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Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life‐threatening cardiovascular disease in North America. Post‐DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease‐4 (mMCP‐4) genetically depleted mice (mMCP‐4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride–endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY‐51469) inhibited mMCP‐4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo . Recombinant chymases, mMCP‐4 (mouse) and CMA‐1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell–generated mMCP‐4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control‐ but not in TY‐51469–treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell–containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP‐4, a granule‐localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

Coronavirus disease 2019 (COVID-19), an acute and highly transmissible infectious disease, has reached a pandemic level since 11 March 2020 and continues to challenge the healthcare system worldwide. The pathogenesis of COVID-19 is a complex process involving mechanisms that suppress the host antiviral and innate immune response, while triggering marked activation of coagulation and hyperinflammation leading to cytokine storm in severe COVID-19. This review summarizes current evidence related to COVID-19-associated cardiovascular severe illness and mortality, which encompasses life-threatening clinical manifestations, including myocardial injury, fulminant myocarditis, cardiac arrhythmia, and ischemic stroke. The onset of hypercoagulable state is consistent with increased venous thromboembolism including deep vein thrombosis and pulmonary embolism. Thromboembolic manifestations include arterial thrombotic events such as stroke, myocardial infarction, and limb ischemia. Several treatment strategies have been investigated to mitigate COVID-19-associated cardiovascular clinical manifestations. The prevalence of thrombo-inflammatory syndrome and subsequent cardiovascular dysfunction prompted the implementation of antithrombotic therapy and strategies targeting major pro-inflammatory cytokines involved in COVID-19 cytokine storm. The development of new guidelines for effective treatment strategies requires concerted efforts to refine our understanding of the mechanisms underlying cardiovascular disease and large-scale clinical trials to reduce the burden of COVID-19 hospitalization and mortality.

Aortic valve stenosis (AVS) is the most common cause of surgical valve replacement worldwide. The vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis and several other cardiovascular diseases; however, its role in the pathogenesis of AVS remains to be determined. Here, we investigated the expression of UII, urotensin-related peptide (URP), and the urotensin receptor (UT) and the role this system plays in AVS.Immunohistochemistry and reverse-transcriptase polymerase chain reaction were used to examine the cellular localization and mRNA expression, of UII, URP, and UT in calcified and noncalcified aortic valves. Human aortic valve interstitial cells were isolated from normal valves and treated with UII or URP, and changes in cell proliferation, cholesterol efflux, calcium deposition, and β-catenin translocation were assessed.The mRNA expression of UII, URP, and UT was significantly greater in patients with AVS. There was abundant presence of UII, URP, and UT immunostaining in diseased compared with nondiseased valves and correlated significantly with presence of calcification (P .0001) and fibrosis (P .0001). Treating human aortic valve interstitial cells with UII or URP significantly increased cell proliferation (P .0001) and decreased cholesterol efflux (P = .0011 and P = .0002, respectively). UII also significantly reduced ABCA1 protein expression (P = .0457) and increased β-catenin nuclear translocation (P .0001) and mineral deposition (P .0001).Together, these data suggest that the urotensin system plays a role in the pathogenesis of AVS and warrants further investigation.

Aortic valve calcification leads to the deposition of calcium phosphate minerals in the extracellular matrix of the aortic valve leaflets. The mineral deposits can severely narrow the opening of the aortic valve, leading to aortic stenosis. There are no therapies to halt or slow down disease progression and the mechanisms governing aortic valve calcification are still poorly understood. Recently, several studies have shown that for the same aortic stenosis severity, women present significantly lower calcification loads than men. The cause of this sex-related difference is unknown. To understand this difference, we analyzed mineral deposits from surgically excised calcified human aortic valves with different material characterization techniques. We find profound differences in mineral composition and morphology between sexes, which strongly suggest that minerals form slower in women than in men and follow a different mineralization pathway. This finding paves the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification. STATEMENT OF SIGNIFICANCE: Aortic valve calcification is a health disorder with increasing prevalence and high morbidity and mortality. Currently there is no approved effective treatment; the only available therapeutic option is invasive valve replacement, to which not all patients are suited. The main reason for such lack of treatment options is our lack of understanding of the calcification mechanism. In this study, we show profound differences in mineral composition and morphology between sexes, suggesting that aortic valve calcification follows different mineralization pathways in men and women. These findings pave the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification.

Stem cell therapy for cardiac regeneration has been gaining traction as a possible intervention for the reduction of the burden associated with MI and heart failure. However, stem cell therapies have several shortcomings, including poor engraftment, limited improvements in cardiac function, and possible teratogenicity. Recently, extracellular vesicles (EVs) from stem cell sources have been explored as a novel therapy to regenerate the injured myocardium in several animal MI trials. In this systematic review and meta-analysis, we investigate the use of stem cell-derived EVs for cardiac repair preclinical trials in animal MI models. Cochrane Library, Medline, Embase, PubMed, Scopus and Web of Science and grey literature (Canadian Agency for Drugs, Technologies in Health, and Google Scholar) were searched through August 20, 2020 and 37 articles were included in the final analysis. The overall effect size observed in EV-treated small animals after MI for ejection fraction (EF) was 10.85 [95 %CI: 8.79, 12.90] and for fractional shortening (FS) was 7.19 [95 %CI: 5.43, 8.96] compared to control-treated animals. The most abundant stem cell source used were mesenchymal stem cells which showed robust improvements in EF and FS (MD = 11.89 [95 % CI: 9.44, 14.34] and MD = 6.96 [95 % CI: 4.97, 8.96], respectively). Significant publication bias was detected for EF and FS outcomes. This study supports the use of EVs derived from stem cells as a novel therapy for cardiac repair after MI. Further investigation in larger animal studies may be necessary before clinical trials.PROSPERO registration number: CRD42019142218.

