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1.  Impact of occult renal impairment on early and late outcomes following coronary artery bypass grafting 
OBJECTIVES
High serum creatinine is considered an independent risk factor for poor outcomes following coronary artery bypass grafting (CABG). However, the impact of occult renal impairment (ORI), defined as an impaired glomerular filtration rate (GFR) with a normal serum creatinine (SCr) level, remains unclear. Thus, we sought to investigate the impact of ORI on outcomes after CABG.
METHODS
Among patients undergoing their first percutaneous coronary intervention (PCI) or CABG enrolled in the CREDO-Kyoto Registry (a registry of first-time PCI and CABG patients in Japan), 1842 patients with normal SCr levels undergoing CABG were enrolled in the study. Patients were divided into two groups based on preoperative estimated GFR calculated by the Cockcroft–Gault equation: 1339 patients with estimated GFR of ≥60 ml/min/1.73 m2 (normal group) and 503 with estimated GFR of <60 ml/min/1.73 m2 (ORI group).
RESULTS
Preoperative estimated GFR differed between the groups (51.3 ± 6.6 vs 85.8 ± 23.0 ml/min/1.73 m2, P < 0.01). ORI was associated with high in-hospital mortality (3.2 vs 1.0%, P < 0.01) and need for dialysis (2.0 vs 0.2%, P < 0.01). In terms of long-term outcomes, ORI was associated with high mortality compared with the normal (hazard ratio [95% confidence interval]: 1.72 [1.16–2.54], P < 0.01) and high incidence of composite cardiovascular events (death, stroke or myocardial infarction: 1.53 [1.16–2.02], P < 0.01).
CONCLUSIONS
ORI was an independent risk factor for early and late death as well as cardiovascular events in patients undergoing CABG with normal SCr levels. A more accurate evaluation of renal function through a combination of SCr and estimated GFR is needed in patients with normal SCr levels.
doi:10.1093/icvts/ivt254
PMCID: PMC3781789  PMID: 23793709
CABG; Renal insufficiency; Long-term outcomes; High-risk populations
2.  MicroRNA-33b knock-in mice for an intron of sterol regulatory element-binding factor 1 (Srebf1) exhibit reduced HDL-C in vivo 
Scientific Reports  2014;4:5312.
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models.
doi:10.1038/srep05312
PMCID: PMC4058878  PMID: 24931346
3.  AMAP1 as a negative-feedback regulator of nuclear factor-κB under inflammatory conditions 
Scientific Reports  2014;4:5094.
NF-κB is a major transcriptional factor regulating many cellular functions including inflammation; therefore, its appropriate control is of high importance. The detailed mechanism of its activation has been well characterized, but that of negative regulation is poorly understood. In this study, we showed AMAP1, an Arf-GTPase activating protein, as a negative feedback regulator for NF-κB by binding with IKKβ, an essential kinase in NF-κB signaling. Proteomics analysis identified AMAP1 as a binding protein with IKKβ. Overexpression of AMAP1 suppressed NF-κB activity by interfering the binding of IKKβ and NEMO, and deletion of AMAP1 augmented NF-κB activity. The activation of NF-κB induced translocation of AMAP1 to cytoplasm from cell membrane and nucleus, which resulted in augmented interaction of AMAP1 and IKKβ. These results demonstrated a novel role of AMAP1 as a negative feedback regulator of NF-κB, and presented it as a possible target for anti-inflammatory treatments.
doi:10.1038/srep05094
PMCID: PMC4035583  PMID: 24865276
4.  Twisted Gastrulation, a BMP Antagonist, Exacerbates Podocyte Injury 
PLoS ONE  2014;9(2):e89135.
Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.
doi:10.1371/journal.pone.0089135
PMCID: PMC3934867  PMID: 24586548
5.  Bmp7 Maintains Undifferentiated Kidney Progenitor Population and Determines Nephron Numbers at Birth 
PLoS ONE  2013;8(8):e73554.
