The clonal hematopoiesis mutation Jak2V617F aggravates endothelial injury and thrombosis in arteries with erosion-like intimas.

BACKGROUND: Superficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2V617F. Neutrophils from mice and humans with JAK2V617F undergo NETosis more readily than Jak2WT (wild-type) cells. We hypothesized that JAK2V617F, by increasing propensity to NETosis, exacerbates aspects of superficial erosion. METHODS AND RESULTS: We generated Jak2V617F and Jak2WT mice with heterozygous Jak2V617F in myeloid cells. We induced areas of denuded endothelium that recapitulate features of superficial erosion and assessed endothelial integrity, cellular composition of the erosion, thrombosis rates, and response to ruxolitinib, a clinically available JAK1/2 inhibitor, in relation to genotype. Following experimental erosion, Jak2V617F mice have greater impairment of endothelial barrier function and increased rates of arterial thrombosis. Neointimas in Jak2V617F mice exhibit increased apoptosis, NETosis, and platelet recruitment. Jak2V617F mice treated with ruxolitinib show increased endothelial continuity and reduced apoptosis in the neointima comparable to levels in Jak2WT. CONCLUSIONS: These observations provide new mechanistic insight into the pathophysiology of superficial erosion, the heightened risk for myocardial infarction in JAK2V617F CHIP, and point the way to personalized therapeutics based on CHIP status.

Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia.

Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA) sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA-KO mice on high-fat diet were generated to explore the impact on CLI. Comparison of dysregulated miRNAs from diabetic individuals with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High-fat-fed, diabetic Mir181a2b2-KO mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes, with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce proangiogenic cytokines. Single-cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice was a result of impaired hematopoiesis, with increased CXCR4 signaling in bone marrow Lineage-Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from nondiabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression was mediated by the miR-181 target, Plac8. Taken together, our results show that MiR-181a/b is a putative mediator of diabetic CLI and contributes to changes in hematopoiesis, monocytosis, and macrophage polarization.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-ß1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600­1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Targeted delivery of protein arginine deiminase-4 inhibitors to limit arterial intimal NETosis and preserve endothelial integrity.

AIMS: Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. METHODS AND RESULTS: We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. CONCLUSIONS: NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Effect of yerba mate and green tea on paraoxonase and leptin levels in patients affected by overweight or obesity and dyslipidemia: a randomized clinical trial.

BACKGROUND: This study aimed to evaluate the effect of the intake of yerba mate (YM) and green tea (GT) on serum levels of leptin and paraoxonase-1 (PON-1), compared to control. METHODS: Controlled, randomized clinical trial (RCT) that evaluated 142 men and women affected by overweight or obesity aged 35-60 years, untreated dyslipidemia and no history of coronary artery disease. Participants were randomized to ingest 1000 mL GT, YM or apple tea (AT, control group) daily, during eight weeks. Serum PON-1 and leptin levels were analyzed by ELISA immunoassay at the beginning (baseline) and after eight weeks of intervention. RESULTS: The intake of 1 l of YM/day resulted in significant increase in serum levels of PON-1 (9.7%; p = 0.005). The consumption of GT induced no significant difference in the levels of PON-1 (p = 0.154) and leptin (p = 0.783). Intergroup analysis showed a significant difference (p = 0.036) in the variation of PON-1 levels in the YM group when compared to GT and AT groups. In addition, the increase in PON-1 levels in the YM group was significantly associated with increased HDL-c (p = 0.004). CONCLUSIONS: The intake of yerba mate increased the antioxidant capacity by increasing serum levels of PON-1 and was positively associated with increased HDL-c, stressing the protective role of this beverage against atherosclerotic diseases. GT intake had no significant effect on serum levels of PON-1 and leptin. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov under protocol number NCT00933647.

Central hemodynamic parameters to predict cardiovascular outcomes and mortality among the elderly: protocol for a systematic review.

