Cardiotonic steroids induce vascular fibrosis via pressure-independent mechanism in NaCl-loaded diabetic rats Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight weeks old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n=16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n=8/group). Blood pressure (BP), MBG, erythrocyte Na/K-ATPase activity, aortic weights and levels of fibrosis markers (Fli1, PKCδ, TGFβ1, SMAD5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic BP were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats vs. control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (EC50=29 nmol/L) vs. control rings (EC50=7 nmol/L) and was restored by anti-MBG mAb (EC50=9 nmol/L). Our results suggest that in salt-loaded diabetic rats MBG stimulates aortic collagen synthesis in a pressure-independent fashion, and that two pro-fibrotic mechanisms, Fli1-dependent and TGFβ-dependent, underlie its effects. Correspondence to Alexei Y. Bagrov, PhD, MD, Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia. Fax: 7-812-552-7901; e-mail: aybagrov@gmail.com Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Drugs to inhibit the NLRP3 inflammasome: not always on-target No abstract available

GPER agonist G1 improves diastolic function and attenuates cardiac RAS activation in estrogen-deficient hypertensive rats This study was aimed to clarify differences in how specific agonists of the three estrogen receptors (ERs) influence diastolic function and the renin angiotensin system (RAS) following ovariectomy (OVX) in twenty-four spontaneously hypertensive (SHR) females undergoing bilateral OVX at 12 weeks of age. Eight weeks postsurgery, rats were randomized (n=6/group) to receive equipotent, daily treatments of one of the ER agonists (ERα agonist, PPT 94 μg/kg; ERβ agonist, DPN 58 μg/kg; GPER agonist, G1 100 μg/kg), or vehicle (peanut oil). Following 4 weeks of treatment, left ventricular (LV) function/structure and systemic/intracardiac pressure measurements were obtained by echocardiography and a fluid-filled catheter attached to a pressure transducer, respectively. Selective ER agonist treatment with G1 or PPT led to improvements in diastolic function after estrogen loss when compared to vehicle treated OVX rats. While mean arterial blood pressure was not overtly different among groups, chronic G1, but not the other ER ligands, enhanced the in vitro vasorelaxant responsiveness to acetylcholine in aortic rings. These favorable effects of G1 were further linked to reductions in cardiac ACE activity, AT1R protein expression and Ang II immunoreactivity. Activation of ERβ had no effect on cardiac function and did not alter components of the canonical cardiac RAS in comparison to vehicle-treated OVX-SHR. These data imply that of the three ERs, GPER has a unique role in preserving diastolic function and favorably modulating the cardiac RAS independent of arterial pressure. Specifically, if GPER is pharmacologically activated, it could provide a therapeutic opportunity to limit the development and/or progression of diastolic dysfunction in hypertensive women after estrogen loss. Corresponding author: Leanne Groban, MD Department of Anesthesiology Wake Forest School of Medicine Medical Center Boulevard Winston-Salem, NC 27157-1009 USA Phone: +1-336-716-4498 Email: lgroban@wakehealth.edu The authors have no conflicts of interest related to this manuscript This work was funded by grants from the National Heart Lung and Blood Institute (CMF and LG) P01-HL051952 and the National Institute on Aging (LG) AG033727 of the National Institute of Health and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Programa de Apoio a Núcleos de Experiência (PRONEX), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Instituto Nacional de Ciência e Tecnologia (INCT-INOFAR, Proc. 465.249/2014-0). Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Role of matrix vesicles in bone-vascular cross-talk Matrix mineralization can be divided into physiological mineralization and pathological mineralization. There is a consensus among existing studies that matrix vesicles (MVs) are the starting sites of bone mineralization, and each component of MVs serves a certain function in mineralization. Additionally, ectopic MVs pathologically promote undesired calcification, the primary focus of which is the promotion of vascular calcification. However, the specific mechanisms of the actions of MVs in bone-vascular axis cross-talk have not been fully elucidated. This review summarizes the latest research in this field and explores the roles of MVs in the bone-vascular axis with the aim of generating new ideas for the prevention and treatment of vascular calcification and bone metabolic disease. Corresponding author at: Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China Tel.: +86 511 85030586. E-mail address: wangtsmc@aliyun.com (Zhongqun Wang). Jiangsu University, Zhenjiang 212001. E-mail address: 459736150@qq.com (Lele Jing). Academic degrees: Dr. Jing, Dr. Li, Dr. Sun, Dr. Bao, Miss Shao, Dr. Yan, Miss Pang, Miss Geng, Miss Zhang, Miss Wang, Dr. Wang. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Association of MMP-1 (rs1799750)-1607 2G/2G and MMP-3 (rs3025058)-1612 6A/6A genotypes with coronary artery disease risk among Iranian Turks This study was conducted to evaluate the association between MMP-1 (rs1799750) -1607 1G/2G and MMP-3 (rs3025058) -1612 5A/6A polymorphisms/haplotypes and coronary artery disease (CAD) risk among Iranian Turks. Totally, 102 patients with CAD and 102 healthy subjects joined the study. Genomic DNA isolation was carried out using “salting out” method from 3-4 ml whole blood samples. The MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) promoter gene polymorphisms were detected via PCR-RFLP. Our results indicated that the frequencies of the MMP-1 (−1607) 2G alleles and 2G/2G genotypes and the MMP-3 (-1612) 6A alleles and 6A/6A genotypes were higher in CAD patients aged over 50 years than healthy controls (P < 0.05). We failed to show statistically significant differences between the CAD patients aged under 50 years and controls concerning MMP1 −1607ins/delG (1G>2G, rs1799750) and MMP-3 -1612ins/delA (5A/6A, rs3025058) polymorphisms (P > 0.05). The frequencies of MMP3/MMP1 haplotypes were not statistically different among tested groups (P > 0.05). This examination as the first study of its own kind in Iranian Turks, reported association between MMP-1 (rs1799750) -1607 2G/2G and MMP-3 (rs3025058) -1612 6A/6A genotypes and CAD risk in patients aged over 50 years. Corresponding author: Dr Morteza Bagheri, Assistant Professor, Cellular and molecular research center, Cellular and molecular medicine institute, Urmia University of Medical Sciences, Urmia, Iran. E-mail: mortezabagheri@umsu.ac.ir, Tel: +98-44-32770969, Fax: +98-44-32234125, P.O. Box: 5756115111. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Infusion of melatonin into the paraventricular nucleus ameliorates myocardial ischemia-reperfusion injury by regulating oxidative stress and inflammatory cytokines Melatonin, the receptors for which are abundant in the hypothalamic paraventricular nucleus (PVN), can protect the heart from myocardial ischemia-reperfusion (MI/R) injury. The aim of this study was to determine whether the infusion of melatonin into the PVN protects the heart from MI/R injury by suppressing oxidative stress or regulating the balance between pro-inflammatory cytokines and anti-inflammatory cytokines in MI/R rats. Male Sprague Dawley (SD) rats were treated with a bilateral PVN infusion of melatonin. MI/R operation was performed one week after infusion. At the end of the 3rd week after the infusion, all the rats were euthanized. This was followed by immunohistochemistry and immunofluorescence studies of the rats. MI/R rats showed larger infarct size, increased left ventricular end-diastolic volume (LVEDV), and decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Moreover, MI/R rats had a higher level of norepinephrine (NE) in the plasma, heart, and PVN; higher PVN levels of reactive oxygen species (ROS), NOX2, NOX4, IL-1β, and NF-κB activity; and lower PVN levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) and IL-10 compared with the sham group. Melatonin infusion in PVN reduced LVEDV, NE, ROS, NOX2, NOX4, IL-1β, and NF-κB activity, and increased LVEF, LVFS, Cu/Zn-SOD, and IL-10. Overall, these results suggest that the infusion of melatonin ameliorates sympathetic nerve activity and MI/R injury by attenuating oxidative stress and inflammatory cytokines in the PVN of MI/R rats. Corresponding author: Wen-Sheng Chen, MD, PhD Department of Cardiovascular Surgery, Xijing Hospital, Air Force Medical