The number of nephrons, the functional units of the kidney, varies among individuals. A low nephron number at birth is associated with a risk of hypertension and the progression of renal insufficiency. The molecular mechanisms determining nephron number during embryogenesis have not yet been clarified. Germline knockout of bone morphogenetic protein 7 (Bmp7) results in massive apoptosis of the kidney progenitor cells and defects in early stages of nephrogenesis. This phenotype has precluded analysis of Bmp7 function in the later stage of nephrogenesis. In this study, utilization of conditional null allele of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at desired time points during kidney development, and to discover the novel function of Bmp7 to inhibit the precocious differentiation of the progenitor cells to nephron. Systemic knockout of Bmp7 in vivo after the initiation of kidney development results in the precocious differentiation of the kidney progenitor cells to nephron, in addition to the prominent apoptosis of progenitor cells. We also confirmed that in vitro knockout of Bmp7 in kidney explant culture results in the accelerated differentiation of progenitor population. Finally we utilized colony-forming assays and demonstrated that Bmp7 inhibits epithelialization and differentiation of the kidney progenitor cells. These results indicate that the function of Bmp7 to inhibit the precocious differentiation of the progenitor cells together with its anti-apoptotic effect on progenitor cells coordinately maintains renal progenitor pool in undifferentiated status, and determines the nephron number at birth.
doi:10.1371/journal.pone.0073554
PMCID: PMC3753328  PMID: 23991197
6.  Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice 
PLoS ONE  2013;8(8):e72173.
Background
Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.
Methods and Results
Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.
Conclusions
Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.
doi:10.1371/journal.pone.0072173
PMCID: PMC3741204  PMID: 23951293
7.  Impact of Diabetes Among Revascularized Patients in Japan and the U.S. 
Diabetes Care  2012;35(3):654-659.
OBJECTIVE
Approximately 25% of patients who undergo percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) have diabetes, and the diagnosis of diabetes roughly doubles the mortality risk associated with coronary artery disease. However, the impact of diabetes may differ according to ethnicity. Our objective was to examine the impact of diabetes on long-term survival among U.S. and Japanese patients who underwent PCI or CABG.
RESEARCH DESIGN AND METHODS
For the current analysis, we included 8,871 patients from a Japanese multicenter registry (Coronary Revascularization Demonstrating Outcome database in Kyoto; median follow-up 3.5 years; interquartile range [IQR] 2.6–4.3) and 7,229 patients from a U.S. multipractice registry (Texas Heart Institute Research Database; median follow-up 5.2 years; IQR 3.8–6.5).
RESULTS
Diabetes was more prevalent among Japanese than U.S. patients (39.2 vs. 31.0%; P < 0.001). However, after revascularization, long-term all-cause mortality was lower in diabetic Japanese patients than in diabetic U.S. patients (85.4 vs. 82.2%; log-rank test P = 0.009), whereas it was similar in nondiabetic Japanese and U.S. patients (89.1 vs. 89.5%; P = 0.50). The national difference in crude mortality was also significant among insulin-using patients with diabetes (80.8 vs. 74.9%; P = 0.023). When long-term mortality was adjusted for known predictors, U.S. location was associated with greater long-term mortality risk than Japanese location among nondiabetic patients (hazard ratio 1.58 [95% CI 1.32–1.88]; P < 0.001) and, especially, diabetic patients (1.88 [1.54–2.30]; P < 0.001).
CONCLUSIONS
Although diabetes was less prevalent in U.S. patients than in Japanese patients, U.S. patients had higher overall long-term mortality risk. This difference was more pronounced in diabetic patients.
doi:10.2337/dc11-1547
PMCID: PMC3322729  PMID: 22301120
8.  Significance of off-pump coronary artery bypass grafting compared with percutaneous coronary intervention: a propensity score analysis 
OBJECTIVE
Although there have been several studies that compared the efficacy of percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG), the impact of off-pump CABG (OPCAB) has not been well elucidated. The objective of the present study was to compare the outcomes after PCI, on-pump CABG (ONCAB), and OPCAB in patients with multivessel and/or left main disease.
METHODS
Among the 9877 patients undergoing first PCI using bare-metal stents or CABG who were enrolled in the CREDO-Kyoto Registry, 6327 patients with multivessel and/or left main disease were enrolled into the present study (67.9 ± 9.8 years old). Among them, 3877 patients received PCI, 1388 ONCAB, and 1069 OPCAB. Median follow-up was 3.5 years.
RESULTS
Comparing PCI with all CABG (ONCAB and OPCAB), propensity-score-adjusted all-cause mortality after PCI was higher than that CABG (hazard ratio (95% confidence interval): 1.37 (1.15–1.63), p < 0.01). The incidence of stroke was lower after PCI than that after CABG (0.75 (0.59–0.96), p = 0.02). CABG was associated with better survival outcomes than PCI in the elderly (interaction p = 0.04). Comparing OPCAB with PCI or ONCAB, propensity-score-adjusted all-cause mortality after PCI was higher than that after OPCAB (1.50 (1.20–1.86), p < 0.01). Adjusted mortality was similar between ONCAB and OPCAB (1.18 (0.93–1.51), p = 0.33). The incidence of stroke after OPCAB was similar to that after PCI (0.98 (0.71–1.34), p > 0.99), but incidence of stroke after ONCAB was higher than that after OPCAB (1.59 (1.16–2.18), p < 0.01).
