Cardiovascular Disease Responses Research Articles

Unlocking the potential of microRNAs: machine learning identifies key biomarkers for myocardial infarction diagnosis

BackgroundMicroRNAs (miRNAs) play a crucial role in regulating adaptive and maladaptive responses in cardiovascular diseases, making them attractive targets for potential biomarkers. However, their potential as novel biomarkers for diagnosing cardiovascular diseases requires systematic evaluation.MethodsIn this study, we aimed to identify a key set of miRNA biomarkers using integrated bioinformatics and machine learning analysis. We combined and analyzed three gene expression datasets from the Gene Expression Omnibus (GEO) database, which contains peripheral blood mononuclear cell (PBMC) samples from individuals with myocardial infarction (MI), stable coronary artery disease (CAD), and healthy individuals. Additionally, we selected a set of miRNAs based on their area under the receiver operating characteristic curve (AUC-ROC) for separating the CAD and MI samples. We designed a two-layer architecture for sample classification, in which the first layer isolates healthy samples from unhealthy samples, and the second layer classifies stable CAD and MI samples. We trained different machine learning models using both biomarker sets and evaluated their performance on a test set.ResultsWe identified hsa-miR-21-3p, hsa-miR-186-5p, and hsa-miR-32-3p as the differentially expressed miRNAs, and a set including hsa-miR-186-5p, hsa-miR-21-3p, hsa-miR-197-5p, hsa-miR-29a-5p, and hsa-miR-296-5p as the optimum set of miRNAs selected by their AUC-ROC. Both biomarker sets could distinguish healthy from not-healthy samples with complete accuracy. The best performance for the classification of CAD and MI was achieved with an SVM model trained using the biomarker set selected by AUC-ROC, with an AUC-ROC of 0.96 and an accuracy of 0.94 on the test data.ConclusionsOur study demonstrated that miRNA signatures derived from PBMCs could serve as valuable novel biomarkers for cardiovascular diseases.

Abstract 327: Gclm-dependent Gsh Synthesis Regulates Efferocytosis During Atherosclerosis

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids, apoptotic cells, and fibrous elements within the arterial wall, can lead to plaque rupture, one of the most common causes of death in cardiovascular disease (CVD). Rupture prone plaques, characterized by large necrotic cores and thin fibrous caps, lead to acute luminal thrombosis resulting in myocardial infarctions (MI) and strokes. Apoptotic cell (AC) accumulation coupled with deficiency in their clearance, results in necrotic core formation. Macrophages are responsible for clearing ACs through efferocytosis; however, as atherosclerosis progresses efferocytosis is impaired. AC accumulation contributes to the chronic inflammatory response in CVD. Within this response, reactive oxygen species production increases disrupting cellular redox balance. Glutathione (GSH), the main antioxidant in the body, maintains redox balance; however, it fails to do so in atherosclerosis. Reports have shown that a single nucleotide polymorphism (SNP) in the promoter region of the modifier subunit of GCL (GCLM), a rate limiting enzyme responsible for de novo synthesis of GSH, limits its transcription under oxidative stress and is associated with increased risk of MI. Moreover, deletion of GCLM shows an increase in atherosclerotic plaque area and necrotic core, however it remains unknown how GCLM contributes to necrotic core formation and increased CVD incidence. Methods: Bone marrow derived macrophages from WT, Het, and KO GCLM mutant mice and peripheral blood mononuclear cells (PBMCs) from blood donors were isolated and used for efferocytosis and protein assays. PBMCs and atherosclerosis plaque sections were used for SNP genotyping and in situ efferocytosis assays. Results: Efferocytosis impairment correlated with decreased GCLM expression in mice. The presence of a SNP in GCLM significantly decreases efferocytosis in vitro as well as in situ in stage 5 human plaques. Conclusion: These results suggest that loss of GCLM in macrophages impairs primary and secondary efferocytosis in mice. This is corroborated with the inhibition of GSH synthesis. We also observed an impairment in efferocytosis in human macrophages with and without the presence of the risk allele in GCLM.

