Publications by authors named "Toru Shimizu"

155 Publications

Slowly melting the urate snow in joints: Explaining gout attacks to patients.

Int J Rheum Dis 2021 Mar 1;24(3):297-299. Epub 2021 Feb 1.

Graduate School of Maritime Sciences, Kobe University, Kobe, Japan.

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http://dx.doi.org/10.1111/1756-185X.13999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126960PMC
March 2021

Portosystemic shunt for portal hypertension after Kasai operation in patients with biliary atresia.

Pediatr Surg Int 2021 Jan 17;37(1):101-107. Epub 2020 Nov 17.

Children's Hospital at Westmead, Sydney, Australia.

Purpose: Many biliary atresia (BA) patients will eventually develop liver failure even after a successful Kasai portoenterostomy. A common complication of long-term BA survivors with their native liver is problematic portal hypertension. The aim of this study was to defend the view that portosystemic shunts can delay or negate the need for transplantation in these children.

Methods: A retrospective single center review of the efficacy of portosystemic shunts in BA patients after a successful Kasai portoenterostomy was conducted.

Results: From 1991 to 2017, 11 patients received portosystemic shunts. Median age of Kasai operation was 48 (36-61) days. Shunts were performed at the median age of 6.2 (4.1-6.8) years. Three of these eleven patients required subsequent liver transplantation. OS at 5 and 10 years were 90.9% and 81.8%, respectively. TFS at 5 and 10 years were 90.9% and 72.7%, respectively. Long-term complications included mild encephalopathy in 2 patients, hypersplenism in 3, and cholestasis in 1.

Conclusion: Portosystemic shunt for the treatment of portal hypertension in carefully selected BA patients is an effective option in delaying or negating the need for liver transplantation.
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http://dx.doi.org/10.1007/s00383-020-04773-2DOI Listing
January 2021

Identification of the Novel Variants in Patients With Chronic Thromboembolic Pulmonary Hypertension.

J Am Heart Assoc 2020 11 24;9(21):e015902. Epub 2020 Oct 24.

Department of Cardiovascular Medicine Tohoku University Graduate School of Medicine Sendai Japan.

Background Although chronic thromboembolic pulmonary hypertension (CTEPH) and acute pulmonary embolism (APE) share some clinical manifestations, a limited proportion of patients with CTEPH have a history of APE. Moreover, in histopathologic studies, it has been revealed that pulmonary vasculature lesions similar to pulmonary arterial hypertension existed in patients with CTEPH. Thus, it remains unknown whether these 3 disorders also share genetic backgrounds. Methods and Results Whole exome screening was performed with DNA isolated from 51 unrelated patients with CTEPH of Japanese ancestry. The frequency of genetic variants associated with pulmonary arterial hypertension or APE in patients with CTEPH was compared with those in the integrative Japanese Genome Variation Database 3.5KJPN. Whole exome screening analysis showed 17 049 nonsynonymous variants in patients with CTEPH. Although we found 6 nonsynonymous variants that are associated with APE in patients with CTEPH, there was no nonsynonymous variant associated with pulmonary arterial hypertension. Patients with CTEPH with a history of APE had nonsynonymous variants of , which encodes factor V. In contrast, patients with CTEPH without a history of APE had a nonsynonymous variant of , which encodes thrombomodulin. Moreover, thrombin-activatable fibrinolysis inhibitor, which is one of the pathogenic proteins in CTEPH, was significantly more activated in those who had the variants of compared with those without it. Conclusions These results provide the first evidence that patients with CTEPH have some variants associated with APE, regardless of the presence or absence of a history of APE. Furthermore, the variants might be different between patients with CTEPH with and without a history of APE.
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http://dx.doi.org/10.1161/JAHA.120.015902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763425PMC
November 2020

Subtype-specific gout susceptibility loci and enrichment of selection pressure on and identified by subtype genome-wide meta-analyses of clinically defined gout patients.

Ann Rheum Dis 2020 05 1;79(5):657-665. Epub 2020 Apr 1.

Department of Preventive Medicine and Public Health, National Defense Medical College, Tokorozawa, Japan.

Objectives: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years.

Methods: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype.