The prevalence of calcification of human aortic valve in populations 65 years old and greater is estimated to be 2–3%. Bicuspid aortic valve disease (BAVD) is a common etiology of aortic stenosis in populations aged 60–75 years of age; 30–50% of operated cases of aortic stenosis were due to calcified BAVD. Dysregulation of the Notch and the canonical Wnt pathway has been well documented to be associated with calcification of the aortic valve. However, recent studies have increased this scope to include the non-canonical pathway where Wnt5a, Wnt5b and Wnt 11 levels were significantly greater in calcified human aortic valves than normal valves, and with Wnt5b specifically being implicated in BAVD pathogenesis. More recently, Lipoprotein(a) [Lp(a)] has been implicated as a key player in the pathogenesis of calcific aortic valve disease. It has been shown that the osteogenic effect of Lp(a) is mediated through the oxidized phospholipid pathway as well as oxidized phospholipid independent pathways involving mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK) and Wnt. Moving forward, further work needs to be conducted in order to elucidate the crosstalk between the different signaling cascades, specifically with regard to BAVD.

Aortic valve stenosis (AVS) is a prevailing and life-threatening cardiovascular disease in adults over 75 years of age. However, the molecular mechanisms governing the pathogenesis of AVS are yet to be fully unraveled. With accumulating evidence that Wnt signaling plays a key role in the development of AVS, the involvement of Wnt molecules has become an integral study target in AVS pathogenesis. Thus, we hypothesized that the Wnt/β-catenin pathway mediators, SFRP2, DVL2, GSK3β and β-catenin are dysregulated in patients with AVS. Using immunohistochemistry, Real-Time qPCR and Western blotting, we investigated the presence of SFRP2, GSK-3β, DVL2, and β-catenin in normal and stenotic human aortic valves. Markedly higher mRNA and protein expression of GSK-3β, DVL2, β-catenin and SFRP2 were found in stenotic aortic valves. This was further corroborated by observation of their abundant immunostaining, which displayed strong immunoreactivity in diseased aortic valves. Proteomic analyses of selective GSK3b inhibition in calcifying human aortic valve interstitial cells (HAVICs) revealed enrichment of proteins involved organophosphate metabolism, while reducing the activation of pathogenic biomolecular processes. Lastly, use of the potent calcification inhibitor, Fetuin A, in calcifying HAVICs significantly reduced the expression of Wnt signaling genes Wnt3a, Wnt5a, Wnt5b, and Wnt11. The current findings of altered expression of canonical Wnt signaling in AVS suggest a possible role for regulatory Wnts in AVS. Hence, future studies focused on targeting these molecules are warranted to underline their role in the pathogenesis of the disease.

Purpose of review Plasma levels of HDL cholesterol are a biomarker of cardiovascular health but not a therapeutic target, as demonstrated by the failure of pharmacological modulation of HDL cholesterol to prevent or treat atherosclerotic cardiovascular disease. In health, HDL particles exert pleiotropic effects against atherosclerosis, including cholesterol removal from foam cells, vasodilatory effects through vascular endothelial cell nitric oxide production, decreased vascular inflammation and oxidative damage, endothelial cell proliferation and antiapoptotic effects. Recent findings These functional effects of HDL are independent of the cholesterol mass and are related to the proteome and lipidome. In disease states and with the ageing process, HDL components are extensively modified and may no longer play a beneficial role but are retained in the atheroma and contribute to atherosclerosis. We have recently shown that desmocollin 1 (DSC1) acts as an apolipoprotein (apo) A-I binding protein that is highly expressed in atherosclerotic plaques and inhibits atheroprotective HDL functions by retaining apoA-I. The apoA-I retention hypothesis proposes that macrophages express DSC1 in a maladaptive process that renders apoA-I inactive and contributes to atherosclerosis. Summary HDL loses their beneficial properties in ageing and disease states. Novel pathways may present new therapeutic avenues to restore their biological functions.