CONCLUSIONS
In patients with multivessel and/or left main disease, CABG, particularly OPCAB, is associated with better survival outcomes than PCI using bare-metal stents. Survival outcomes are similar between ONCAB and OPCAB.
doi:10.1016/j.ejcts.2011.04.004
PMCID: PMC3241114  PMID: 21676626
Coronary artery bypass grafting; Percutaneous coronary intervention; Off-pump
9.  MicroRNA-33 Deficiency Reduces the Progression of Atherosclerotic Plaque in ApoE−/− Mice 
Background
Cholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo.
Methods and Results
We assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33−/−Apoe−/−) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33+/+Apoe−/− mice. Peritoneal macrophages from miR-33−/−Apoe−/− mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33+/+Apoe−/− macrophages. Consistent with these results, miR-33−/−Apoe−/− mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33−/−Apoe−/− bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33+/+Apoe−/− bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33−/−Apoe−/− mice compared with miR-33+/+Apoe−/− mice, whereas CPT1a and AMPKα were not.
Conclusions
These data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.
doi:10.1161/JAHA.112.003376
PMCID: PMC3540673  PMID: 23316322
ABCA1; ABCG1; atherosclerosis; HDL-C; microRNA
11.  Protein Inhibitor of Activated STAT, PIASy Regulates α-Smooth Muscle Actin Expression by Interacting with E12 in Mesangial Cells 
PLoS ONE  2012;7(7):e41186.
Phenotypic transformation of mesangial cells (MCs) is implicated in the development of glomerular disease; however, the mechanisms underlying their altered genetic program is still unclear. α-smooth muscle actin (α-SMA) is known to be a crucial marker for phenotypic transformation of MCs. Recently, E-boxes and the class I basic helix-loop-helix proteins, such as E12 have been shown to regulateα-SMA expression. Therefore, we tried to identify a novel E12 binding protein in MCs and to examine its role in glomerulonephritis. We found that PIASy, one of the protein inhibitors of activated STAT family protein, interacted with E12 by yeast two-hybrid screens and coimmunopreciptation assays. Overexpression of E12 significantly enhanced theα-SMA promoter activity, and the increase was blocked by co-transfection of PIASy, but not by a PIASy RING mutant. In vivo sumoylation assays revealed that PIASy was a SUMO E3 ligase for E12. Furthermore, transforming growth factor-β (TGF-β) treatment induced expression of both PIASy and E12, consistent with α-SMA expression. Moreover, reduced expression of PIASy protein by siRNA specific for PIASy resulted in increased TGF-β-mediated α-SMA expression. In vivo, PIASy and E12 were dramatically upregulated along with α-SMA and TGF-β in the proliferative phase of Thy1 glomerulonephritis. Furthermore, an association between PIASy and E12 proteins was observed at day 6 by IP-western blotting, but not at day 0. These results suggest that TGF-β up-regulates PIASy expression in MCs to down-regulateα-SMA gene transcription by the interaction with E12.
doi:10.1371/journal.pone.0041186
PMCID: PMC3400623  PMID: 22829926
12.  Left atrial volume predicts adverse cardiac and cerebrovascular events in patients with hypertrophic cardiomyopathy 
Aims
To prospectively evaluate the relationship between left atrial volume (LAV) and the risk of clinical events in patients with hypertrophic cardiomyopathy (HCM).
Methods
We enrolled a total of 141 HCM patients with sinus rhythm and normal pump function, and 102 patients (73 men; mean age, 61 ± 13 years) who met inclusion criteria were followed for 30.8 ± 10.0 months. The patients were divided into two groups with or without major adverse cardiac and cerebrovascular events (MACCE), a composite of stroke, sudden death, and congestive heart failure. Detailed clinical and echocardiographic data were obtained.
Results
MACCE occurred in 24 patients (18 strokes, 4 congestive heart failure and 2 sudden deaths). Maximum LAV, minimum LAV, and LAV index (LAVI) corrected for body surface area (BSA) were significantly greater in patients with MACCE than those without MACCE (maximum LAV: 64.3 ± 25.0 vs. 51.9 ± 16.0 ml, p = 0.005; minimum LAV: 33.9 ± 15.1 vs. 26.2 ± 10.9 ml, p = 0.008; LAVI: 40.1 ± 15.4 vs. 31.5 ± 8.7 ml/mm2, p = 0.0009), while there were no differences in the other echocardiographic parameters.