Serine/threonine kinase 3 promotes oxidative stress and mitochondrial damage in septic cardiomyopathy through inducing Kelch-like ECH-associated protein 1 phosphorylation and nuclear factor erythroid 2-related factor 2 degradation.

Serine/threonine kinases (STK3) is a core component of the Hippo pathway and modulates oxidative stress and inflammatory responses in cardiovascular diseases. However, its potential role in septic cardiomyopathy remains undefined. STK3-mediated phosphorylation of Kelch-like ECH-associated protein 1 (KEAP1) was shown to suppress antioxidant gene transcription controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages. To explore whether STK3 induces KEAP1-mediated suppression of Nrf2 in septic cardiomyopathy, wild-type and global STK3 knockout (STK3 -/- ) mice were treated with LPS. LPS treatment upregulated cardiac STK3 expression. STK3 deletion attenuated myocardial inflammation and cardiomyocyte death, and improved myocardial structure and function. In LPS-challenged HL-1 cardiomyocytes, shRNA-mediated STK3 knockdown normalized mitochondrial membrane potential and ATP production, attenuated apoptosis, and rescued antioxidant gene expression by preventing Nrf2 downregulation. Co-IP, docking analysis, western blotting, and immunofluorescence assays further showed that STK3 binds and phosphorylates KEAP1, promoting Nrf2 downregulation. Accordingly, transfection of phosphodefective KEAP1 mutant protein in cardiomyocyte restored Nrf2 expression and mitochondrial performance upon LPS, while expression of a phosphomimetic KEAP1 mutant abolished the mitochondria-protective and pro-survival effects of STK3 deletion. These findings suggest that STK3 upregulation contributes to septic cardiomyopathy by phosphorylating KEAP1 to promote Nrf2 degradation and suppression of the antioxidant response.

Potential role of ghrelin in the regulation of inflammation.

Several diseases are caused or progress due to inflammation. In the past few years, accumulating evidence suggests that ghrelin, a gastric hormone of 28-amino acid residue length, exerts protective effects against inflammation by modulating the related pathways. This review focuses on ghrelin's anti-inflammatory and potential therapeutic effects in neurological, cardiovascular, respiratory, hepatic, gastrointestinal, and kidney disorders. Ghrelin significantly alleviates excessive inflammation and reduces damage to different target organs mainly by reducing the secretion of inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), and inhibiting the nuclear factor kappa-B (NF-κB) and NLRP3 inflammasome signaling pathways. Ghrelin also regulates inflammation and apoptosis through the p38 MAPK/c-Jun N-terminal kinase (JNK) signaling pathway; restores cerebral microvascular integrity, and attenuates vascular leakage. Ghrelin activates the phosphoInositide-3 kinase (PI3K)/protein kinase B (Akt) pathway and inhibits inflammatory responses in cardiovascular diseases and acute kidney injury. Some studies show that ghrelin exacerbates colonic and intestinal manifestations of colitis. Interestingly, some inflammatory states, such as non-alcoholic steatohepatitis, inflammatory bowel diseases, and chronic kidney disease, are often associated with high ghrelin levels. Thus, ghrelin may be a potential new therapeutic target for inflammation-related diseases.

Neutrophils and Neutrophil Extracellular Traps in Cardiovascular Disease: An Overview and Potential Therapeutic Approaches.

Recent advances in pharmacotherapy have markedly improved the prognosis of cardiovascular disease (CVD) but have not completely conquered it. Therapies targeting the NOD-like receptor family pyrin domain containing 3 inflammasome and its downstream cytokines have proven effective in the secondary prevention of cardiovascular events, suggesting that inflammation is a target for treating residual risk in CVD. Neutrophil-induced inflammation has long been recognized as important in the pathogenesis of CVD. Circadian rhythm-related and disease-specific microenvironment changes give rise to neutrophil diversity. Neutrophils are primed by various stimuli, such as chemokines, cytokines, and damage-related molecular patterns, and the activated neutrophils contribute to the inflammatory response in CVD through degranulation, phagocytosis, reactive oxygen species generation, and the release of neutrophil extracellular traps (NETs). In particular, NETs promote immunothrombosis through the interaction with vascular endothelial cells and platelets and are implicated in the development of various types of CVD, such as acute coronary syndrome, deep vein thrombosis, and heart failure. NETs are promising candidates for anti-inflammatory therapy in CVD, and their efficacy has already been demonstrated in various animal models of the disease; however, they have yet to be clinically applied in humans. This narrative review discusses the diversity and complexity of neutrophils in the trajectory of CVD, the therapeutic potential of targeting NETs, and the related clinical issues.