Aortic valve stenosis (AVS) affects a significant percentage of our elderly population and younger subjects with familial hypercholesterolemia. Lipoprotein(a) [Lp(a)] has been associated with AVS in recent genetic studies. The purpose of this study was to determine the effects of Lp(a) on human aortic valve interstitial cells (HAVICs), and to identify apolipoproteins and phospholipids in diseased human aortic valves.We examined the effects of Lp(a) on HAVICs mineralization and oxidant formation. Proteomic analyses were used to determine the effects of Lp(a) on downstream intracellular markers. We also used mass spectroscopy to identify the different lipoproteins and oxidized phospholipids in calcified aortic valves.HAVICs incubated with either LDL or Lp(a) had significantly higher calcium deposition, compared to control (p0.001), with Lp(a) having the most significant effect (p0.01) compared to LDL. Proteomic analysis after 10 days of treatment with Lp(a) resulted in enrichment of proteins involved in calcium deposition and vesicle biogenesis. Treatment of HAVICs with Lp(a) significantly increased ROS formation (p0.05). Patients with calcific aortic stenosis had higher plasma Lp(a) concentrations compared to non-CAD individuals (p0.001). LC-MS/MS revealed the presence of apolipoproteins and phospholipids in calcified human aortic valves.The present study outlines an association between Lp(a) and AVS, and suggests that Lp(a) may serve as a potential target for therapeutic purposes to manage the progression of AVS.

Purpose of review The major cardio-protective function of HDL is to remove excess cellular cholesterol in the process of HDL particle formation and maturation. The HDL biogenic procedure requiring protein-lipid interactions has been incompletely understood, and here we discuss recent progress and insights into the mechanism of HDL biogenesis. Recent findings The initial and rate-limiting step of HDL biogenesis is the interaction between apoA-I and plasma membrane microdomains created by ATP-binding cassette transporter A1 (ABCA1) transporter. Computer simulation of molecular dynamics suggests that ABCA1 translocates phospholipids from the inner to the outer leaflet of the plasma membrane to create a transbilayer density gradient leading to the formation of an exovesiculated plasma membrane microdomain. The cryo-electron microscopy structure of ABCA1 suggests that an elongated hydrophobic tunnel formed by the extracellular domain of ABCA1 may function as a passageway to deliver lipids to apoA-I. In contrast to ABCA1-created plasma membrane microdomains, desmocollin 1 (DSC1) contained in a cholesterol-rich plasma membrane microdomain binds apoA-I to prevent HDL biogenesis. The identification of DSC1-containing plasma membrane microdomains as a negative regulator of HDL biogenesis may offer potential therapeutic avenues. Summary Isolation and characterization of plasma membrane microdomains involved in HDL biogenesis may lead to a better understanding of the molecular mechanism of HDL biogenesis.

Aims The biogenesis of high-density lipoprotein (HDL) particles by cholesterol-laden foam cells in atherosclerotic lesions is crucial for the removal of excess cholesterol from the lesions. Impairment in the HDL biogenic process contributes to the progression of atherosclerosis. The aim of this study is to identify novel cellular factors regulating HDL biogenesis. Methods and results HDL biogenesis is a process of apolipoprotein (apo)-mediated solubilization of specific plasma membrane (PM) microdomains generated in cholesterol-accumulated cells. We established a new method to isolate PM microdomains interacting with the major HDL protein constituent, apoA-I. Lipidomic and proteomic analyses of an isolated PM microdomain revealed that apoA-I binds to cholesterol-rich and desmocollin 1 (DSC1)-containing microdomains. In this novel apoA-I binding microdomain, DSC1 binds and prevents apoA-I from interacting with another PM microdomain created by adenosine triphosphate-binding cassette transporter A1 (ABCA1) for the formation of HDL. Inhibition of apoA-I-DSC1 binding by silencing DSC1 expression or using DSC1 blocking antibodies increases apoA-I accessibility to ABCA1-created microdomains and thus enhances HDL biogenesis. Importantly, DSC1 is abundantly expressed in macrophages and human atherosclerotic lesions, suggesting that DSC1 may contribute to cholesterol accumulation in atherosclerotic lesions by sequestering apoA-I and impairing HDL biogenesis. Conclusions The binding of apoA-I to two functionally opposing PM microdomains, ABCA1 and DSC1 domains, suggests that HDL biogenesis and PM cholesterol levels may be regulated by the relative abundance of the two domains and that novel HDL biogenic therapies may be developed by targeting DSC1.

Objective— The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, the crucial roles of noncanonical Wnt ligands WNT5a, WNT5b, and WNT11 in osteogenesis make them critical targets in the study of aortic valve calcification. Approach and Results— Using immunohistochemistry, real-time qPCR, Western blotting, and tissue culture, we examined the tissue distribution of WNT5a, WNT5b, and WNT11 in noncalcified and calcified aortic valves and their effects on human aortic valve interstitial cells (HAVICs). Only focal strong immunostaining for WNT5a was seen in and around areas of calcification. Abundant immunostaining for WNT5b and WNT11 was seen in inflammatory cells, fibrosis, and activated myofibroblasts in areas of calcified foci. There was significant correlation between WNT5b and WNT11 overall staining and presence of calcification, lipid score, fibrosis, and microvessels ( P P P P Conclusions— These findings suggest a potential role for noncanonical Wnt signaling in the pathogenesis of aortic valve calcification.

