator of transcription 3, and activation of the calcineurin-nuclear factor of the activated T cells pathway were observed. Pharmacologic inhibition of the mineralocorticoid receptor or neuron-specific deletion of the mineralocorticoid receptor gene significantly ameliorated cardiac hypertrophy in lactating Npr1-knockout mice. Anti-interleukin-6 receptor antibody administration tended to reduce cardiac hypertrophy in lactating Npr1-knockout mice. CONCLUSIONS: These results suggest that the characteristics of lactation-induced cardiac hypertrophy in wild-type mice are different from exercise-induced cardiac hypertrophy, and that the endogenous atrial natriuretic peptide/brain natriuretic peptide-NPR1 system plays an important role in protecting the maternal heart from interleukin-6-induced inflammation and remodeling in the lactation period, a condition mimicking peripartum cardiomyopathy.

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Cell competition is involved in mammalian embryogenesis and tumor elimination and progression. It was previously shown that, whereas NIH3T3 mouse fibroblasts expressing high levels of the yes-associated protein 1(YAP1) target TEA domain family (TEAD) transcription factors become "winners" in cell competitions, Madin-Darby canine kidney cells expressing activated YAP1 become "losers" and are eliminated from culture monolayers. Thus, YAP1's role in cell competitions is clearly context dependent. Here, we show that keratinocytes overexpressing a constitutively activated YAP1 mutant lose in in vitro competitions with control cells conducted in standard tissue culture dishes and undergo apical extrusion. Similarly, cells in which endogenous YAP1 is activated by NF2 knockdown become losers. The YAP1-overexpressing cells exhibit a decrease in cell-matrix adhesion because of defective expression of adhesion molecules such as fibronectin-1. Cell adhesion-mediated proliferation is also impaired. However, because of intrinsic factors, YAP1-expressing cells proliferate faster than control cells when cocultured in dishes impeding cell adhesion. In vivo, Mob1a/b-deficient (YAP1-activated) epidermis, which shows decreased expression of type XVII collagen, cannot be engrafted successfully onto donor mice. YAP1-activated skin grafts shrink away from surrounding control skin, and the epidermis peels off the basement membrane. Our data show that YAP1 activation controls cell competition in part by decreasing cell adhesion.-Nishio, M., Miyachi, Y., Otani, J., Tane, S., Omori, H., Ueda, F., Togashi, H., Sasaki, T., Mak, T. W., Nakao, K., Fujita, Y., Nishina, H., Maehama, T., Suzuki, A. Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

C-type natriuretic peptide (CNP)/guanylate cyclase B (GC-B) system and endothelin-1(ET-1)/ET receptor A and B system in human vasculature.

To assess the physiological and clinical implications of the C-type natriuretic peptide (CNP)/guanylyl cyclase B (GC-B) system in the human vasculature, we have examined gene expressions of CNP and its receptor, GC-B, in human vascular endothelial cells (ECs) and smooth muscle cells (SMCs) and have also compared the endothelin-1(ET-1)/endothelin receptor-A (ETR-A) and endothelin receptor-B (ETR-B) system in human aortic ECs (HAECs) and vascular SMCs (HSMCs) in vitro. We also examined these gene expressions in human embryonic stem (ES)/induced pluripotent stem cell (iPS)-derived ECs and mural cells (MCs). A little but significant amount of mRNA encoding CNP was detected in both human ES-derived ECs and HAECs. A substantial amount of GC-B was expressed in both ECs (iPS-derived ECs and HAECs) and SMCs (iPS-derived MCs and HSMCs). ET-1 was expressed solely in ECs. ETR-A was expressed in SMCs, while ETR-B was expressed in ECs. These results indicate the existence of a vascular CNP/GC-B system in the human vascular wall, indicating the evidence for clinical implication of the CNP/GC-B system in concert with the ET-1/ETR-A and ETR-B system in the human vasculature.

The association between uric acid levels and renal function of CKD patients with hyperlipidemia: a sub-analysis of the ASUCA trial.