BACKGROUND: Central blood pressure is a factor that may predict cardiovascular events. However, its use in clinical practice is not well consolidated. Therefore, the aim of our study will be to summarize the use of central hemodynamic parameters to predict cardiovascular-related outcomes and all-cause mortality. DESIGN AND SETTING: Protocol for systematic review of longitudinal observational studies conducted in healthcare institutions, as presented in the studies included. METHODS: We will perform a systematic search in the electronic databases MEDLINE (via PubMed), EMBASE and LILACS (via Virtual Health Library (VHL)), using health descriptors terms for elderly people and for hemodynamic indices of central blood pressure. We will include articles that evaluated hemodynamic indices and at least one of the following outcomes: all-cause mortality, total cardiovascular death, total non-cardiovascular death, myocardial infarction, stroke, coronary artery restenosis after percutaneous coronary intervention, revascularization and aortic syndromes. Two independent reviewers will conduct analysis on the abstracts selected and on the full-text articles. Two reviewers will independently perform data extraction and evaluate the methodological quality of the articles selected, and a third reviewer will evaluate any divergences. The methodological quality of the studies will be assessed in accordance with the ROBINS-I tool (Risk Of Bias In Non-randomized Studies of Interventions). RESULTS AND CONCLUSIONS: Through this systematic review, we intend to summarize evidence that supports the use of central hemodynamic parameters for central blood pressure to diagnose and perform prognostics on arterial hypertension in elderly patients within clinical practice and predict future cardiovascular events in this population. REGISTRATION: Prospero - CRD42018085264.

Statin Treatments And Dosages In Children With Familial Hypercholesterolemia: Meta-Analysis / Tratamentos e Dosagens de Estatinas em Crianças com Hipercolesterolemia Familiar: Uma Meta-Análise

Abstract Background: Children with familial hypercholesterolemia may develop early endothelial damage leading to a high risk for the development of cardiovascular disease (CVD). Statins have been shown to be effective in lowering LDL cholesterol levels and cardiovascular events in adults. The effect of statin treatment in the pediatric population is not clearly demonstrated. Objective: To systematically review the literature to evaluate the effects of different statins and dosages in total cholesterol levels in children and adolescents with familial hypercholesterolemia. We also aimed to evaluate statin safety in this group. Methods: PubMed, EMBASE, Bireme, Web of Science, Cochrane Library, SciELO and LILACS databases, were searched for articles published from inception until February 2016. Two independent reviewers performed the quality assessment of the included studies. We performed a meta-analysis with random effects and inverse variance, and subgroup analyses were performed. Results: Ten trials involving a total of 1543 patients met the inclusion criteria. Our study showed reductions in cholesterol levels according to the intensity of statin doses (high, intermediate and low): (-104.61 mg/dl, -67.60 mg/dl, -56.96 mg/dl) and in the low-density lipoprotein cholesterol level: [-105.03 mg/dl (95% CI -115.76, -94.30), I2 19.2%], [-67.85 mg/dl (95% CI -83.36, -52.35), I2 99.8%], [-58.97 mg/dl (95% CI -67.83, -50.11), I2 93.8%. The duration of statin therapy in the studies ranged from 8 to 104 weeks, precluding conclusions about long-term effects. Conclusion: Statin treatment is efficient in lowering lipids in children with FH. There is need of large, long-term and randomized controlled trials to establish the long-term safety of statins.

Resumo Fundamentos: Crianças com hipercolesterolemia familiar podem desenvolver dano endotelial precoce, aumentando o risco de desenvolver doenças cardiovasculares. As estatinas tiveram sua eficácia em diminuir níveis de colesterol LDL e eventos cardiovasculares em adultos comprovada. O efeito das estatinas na população pediátrica não está claramente demonstrado. Objetivo: Revisar sistematicamente a literatura para avaliar os efeitos e a segurança de diferentes estatinas e suas dosagens nos níveis de colesterol total em crianças e adolescentes com hipercolesterolêmica familiar. Métodos: Artigos publicados desde o início até fevereiro de 2016 foram pesquisados nas bases PubMed, EMBASE, Bireme, Web of Science, Cochrane Library, SciELO e LILACS. Dois revisores independentes avaliaram a qualidade dos estudos incluídos. Realizamos meta-análise com efeitos aleatórios e variância inversa. Análises de subgrupos foram realizadas. Resultados: Dez ensaios envolvendo 1.543 pacientes preencheram os critérios de inclusão. Em nosso estudo, as análises demostraram reduções nos níveis de colesterol, de acordo com a intensidade das doses de estatina (alta, intermediária e baixa): (-104,61 mg/dl, -67,60 mg/dl, -56,96 mg/dl) e no nível de lipoproteínas de baixa densidade: [-105,03 mg/dl (IC95% -115.76, -94.30), I2 19.2%], [-67.85 mg/dl (IC95% -83.36, -52.35), I2 99.8%], [-58.97 mg/dl (IC95% -67.83, -50.11), I2 93,8%. A duração da terapia com estatina variou de 8 a 104 semanas, impedindo conclusões sobre os efeitos a longo prazo. Conclusão: O tratamento com estatinas é eficiente na redução de lipídios em crianças com hipercolesterolemia familiar. É necessário realizar ensaios controlados randomizados de longo prazo para estabelecer a segurança do uso de estatinas a longo prazo.