High-density lipoproteins (HDLs) have multiple pleiotropic effects against arteriosclerosis. Most are independent of the cholesterol mass within HDL particles. Yet, HDL cholesterol (HDL-C) remains a biomarker to assess cardiovascular risk. Whereas the epidemiological association between HDL-C and cardiovascular risk is strong, graded and coherent across populations, Mendelian randomization studies cast doubt on whether HDL-C is causally related to atherosclerotic cardiovascular disease. The apparent failure of HDL-C-raising therapies (fibrates, niacin, and cholesteryl ester transfer protein inhibitors) raises questions about the HDL-C hypothesis. HDL particles are heterogeneous in lipid and protein composition, and thus in size and function. Multiple factors related to oxidation and inflammation might render HDL particles malfunctional or proatherogenic. HDL functionality might be a preferred biomarker and therapeutic target. However, most of the beneficial events of HDL particles occur in the subendothelial layer of arteries and not in plasma. In this report, we review the complexity and controversies surrounding HDL and atherosclerotic cardiovascular disease. Importantly, intimal HDL biogenesis, function, and egress from the arterial wall might hold the key to unlocking the therapeutic potential of HDL.

The Hippo-signaling pathway is a mechanism implicated in cardiomyocyte cytoprotection and regeneration after a myocardial infarction. Yes-associated protein 1, the main effector protein of this pathway, acts as a co-transcriptional activator to promote cardiomyocyte proliferation and survival. However, the biological mechanisms by which yes-associated protein 1 protects the heart post-MI are currently unknown. Here, we propose that yes-associated protein 1 plays a critical role in cardiomyocyte cytoprotection after simulated ischemia-reperfusion injury. AC16 human cardiomyocytes were infected with lentiviral plasmids containing normal human yes-associated protein 1 and a constitutively active form of YAP, YAP1S127A. Cells were exposed to ischemia-reperfusion injury using a hypoxic chamber. Hippo-signaling characterization after ischemia-reperfusion injury was performed via Western blotting and reverse transcriptase polymerase chain reaction. Cell viability, apoptosis, and cellular hypertrophy were assessed as a measure of cytoprotection. The GSK3β inhibitor CHIR99021 was used to investigate cross-talk between Hippo and Wnt-signaling and their role in cytoprotection after ischemia-reperfusion-injury. Ischemia-reperfusion injury resulted in significant decreased expression of the non-phosphorylated Hippo signaling kinases MST1 and LATS1, along with decreased expression of YAP/TAZ. Overexpression of yes-associated protein 1 improved cellular viability, while reducing hypertrophy and apoptosis via the ATM/ATR DNA damage response pathway. Activation of β-catenin in YAP-infected cardiomyocytes synergistically reduced cellular hypertrophy after ischemia-reperfusion-injury. Our findings indicate that yes-associated protein 1 is cytoprotective in AC16 human cardiomyocytes after ischemia-reperfusion injury, which may be mediated by co-activation of the canonical Wnt/β-catenin pathway. Thus, activation of yes-associated protein 1 may be a novel therapeutic to repair the infarcted myocardium.Impact statementGenetically engineering the cells of the heart after myocardial infarction to display a more regenerative phenotype is a promising therapy for heart failure patients. Here, we support a regenerative role for yes-associated protein 1, the main effector protein of the Hippo signaling pathway, in AC16 human cardiomyocytes as a potential therapeutic gene target for cardiac repair after myocardial infarction.

Adult cardiac myocytes are understood to be terminally differentiated. Although several reports have highlighted the presence of endogenous cardiac progenitor cells, an ongoing debate exits on their origin and role in myocardial regeneration [1,2].

// Alexander A. Goldberg 1, 2 , Kwang-Bo Joung 1, 2 , Asma Mansuri 1, 2 , Yujin Kang 1, 2 , Raquel Echavarria 1, 2 , Ljiljana Nikolajev 3, 4 , Yang Sun 5 , Jane J. Yu 5 , Stephane A. Laporte 3, 4 , Adel Schwertani 6 , Arnold S. Kristof 1, 2 1 Meakins-Christie Laboratories, Translational Research in Respiratory Diseases Program, Montreal, Quebec, Canada 2 Department of Critical Care Medicine, McGill University Health Centre Research Institute, Montreal, Quebec, Canada 3 Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada 4 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada 5 College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA 6 Division of Cardiology, Montreal General Hospital, Montreal, Quebec, Canada Correspondence to: Arnold S. Kristof, email: arnold.kristof@mcgill.ca Keywords: TSC2, mTOR, urotensin, lymphangioleiomyomatosis, angiomyolipoma Received: May 20, 2016 Accepted: July 01, 2016 Published: July 21, 2016 ABSTRACT Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors (‘LAM nodules’) in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein ‘tuberous sclerosis complex-2’ (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor.