BACKGROUND: The influence of uric acid (UA) on renal function and the significance of UA-lowering therapy are unclear. The purpose of the sub-analysis of the Assessment of Clinical Usefulness in chronic kidney disease patients with Atorvastatin (ASUCA) trial was to evaluate the influence of serum UA levels on renal function in Japanese chronic kidney disease patients with hyperlipidemia. METHODS: Of 344 participants in the ASUCA trial, 279 participants whose UA levels at both baseline and 24 months were available were included. Based on UA level at baseline or mean UA level during the trial period, they were divided into four groups: < 5.0, 5.0-6.0, 6.0-7.0, or ≥ 7.0 mg/dL, irrespective of allocation. Changes in the estimated glomerular filtration rate (eGFR) after 24 months were compared among the groups in relation to baseline or mean UA levels. RESULTS: For baseline UA levels (< 5.0, 5.0-6.0, 6.0-7.0, or ≥ 7.0 mg/dL), the change in eGFR after 24 months was - 1.32 ± 10.3, - 1.74 ± 8.94, - 2.53 ± 7.34, and - 3.51 ± 9.10 mL/min/1.73 m2, respectively. A negative correlation between changes in eGFR after 24 months and baseline UA level was observed with adjustment for confounding factors. The relationship between changes in eGFR and mean UA levels during trial period showed a similar trend. CONCLUSION: In CKD patients with dyslipidemia, hyperuricemia was an independent risk factor for CKD progression. An ongoing clinical trial (TARGET-UA, UMIN-ID 000,026,741) may reveal the significance of strict UA-lowering therapy in CKD patients.

Achieving LDL cholesterol target levels <1.81 mmol/L may provide extra cardiovascular protection in patients at high risk: Exploratory analysis of the Standard Versus Intensive Statin Therapy for Patients with Hypercholesterolaemia and Diabetic Retinopathy study.

AIMS: To assess the benefits of intensive statin therapy on reducing cardiovascular (CV) events in patients with type 2 diabetes complicated with hyperlipidaemia and retinopathy in a primary prevention setting in Japan. In the intension-to-treat population, intensive therapy [targeting LDL cholesterol <1.81 mmol/L (<70 mg/dL)] was no more effective than standard therapy [LDL cholesterol ≥2.59 to <3.10 mmol/L (≥100 to <120 mg/dL)]; however, after 3 years, the intergroup difference in LDL cholesterol was only 0.72 mmol/L (27.7 mg/dL), and targeted levels were achieved in <50% of patients. We hypothesized that the intergroup difference in CV events would have been statistically significant if more patients had been successfully treated to target. MATERIALS AND METHODS: This exploratory post hoc analysis focused on intergroup data from patients who achieved their target LDL cholesterol levels. The primary endpoint was the composite incidence of CV events. A Cox proportional hazards model was used to estimate hazard ratios (HRs) for incidence of the primary endpoint in patients who achieved target LDL cholesterol levels in each group. RESULTS: Data were analysed from 1909 patients (intensive: 703; standard: 1206) who achieved target LDL cholesterol levels. LDL cholesterol at 36 months was 1.54 ± 0.30 mmol/L (59.7 ± 11.6 mg/dL) in the intensive group and 2.77 ± 0.46 mmol/L (107.1 ± 17.8 mg/dL) in the standard group (P < 0.05). After adjusting for baseline prognostic factors, the composite incidence of CV events or deaths associated with CV events was significantly lower in the intensive than the standard group (HR 0.48; 95% confidence interval 0.28-0.82; P = 0.007). CONCLUSIONS: This post hoc analysis suggests that achieving LDL cholesterol target levels <1.81 mmol/L may more effectively reduce CV events than achieving target levels ≥2.59 to <3.10 mmol/L in patients with hypercholesterolaemia and diabetic retinopathy.

Dysregulated YAP1/TAZ and TGF-ß signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice.

Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-ß)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-ß pathway may be effective treatment for cHC-CCs and ICCs.

Translational science: Newly emerging science in biology and medicine - Lessons from translational research on the natriuretic peptide family and leptin.