Statin Treatments And Dosages In Children With Familial Hypercholesterolemia: Meta-Analysis.

BACKGROUND: Children with familial hypercholesterolemia may develop early endothelial damage leading to a high risk for the development of cardiovascular disease (CVD). Statins have been shown to be effective in lowering LDL cholesterol levels and cardiovascular events in adults. The effect of statin treatment in the pediatric population is not clearly demonstrated. OBJECTIVE: To systematically review the literature to evaluate the effects of different statins and dosages in total cholesterol levels in children and adolescents with familial hypercholesterolemia. We also aimed to evaluate statin safety in this group. METHODS: PubMed, EMBASE, Bireme, Web of Science, Cochrane Library, SciELO and LILACS databases, were searched for articles published from inception until February 2016. Two independent reviewers performed the quality assessment of the included studies. We performed a meta-analysis with random effects and inverse variance, and subgroup analyses were performed. RESULTS: Ten trials involving a total of 1543 patients met the inclusion criteria. Our study showed reductions in cholesterol levels according to the intensity of statin doses (high, intermediate and low): (-104.61 mg/dl, -67.60 mg/dl, -56.96 mg/dl) and in the low-density lipoprotein cholesterol level: [-105.03 mg/dl (95% CI -115.76, -94.30), I2 19.2%], [-67.85 mg/dl (95% CI -83.36, -52.35), I2 99.8%], [-58.97 mg/dl (95% CI -67.83, -50.11), I2 93.8%. The duration of statin therapy in the studies ranged from 8 to 104 weeks, precluding conclusions about long-term effects. CONCLUSION: Statin treatment is efficient in lowering lipids in children with FH. There is need of large, long-term and randomized controlled trials to establish the long-term safety of statins.

Secondary Dyslipidemia In Obese Children - Is There Evidence For Pharmacological Treatment?

BACKGROUND: Long-term safety, effectiveness and criteria for treatment with statins in children are still unclear in clinical practice. There is very limited evidence for the use of medication to treat children with dyslipidemia secondary to obesity who do not respond well to lifestyle modification. OBJECTIVE: Systematic review of randomized clinical trials of statin use to treat children and adolescents with dyslipidemia secondary to obesity. METHODS: We performed a search in PubMed, EMBASE, Bireme, Web of Science, Cochrane Library, SciELO, and LILACS for data to evaluate the effect of statins on: improvement of surrogate markers of coronary artery disease in clinical outcomes of adulthood; increased serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apolipropotein B (APOB); and decreased serum levels of high-density lipoprotein cholesterol (HDL-C) from inception to February 2016. Participants were children and adolescents. RESULTS: Of the 16793 potentially relevant citations recovered from the electronic databases, no randomized clinical trials fulfilled the inclusion criteria for children with dyslipidemia secondary to obesity. CONCLUSIONS: We found no specific evidence to consider statins in the treatment of hypercholesterolemia secondary to obesity in children. The usual practice of extrapolating findings from studies in genetic dyslipidemia ignores the differences in long-term cardiovascular risks and the long-term drug treatment risks, when compared to recommendation of lifestyle changes. Randomized clinical trials are needed to understand drug treatment in dyslipidemia secondary to obesity.

Mesenchymal stem cells from sternum: the type of heart disease, ischemic or valvular, does not influence the cell culture establishment and growth kinetics.

BACKGROUND: In an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results. METHODS: To study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 107 cells/mL. RESULTS: Morphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively). CONCLUSIONS: Together, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

RATIONALE: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. OBJECTIVE: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. METHODS AND RESULTS: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). CONCLUSIONS: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.

Cell Therapy in Ischemic Heart Disease: Interventions That Modulate Cardiac Regeneration.

The incidence of severe ischemic heart disease caused by coronary obstruction has progressively increased. Alternative forms of treatment have been studied in an attempt to regenerate myocardial tissue, induce angiogenesis, and improve clinical conditions. In this context, cell therapy has emerged as a promising alternative using cells with regenerative potential, focusing on the release of paracrine and autocrine factors that contribute to cell survival, angiogenesis, and tissue remodeling. Evidence of the safety, feasibility, and potential effectiveness of cell therapy has emerged from several clinical trials using different lineages of adult stem cells. The clinical benefit, however, is not yet well established. In this review, we discuss the therapeutic potential of cell therapy in terms of regenerative and angiogenic capacity after myocardial ischemia. In addition, we addressed nonpharmacological interventions that may influence this therapeutic practice, such as diet and physical training. This review brings together current data on pharmacological and nonpharmacological approaches to improve cell homing and cardiac repair.