Background Acute myocardial infarction (MI) remains one of the leading causes of death worldwide with no curative therapy available. Stem cell therapies have been gaining interest as a means to repair the cardiac tissue after MI and prevent the onset of heart failure. Many in vivo reports suggest that the use of stem cells is promising, yet clinical trials suggest that the cells fail to integrate into the native tissue, resulting in limited improvements in cardiac function and repair. To battle this limitation, the combination of using stem cells embedded in a bioactive scaffold that promotes cell retention is growing in interest. Yet, a systematic review of the literature on the use of stem cells embedded in bioactive scaffolds for cardiac repair has not yet been performed. In this protocol, we outline a systematic review and meta-analysis of preclinical trials in animal MI models that utilize stem cell-embedded scaffolds for cardiac repair and compare their effects to stem cell-treated animals without the use of a scaffold. Methods/design We will search the following electronic databases: Cochrane Library, MEDLINE, Embase, PubMed, Scopus and Web of Science, and gray literature: Canadian Agency for Drugs and Technologies in Health and Google Scholar. We will only include randomly controlled preclinical trials that have directly investigated the effects of stem cells embedded in a scaffold for cardiac repair in an animal MI model. Two investigators will independently review each article included in the final analysis. The primary endpoint that will be investigated is left ventricular ejection fraction. Secondary endpoints will include infarct size, end systolic volume, end diastolic volume, fractional shortening and left ventricular wall thickness. Pooled analyses will be conducted using the DerSimonian-Laird random effects and Mantel-Haenszel fixed-effect models. Between-studies heterogeneity will be quantified and determined using the Tau2 and I2 statistics. Publication bias will be assessed using visual inspection of funnel plots and complemented by Begg’s and Egger’s statistical tests. Possible sources of heterogeneity will be assessed using subgroup-meta analysis and meta-regression. Discussion To date, the use of scaffolds in myocardial repair has not yet been systematically reviewed. The results of this meta-analysis will aid in determining the efficacy of stem cell-embedded scaffolds for cardiac repair and help bring this therapy to the clinic. Electronic supplementary material The online version of this article (10.1186/s13643-018-0845-z) contains supplementary material, which is available to authorized users.

Cardiovascular diseases remain the primary cause of death.1 Myocardial infarction (MI), angina, and stroke take >16 million lives every year, but the underlying mechanisms by which these often‐fatal cardiovascular events occur tend to escape attention.2 Atherosclerosis is the underlying pathological inflammatory vascular disease not only responsible for most MIs and strokes, but it accounts for 29% of deaths worldwide.1, 3 Once believed to be a passive process, it is now understood that atherosclerosis takes an active route involving several cell types, with contributions from a multitude of organ systems, molecular mechanisms, and other pathological conditions, including, but not limited to, hypertension, hyperlipidemia, and type 2 diabetes mellitus.1, 3, 4, 5 Atherosclerosis is characterized by the accumulation of lipids, fibrous elements, and inflammatory cells within the vascular wall of medium and large muscular and elastic arteries.1, 3, 6 Atherosclerotic plaque formation leads to intimal thickening and luminal stenosis.1 Reduction of blood flow ensues, leading to ischemia of the brain, heart, and extremities and clinically manifesting as MI, angina, or stroke. The main features of atherosclerosis are endothelial dysfunction, intimal thickening, inflammation, and vascular calcification (VC).4, 7, 8 These processes are considered a consequence to vascular injury.1 VC is characterized by calcium deposition in the walls of the vasculature.5 Expansion of the calcified lesion leads to thrombus formation and, if significant enough, vascular occlusion. Although the exact mechanisms for VC are unknown, there has been an increase in interest about the role Wingless (Wnt) signaling plays in disease pathogenesis. Briefly, the Wnt signaling pathway is an evolutionarily conserved pathway across kingdom Animalia and plays a crucial role in pattern formation during embryogenesis.9 The Wnt signal transduction pathway plays a crucial role in organ formation in embryonic development, cell proliferation, polarity, migration, and differentiation.10 Thus, deregulated Wnt signaling is associated with many human diseases.11 Therefore, it is justifiable to suggest that research delineating atherogenesis, with focus on the contribution of Wnt signaling in the pathogenesis of atherosclerotic calcification, is warranted.

Aortic valve stenosis is one of the most common valve diseases in the world for which there is currently no pharmacological treatment to prevent or halt disease progression. Recent genetic research has demonstrated a causal association between elevated blood levels of lipoprotein (a) (Lp (a)) and aortic valve calcification, however, the mechanisms by which Lp (a) contributes to aortic valve calcification and stenosis, is unknown. In the present study, we aimed at determining Lp (a)-induced changes in human aortic valve interstitial cells using an integrated bioinformatics approach. The Lp (a)-induced cellular pathways were analysed using microarray gene expression and proteomic data from non-stenotic human aortic valve interstitial cells. Lp (a) treatment induced osteogenic differentiation, extracellular remodeling, extracellular vesicles biogenesis, and apoptosis of human aortic valve interstitial cells. In particular, the Wnt/ β-catenin signalling pathway, a known calcification pathway contributing to aortic valve stenosis, was differentially expressed compared to non-treated cells. Lp (a) also induced the expression of 14-3-3 proteins known to regulate various signalling pathway relevant to aortic valve disease. Elucidating the mechanisms and molecular players that Lp (a) induces in the early stages of the disease to initiate aortic valve calcification could provide insight into potential pharmacological targets for the treatment of this debilitating disease.

