Mechanisms Of Atherosclerosis Research Articles

Preface

As Editor-in-Chief of GenoMed Connect, it is my great pleasure to introduce the inaugural issue of this journal, dedicated to bridging the fields of genomics and medicine. Our vision for GenoMed Connect is to serve as a platform for the latest research that informs precision medicine and promotes integrative healthcare approaches. This inaugural issue features a diverse collection of seven articles, showcasing the journal's commitment to interdisciplinary research and its impact on advancing medical science. Each study addresses a unique aspect of genomics and medical research: 1. Evaluation of IL-12 Gene Expression in Coronary Artery Disease examines the role of IL-12 in atherosclerosis, shedding light on inflammatory pathways that may serve as therapeutic targets in coronary artery disease. 2. A Case Report on Epidermolytic Hyperkeratosis Type 1 introduces a novel mutation in the KRT1 gene, expanding the understanding of genetic dermatological conditions and offering insights into the molecular underpinnings of epidermolytic hyperkeratosis. 3. Identification of Prognostic Biomarkers in Uveal Melanoma explores tumor immune microenvironments, identifying genetic markers that could improve the prediction and treatment strategies for this rare but aggressive form of eye cancer. 4. HLA Class I and II Gene Diversity in North India provides an extensive genetic analysis that is valuable for transplantation immunology and population genetics, enhancing the knowledge of HLA variability and its implications in disease association. 5. Investigation of TLR and CD36 Expression in Coronary Artery Disease delves into the immune response mechanisms in coronary artery disease, examining the role of toll-like receptors and their potential as markers for disease progression. 6. Ethical Approaches in Reporting Secondary Findings from Exome Sequencing reviews current guidelines and ethical considerations, addressing challenges in reporting incidental findings and highlighting pathways for more responsible genomic data sharing. 7. The Role of Microbes in Endocrine Health presents an intriguing review on microbial endocrinology, exploring the influence of the gut microbiome on hormone regulation and its implications for metabolic health. These articles embody the interdisciplinary nature of GenoMed Connect, contributing to a deeper understanding of genomics in disease diagnosis, treatment, and ethical considerations. I extend my gratitude to our dedicated editorial board, the reviewers, and the team at Scifiniti for their hard work in bringing this issue to life. I look forward to witnessing the journal’s growth as it continues to foster collaboration and drive innovation in genomic medicine. I hope that the research presented in this issue inspires readers and advances our collective efforts to improve healthcare through genomic insights.

Abstract 4137846: Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Our data implicates MDA5 activation to occur in SMC phenotypic modulation and accelerates formation of chondromyocytes in a distinct cellular lineage trajectory — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA in a cell type and context specific mechanism mediates disease progression, bridging our understanding of human genetics and disease causality.

A Comprehensive Genetic and Bioinformatic Analysis Provides Evidence for the Engagement of COVID-19 GWAS-Significant Loci in the Molecular Mechanisms of Coronary Artery Disease and Stroke

Cardiovascular diseases (CVDs) significantly exacerbate the severity and mortality of COVID-19. We aimed to investigate whether GWAS-significant SNPs correlate with CVDs in severe COVID-19 patients. DNA samples from 199 patients with severe COVID-19 hospitalized in intensive care units were genotyped using probe-based PCR for 10 GWAS SNPs previously implicated in severe COVID-19 outcomes. SNPs rs17713054 SLC6A20-LZTFL1 (risk allele A, OR = 2.14, 95% CI 1.06–4.36, p = 0.03), rs12610495 DPP9 (risk allele G, OR = 1.69, 95% CI 1.02–2.81, p = 0.04), and rs7949972 ELF5 (risk allele T, OR = 2.57, 95% CI 1.43–4.61, p = 0.0009) were associated with increased risk of coronary artery disease (CAD). SNPs rs7949972 ELF5 (OR = 2.67, 95% CI 1.38–5.19, p = 0.003) and rs61882275 ELF5 (risk allele A, OR = 1.98, 95% CI 1.14–3.45, p = 0.01) were linked to a higher risk of cerebral stroke (CS). No associations were observed with AH. Bioinformatics analysis revealed the involvement of GWAS-significant loci in atherosclerosis, inflammation, oxidative stress, angiogenesis, and apoptosis, which provides evidence of their role in the molecular mechanisms of CVDs. This study provides novel insights into the associations between GWAS-identified SNPs and the risk of CAD and CS.