Translation is the process of turning observations in the laboratory, clinic, and community into interventions that improve the health of individuals and the public, ranging from diagnostics and therapeutics to medical procedures and behavioral changes. Translational research is defined as the effort to traverse a particular step of the translation process for a particular target or disease. Translational science is a newly emerging science, distinct from basic and clinical sciences in biology and medicine, and is a field of investigation focused on understanding the scientific and operational principles underlying each step of the translational process. Advances in translational science will increase the efficacy and safety of translational research in all diagnostic and therapeutic areas. This report examines translational research on novel hormones, the natriuretic peptide family and leptin, which have achieved clinical applications or for which studies are still ongoing, and also emphasizes the lessons that translational science has learned from more than 30 years' experience in translational research.

CRISPR/Cas9-mediated Angptl8 knockout suppresses plasma triglyceride concentrations and adiposity in rats.

Angiopoietin-like protein (ANGPTL)8 is a liver- and adipocyte-derived protein that controls plasma triglyceride (TG) levels. Most animal studies have used mouse models. Here, we generated an Angptl8 KO rat model using a clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system to clarify the roles of ANGPTL8 in glucose and lipid metabolism. Compared with WT rats, Angptl8 KO rats had lower body weight and fat content, associated with impaired lipogenesis in adipocytes; no differences existed between the groups in food intake or rectal temperature. Plasma TG levels in both the fasted and refed states were significantly lower in KO than in WT rats, and an oral fat tolerance test showed decreased plasma TG excursion in Angptl8 KO rats. Higher levels of lipase activity in the heart and greater expression of genes related to ß-oxidation in heart and skeletal muscle were observed in Angptl8 KO rats. However, there were no significant differences between KO and WT rats in glucose metabolism or the histology of pancreatic ß-cells on both standard and high-fat diets. In conclusion, we demonstrated that Angptl8 KO in rats resulted in lower body weight and plasma TG levels without affecting glucose metabolism. ANGPTL8 might be an important therapeutic target for obesity and dyslipidemia.

Increase of Total Nephron Albumin Filtration and Reabsorption in Diabetic Nephropathy.

The amount of albumin filtered through the glomeruli and reabsorbed at the proximal tubules in normal and in diabetic kidneys is debated. The megalin/cubilin complex mediates protein reabsorption, but genetic knockout of megalin is perinatally lethal. To overcome current technical problems, we generated a drug-inducible megalin-knockout mouse line, megalin(lox/lox);Ndrg1-CreERT2 (iMegKO), in which megalin expression can be shut off at any time by administration of tamoxifen (Tam). Tam administration in adult iMegKO mice decreased the expression of renal megalin protein by 92% compared with that in wild-type C57BL/6J mice and almost completely abrogated renal reabsorption of intravenously injected retinol-binding protein. Furthermore, urinary albumin excretion increased to 175 µg/d (0.46 mg albumin/mg creatinine) in Tam-treated iMegKO mice, suggesting that this was the amount of total nephron albumin filtration. By comparing Tam-treated, streptozotocin-induced diabetic iMegKO mice with Tam-treated nondiabetic iMegKO mice, we estimated that the development of diabetes led to a 1.9-fold increase in total nephron albumin filtration, a 1.8-fold increase in reabsorption, and a significant reduction in reabsorption efficiency (86% efficiency versus 96% efficiency in nondiabetic mice). Insulin treatment normalized these abnormalities. Akita;iMegKO mice, another model of type 1 diabetes, showed equivalent results. Finally, nondiabetic iMegKO mice had a glomerular sieving coefficient of albumin of 1.7×10-5, which approximately doubled in diabetic iMegKO mice. This study reveals actual values and changes of albumin filtration and reabsorption in early diabetic nephropathy in mice, bringing new insights to our understanding of renal albumin dynamics associated with the hyperfiltration status of diabetic nephropathy.

Seipin is necessary for normal brain development and spermatogenesis in addition to adipogenesis.