Results: In addition to the eight loci we reported previously, two novel loci, and , were identified at a genome-wide significance level (p<5.0×10) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, and , from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on in addition to loci for all subtypes except for normal type gout.

Conclusions: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.
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http://dx.doi.org/10.1136/annrheumdis-2019-216644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213308PMC
May 2020

Brain activity underlying American crow processing of encounters with dead conspecifics.

Behav Brain Res 2020 05 6;385:112546. Epub 2020 Feb 6.

Department of Radiology and Imaging Sciences, University of Utah, Salt Lake City, UT, United States.

Animals utilize a variety of auditory and visual cues to navigate the landscape of fear. For some species, including corvids, dead conspecifics appear to act as one such visual cue of danger, and prompt alarm calling by attending conspecifics. Which brain regions mediate responses to dead conspecifics, and how this compares to other threats, has so far only been speculative. Using 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) we contrast the metabolic response to visual and auditory cues associated with a dead conspecific among five a priori selected regions in the American crow (Corvus brachyrhynchos) brain: the hippocampus, nidopallium caudolaterale, striatum, amygdala, and the septum. Using a repeated-measures, fully balanced approach, we exposed crows to four stimuli: a dead conspecific, a dead song sparrow (Melospiza melodia), conspecific alarm calls given in response to a dead crow, and conspecific food begging calls. We find that in response to observations of a dead crow, crows show significant activity in areas associated with higher-order decision-making (NCL), but not in areas associated with social behaviors or fear learning. We do not find strong differences in activation between hearing alarm calls and food begging calls; both activate the NCL. Lastly, repeated exposures to negative stimuli had a marginal effect on later increasing the subjects' brain activity in response to control stimuli, suggesting that crows might quickly learn from negative experiences.
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http://dx.doi.org/10.1016/j.bbr.2020.112546DOI Listing
May 2020

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor GcHK abolishes bacterial signal transduction.

J Biol Chem 2020 02 30;295(6):1587-1597. Epub 2019 Dec 30.

Department of Biochemistry, Faculty of Science, Charles University, Prague 2, 128 43 Czech Republic. Electronic address:

The heme-based oxygen sensor protein GcHK is a globin-coupled histidine kinase in the soil bacterium sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in GcHK. We present a crystal structure of a monomeric imidazole-bound GcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length GcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of GcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase GcHK from .
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http://dx.doi.org/10.1074/jbc.RA119.011574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008379PMC
February 2020

Identification of Adipsin as a Novel Prognostic Biomarker in Patients With Coronary Artery Disease.

J Am Heart Assoc 2019 12 22;8(23):e013716. Epub 2019 Nov 22.

Department of Cardiovascular Medicine Tohoku University Graduate School of Medicine Sendai Japan.

Background Circulating proteins are exposed to vascular endothelial layer and influence their functions. Among them, adipsin is a member of the trypsin family of peptidases and is mainly secreted from adipocytes, monocytes, and macrophages, catalyzing the rate-limiting step of the alternative complement pathway. However, its pathophysiological role in cardiovascular disease remains to be elucidated. Here, we examined whether serum adipsin levels have a prognostic impact in patients with coronary artery disease. Methods and Results In 370 consecutive patients undergoing diagnostic coronary angiography, we performed a cytokine array analysis for screening serum levels of 50 cytokines/chemokines and growth factors. Among them, classification and regression analysis identified adipsin as the best biomarker for prediction of their long-term prognosis (median 71 months; interquartile range, 55-81 months). Kaplan-Meier curve showed that higher adipsin levels (≥400 ng/mL) were significantly associated with all-cause death (hazard ratio [HR], 4.2; 95% CI, 1.7-10.6 [<0.001]) and rehospitalization (HR, 2.4; 95% CI, 1.7-3.5 [<0.001]). Interestingly, higher high-sensitivity C-reactive protein levels (≥1 mg/L) were significantly correlated with all-cause death (HR, 3.2; 95% CI, 1.7-5.9 [<0.001]) and rehospitalization (HR, 1.5, 95% CI, 1.1-1.9 [<0.01]). Importantly, the combination of adipsin (≥400 ng/mL) and high-sensitivity C-reactive protein (≥1 mg/L) was more significantly associated with all-cause death (HR, 21.0; 95% CI, 2.9-154.1 [<0.001]). Finally, the receiver operating characteristic curve demonstrated that serum adipsin levels predict the death caused by acute myocardial infarction in patients with coronary artery disease (C-statistic, 0.847). Conclusions These results indicate that adipsin is a novel biomarker that predicts all-cause death and rehospitalization in patients with coronary artery disease, demonstrating the novel aspects of the alternative complementary system in the pathogenesis of coronary artery disease.
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http://dx.doi.org/10.1161/JAHA.119.013716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912964PMC
December 2019