The Intriguing Role of TLR Accessory Molecules in Cardiovascular Health and Disease.

Activation of Toll like receptors (TLR) plays an important role in cardiovascular disease development, progression and outcomes. Complex TLR mediated signaling affects vascular and cardiac function including tissue remodeling and repair. Being central components of both innate and adaptive arms of the immune system, TLRs interact as pattern recognition receptors with a series of exogenous ligands and endogenous molecules or so-called danger associated molecular patterns (DAMPs) that are released upon tissue injury and cellular stress. Besides immune cells, a number of structural cells within the cardiovascular system, including endothelial cells, smooth muscle cells, fibroblasts and cardiac myocytes express TLRs and are able to release or sense DAMPs. Local activation of TLR-mediated signaling cascade induces cardiovascular tissue repair but in a presence of constant stimuli can overshoot and cause chronic inflammation and tissue damage. TLR accessory molecules are essential in guiding and dampening these responses toward an adequate reaction. Furthermore, accessory molecules assure specific and exclusive TLR-mediated signal transduction for distinct cells and pathways involved in the pathogenesis of cardiovascular diseases. Although much has been learned about TLRs activation in cardiovascular remodeling, the exact role of TLR accessory molecules is not entirely understood. Deeper understanding of the role of TLR accessory molecules in cardiovascular system may open therapeutic avenues aiming at manipulation of inflammatory response in cardiovascular disease. The present review outlines accessory molecules for membrane TLRs that are involved in cardiovascular disease progression. We first summarize the up-to-date knowledge on TLR signaling focusing on membrane TLRs and their ligands that play a key role in cardiovascular system. We then survey the current evidence of the contribution of TLRs accessory molecules in vascular and cardiac remodeling including myocardial infarction, heart failure, stroke, atherosclerosis, vein graft disease and arterio-venous fistula failure.

A Vicious Circle of Clonal Haematopoiesis of Indeterminate Potential and Cardiovascular Disease.

Clonal haematopoiesis of indeterminate potential (CHIP) represents a recently identified overlap between cancer and cardiovascular disease (CVD). CHIP develops as a result of certain acquired somatic mutations that predispose to leukaemia, but clinically even more prevalent, associate with increased risk for CVD and CVD-related death. Experimental studies suggest a causal role for CHIP aggravating inflammatory processes in CVD, and recent epidemiologic and genetic studies indicate that classical CVD risk factors may increase the risk of acquiring CHIP driver mutations, thus fuelling a vicious circle. The potential mechanism underlying the associative link between CHIP and CVD and mortality has been the focus of a few recent excellent experimental and observational studies which are summarized and discussed in this concise non-systematic review article. These data support a pathomechanistic view of a spiralling vicious circle in which CHIP aggravates the inflammatory immune response in CVD, and CVD-driven elevated haematopoietic activity promotes CHIP development.

Upregulation of MicroRNA-125b Leads to the Resistance to Inflammatory Injury in Endothelial Progenitor Cells.