Genetic association with autoimmune diseases identifies molecular mechanisms of coronary artery disease

Autoimmune patients have a significantly increased risk of developing coronary artery disease (CAD) compared to the general population. However, autoimmune patients often lack traditional risk factors for CAD and there is increasing recognition of inflammation in CAD development. In this study, we leveraged genome-wide association study (GWAS) data to understand whether there is a genetic relationship between CAD and autoimmunity. Statistical genetic comparison methods were used to identify correlated and causal SNPs between various autoimmune diseases and CAD. Pleiotropic SNPs were identified by cross-phenotype association analysis (CPASSOC) and overlap between GWAS. Causal SNPs were identified using Mendelian Randomization (MR) and Colocalization (COLOC). Using SNP-to-gene mapping, we additionally identified pleiotropic and causal genes and pathways associated between autoimmunity and CAD, which were contextualized by documentation of enrichment in individual cell types identified from coronary atherosclerotic plaques by single-cell RNA sequencing. These results provide insight into potential inflammatory therapeutic targets for CAD.

Abstract 3074: RNA Editing Enzyme ADAR1 In Vascular Smooth Muscle Controls Vascular Integrity And Progression Of Atherosclerosis

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism for coronary artery disease (CAD) risk. Here, we show that human atherosclerotic smooth muscle cells (SMCs) express immunogenic RNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC) and loss of RNA editing causes activation of MDA5, characterized by upregulation of ISG genes (i.e. ISG15 ) and others with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro . In a high fat diet atherosclerosis model, scRNAseq analysis revealed SMC to have increasing ISG expression with phenotypic modulation. To evaluate SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observed 50% mortality with a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. scRNAseq of aortic tissue showed that SMC- Adar1 KD causes ISG activation and coordinates a complex transcriptional response throughout the vessel wall with robust macrophage cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Through study of SMC- Adar1 haploinsufficiency in the atherosclerosis mouse model, we further reveal that MDA5 activation occurs in SMC phenotypic modulation and accelerates formation of chondromyocytes — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA mediates disease progression, promoting our understanding of disease causality and CAD genetic risk.

Elevated white blood cell count and long-term clinical outcomes of patients with vasospastic angina.

Inflammation is known as one of key pathophysiologic mechanisms of coronary artery disease. We aimed to investigate the relationship between white blood cell (WBC) count and long-term clinical outcomes of patients with vasospastic angina (VA). A total of 823 patients who were diagnosed as VA without significant coronary lesion by coronary angiography with ergonovine provocation test were enrolled for analysis. Patients were divided according to WBC count tertile at the time of diagnosis: group I, tertile 1 and 2 (n = 546, <7490/ml); group II, tertile 3 (n = 277, ≥7490/ml). Primary outcome was defined as major adverse cardiovascular events (MACE), a composite outcome of all-cause death, cardiac death, myocardial infarction (MI), readmission due to cardiac symptoms, and revascularization. Median follow-up duration was 4.3 years. No significant difference of primary outcome was observed between group I and group II (14.7% vs. 20.2%, hazard ratio (HR) 1.29, confidence interval (CI) 0.90-1.83, P = 0.162), while incidence of cardiac death and MI was significantly higher in group II (1.5% vs. 4.3%, HR 2.86, CI 1.14-7.17), P = 0.025). In multivariate Cox regression model, elevated WBC count at the time of diagnosis of VA was an independent predictor of MI (HR 3.43, CI 1.02-11.59, P = 0.047). Elevated WBC count at the time of diagnosis was associated with a significantly increased risk of cardiac death and MI during long-term follow-up in VA patients.

Identification and validation of diagnostic biomarkers of coronary artery disease progression in type 1 diabetes via integrated computational and bioinformatics strategies