Heme: emergent roles of heme in signal transduction, functional regulation and as catalytic centres.

Chem Soc Rev 2019 Dec;48(24):5624-5657

Department of Biochemistry, Faculty of Science, Charles University, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.

Protoporphyrin IX iron complex (heme) is an important cofactor for oxygen transfer, oxygen storage, oxygen activation, and electron transfer when bound to the heme proteins hemoglobin, myoglobin, cytochrome P450 and cytochrome c, respectively. In addition to these prototypical heme proteins, there are emergent, critical roles of exchangeable/labile heme in signal transduction. Specifically, it has been shown that association/dissociation of heme to/from heme-responsive sensors regulates numerous functions, including transcription, DNA binding, microRNA splicing, translation, protein kinase activity, protein degradation, heme degradation, K+ channel function, two-component signal transduction, and many other functions. In this review, we provide a comprehensive overview of structure-function relationships of heme-responsive sensors and describe new, additional roles of exchangeable/labile heme as functional inhibitors and activators. In order to complete the description of the various roles of heme in heme-bound proteins, we also mention heme as a novel chemical reaction centre for aldoxime dehydratase, cis-trans isomerase, N-N bond formation, hydrazine formation and S-S formation, and other functions. These unprecedented functions of exchangeable/labile heme and heme proteins should be of interest to biological chemists. Insight into underlying molecular mechanisms is essential for understanding the new role of heme in important physiological and pathological processes.
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http://dx.doi.org/10.1039/c9cs00268eDOI Listing
December 2019

Cyclophilin A as a biomarker for the therapeutic effect of balloon angioplasty in chronic thromboembolic pulmonary hypertension.

J Cardiol 2020 04 12;75(4):415-423. Epub 2019 Oct 12.

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.

Background: Although cardiac troponin and natriuretic peptide have been shown to decrease after balloon pulmonary angioplasty (BPA) with improved right ventricular afterload in chronic thromboembolic pulmonary hypertension (CTEPH), biomarkers to evaluate the effects of BPA independently of heart failure status remain to be developed.

Methods: In 39 consecutive CTEPH patients including 31 who underwent BPA, we measured plasma levels of cyclophilin A (CyPA), which we demonstrated is secreted from pulmonary vascular smooth muscle cells in response to mechanical stretch and hypoxia.

Results: CyPA levels were elevated in CTEPH patients (12.7, IQR: 7.6-16.0) compared with 8 thromboembolic controls with a history of venous thromboembolism (4.9, IQR: 2.4-11.2) or 18 healthy controls (4.1, IQR: 2.4-6.8) (both p< 0.05) and were linearly correlated with mean pulmonary arterial pressure (r=0.50, p = 0.0003) and pulmonary vascular resistance (r=0.32, p= 0.026). BPA reduced CyPA levels and tended to lower brain-type natriuretic peptide (BNP) levels (p< 0.01 and p = 0.07). When comparing the changes in CyPA before and after BPA in the two subgroups with higher (≥35pg/mL) and normal (<35pg/mL) BNP at baseline, CyPA decreased both in patients with higher BNP and those with normal BNP (both p< 0.05). In contrast, BNP decreased only in patients with higher BNP (p< 0.05). Also, CyPA decreased both in patients with lower (<25 kg/m) and higher (≥25kg/m) body mass index (BMI) at baseline (both p<0.05), whereas BPA tended to reduce BNP in patients with lower BMI (p = 0.12) but not in those with higher BMI (p = 0.55).