University, Xi'an 710032, China Phone: +86 2984775576 Fax: +86 2984775576 Email: drchenwensheng@163.com Conflicts of interest: The authors declare no conflicts of interest. a These authors contributed equally to this work. Funding: This work was supported by grants from the National Natural Science Foundation of China (grant nos. 81470411, 81370356, 91439120, 81770426, 81600333 and 81800373) and Shaanxi Provincial Natural Science Foundation (grant no. 2014JM4106). This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Alirocumab in Acute Myocardial Infarction: Results from the Virginia Commonwealth University Alirocumab Response Trial (VCU-AlirocRT) Alirocumab improves outcomes in patients with a history of recent acute coronary syndrome, but treatment acutely at the time of myocardial infarction (MI) is untested. We present the results of a randomized, placebo-controlled, double-blinded pilot study of alirocumab treatment at the time of non-ST elevation myocardial infarction (NSTEMI). Twenty patients with type 1 NSTEMI and low-density lipoprotein cholesterol (LDL-C) >70 mg/dL despite high intensity statin therapy were randomized 1:1 to one dose of alirocumab 150 mg subcutaneously or placebo within 24 hours of presentation. LDL-C and inflammatory biomarkers – including C-reactive protein (CRP) – were obtained at baseline, 72 hours, and 14 days. Median (IQR) were: age 59 (49,65) years, 7 (35%) male, 16 (80%) black; baseline characteristics were similar between groups. Alirocumab significantly reduced LDL-C from baseline to 14 days by 64 mg/dL (-96,-47) compared to placebo (+1 mg/dL [-25,+16])[primary endpoint]. There were no significant between-group differences in CRP changes at any time point (all p>0.2) or serious adverse events attributable to the study treatment. In conclusion, alirocumab administration at the time of NSTEMI significantly reduced LDL-C levels at 14 days, was safe, and had neutral effects on inflammatory biomarkers. Further studies are warranted to explore the effects on clinical outcomes. Address for Correspondence: Cory Trankle, MD VCU Pauley Heart Center Virginia Commonwealth University PO Box 980053 1200 E Marshall Street Richmond, VA Email: cory.trankle@vcuhealth.orgClinicalTrials.gov Identification Number: NCT02938949 Potential Conflicts of Interest: None Sources of Funding: The study was funded by an investigator-initiated grant from Regeneron/Sanofi. Regeneron laboratory services performed the PCSK9 level analysis, but otherwise neither company had a role in the study design, conduct, analysis, or reporting. It was also supported by a Clinical and Translational Science Award (UL1TR000058 from the National Center for Research Resources) to the Virginia Commonwealth University Center for Clinical and Translational Research. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Inhibiting NLRP3 Inflammasome Activity in Acute Myocardial Infarction: A Review of Pharmacologic Agents and Clinical Outcomes The NLRP3 inflammasome is an intracellular, multimeric protein complex that initiates a potent inflammatory response to danger signals. After acute myocardial infarction, NLRP3 inflammasome-dependent inflammation promotes adverse left ventricular remodeling and recurrent atherosclerotic events. Selective and non-selective inhibitors of the NLRP3 inflammasome or its downstream effects (interleukin-1β and interleukin-18) may prevent adverse left ventricular remodeling and recurrent atherosclerotic events. In this review, we highlight strategies to inhibit NLRP3 inflammasome activity and their potential roles in the management of acute myocardial infarction. Address for Correspondence: Leo F. Buckley PharmD Department of Pharmacy Services Brigham and Women’s Hospital 75 Francis Street PB-AB-314 Boston MA 02120 LFBuckley@bwh.harvard.edu LFB has nothing to disclose. PL is an unpaid consultant to, or involved in clinical trials for Amgen, AstraZeneca, Esperion Therapeutics, Ionis Pharmaceuticals, Kowa Pharmaceuticals, Novartis, Pfizer, Sanofi-Regeneron, and XBiotech, Inc. PL is a member of scientific advisory board for Amgen, Corvidia Therapeutics, DalCor Pharmaceuticals, IFM Therapeutics, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, and XBiotech, Inc. PL’s laboratory has received research funding in the last 2 years from Novartis. PL is supported by the National Heart, Lung, and Blood Institute: (R01HL080472); American Heart Association (18CSA34080399); RRM Charitable Fund. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Effects of oral anticoagulants on ≥90-year-old atrial fibrillation patients: comparison among direct oral anticoagulant, warfarin anticoagulant, and non-anticoagulation This study aimed to investigate the effects of anticoagulants on ultra-aged patients with non-valvular atrial fibrillation (AF). We retrospectively studied 320 consecutive AF patients (median age, 91 years; range 90-100.1 years). Patients were categorized as follows: patients taking direct oral anticoagulant (DOAC group, n=93), those taking warfarin (Warfarin group, n=147), and those not taking oral anticoagulants (non-OAC group, n=80). During the follow-up periods (median 3.00 years; 1st and 4th quantiles, 1.13 and 4.56 years, respectively), in thromboembolic events, the DOAC, Warfarin, and non-OAC groups showed the lowest (0%, 0/93; 0%/year), intermediate (4.7%, 7/149; 1.43%/year), and highest (5%, 4/80; 2.65%/year) incidence rates, respectively. In major bleeding events, the DOAC, Warfarin, and non-OAC groups showed the highest (9.67%, 9/96; 5.00%/year), intermediate (8.1%, 12/149; 2.46%/year), and lowest (0%, 0/80; 0%/year) incidence rates, respectively. These differences in the relationships of the three groups were statistically significant. Confounding factors did not affect these results. Bruises associated with impairment of motor function with aging caused major bleeding in approximately 60% of major bleeding cases. Cox proportional hazard model revealed that warfarin decreased mortality, whereas antiplatelet drugs increased mortality. In conclusion, DOACs had considerably high incidence of major bleeding events, whereas absence of OAC treatment was associated with substantially high thromboembolic events. Warfarin showed acceptable incidence ratios of both events. At present, warfarin is thus thought to be adequate for ultra-aged (≥90 years) non-valvular AF patients. Avoidance of bruises was important to prevent major bleeding events. Antiplatelet drugs were suggested not to be adequate for these patients. Corresponding Author: Hirosuke Yamaji, M.D. Heart Rhythm Center, Okayama Heart Clinic Takeda 54-1, Naka-Ku, Okayama 703-8251, Okayama, Japan Tel: +81-86-271-8101; Fax: +81-86-271-8102 E-mail: yamaji2@mac.com; secondary E-mail address: yamaji@okayama-heart.com Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Carthamin yellow protects heart against ischemia/reperfusion injury with reduced ROS release and inflammatory response Carthamin yellow is a flavonoid compound isolated from safflower that is widely used clinically in China. It has various pharmacological effects including promoting blood circulation to remove blood stasis and alleviating pain. Ischemic heart disease is one of the main culprits of illness and death. Here in the present study, ex vivo and in vivo and models were used to investigate whether CY reduces ischemia/reperfusion injury. In vitro experiments further verify and explain the potential mechanisms of CY cardioprotective function. Isolated hearts from male rats with or without CY pretreatment before ischemia underwent 30 min ischemia followed by 60 min reperfusion showed that CY pretreatment significantly reduced the infarct size and lactate dehydrogenase release. The in vivo experiments also indicated CY pre-administration (i.v.) reduced infarct size and improved the heart function which was impaired by myocardial ischemia/reperfusion injury. The in vitro model on myocardial cell also showed that CY reduced ischemia/reperfusion injury with reducing the LDH and ROS releasing. Eliminate ROS with N-acetylcysteine or pre inject CY into rat jugular vein reduces the expression of IL-6, TNF-a, and especially IL-1b in vivo I/R model. What’s more, CY pretreatment strongly reduced ischemia/reperfusion induced NLRP3 up expression and caspase-1 activation. Our results indicated carthamin yellow reduced ischemia-reperfusion injury when administered prior to reperfusion. The reduction in injury is accompanied by a reduced ROS release and decreased inflammatory response. Corresponding author: Gui min, Zhang, Professor of Kunming Medical University. Email: zguimin2006@outlook.com # These authors contribute equally to this work Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.