Objectives MicroRNA-125b (miR-125b) has been recognized as one of the key regulators of the inflammatory responses in cardiovascular diseases recently. This study sought to dissect the role of miR-125b in modulating the function of endothelial progenitor cells (EPCs) in the inflammatory environment of ischemic hearts. Methods EPCs were cultured and transfected with miR-125b mimic and negative control mimic. Cell migration and adhesion assays were performed after tumor necrosis factor-α (TNF-α) treatment to determine EPC function. Cell apoptosis was analyzed by flow cytometry. The activation of the NF-κB pathway was measured by western blotting. EPC-mediated neovascularization in vivo was studied by using a myocardial infarction model. Results miR-125b-overexpressed EPCs displayed improved cell migration, adhesion abilities, and reduced cell apoptosis compared with those of the NC group after TNF-α treatment. miR-125b overexpression in EPCs ameliorated TNF-α-induced activation of the NF-κB pathway. Mice transplanted with miR-125b-overexpressed EPCs showed improved cardiac function recovery and capillary vessel density than the ones transplanted with NC EPCs. Conclusions miR-125b protects EPCs against TNF-α-induced inflammation and cell apoptosis by attenuating the activation of NF-κB pathway and consequently improves the cardiac function recovery and EPC-mediated neovascularization in the ischemic hearts.

βIV-Spectrin/STAT3 complex regulates fibroblast phenotype, fibrosis, and cardiac function.

Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CFs) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we tested the hypothesis that a βIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal nonactivated state) in CFs. We reported increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of βIV-spectrin deficiency. Loss of βIV-spectrin function promoted STAT3 nuclear accumulation and transcriptional activity, and it altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in βIV-spectrin-deficient fibroblasts by expressing a βIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We found that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global βIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of βIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the βIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease (CVD).

Sitagliptin inhibits vascular inflammation via the SIRT6-dependent signaling pathway

Sitagliptin has recently been shown to inhibit inflammatory response in cardiovascular disease. Sirtuin6 (SIRT6), a NAD+-dependent class III histone deacetylase, participates in the regulation of cellular inflammation. We hypothesized that sitagliptin could attenuate vascular inflammation via modulation of SIRT6 pathway. It was found that sitagliptin decreased the expression of monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-6 and IL-1β, but up-regulated SIRT6 expression, both in mice and in TNF-α-stimulated endothelial cells. Moreover, knockdown of SIRT6 reversed the inhibitory effect of sitagliptin on MCP-1, IL-6 and IL-1β expression. Further study revealed that sitagliptin also decreased the expression of MCP-1, IL-6 and IL-1β partly through suppression of reactive oxygen species (ROS). In vivo, hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 for 1 month. Hyperlipidemia-induced production of MCP-1, IL-6 and IL-1β was significantly suppressed in the sitagliptin-supplemented animals, but the effect of was abolished by SIRT6 knockout in endothelium. These results indicate that sitagliptin protects against vascular inflammation via the SIRT6/ROS-dependent signaling pathway.

Role of thrombin in the pathogenesis of atherosclerosis.

Atherosclerosis is an arterial disease associated with inflammation. Thrombin is a procoagulant and proinflammatory serine protease that contributes to the pathology of atherosclerosis by enhancing the expression of cell adhesion molecules, inducing the secretion of proinflammatory cytokines, activating inflammatory responses in atherosclerotic plaques, stimulating proliferation of aortic smooth muscle cells, and exacerbating vascular lesions at sites of injury. Hence, thrombin appears to be an important target for treatment of atherosclerosis and thrombin pharmacological inhibitors have significant therapeutic potency for suppressing inflammatory responses in cardiovascular diseases. This review summarizes the proinflammatory signaling functions of thrombin as well as the therapeutic potency of thrombin inhibitors in the pathogenesis of atherosclerosis and hence their potential therapeutic value in this condition.

Anti-High-Density Lipoprotein Antibodies and Antioxidant Dysfunction in Immune-Driven Diseases.