LAV/BSA of ≥ 40.4 ml/m2 to identify patients with cardiovascular complications with a sensitivity of 73% and a specificity of 88%.
Conclusion
LAVI may be an effective marker for detecting the risk of MACCE in patients with HCM and normal pump function.
doi:10.1186/1476-7120-9-34
PMCID: PMC3229541  PMID: 22099329
hypertrophic cardiomyopathy; left atrial volume; cardiac and cerebrovascular events; paroxysmal atrial fibrillation
13.  Prognostic utility of T-wave alternans in a real-world population of patients with left ventricular dysfunction: the PREVENT-SCD study 
Clinical Research in Cardiology  2011;101(2):89-99.
Background
The predictive value of T-wave alternans (TWA) for lethal ventricular tachyarrhythmia in patients with left ventricular (LV) dysfunction is controversial. Also, long-term arrhythmia risk of patients ineligible for the TWA test is unknown.
Methods
This was a multicenter, prospective observational study of patients with LV ejection fraction ≤40% due to ischemic or non-ischemic cardiomyopathies, designed to evaluate the prognostic value of TWA for lethal ventricular tachyarrhythmia. The primary end point was a composite of sudden cardiac death, sustained rapid ventricular tachycardia (VT) or ventricular fibrillation (VF), and appropriate defibrillator therapy for rapid VT or VF.
Results
Among 453 patients enrolled in the study, 280 (62%) were eligible for the TWA test. TWA was negative in 82 patients (29%), who accounted for 18% of the total population. The median of follow-up was 36 months. The 3-year event-free rate for the primary end point was significantly higher in TWA-negative patients (97.0%) than in TWA non-negative patients (89.5%, P = 0.037) and those ineligible for the TWA test (84.4%, P = 0.003). Multivariable analysis identified both non-negative TWA [hazard ratio (HR) 4.43; 95% confidence interval (CI) 1.02–19.2; P = 0.047) and ineligibility for the TWA test (HR 6.89; 95% CI 1.59–29.9; P = 0.010) to be independent predictors of the primary end point.
Conclusions
TWA showed high negative predictive ability for lethal ventricular tachyarrhythmia in patients with LV dysfunction, although the TWA-negative patients accounted for only 18% of the entire population. Those ineligible for the TWA test had the highest risk for lethal ventricular tachyarrhythmia.
Electronic supplementary material
The online version of this article (doi:10.1007/s00392-011-0368-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00392-011-0368-2
PMCID: PMC3268016  PMID: 21960418
Sudden death; Ventricular arrhythmia; Cardiomyopathy; T-wave alternans
14.  Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice 
The Journal of Clinical Investigation  2011;121(10):3981-3990.
In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero–Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia.
doi:10.1172/JCI57301
PMCID: PMC3195468  PMID: 21911936
15.  MicroRNA-27a Regulates Beta Cardiac Myosin Heavy Chain Gene Expression by Targeting Thyroid Hormone Receptor β1 in Neonatal Rat Ventricular Myocytes▿  
Molecular and Cellular Biology  2010;31(4):744-755.
MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene expression and play important roles in gene regulation in the heart. To examine the role of miRNAs in the expression of the two isoforms of the cardiac myosin heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility, endogenous miRNAs were downregulated in neonatal rat ventricular myocytes (NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer, an essential enzyme for miRNA biosynthesis, and MHC expression levels were examined. As a result, Dicer siRNA could downregulate endogenous miRNAs simultaneously and the β-MHC gene but not α-MHC, which implied that specific miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC protein was downregulated by silencing of endogenous miR-27a. Through a bioinformatics screening using TargetScan, we identified thyroid hormone receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation via thyroid hormone signaling and to be upregulated during the differentiation of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC gene expression by targeting TRβ1 in cardiomyocytes.
doi:10.1128/MCB.00581-10
PMCID: PMC3028640  PMID: 21149577
16.  Activation of Src Mediates PDGF-Induced Smad1 Phosphorylation and Contributes to the Progression of Glomerulosclerosis in Glomerulonephritis 
PLoS ONE  2011;6(3):e17929.