The cellular mechanisms that induce calcific aortic stenosis are yet to be unraveled. Wnt signaling is increasingly being considered as a major player in the disease process. However, the presence of Wnt Frizzled (Fzd) receptors and co-receptors LRP5 and 6 in normal and diseased human aortic valves remains to be elucidated. Immunohistochemistry and quantitative polymerase chain reaction were used to determine Fzd receptor expression in normal and calcified human aortic valve tissue, as well as human aortic valve interstitial cells (HAVICs) isolated from calcified and normal human aortic valves. There was significantly higher mRNA expression of 4 out of the 10 Fzd receptors in calcified aortic valve tissues and 8 out of the 10 in HAVICs, and both LRP5/6 co-receptors in calcified aortic valves (P < 0.05). These results were confirmed by immunohistochemistry, which revealed abundant increase in immunoreactivity for Fzd3, 7, and 8, mainly in areas of lipid core and calcified nodules of diseased aortic valves. The findings of abundant expression of Fzd and LRP5/6 receptors in diseased aortic valves suggests a potential role for both canonical and noncanonical Wnt signaling in the pathogenesis of human aortic valve calcification. Future investigations aimed at targeting these molecules may provide potential therapies for aortic valve stenosis.

Urotensin II (UII) is a vasoactive peptide with various roles in cardiovascular physiology and pathophysiology. There is an accumulating evidence implicating UII in atherosclerosis and coronary artery disease, making it an important target in acute coronary syndrome (ACS). In this study, we sought to determine the plasma levels of UII in ACS patients within 48 h of clinical presentation and after a 12-week recovery period. We compared them to patients with stable coronary artery disease (CAD) and a control group of normolipidemic subjects without known CAD. Using a highly sensitive ELISA technique, we measured plasma UII in 27 ACS patients, 26 stable CAD patients and 22 age-matched controls. ACS patients had significantly elevated plasma UII during the first 48 h of clinical presentation compared to stable CAD patients and controls. We also found significant positive correlations between UII and CRP and with triglycerides and a significant negative correlation between UII and EF. There was no correlation with LDL-C. In conclusion, plasma UII levels were elevated in patients with acute coronary syndrome, particularly immediately after clinical presentation. This suggests an upregulation of UII expression in ACS.

Aortic valve stenosis (AVS) is one of the most common heart valve diseases, and surgical intervention remains the only viable treatment option. Thus, there is a need for novel and innovative treatments. One approach is to study the biological pathogenesis of this disease in hopes of finding new targets for drug therapy. Although this may be a viable option, it faces some methodological concerns. Many studies attempted to address the underlying mechanisms of this disease using aortic valves from bovine models. Although these may be viable models in certain diseased conditions, this may not be the case when studying calcification in AVS. Thus, the aim of our study was to assess the significance of drawing conclusions from bovine models to humans in the context of AVS, and investigate the role of alkaline phosphatase (ALP), an enzyme that increases calcium mineralization and deposition in aortic valve calcification. We also wanted to identify any differences in calcification when using different osteogenic media. We used human and bovine valve interstitial cells (HAVICs and BAVICs, respectively), which are the most commonly used when study calcification in AVS, and cultured them in osteogenic media or DMEM as control media. We found that ALP activity differs widely between the two models, with bovine samples having approximately ten times more ALP activity. Our data also suggests that the degree of calcification and ALP activity differs between the different osteogenic media used. Careful consideration should be taken when experimenting with bovine valves and drawing conclusions to human AVS, as they may not exhibit the same mechanisms of action. Furthermore, it may be important to identify a single standard osteogenic medium to use when studying calcification in AVS.

Background and Aims: The aims of the present study were to determine the expression of urotensin II (UII), urotensin-II related peptide (URP), and their receptor (UT) in stable and unstable carotid atherosclerosis, and determine the effects of UII on human aortic smooth muscle cell (SMCs) calcification. Methods and Results: We examined UII, URP, and UT protein expression in 88 carotid endarterectomy specimens using immunohistochemistry. Expression of UII, URP, and UT was more evident in unstable compared to stable plaques (P < 0.05). Multivariate Spearman correlation analyses revealed significant positive correlations between UII, URP and UT overall staining and presence of calcification, severity of stenosis and inflammation (P < 0.05). Subjects undergoing carotid endarterectomy had significantly higher plasma UII levels, as assessed by ELISA, when compared with normolipidemic healthy control subjects (P < 0.05). Incubation of human aortic SMCs cultured in phosphate media with varying concentrations of UII resulted in a significant increase in calcium deposition and alkaline phosphatase activity. UII also significantly increased β-catenin translocation and expression of ALPL, BMP2, ON, and SOX9 (P < 0.05). Incubation of cells with phosphate medium alone increased the expression of the pre-UT and mature UT (P < 0.01), and addition of UII had a synergistic effect on pre-UT protein expression (P < 0.001) compared to phosphate medium alone. Conclusions: Upregulation of UII, URP, and UT in unstable carotid endarterectomy plaques and plasma, and the stimulatory effect of UII on vascular smooth muscle cell calcification suggest that the UII system may play a role in the pathogenesis of vascular calcification and stability of atherosclerosis.