Impaired high-density lipoprotein (HDL) levels and antioxidant functionality of HDL, mainly attributed to a decreased paraoxonase-1 (PON1) functionality, have been described in autoimmune conditions. In this setting, a role for humoral response in cardiovascular disease is emerging. This study evaluates the role of immunoglobulin G (IgG) antibodies against HDL and disease-related autoantibodies on HDL dysfunction in immune-driven diseases. Serum IgG anti-HDL antibodies, PON1 activity, and total antioxidant capacity (TAC) were quantified in 381 patients with different immune-driven diseases [18 mixed connective tissue disease (MCTD), 35 primary Sjögren syndrome (pSS), 38 systemic sclerosis (SSc), 33 ANCA-associated vasculitis (AAV), 60 diabetes mellitus 1, 29 autoimmune B12 deficiency/pernicious anemia, 29 primary biliary cirrhosis, 46 IBD/Crohn, 54 IBD/UC, and 39 celiac disease (CD)] and 138 healthy controls. IgG anti-HDL antibodies were increased in MCTD, pSS, AAV, and inflammatory bowel disease (IBD) [Crohn and ulcerative colitis (UC)], even after correcting for total IgG levels, but not in organ-specific autoimmune diseases. Anti-HDL antibodies were negatively associated with PON1 activity in MCTD (r = -0.767, p < 0.001) and AAV (r = -0.478, p = 0.005), whereas both anti-HDL and anti-neutrophil cytoplasm antibod levels were related to an impaired PON1 activity and TAC in IBD/UC. In SSc, anti-centromere antibodies correlated PON1 activity. anti-Saccharomyces cerevisiae antibodies levels were negatively associated with PON1 activity (r = -0.257, p = 0.012) and PON1/TAC ratio (r = -0.261, p = 0.009) in IBD/Crohn. HDL dysfunction in CD was only related to anti-transglutaminase levels. IgG anti-HDL antibodies and HDL dysfunction are common hallmarks of systemic autoimmunity. Anti-HDL and disease-related autoantibodies account for the HDL antioxidant dysfunction in immune-driven conditions, mainly in systemic autoimmune disorders.

Abstract 15173: Empowering Human Cardiac Progenitor Cell-mediated Repair of Injured Myocardium by Overexpressing P2Y14 Nucleotide Receptor

Heart failure is a leading cause of death in the US due to the limited capability of adult mammalian heart to regenerate following injury. Autologous stem cell therapy holds promise for regeneration of injured myocardium after myocardial infarction. However, stem cells derived from diseased organs exhibit impaired proliferation and migration and increased susceptibility to cell death. Empowering stem cells from diverse origins, including cardiac progenitor cells (CPCs), with pro-survival genes has been attempted. Despite the well-established roles of purinergic signaling mediated by extracellular nucleotides in regulating diverse cellular responses in cardiovascular diseases, it has not been well-defined in CPCs. Our preliminary data show, for the first time, that the majority of P2 purinergic receptors are expressed in human CPCs (hCPCs) isolated from patients undergoing left ventricular assist device (LVAD) implantation surgery. The G protein-coupled UDP-sugar-sensing P2Y14 receptor (P2Y14R) has been shown to stimulate keratinocyte proliferation and migration, neutrophil and hematopoietic stem cell (HSC) chemotaxis in addition to increasing HSC resistance to stress-induced senescence. We aim to determine whether P2Y14R plays similar regenerative roles in cardiac tissue where the P2Y14R-mediated physiological responses haven’t been previously addressed. Our preliminary data show that the P2Y14R selective agonist UDP-Glucose enhances hCPC proliferation, migration and survival. Interestingly, hCPCs that exhibit relatively slower growth kinetics and enhanced senescence show a dramatic decrease in P2Y14R expression compared to fast-growing hCPCs consistent with our hypothesis that overexpressing P2Y14R participates in rejuvenating hCPCs and improving their growth capabilities. This hypothesis will be tested in vivo by determining whether P2Y14R overexpression in hCPCs improves their reparative potential for injured mouse myocardium. Mechanistically, we show for the first time that UDP-Glu induces downstream activation of YAP linking the extracellular nucleotides released during cardiac ischemia to extracellular matrix sensing and Hippo signaling that have been recently implicated in cardiac regeneration.