Platelet-derived growth factor (PDGF) plays critical roles in mesangial cell (MC) proliferation in mesangial proliferative glomerulonephritis. We showed previously that Smad1 contributes to PDGF-dependent proliferation of MCs, but the mechanism by which Smad1 is activated by PDGF is not precisely known. Here we examined the role of c-Src tyrosine kinase in the proliferative change of MCs. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, MC proliferation and type IV collagen (Col4) expression peaked on day 6. Immunohistochemical staining for the expression of phospho-Src (pSrc), phospho-Smad1 (pSmad1), Col4, and smooth muscle α-actin (SMA) revealed that the activation of c-Src and Smad1 signals in glomeruli peaked on day 6, consistent with the peak of mesangial proliferation. When treated with PP2, a Src inhibitor, both mesangial proliferation and sclerosis were significantly reduced. PP2 administration also significantly reduced pSmad1, Col4, and SMA expression. PDGF induced Col4 synthesis in association with increased expression of pSrc and pSmad1 in cultured MCs. In addition, PP2 reduced Col4 synthesis along with decreased pSrc and pSmad1 protein expression in vitro. Moreover, the addition of siRNA against c-Src significantly reduced the phosphorylation of Smad1 and the overproduction of Col4. These results provide new evidence that the activation of Src/Smad1 signaling pathway plays a key role in the development of glomerulosclerosis in experimental glomerulonephritis.
doi:10.1371/journal.pone.0017929
PMCID: PMC3062564  PMID: 21445358
17.  Periportal and sinusoidal liver dendritic cells suppressing T helper type 1‐mediated hepatitis 
Gut  2007;56(10):1445-1451.
Background
Recently, we found that portal vein tolerance is associated with generation of Th2 cells and apoptosis of Th1 cells in the liver, which is regulated by antigen (Ag)‐presenting dendritic cells (DCs) in the periportal area and sinusoids.
Aim
In this study, we tested whether the periportal and sinusoidal DCs, which were loaded with an Ag in vivo, can inhibit liver injury caused by Th1 cells activated by the Ag administered systemically.
Methods
Ag‐specific hepatitis model was created by adoptively transferring ovalbumin (OVA)‐specific CD4+ T cells to BALB/c mice and venous injection of OVA‐containing liposomes. Liver CD11c+ cells obtained from mice fed OVA were then transferred into these mice.
Results
The transfer of liver CD11c+ cells from OVA‐fed mice completely inhibited hepatic injury, which was associated with apoptosis of OVA‐specific CD4+ T cells and emergence of Th2 cells in the liver. Transfer of CD11c+ cells and subcutaneous OVA challenge led to enhancement of OVA‐specific IgE Ab as well as Th2 cytokine responses in the recipient mice.
Conclusions
Periportal and sinusoidal DCs loaded with an Ag in the portal vein can induce Th2 response in the liver and prevent hepatic injury caused by Th1 cells.
doi:10.1136/gut.2007.121251
PMCID: PMC2000248  PMID: 17872571
18.  Acute doxorubicin cardiotoxicity is associated with miR-146a-induced inhibition of the neuregulin-ErbB pathway 
Cardiovascular Research  2010;87(4):656-664.
Aims
A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity.
Methods and results
Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3′UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed ‘decoy’ genes that had tandem complementary sequences for miR-146a in the 3′UTR of a luciferase gene. When miR-146a ‘decoy’ genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced.
Conclusion
These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.
doi:10.1093/cvr/cvq148
PMCID: PMC2920811  PMID: 20495188
MicroRNA; Neuregulin; Cardiomyocyte
19.  Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase* 
The Journal of Biological Chemistry  2010;285(21):16231-16238.
Eukaryotic cells dynamically reorganize the actin cytoskeleton to regulate various cellular activities, such as cell shape change, cell motility, cytokinesis, and vesicular transport. Diaphanous-related formins (DRFs), such as Daam1 and mDia1, play central roles in actin dynamics through assembling linear actin filaments. It has been reported that the GTP-bound active Rho binds directly to DRFs and partially unleashes the intramolecular autoinhibition of DRFs. However, whether proteins other than Rho involve the regulation of the actin assembly activity of DRFs has been unclear. Here, we show that Flightless-I (Fli-I), a gelsolin family protein essential for early development, binds directly to Daam1 and mDia1. Fli-I enhances the intrinsic actin assembly activity of Daam1 and mDia1 in vitro and is required for Daam1-induced actin assembly in living cells. Furthermore, Fli-I promotes the GTP-bound active Rho-mediated relief of the autoinhibition of Daam1 and mDia1. Thus, Fli-I is a novel positive regulator of Rho-induced linear actin assembly mediated by DRFs.