Objectives: High plasma levels of lipoprotein (a) (Lp(a)) are associated with increased risk of aortic valve stenosis. The purpose of this study was to determine ability of human aortic valve interstitial cells (HAVICs) to generate HDL particles from plasma of patients with high Lp(a), and understand the mechanism involved. Methods: HAVICs were isolated from consented participants and cultured in DMEM, 10% FBS, 5% glucose. ABCA1 proteins expression was tested in HAVICs lyses after detection by polyclonal anti-ABCA1 antibody. Cholesterol efflux was determined in HAVICs against HDL isolated by PEG from patients with aortic valve stenosis exhibiting mild or severe plasma Lp(a), compared to normolipidemic controls. FPLC and 2D-PAGGE were used to illustrate HDL profiles from these patients after incubation with HAVICs. Results: Our data showed abundant expression of ABCA1, and a significant decrease in HAVICs cholesterol efflux (5%, for 8h) in response to HDL from plasma of patients with high Lp(a) and aortic valve stenosis (mild 16±0.01%cpm, severe 23.4±0.21%cpm) compared to normolipidemic HDL (29.57cpm; P Conclusion: These findings demonstrate a defect in HAVICs ABCA1 cholesterol removal associated a loss of α1 species of nHDL in patient with high Lp(a) and aortic valve stenosis. Therefore, the HAVICs cholesterol efflux defect associated with the elevated plasma Lp(a) may explain the pathological changes associated aortic valve calcification.

Cardiomyocytes are understood to possess a limited regenerative capacity. Any myocardial insult leads to an irreversible injury. Mesenchymal stem cell differentiation into cardiomyocyte-like cells stands as one of the leading experimental therapies. However, a candidate cell source has yet to be defined. Here, we examined the in vitro and in vivo cardiac differentiation potential of human placenta derived stem cells (hPDSCs); a unique, abundant, and non-immunogenic cell source.H9c2 cell culture medium was applied to hPDSCs at different ratios for a period of 4weeks. In parallel, hPDSCs, human bone marrow stem cells, or cell free culture medium was injected in peri-infarcted regions induced in rat hearts.In vitro, hPDSCs pre-conditioned with H9c2 cell culture medium proportionally over-expressed alpha sarcoplasmic actinin and displaced connexin 43 from the cytoplasm to the cell membrane. Additionally, pre-conditioning promoted hPDSCs survival and triggered vascular endothelial growth factor (VEGF) dependent angiogenesis by activating the pAkt and p38MAPK/pSTAT3 pathways. In vivo, echocardiography analysis showed a significant improvement in cardiac parameters in the rats injected with hPDSCs, similar to the human bone marrow stem cells injected group. Moreover, hPDSCs detected within rat cardiac tissues expressed troponin I and myosin heavy chain. In accordance with the pre-conditioning findings, VEGF positive neovessels were observed in hearts injected with hPDSCs.hPDSCs have the potential to differentiate into cardiac-like cells and induce angiogenesis via paracrine effects. With the advantages of easy availability and young age, these cells could be more suitable for clinical translation.

Aims To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. Materials and methods Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52 weeks. Macitentan (30 mg/kg/day), a dual ET A /ET B receptor antagonist, was given orally for 6 weeks (27 week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Masson's trichrome stain and α-smooth muscle actin labeling. Key findings Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. Significance These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent.

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in μM(-1)s(-1)): 2.29 × 10(-4)vs. 6.41 × 10(-6); ET-1 (1-31) production: 2.19 × 10(-3)vs. 6.57 × 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.

Introduction: Atherosclerosis is a leading cause of death in Western societies. Vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis and several other cardiovascular diseases however further research is required to develop a complete understanding of UII’s role in the pathogenesis of atherosclerosis. Hypothesis: We hypothesized that UII stimulates calcification in vascular smooth muscle cells and that UII, urotensin II related peptide (URP) and UT receptor expression are upregulated in calcified aortic valves. Methods and Results: Human aortic smooth muscle cells (HASMC) were cultured in phosphate media (2.6mmol/L) for 13 days in the presence of varying concentrations of UII (0, 10, 50, 100nm) and the amount of calcium was measured with a calcium assay kit. Protein was extracted and measured with a protein assay kit. HASMC calcification was assessed as the ratio of calcium (μg)/protein (mg). HASMC calcification increased with increasing UII concentration and was significantly elevated in 100nm of UII (N=6, P Conclusion: The stimulatory effect of UII on vascular smooth muscle cell calcification as well as the upregulated expression of UII, URP and UT in calcified aortic valves suggests that the UT receptor system plays a key role in the pathogenesis of atherosclerosis and valve calcification.