Nicotinic acid inhibits vascular inflammation via the SIRT1-dependent signaling pathway

Nicotinic acid (NA) has recently been shown to inhibit inflammatory response in cardiovascular disease. Sirtuin1 (SIRT1), a NAD+-dependent class III histone deacetylase, participates in the regulation of cellular inflammation. We hypothesized that dietary supplementation of NA could attenuate vascular inflammation via modulation of SIRT1 pathway. New Zealand White rabbits received chow or chow supplemented with 0.6% (wt/wt) NA for 2 weeks. Acute vascular inflammation was induced in the animals by placing a non-occlusive silastic collar around the left common carotid artery. At 24 h after collar implantation, the collar-induced production of C-reactive protein and monocyte chemotactic protein-1 was significantly suppressed in the NA-supplemented animals. Meanwhile, NA also decreased the expression of cluster of differentiation 40 (CD40) and CD40 ligand, but up-regulated SIRT1 expression, both in rabbits and in lipopolysaccharide-stimulated endothelial cells. Moreover, knockdown of SIRT1 reversed the inhibitory effect of NA on CD40 expression. Further study revealed that NA also decreased the expression of CD40 partly through mammalian target of rapamycin. These results indicate that NA protects against vascular inflammation via the SIRT1/CD40-dependent signaling pathway.

Abstract 159: Empowering Cardiac Progenitor Cell-mediated Repair of Injured Myocardium by Overexpressing P2Y2 Nucleotide Receptor

Heart failure is a leading cause of death in the US due to the limited capability of adult mammalian heart to regenerate following injury. Autologous stem cell therapy holds promise for regeneration of injured myocardium after myocardial infarction. However, stem cells derived from diseased organs exhibit impaired proliferative and migratory capabilities and increased susceptibility to cell death. Empowering stem cells from diverse origins, including cardiac progenitor cells (CPCs), with pro-survival genes has been attempted. Despite the well-established roles of purinergic signaling mediated by extracellular nucleotides in regulating diverse cellular responses in cardiovascular diseases, it has not been well-defined in CPCs. Our preliminary data show, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in mouse and human CPCs. Since previous findings have shown that the G protein-coupled P2Y 2 receptor (P2Y 2 R) induces cardioprotective responses in animal models as well as human cardiomyocytes and regulates a wide range of signaling pathways that are crucial to tissue repair in various experimental models and in stem cells from diverse origins, we aim to determine whether P2Y 2 R plays similar roles in CPCs. Our preliminary data show that the P2Y 2 R agonists ATP and UTP enhance human CPC (hCPC) proliferation, migration and survival. Interestingly, hCPCs that exhibit relatively slower growth kinetics and higher levels of senescence markers show a dramatic decrease in P2Y 2 R expression as compared to fast-growing hCPCs consistent with our hypothesis that overexpressing P2Y 2 R participates in rejuvenating hCPCs and improving their growth capabilities. This hypothesis will be tested in vivo by determining whether P2Y 2 R overexpression in hCPCs improves their reparative potential for injured mouse myocardium. We also introduce the novel hypothesis that P2Y 2 R-induced regenerative responses in hCPCs involve the activation of Hippo signaling that is known to be regulated by different GPCRs, which links the extracellular nucleotides released during cardiac ischemia to extracellular matrix sensing and Hippo signaling that have been recently implicated in cardiac regeneration.

Abstract 281: Local MicroRNA Modulation Using a Novel Anti-mir-21-eluting Stent Effectively Prevents In-stent Restenosis

The major limitation of vascular angioplasty remains the occurrence of in-stent restenosis (ISR). The central role of miRNAs in the pathophysiology of the proliferative response in cardiovascular disease offers attractive concepts for possible therapeutic interventions, with systemic miRNA modulation however potentially causing substantial off-target effects. Aim of the current study was to test the feasibility of local miRNA modulation using drug eluted stent (DES) technology. Utilizing human ISR tissue samples, as well as a humanized rodent model of balloon-injured human internal mammary arteries with subsequent stent implantation in RNU rats, we were able to identify several miRNAs dys-regulated, including the well-characterized miR-21. We inhibited miR-21 (with a FAM-tagged-LNA-anti-miR-21), using either systemic or local delivery via DES. Systemic suppression of miR-21 showed a dose-dependent reduction of luminal obliteration, while not impeding vascular re-endothelialization. However, miR-21 expression in liver, heart, lung, and kidney appeared significantly reduced, with increased levels of serum creatinine, pointing towards the expected off-target effects of systemic miRNA modulation. Local mir-21 suppression, using the anti-miR-21-DES, effectively reduced myointimal hyperplasia and ISR compared to bare metal stents, while not exerting detectable off-target effects on any other organ we investigated. Additional in vitro experiments in cultured human coronary artery smooth muscle and endothelial cells further confirmed the ability of anti_miR-21 to limit SMC proliferation and migration by repressing mRNA and protein levels of its established target PTEN, while not affecting the proliferative response in the ECs. This is the first experimental study to demonstrate the efficacy, feasibility and suitability of an anti-miRNA-eluting stent (anti-miR-21-DES) for the reduction of ISR through dominant inhibition of SMC proliferation.

Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells.

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), and β-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.

Pharmacogenetics of heart failure.

Novel medical approaches and personalized medicine seek to use genetic information to 'individualize' and improve diagnosis, prevention, and therapy. The personalized management of cardiovascular disease involves a large spectrum of potential applications, from diagnostics of monogenic disorders, to prevention and management strategies based on modifier genes, to pharmacogenetics, in which individual genetic information is used to optimize the pharmacological treatments. Evidence suggests that the common polymorphic variants of modifier genes could influence drug response in cardiovascular disease in a variety of areas, including heart failure, arrhythmias, dyslipidemia, and hypertension. In heart failure, common genetic variants of β-adrenergic receptors, α-adrenergic receptors, and endothelin receptors (among others) have been associated with variable response to heart failure therapies. The challenge remains to develop strategies to leverage this information in ways that personalize and optimize cardiovascular therapy based on a patient's genetic profile. Although advances in technologies will continue to transition personalized medicine from the research to the clinical setting, healthcare providers will need to reshape the clinical diagnostic paradigms. Ultimately, pharmacogenetics will give providers the options for improving patient management on the basis of pharmacogenetic data.

Cholesterol Crystals and Inflammation

Chronic vascular inflammation is regarded as a crucial aspect of cardiovascular disease. However, the elicitors of this inflammatory response in the vessel wall are currently not well understood. Excessive amounts of cholesterol, an abundant and fundamental lipid molecule in mammalian cells, can initiate the development and progression of atherosclerosis. Accumulation of cholesterol in early atherosclerotic lesions results in the formation of macrophage foam cells, and crystalline cholesterol is found as a characteristic of advanced atherosclerotic plaques. Cholesterol crystals can activate the NLRP3 inflammasome, a multimolecular signaling complex of the innate immune system, resulting in caspase-1 mediated activation and secretion of proinflammatory interleukin-1 family cytokines. Furthermore, crystalline cholesterol is believed to induce plaque rupture by physical disruption of the fibrous cap covering atherosclerotic lesions. Here we review the effect of cholesterol deposition and crystallization on inflammatory responses in cardiovascular diseases.

MicroRNA signatures in total peripheral blood as novel biomarkers for acute myocardial infarction

MicroRNAs (miRNAs) are important regulators of adaptive and maladaptive responses in cardiovascular diseases and hence are considered to be potential therapeutical targets. However, their role as novel biomarkers for the diagnosis of cardiovascular diseases still needs to be systematically evaluated. We assessed here for the first time whole-genome miRNA expression in peripheral total blood samples of patients with acute myocardial infarction (AMI). We identified 121 miRNAs, which are significantly dysregulated in AMI patients in comparison to healthy controls. Among these, miR-1291 and miR-663b show the highest sensitivity and specificity for the discrimination of cases from controls. Using a novel self-learning pattern recognition algorithm, we identified a unique signature of 20 miRNAs that predicts AMI with even higher power (specificity 96%, sensitivity 90%, and accuracy 93%). In addition, we show that miR-30c and miR-145 levels correlate with infarct sizes estimated by Troponin T release. The here presented study shows that single miRNAs and especially miRNA signatures derived from peripheral blood, could be valuable novel biomarkers for cardiovascular diseases.