Conclusions: CyPA could be a useful biomarker to evaluate the effects of BPA even in patients with normal BNP or high BMI.
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http://dx.doi.org/10.1016/j.jjcc.2019.09.010DOI Listing
April 2020

Review of pediatric abdominal trauma: operative and non-operative treatment in combined adult and pediatric trauma center.

Acute Med Surg 2019 Oct 7;6(4):358-364. Epub 2019 Apr 7.

Department of Pediatric Surgery Okinawa Prefectural Nanbu Children's Medical Center Haebaru-cho Japan.

Aim: More than 90% of pediatric solid organ abdominal injuries are treated non-operatively. It remains difficult to decide who should graduate to surgical management, more so if adult physicians must make these decisions on pediatric patients. The purpose of this study was to examine outcomes of all pediatric abdominal trauma cases in a single center, focusing on the decision-making algorithm for operative or non-operative treatment by pediatric and adult physicians.

Methods: We undertook a retrospective review of a pediatric trauma database from April 2006 to March 2016. Groups were divided into operative and non-operative, single or multi-organ injury, and adult or pediatric physician. Operative treatments included laparotomy or interventional radiology procedures. Primary outcome was survival within 30 days.

Results: There were 53 abdominal trauma cases; among them, 48 (90.6%) survived and 5 (9.4%) died within 30 days. The probability of survival for mortalities was less than 11%. Forty-two cases were treated non-operatively and 11 operatively. Injury Severity Score was higher in operative group (17 [9, 41]/9 [4, 16.3]). Adult physicians saw 33 patients including seven operative, whereas pediatric physicians saw 20 including four operative cases. There was no statistical difference for the management decision between adult and pediatric physicians.

Conclusion: Our decisions for intervention were within acceptable rates. Adult physicians did not tend to operate more, but there were cases that did not fit the criteria of the algorithm. Further investigation is needed to look at which factors should be focused on to determine whether or not operative treatments are indicated.
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http://dx.doi.org/10.1002/ams2.421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773634PMC
October 2019

Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis.

J Inorg Biochem 2019 12 6;201:110833. Epub 2019 Sep 6.

Department of Biochemistry, Faculty of Science, Charles University, Hlavova (Albertov) 2030/8, Prague 2, Czech Republic. Electronic address:

Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5-9.0, and catalysis required either Mg or Mn; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were K 84 ± 21 μM and k 1.2 min. YddV-MBP with heme Fe(II), heme Fe(II)-O, and heme Fe(II)-CO complexes had k values of 0.3 min, 0.95 min, and 0.3 min, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The k values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min, 0.26 min and 0.54 min, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.
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http://dx.doi.org/10.1016/j.jinorgbio.2019.110833DOI Listing
December 2019

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout.

Ann Rheum Dis 2019 10 8;78(10):1430-1437. Epub 2019 Jul 8.

Laboratory for Mathematics, National Defense Medical College, Tokorozawa, Saitama, Japan.

Objective: The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout.

Methods: We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia).

Results: This new approach enabled us to identify two novel gout loci (rs7927466 of and rs9952962 of ) and one suggestive locus (rs12980365 of ) at the genome-wide significance level (p<5.0×10 ). The present study also identified the loci of , and . One of them, rs671 of , was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three 'gout vs AHUA GWAS'-specific loci (, and ) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level.

Conclusions: This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals.
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http://dx.doi.org/10.1136/annrheumdis-2019-215521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788923PMC
October 2019

Beneficial Effects of Imatinib in a Patient with Suspected Pulmonary Veno-Occlusive Disease.

Tohoku J Exp Med 2019 02;247(2):69-73

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension (PH). The prognosis of PVOD patients remains poor, since no effective medical therapy is yet available. Imatinib is a tyrosine kinase inhibitor specific for platelet-derived growth factor receptor and is expected as a treatment option for pulmonary arterial hypertension (PAH). Recently, it has been reported that imatinib improved functional capacity of a patient with PVOD. We here report a patient with suspected PVOD who has been successfully treated with imatinib and is alive for 6 years after diagnosis. A 57-year-old woman was admitted to a hospital for severe dyspnea. Echocardiography suggested the presence of PH, because tricuspid regurgitation pressure gradient was elevated. The patient was then transferred to our hospital by an ambulance ahead of schedule due to fever and worsening dyspnea. Because the patient had no left heart disease, we diagnosed that she had PAH associated with severe right heart failure. We immediately started treatment with nitric oxide (NO) for her severe hypoxia; however, it caused pulmonary edema. We suspected PVOD from CT characteristics and pulmonary edema after PAH-targeted vasodilator therapy, and then started oral imatinib treatment. In response to imatinib, her pulmonary edema gradually improved. Since then, the patient has been alive for 6 years with imatinib and pulmonary vasodilators. At present, lung transplantation is the only effective therapy for PVOD with limited availability. We therefore propose that imatinib may be a treatment option for PVOD and a bridge to lung transplantation.
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http://dx.doi.org/10.1620/tjem.247.69DOI Listing
February 2019