Introduction: The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, the crucial roles of non-canonical Wnt ligands Wnt5b and Wnt11 in osteogenesis make them critical targets in the study of aortic valve calcification. Hypothesis: We hypothesized that Wnt signaling mediated by ligands Wnt5b and Wnt11 is upregulated and the expression of these molecules may be associated with the pathological calcium deposition in aortic valves. Methods and Results: Mitral, non-calcified and calcified aortic valves were collected from patients with valve replacement. Total RNA was isolated, first strand cDNAs were synthesized and gene specific qPCRs were performed. The relative mRNA expression of Wnt5b and Wnt11 were calculated using the ΔΔCT method against GAPDH control and are expressed as fold change ± SEM compared to expression in non-calcified valves. We found significant increases in both Wnt5b (15.14±4.08) and Wnt11 (4.09±0.63) expression in calcified compared to non-calcified aortic or mitral valves (P Conclusion: The upregulated Wnt5b and Wnt11 expression in calcified aortic valves, particularly in areas of calcification, fibrosis and activated myointimal cells suggests a potential involvement in the pathogenesis of aortic calcification.

The metabolic syndrome is defined by the presence of hyperlipidemia, obesity, hypertension, and diabetes. The syndrome is associated with significant cardiovascular morbidity and mortality. The aim of the present study was to determine the role of the vasoactive peptide urotensin II (UII) in the pathogenesis of the metabolic syndrome. We used obese mice (ob/ob) to determine the effect of UII receptor (UT) blockage on the different facets of the metabolic syndrome with special emphasis on cardiac function. Our data demonstrate a significant increase in UII and UT expression in the myocardium of obese mice accompanied by a significant decrease in sarco/endoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression, as well as intracellular Na(+) and Ca(2+) compared with wild-type mice (P0.05). Treatment of ob/ob mice with the UII receptor antagonist SB657510 significantly improved glucose levels, blood pressure, hyperlipidemia, expression of myocardial SERCA2a, intracellular Na(+) and Ca(2+) and cardiac function in association with a decrease in weight gain, and mammalian target of rapamycin (mTOR) and sodium/hydrogen exchanger 1 (NHE-1) protein expression compared with vehicle (P0.05). These findings demonstrate an important role for UII in the pathogenesis of the metabolic syndrome and suggest that the use of UT receptor antagonists may provide a new therapeutic tool for the treatment of this syndrome.

IL-15 is an inflammatory cytokine secreted by many cell types. IL-15 is also produced during physical exercise by skeletal muscle and has been reported to reduce weight gain in mice. Contrarily, our findings on IL-15 knockout (KO) mice indicate that IL-15 promotes obesity. The aim of this study is to investigate the mechanisms underlying the pro-obesity role of IL-15 in adipose tissues.Control and IL-15 KO mice were maintained on high fat diet (HFD) or normal control diet. After 16 weeks, body weight, adipose tissue and skeletal mass, serum lipid levels and gene/protein expression in the adipose tissues were evaluated. The effect of IL-15 on thermogenesis and oxygen consumption was also studied in primary cultures of adipocytes differentiated from mouse preadipocyte and human stem cells.Our results show that IL-15 deficiency prevents diet-induced weight gain and accumulation of lipids in visceral and subcutaneous white and brown adipose tissues. Gene expression analysis also revealed elevated expression of genes associated with adaptive thermogenesis in the brown and subcutaneous adipose tissues of IL-15 KO mice. Accordingly, oxygen consumption was increased in the brown adipocytes from IL-15 KO mice. In addition, IL-15 KO mice showed decreased expression of pro-inflammatory mediators in their adipose tissues.Absence of IL-15 results in decreased accumulation of fat in the white adipose tissues and increased lipid utilization via adaptive thermogenesis. IL-15 also promotes inflammation in adipose tissues that could sustain chronic inflammation leading to obesity-associated metabolic syndrome.

Objective— Urotensin II (UII) is a potent vasoactive peptide that binds to the urotensin receptor–coupled receptor-14 (known as UT) and exerts a wide range of actions in humans and experimental animals. We tested the hypothesis that UII gene deletion or UT blockade ameliorate experimental atherosclerosis. Methods and Results— We observed a significant reduction in weight gain, visceral fat, blood pressure, circulating plasma lipids, and proatherogenic cytokines and improvement of glucose tolerance in UII knockout mice compared with wild type ( P P P Conclusion— These findings demonstrate for the first time the role of UII gene deletion in atherosclerosis and suggest that the use of pharmaceutical agents aimed at blocking the UII pathway may provide a novel approach in the treatment of atherosclerosis and its associated precursors such as obesity, hyperlipidemia, diabetes mellitus, and hypertension.

Urotensin II (UII) is a vasoactive peptide that was first discovered in the teleost fish, and later in mammals and humans. UII binds to the G protein coupled receptor GPR14 (now known as UT). UII mediates important physiological and pathological actions by interacting with its receptor. The metabolic syndrome (MetS) is described as cluster of factors such as obesity, dyslipidemia, hypertension and insulin resistance, further leading to development of type 2 diabetes mellitus and cardiovascular diseases. UII levels are upregulated in patients with the MetS. Evidence directly implicating UII in every risk factor of the MetS has been accumulated. The mechanism that links the different aspects of the MetS relies primarily on insulin resistance and inflammation. By directly modulating both of these factors, UII is thought to play a central role in the pathogenesis of the MetS. Moreover, UII also plays an important role in hypertension and hyperlipidemia thereby contributing to cardiovascular complications associated with the MetS.