Seipin, encoded by BSCL2 gene, is a protein whose physiological functions remain unclear. Mutations of BSCL2 cause the most-severe form of congenital generalized lipodystrophy (CGL). BSCL2 mRNA is highly expressed in the brain and testis in addition to the adipose tissue in human, suggesting physiological roles of seipin in non-adipose tissues. Since we found BSCL2 mRNA expression pattern among organs in rat is similar to human while it is not highly expressed in mouse brain, we generated a Bscl2/seipin knockout (SKO) rat using the method with ENU (N-ethyl-N-nitrosourea) mutagenesis. SKO rats showed total lack of white adipose tissues including mechanical fat such as bone marrow and retro-orbital fats, while physiologically functional brown adipose tissue was preserved. Besides the lipodystrophic phenotypes, SKO rats showed impairment of spatial working memory with brain weight reduction and infertility with azoospermia. We confirmed reduction of brain volume and number of sperm in human patients with BSCL2 mutation. This is the first report demonstrating that seipin is necessary for normal brain development and spermatogenesis in addition to white adipose tissue development.

Effects of atorvastatin on renal function in patients with dyslipidemia and chronic kidney disease: assessment of clinical usefulness in CKD patients with atorvastatin (ASUCA) trial.

BACKGROUND: Dyslipidemia is a risk factor for the progression of chronic kidney disease (CKD). While conventional lipid lowering therapy provides a benefit to CKD management, the effect of statins on eGFR remains unclear. METHODS: A prospective, multi-center, open-labeled, randomized trial. Total of 349 CKD patients with hyperlipidemia were randomized into 2 groups, and followed for 2 years. Group A included patients who were treated with atorvastatin. Group C were treated with conventional lipid lowering drugs other than statin. Primary endpoint was changes in eGFR. Secondary endpoints included changes in urinary albumin excretion, serum LDL-C, serum triglyceride, cardio-vascular events and all-cause mortality. RESULTS: As the primary endpoint, eGFR decreased by 2.3 ml/min/1.73 m2 in Group A and by 2.6 ml/min/1.73 m2 in Group C, indicating that there was no difference in change of eGFR between the two groups. As secondary endpoints, atorvastatin succeeded to reduce serum LDL-C level significantly and rapidly, but conventional therapy did not. In fact, mean LDL-C level did not reach the target level of 100 mg/dl in Group C. Serum triglyceride was lowered only by atorvastatin, but not conventional drugs. The number of cardiovascular events and all-cause mortality did not differ between in two groups. CONCLUSION: The ASUCA (Assessment of Clinical Usefulness in CKD Patients with Atorvastatin) trial demonstrated that atorvastatin failed to exhibit reno-protections compared to conventional therapy in Japanese patients with dyslipidemia and CKD. It would be due in part to the ability of atorvastatin to more potently reduce serum LDL and triglycerides compared to conventional therapy.

Development of ghrelin transgenic mice for elucidation of clinical implication of ghrelin.

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of ß-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

Reciprocal expression of MRTF-A and myocardin is crucial for pathological vascular remodelling in mice.

Myocardin-related transcription factor (MRTF)-A is a Rho signalling-responsive co-activator of serum response factor (SRF). Here, we show that induction of MRTF-A expression is key to pathological vascular remodelling. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE(-/-) mice than in healthy control tissues, whereas myocardin expression was significantly lower. Both neointima formation in wire-injured femoral arteries in MRTF-A knockout (Mkl1(-/-)) mice and atherosclerotic lesions in Mkl1(-/-); ApoE(-/-) mice were significantly attenuated. Expression of vinculin, matrix metallopeptidase 9 (MMP-9) and integrin ß1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1(-/-) mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.

Overall safety and efficacy of high-dose and low-dose intravenous glucocorticoid therapy in patients with moderate-to-severe active Graves' ophthalmopathy.