Characteristics of gout patients according to the laterality of nephrolithiasis: A cross-sectional study using helical computed tomography.

Int J Rheum Dis 2019 Apr 28;22(4):567-573. Epub 2018 Nov 28.

Department of Internal Medicine, Midorigaoka Hospital, Takatsuki-shi, Japan.

Objective: To clarify the clinical and laboratory characteristics of nephrolithiasis in gout by computed tomography (CT).

Methods: In 350 gout patients, unenhanced CT was performed at the 1st visit to hospital. Calculus density spots exceeding 1 mm in diameter with a CT value >120 Hounsfield units in the kidneys were defined as kidney stones. The association between laterality and the number of stones was investigated in each stone carrier. The 350 patients were classified into three groups (bilateral, unilateral and non-stone carriers). Then serum urate (Sua), renal function, uric acid metabolism, and the prevalence of metabolic syndrome (Mets) were compared among these groups by the Tukey-Kramer test or Fisher's exact test.

Results: Kidney stone(s) were detected in 108 (31%) of the 350 patients (bilateral in 58 and unilateral in 50). In 64 of the 108 patients (59%), there was no history of urolithiasis. Sua, serum creatinine and uric acid clearance were significantly higher (P = 0.001, P < 0.001, P = 0.043, respectively), while the estimated glomerular filtration rate was significantly lower (P = 0.039) in bilateral stone carriers than in non-stone carriers. No significant differences of uric acid metabolism or the prevalence of Mets were noted among the three groups.

Conclusions: Approximately one-third of gout patients had kidney stones and more than half of the patients with stones were bilateral and multiple stone carriers. Elevation of Sua might increase the stone burden in gout, leading to more severe renal dysfunction. An association between nephrolithiasis and Mets was not demonstrated in gout patients.
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http://dx.doi.org/10.1111/1756-185X.13443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587737PMC
April 2019

Successful Mesoporous Silica Encapsulation of Optimally Functional EcDOS (E. coli Direct Oxygen Sensor), a Heme-based O-Sensing Phosphodiesterase.

Anal Sci 2019 Mar 16;35(3):329-335. Epub 2018 Nov 16.

National Institute of Advanced Industrial Science and Technology (AIST).

The heme-based O sensor from Escherichia coli, EcDOS, exerts phosphodiesterase activity towards cyclic-di-GMP (c-di-GMP), an important second messenger that regulates biofilm formation, virulence, and other important functions necessary for bacterial survival. EcDOS is a two-domain protein composed of an N-terminal heme-bound O-sensing domain and a C-terminal functional domain. O binding to the heme Fe(II) complex in the O-sensing domain substantially enhances the catalytic activity of the functional domain, a property with potentially promising medical applications. Mesoporous silica is a useful material with finite-state machine-like features suitable for mediating numerous enzymatic functions. Here, we successfully encapsulated EcDOS into mesoporous silica, and demonstrated that encapsulated EcDOS was substantially activated by CO, an alternative signaling molecule used in place of O, exhibiting the same activity as the native enzyme in aqueous solution. Encapsulated EcDOS was sufficiently stable to exert its enzymatic function over several experimental cycles under aerobic conditions at room temperature. Thus, the present study demonstrates the successful encapsulation of the heme-based O sensor EcDOS into mesoporous silica and shows that the native gas-stimulated function of EcDOS is well conserved. As such, this represents the first application of mesoporous silica to an oxygen-sensing-or any gas-sensing-enzyme.
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http://dx.doi.org/10.2116/analsci.18P449DOI Listing
March 2019

Simulated viral infection in early-life alters brain morphology, activity and behavior in zebra finches (Taeniopygia guttata).