ObjectiveOur study aimed to identify early peripheral blood diagnostic biomarkers and elucidate the immune mechanisms of coronary artery disease (CAD) progression in patients with type 1 diabetes mellitus (T1DM). MethodsThree transcriptome datasets were retrieved from the Gene Expression Omnibus (GEO) database. Gene modules associated with T1DM were selected with weighted gene co-expression network analysis. Differentially expressed genes (DEGs) between CAD and acute myocardial infarction (AMI) peripheral blood tissues were identified using limma. Candidate biomarkers were selected with functional enrichment analysis, node gene selection from a constructed protein-protein interaction (PPI) network, and 3 machine learning algorithms. Candidate expression was compared, and the receiver operating characteristic curve (ROC) and nomogram were constructed. Immune cell infiltration was assessed with the CIBERSORT algorithm. ResultsA total of 1283 genes comprising 2 modules were detected as the most associated with T1DM. In addition, 451 DEGs related to CAD progression were identified. Among them, 182 were common to both diseases and mainly enriched in immune and inflammatory response regulation. The PPI network yielded 30 top node genes, and 6 were selected using the 3 machine learning algorithms. Upon validation, 4 genes (TLR2, CLEC4D, IL1R2, and NLRC4) were recognized as diagnostic biomarkers with the area under the curve (AUC) > 0.7. All 4 genes were positively correlated with neutrophils in patients with AMI. ConclusionWe identified 4 peripheral blood biomarkers and provided a nomogram for early diagnosing CAD progression to AMI in patients with T1DM. The biomarkers were positively associated with neutrophils, indicating potential therapeutic targets.

Abstract 9935: Culprit Plaque Morphology in Young versus Elder Acute Coronary Syndrome Patients: An Optical Coherence Tomography Study

Objective: Culprit plaque morphology in young acute coronary syndrome (ACS) patients differs from that of elder patients. The underlying mechanisms responsible for ACS in younger compared to elder patients are yet to be explored with optical coherence tomography. Methods: This was a prospective, single-center, investigational study. Patients were divided into groups according to age, ≤35 (N=43) and &gt;35 years (N=50) and further subdivided according to the underlying mechanism i.e. plaque rupture (PR) and plaque erosion (PE). Results: A total of 93 patients were included in this analysis. Thin-cap fibroatheroma (TCFA) was significantly higher among elder than younger patients for both PR (80.0% vs. 31.8%, p=0.002) and PE (66.7% vs. 6.3%, p&lt;0.001) groups. Microchannels were also significantly more prevalent among elder than younger patients for both PR (65.0% vs. 18.2%, p=0.004) and PE groups (55.6% vs.12.5%, p=0.013). Macrophages were significantly higher in elder than younger patients for both PR (25.0% vs. 0%, p=0.018) and PE (44.4% vs. 0%, p=0.003) groups. In contrast, fibrous cap thickness was greater in younger than elder patients for both PR (105.71±48.02 μm vs. 58.00±15.76 μm, p&lt;0.001) and PE (126.67±48.22 μm vs. 54.38±24.21 μm, p&lt;0.001) groups. Intimal thickness was greater in elder than younger patients for both PR (728.00±313.92 mm 2 vs. 342.27±142.02 mm 2 , p&lt;0.001) and PE (672.78±334.57 mm 2 vs. 295.00±99.60 mm 2 , p&lt;0.001) groups. Plaque type, thrombus formation, and minimal luminal diameter did not differ significantly according to age. Conclusion: We conclude that the high resolution of OCT is able to define the detailed microstructure of the culprit lesion and allows a greater understanding of the mechanisms of coronary artery disease. The frequency of TCFA, microchannels, macrophages, and intimal thickness was significantly higher in elder ACS patients compared to younger patients. However, fibrous cap thickness was significantly greater in younger ACS patients compared to elder patients.

Abstract 11177: Uncontrolled Systolic Hypertension Exaggerates the Effect of Water Hammer Shock from a Retrograde Direction While Diastolic Hypertension Aggravates the Injury from an Antegrade Direction: New Mechanism of Coronary Artery Disease by Angiographic and Machine Learning Investigation