The objective of this study was to compare the safety and efficacy of high-dose and low-dose intravenous (iv) glucocorticoid (GC) therapy in patients with Graves' ophthalmopathy (GO) and to investigate which factors may help determine appropriate iv GC doses. The medical records of 43 patients who received different doses of iv GCs for GO were retrospectively reviewed. Twenty patients received high-dose iv GCs (HD group, cumulative dose 9.0-12.0 g) and 18 received low-dose iv GCs (LD group, cumulative dose 4.5 g). Five patients with previous treatment for GO were excluded. Changes in ophthalmic parameters after treatment and frequencies of adverse effects due to GCs of the 2 groups were compared. We also reviewed the incidence of GO progression and hepatic dysfunction after patients were discharged. We evaluated correlations among pretreatment (before treatment) ophthalmic parameters and investigated useful predictive factors for determining iv GC doses. There were no significant differences in ophthalmic parameters reflecting treatment efficacy or overall safety between the groups. Among baseline ophthalmic parameters, corrected signal intensity ratio (cSIR) correlated well with magnetic resonance imaging findings and were more strongly associated with changes in ophthalmic parameters after treatment in the HD group than in the LD group, indicating that pretreatment cSIR might be useful for determining iv GC doses. In conclusion, there were no significant differences in overall safety and efficacy between high-dose and low-dose iv GC therapy in patients with active GO. Further randomized clinical trials with longer observation periods are required to establish the optimal treatment regimen of GO.

Ectopic automaticity induced in ventricular myocytes by transgenic overexpression of HCN2.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are expressed in the ventricles of fetal hearts but are normally down-regulated as development progresses. In the hypertrophied heart, however, these channels are re-expressed and generate a hyperpolarization-activated, nonselective cation current (Ih), which evidence suggests may increase susceptibility to arrhythmia. To test this hypothesis, we generated and analyzed transgenic mice overexpressing HCN2 specifically in their hearts (HCN2-Tg). Under physiological conditions, HCN2-Tg mice exhibited no discernible abnormalities. After the application of isoproterenol (ISO), however, ECG recordings from HCN2-Tg mice showed intermittent atrioventricular dissociation followed by idioventricular rhythm. Consistent with this observation, 0.3 µmol/L ISO-induced spontaneous action potentials (SAPs) in 76% of HCN2-Tg ventricular myocytes. In the remaining 24%, ISO significantly depolarized the resting membrane potential (RMP), and the late repolarization phase of evoked action potentials (APs) was significantly longer than in WT myocytes. Analysis of membrane currents revealed that these differences are attributable to the Ih tail current. These findings suggest HCN2 channel activity reduces the repolarization reserve of the ventricular action potential and increases ectopic automaticity under pathological conditions such as excessive ß-adrenergic stimulation.

MicroRNA-26a inhibits TGF-ß-induced extracellular matrix protein expression in podocytes by targeting CTGF and is downregulated in diabetic nephropathy.

AIMS/HYPOTHESIS: The accumulation of extracellular matrix (ECM) is a characteristic of diabetic nephropathy, and is partially caused by profibrotic proteins TGF-ß and connective tissue growth factor (CTGF). We aimed to identify microRNAs (miRNAs) targeting CTGF on podocytes in diabetic nephropathy. METHODS: We investigated miRNAs targeting CTGF on podocytes with miRNA array analysis and identified a candidate miRNA, miR-26a. Using overexpression and silencing of miR-26a in cultured podocytes, we examined changes of ECM and its host genes. We further investigated glomerular miR-26a expression in humans and in mouse models of diabetic nephropathy. RESULTS: miR-26a, which was downregulated by TGF-ß1, was expressed in glomerular cells including podocytes and in tubules by in situ hybridisation. Glomerular miR-26a expression was downregulated by 70% in streptozotocin-induced diabetic mice. Transfection of miR-26a mimics in cultured human podocytes decreased the CTGF protein level by 50%, and directly inhibited CTGF expression in podocytes, as demonstrated by a reporter assay with the 3'-untranslated region of the CTGF gene. This effect was abolished by a mutant plasmid. miR-26a mimics also inhibited TGF-ß1-induced collagen expression, SMAD-binding activity and expression of its host genes CTDSP2 and CTDSPL. Knockdown of CTDSP2 and CTDSPL increased collagen expression in TGF-ß-stimulated podocytes, suggesting that host genes also regulate TGF-ß/SMAD signalling. Finally, we observed a positive correlation between microdissected glomerular miR-26a expression levels and estimated GFR in patients with diabetic nephropathy. CONCLUSIONS/INTERPRETATION: The downregulation of miR-26a is involved in the progression of diabetic nephropathy both in humans and in mice through enhanced TGF-ß/CTGF signalling.