Physiol Behav 2018 11 19;196:36-46. Epub 2018 Aug 19.

Department of Psychology, University of South Florida, Tampa, FL 33620, United States.

Early-life immune challenges (ELIC) have long-term effects on adult behavior and brain development. ELIC studies on birds are still few, but they are epidemiologically crucial since birds are important hosts of many mosquito-borne viruses. In this study, we administered a viral infection mimicking agent, Polyinosinic: polycytidylic acid (Poly I:C), to nestling zebra finches on post-hatch day 14. When birds became sexually mature, their general activity (i.e., hopping, feeding behavior) and mosquito defense behaviors (i.e., hops, head movements, pecks, wing movements, foot movements, and scratches) were measured. Following behavioral trials, brains of male birds were collected for anatomical and histochemical analyses. Poly I:C challenge had sex-dependent effects on general activity and mosquito defense behaviors. When compared to control females, Poly I:C challenged females hopped and fed less often in their general activities, but hopped more often in the presence of mosquitoes. Poly I:C challenged males did not differ from control males in any behaviors. Brain analysis revealed that the nucleus taeniae of the amygdala (TnA) of Poly I:C challenged males were smaller in volume yet had more neurons expressing immediate-early gene proteins compared with controls, suggesting a more active TnA. These results suggest that immune challenges early in the life could have long-term effects on behaviors and brains of zebra finches, which may influence disease spread and fitness of individual birds.
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http://dx.doi.org/10.1016/j.physbeh.2018.08.004DOI Listing
November 2018

[Explanation of JIS T 62570 Standard Practice for Marking Medical Devices and Other Items for Safety in the Magnetic Resonance Environment].

Authors:
Toru Shimizu

Nihon Hoshasen Gijutsu Gakkai Zasshi 2018 ;74(7):739-741

Japan Medical Imaging and Radiological Systems Industries Association, SC-4402 Former Convener (JIS T 62570 Working Group Convener) Standardization Committee, Standardization Division.

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http://dx.doi.org/10.6009/jjrt.2018_JSRT_74.7.739DOI Listing
March 2019

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice.

Proc Natl Acad Sci U S A 2018 07 9;115(30):E7129-E7138. Epub 2018 Jul 9.

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Miyagi, 980-8574, Japan;

Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient () and ROCK2-deficient () mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in mice compared with controls, whereas cardiac hypertrophy was attenuated in mice after TAC. Consistently, the levels of oxidative stress were up-regulated in hearts and down-regulated in hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in mice, whereas their expressions were significantly lower in mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.
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http://dx.doi.org/10.1073/pnas.1721298115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064988PMC
July 2018

Sex differences in hemodynamic responses and long-term survival to optimal medical therapy in patients with pulmonary arterial hypertension.

Heart Vessels 2018 Aug 13;33(8):939-947. Epub 2018 Feb 13.

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.

It is widely known that the incidence of pulmonary arterial hypertension (PAH) is higher in female, whereas prognosis is poorer in male patients. However, sex differences in hemodynamic response to and long-term prognosis with PAH-targeted treatment in the modern era remain to be fully elucidated. We examined the long-term prognosis of 129 consecutive PAH patients (34 males and 95 females) diagnosed in our hospital from April 1999 to October 2014, and assessed hemodynamic changes in response to PAH-targeted therapy. Female patients had better 5-year survival compared with male patients (74.0 vs. 53.4%, P = 0.003); however, higher age quartiles in females were associated with poor outcome. Follow-up examination after medical treatment showed significant decreases in mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), and pulmonary arterial capacitance (PAC) in both sexes (both P < 0.05), whereas only females had a significant improvement in right ventricular end-diastolic pressure (RVEDP), right atrial pressure (RAP), cardiac index, and mixed venous oxygen saturation (SvO) (all P < 0.05). Baseline age significantly correlated with the hemodynamic changes only in female patients; particularly, there were significant sex interactions in RVEDP and RAP (both P < 0.10). The multivariable analysis showed that SvO at baseline and mPAP and SvO at follow-up were significant prognostic factors in males, whereas the changes in mPAP, PVR, and PAC and use of endothelin-receptor antagonist in females. These results indicate that female PAH patients have better long-term prognosis than males, for which better improvements of right ventricular functions and hemodynamics may be involved.
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http://dx.doi.org/10.1007/s00380-018-1140-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060798PMC
August 2018

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor GcHK associated with intraprotein signal transduction.