doi:10.1074/jbc.M109.079236
PMCID: PMC2871490  PMID: 20223827
Cytoskeleton/Actin; G Proteins/Low Molecular Weight; Signal Transduction/G-proteins; Subcellular Organelles/Cytoskeleton; Rho; Cell Shape; Cytoskeletal Reorganization; Formin
20.  Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome 
The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization–associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3–/– mice, which model Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3–/– mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.
doi:10.1172/JCI39569
PMCID: PMC2827946  PMID: 20197625
21.  Variation in the Lectin-like Oxidized LDL Receptor 1 (LOX-1) Gene Is Associated With Plasma Soluble LOX-1 Levels 
Experimental physiology  2008;93(9):1085-1090.
The lectin-like ox-LDL receptor 1 (LOX-1) expressed on vascular cells plays a major role in atherogenesis by internalizing and degrading oxidized LDL. LOX-1 can be cleaved from the cell surface and released as soluble LOX-1 (sLOX-1), and elevated sLOX-1 levels may be indicative of atherosclerotic plaque instability. We examined associations between the LOX-1 3′UTR-C/T and G501C polymorphisms and plasma sLOX-1 levels in 97 healthy older men and women. The frequencies for the 3′UTR-T and 501C alleles were 46% and 10%, respectively. Plasma sLOX-1 levels were significantly higher in the 3′UTR CC genotype group compared to both the CT (p=0.02) and TT (p=0.002) genotype groups. Plasma sLOX-1 were also significantly higher in the 501GC genotype group compared to the GG genotype group (p=0.004). In univariate analyses, sLOX-1 levels were significantly associated with both the 3′UTR-C/T and G501 C polymorphisms. These associations remained significant after adjusting for age, gender, race, and BMI. In conclusion, variation in the LOX-1 gene is associated with plasma sLOX-1 levels in older men and women.
doi:10.1113/expphysiol.2008.042267
PMCID: PMC2652129  PMID: 18469066
receptor; cardiovascular; gene expression
22.  The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats 
Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure–induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans.
doi:10.1172/JCI33160
PMCID: PMC2248328  PMID: 18292809
23.  Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo 
The Journal of Cell Biology  2007;176(7):1061-1071.
Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.
doi:10.1083/jcb.200611026
PMCID: PMC2064089  PMID: 17371835
24.  Effects of exercise on the development of atherosclerosis in apolipoprotein E-deficient mice 
It has already been shown that mild to moderate exercise training may protect against the development of atherosclerosis. However, the precise mechanisms behind this protection are still unknown. The hypothesis that exercise training reduces the severity of experimental atherosclerosis in apolipoprotein (apo) E-deficient mice was assessed. Swimming training was conducted three times per week for 20 min on apo E-deficient mice fed a high-fat diet for eight or 16 weeks. Atherosclerotic lesions were evaluated. Fatty streak formation and fibrofatty plaques developed in apo E-deficient mice fed the high-fat diet, and were markedly suppressed in mice that received exercise for eight or 16 weeks compared with in nonexercise mice. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. Thus, exercise therapy markedly suppressed experimental atherosclerosis.
PMCID: PMC2274844  PMID: 18651017
Apolipoprotein E-deficient mice; Atherosclerosis; Exercise; Lipids
25.  Uterine sensitization-associated gene–1 (USAG-1), a novel BMP antagonist expressed in the kidney, accelerates tubular injury 
Dialysis dependency is one of the leading causes of morbidity and mortality in the world, and once end-stage renal disease develops, it cannot be reversed by currently available therapy. Although administration of large doses of bone morphogenetic protein–7 (BMP-7) has been shown to repair established renal injury and improve renal function, the pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. Here we show that the product of uterine sensitization-associated gene–1 (USAG1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney and that mice lacking USAG-1 (USAG1–/– mice) are resistant to renal injury. USAG1–/– mice exhibited prolonged survival and preserved renal function in acute and chronic renal injury models. Renal BMP signaling, assessed by phosphorylation of Smad proteins, was significantly enhanced in USAG1–/– mice with renal injury, indicating that the preservation of renal function is attributable to enhancement of endogenous BMP signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG1–/– mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP and that inhibition of USAG-1 is a promising means of development of novel treatment for renal diseases.
doi:10.1172/JCI25445
PMCID: PMC1307562  PMID: 16341262

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