J Biol Chem 2017 12 1;292(51):20921-20935. Epub 2017 Nov 1.

From the Department of Biochemistry, Faculty of Science, Charles University, Hlavova (Albertov) 2030/8, Prague 2, 128 43 Czech Republic,

The heme-based oxygen sensor histidine kinase GcHK is part of a two-component signal transduction system in bacteria. O binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH and -CN complexes of GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of GcHK. We conclude that GcHK functions as an ensemble of molecules sampling at least two conformational states.
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http://dx.doi.org/10.1074/jbc.M117.817023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743068PMC
December 2017

Subarachnoid Block-Induced Deafferentation Pain Successfully Treated with Pentazocine.

J Nippon Med Sch 2017 ;84(4):183-185

Department of Anesthesiology, Nippon Medical School.

Deafferentation pain induced by subarachnoid block (SAB) is rare, but it can appear in the form of recurrent phantom lower limb pain, new acute-onset stump pain in amputees, lower limb pain in patients with tabes dorsalis, and neuropathic pain. We have previously reported that thiopental is an effective treatment for deafferentation pain induced by therapeutic SAB applied to treat neuropathic pain of central origin. Here, we report the case of an amputee who developed new stump pain in his lower limb immediately after subarachnoid tetracaine was administered prior to appendectomy. A 51-year-old man who had previously undergone right below-knee amputation for acute arterial thrombosis, and who had not previously experienced chronic phantom limb or stump pain, was scheduled for emergency open appendectomy. For anesthesia, we induced SAB with a hyperbaric tetracaine solution. No paresthesia occurred during administration. However, the patient immediately complained of severe, lightning-bolt pain in the right lower limb stump after the SAB was established. He was given intravenous pentazocine, which promptly resolved the pain. Appendectomy was then performed under sedation using intravenous midazolam. The patient did not experience further deafferentation pain during his hospital stay and has reported no stump pain since discharge from the hospital. This case report suggests that SAB induces deafferentation pain in some patients and that this unusual pain can be treated with pentazocine.
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http://dx.doi.org/10.1272/jnms.84.183DOI Listing
February 2018

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system.

J Liposome Res 2018 Dec 5;28(4):275-284. Epub 2017 Sep 5.

b Department of Applied Chemistry, Graduate School of Engineering , Osaka Prefecture University , Osaka , Japan.

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3 D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.
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http://dx.doi.org/10.1080/08982104.2017.1369995DOI Listing
December 2018

Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction.

JCI Insight 2017 Jul 6;2(13). Epub 2017 Jul 6.

Although left ventricular (LV) diastolic dysfunction is often associated with hypertension, little is known regarding its underlying pathophysiological mechanism. Here, we show that the actin cytoskeletal regulator, Rho-associated coiled-coil containing kinase-2 (ROCK2), is a critical mediator of LV diastolic dysfunction. In response to angiotensin II (Ang II), mutant mice with fibroblast-specific deletion of ROCK2 (ROCK2Postn-/-) developed less LV wall thickness and fibrosis, along with improved isovolumetric relaxation. This corresponded with decreased connective tissue growth factor (CTGF) and fibroblast growth factor-2 (FGF2) expression in the hearts of ROCK2Postn-/- mice. Indeed, knockdown of ROCK2 in cardiac fibroblasts leads to decreased expression of CTGF and secretion of FGF2, and cardiomyocytes incubated with conditioned media from ROCK2-knockdown cardiac fibroblasts exhibited less hypertrophic response. In contrast, mutant mice with elevated fibroblast ROCK activity exhibited enhanced Ang II-stimulated cardiac hypertrophy and fibrosis. Clinically, higher leukocyte ROCK2 activity was observed in patients with diastolic dysfunction compared with age- and sex-matched controls, and correlated with higher grades of diastolic dysfunction by echocardiography. These findings indicate that fibroblast ROCK2 is necessary to cause cardiac hypertrophy and fibrosis through the induction CTGF and FGF2, and they suggest that targeting ROCK2 may have therapeutic benefits in patients with LV diastolic dysfunction.
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http://dx.doi.org/10.1172/jci.insight.93187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499369PMC
July 2017

Independent effects of ADH1B and ALDH2 common dysfunctional variants on gout risk.

Sci Rep 2017 05 31;7(1):2500. Epub 2017 May 31.

Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan.

Gout is caused by hyperuricemia, with alcohol consumption being an established risk factor. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are crucial enzymes for alcohol metabolism. We recently performed a genome-wide association study of gout and a subsequent fine-mapping study which identified rs671 of ALDH2 as a gout locus. However, the association between gout and common variants of ADH1B has hitherto remained unreported, prompting us to investigate the association between gout and common dysfunctional variants of ADH1B (rs1229984) and ALDH2 (rs671). We used 1,048 clinically defined gout cases and 1,334 controls of Japanese male. The "His carrier" (His/His or His/Arg) of rs1229984 (His48Arg) of ADH1B significantly increased gout risk (P = 4.3 × 10, odds ratio = 1.76), as did the "non-Lys carrier (Glu/Glu)" of rs671 (Glu504Lys) of ALDH2. Furthermore, common variants of ADH1B and ALDH2 are independently associated with gout. Our findings likewise suggest that genotyping these variants can be useful for the evaluation of gout risk.
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http://dx.doi.org/10.1038/s41598-017-02528-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451470PMC
May 2017

GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes.

Ann Rheum Dis 2017 05 29;76(5):869-877. Epub 2016 Nov 29.

Department of Medicine, University of Otago, Christchurch, New Zealand.

Objective: A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific.

Methods: Putative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study.

Results: In addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10): urate transporter genes ( and ) and for all gout cases, and and for the renal underexcretion gout subtype. While encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and was associated with gout in all cases. A meta-analysis of the three populations revealed to be associated with gout at a genome-wide level of significance (p =3.58×10).

Conclusions: Our findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia.
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http://dx.doi.org/10.1136/annrheumdis-2016-209632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530361PMC
May 2017

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK.

Biometals 2016 08 9;29(4):715-29. Epub 2016 Jul 9.

Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, Prague 2, 128 43, Czech Republic.

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.
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http://dx.doi.org/10.1007/s10534-016-9947-zDOI Listing
August 2016

Structural characterization of the heme-based oxygen sensor, AfGcHK, its interactions with the cognate response regulator, and their combined mechanism of action in a bacterial two-component signaling system.

Proteins 2016 10 23;84(10):1375-89. Epub 2016 Jun 23.

Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25083DOI Listing
October 2016

Rho Kinases and Cardiac Remodeling.

Circ J 2016 Jun 1;80(7):1491-8. Epub 2016 Jun 1.

Section of Cardiology, Department of Medicine, University of Chicago.

Hypertensive cardiac remodeling is characterized by left ventricular hypertrophy and interstitial fibrosis, which can lead to heart failure with preserved ejection fraction. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the serine/threonine protein kinase family, which mediates the downstream effects of the small GTP-binding protein RhoA. There are 2 isoforms: ROCK1 and ROCK2. They have different functions in different types of cells and tissues. There is growing evidence that ROCKs contribute to the development of cardiovascular diseases, including cardiac fibrosis, hypertrophy, and subsequent heart failure. Recent experimental studies using ROCK inhibitors, such as fasudil, have shown the benefits of ROCK inhibition in cardiac remodeling. Mice lacking each ROCK isoform also exhibit reduced myocardial fibrosis in a variety of pathological models of cardiac remodeling. Indeed, clinical studies with fasudil have suggested that ROCKs could be potential novel therapeutic targets for cardiovascular diseases. In this review, we summarize the current understanding of the roles of ROCKs in the development of cardiac fibrosis and hypertrophy and discuss their therapeutic potential for deleterious cardiac remodeling. (Circ J 2016; 80: 1491-1498).
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http://dx.doi.org/10.1253/circj.CJ-16-0433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563468PMC
June 2016
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