Introduction: Hypertension (HTN) is a strong risk factor of coronary artery disease. Hypothesis: Based on the damages induced by flow abnormalities in pipes, our research is built on the hypothesis that flow disturbances start the atherosclerotic process. Our strategy is to use today’s practice of fluid mechanics to unlock the mechanism of HTN on coronary plaques. Methods: All patients with uncontrolled HTN and unstable angina underwent coronary angiography and were selected if they had 1 or 2 moderate lesions. At first. contrast was injected until the index artery was completely opacified. As the injection stopped, the blood moved in and displaced the contrast. The movements and interactions of the blood flow in white color above a black background could be clearly analyzed. At the same time, Artificial Intelligence (Machine Learning (ML algorithms) program had 2 models built on Python. Model 1 was based on U-net and Densenet-121 for vessel segmentation. Model 2 was used for classification and movement of flow. Model 2 was trained based on the convolutional neural network. The main measurements were the types, durations and directions of flows. Results: 20 patients were enrolled. 80% of lesions happened at the mid right coronary artery, near the bifurcation of the left anterior descending artery and diagonal, and of the circumflex artery and obtuse marginal. At the locations of lesions, 100% patients had turbulence caused by collision of the antegrade against the retrograde flow (water hammer shock). If there was excessive diastolic HTN &gt;100mmHg, the duration of retrograde flow was short (&lt;0.18 sec) and 75% turbulence happened DISTAL to the collision/bifurcation site. It happened due to higher gradient from proximal to distal. In severe systolic HTN, the duration of retrograde flow was longer (&gt; 0.30 sec) and 80% turbulence happened PROXIMAL to the collision/bifurcation site due to higher gradient from distal to proximal. Conclusions: Uncontrolled diastolic HTN exaggerated the strength of antegrade flow and turbulence DISTAL to the collision/bifurcation site while uncontrolled systolic HTN intensified the retrograde flow and turbulence PROXIMAL to the collision/bifurcation site. Both inflicted damage on the intima and started the atheroslerotic process.

Platelet-Derived PCSK9 Is Associated with LDL Metabolism and Modulates Atherothrombotic Mechanisms in Coronary Artery Disease.

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9’s role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.

Cytokine status in middle-aged men with acute coronary syndrome after coronary artery stenting

Background. Inflammatory cytokines and growth factors are involved in various mechanisms of coronary artery disease. Clinical studies have shown the correlation between the increase in the level of proinflammatory cytokines and the severity of coronary artery disease, while the data on the role of proinflammatory interleukin IL-8 and anti-inflammatory interleukin IL-4 are contradictory. The aim of the study is to assess the levels of proinflammatory cytokines (IL-8, TNF-) and anti-inflammatory interleukin (IL-4) in patients with various forms of coronary artery disease who underwent coronary artery stenting. Materials and methods. By the method of enzyme-linked immunosorbent assay, the levels of cytokines were determined in 30 patients with acute coronary syndrome who underwent primary stenting of the coronary arteries and in 24 patients with chronic coronary syndrome who had previously had myocardial infarction with stenting of an infarction-associated artery, who were admitted to the clinic for staged stenting of the coronary arteries. Results. In patients with chronic coronary syndrome the levels of IL-4 a do not exceed the reference values, in patients with acute coronary syndrome the levels of IL-4 there was an increase 3,70 0,24 and 240,85 49,25 pg/ml, р 0,001. In patients with chronic coronary syndrome the levels of IL-8 a do not exceed the reference values, in patients with acute coronary syndrome the levels of IL-8 there was an increase 7,34 1,29 and 110,33 27,67 pg/ml, р 0,001. Conclusion. Most likely the increase in the level of IL-4 has a compensatory character and, along with a slight increase in TNF-, can be considered as a positive factor stabilizing the course of the disease. There may be some relationship between of the increase in the level of interleukins in patients with acute coronary syndrome on the degree of stenosis of the coronary arteries (9095%) and impaired myocardial contractility was established.

Evolution of antithrombotic therapy in patients undergoing percutaneous coronary intervention: a 40-year journey.

Since its introduction in 1977, percutaneous coronary intervention has become one of the most commonly performed therapeutic procedures worldwide. Such widespread diffusion, however, would have not been possible without a concomitant evolution of the pharmacotherapies associated with this intervention. Antithrombotic agents are fundamental throughout the management of patients undergoing coronary stent implantation, starting from the procedure itself to the long-term prevention of cardiovascular events. The last 40 years of interventional cardiology have seen remarkable improvements in both drug therapies and device technologies, which largely reflected a progressive understanding of the pathophysiological mechanisms of coronary artery disease, as well as procedure- and device-related adverse events. The purpose of this article is to provide an overview of the important milestones in antithrombotic pharmacology that have shaped clinical practice of today while also providing insights into knowledge gaps and future directions.

Abstract 17182: Coronary Endothelial Dysfunction in Humans is Associated With Elevated Expression of Clonal Hematopoiesis of Indeterminate Potential

Introduction: Recent evidence has demonstrated that Clonal Hematopoiesis of Indeterminate Potential (CHIP) increases the risk of cardiovascular events. However, the exact role played by CHIP in early stages of coronary atherosclerosis, characterized by coronary endothelial dysfunction remains to be elucidated. Hypothesis: The current study was designed to test the hypothesis that the presence of CHIP in peripheral blood cells is associated with coronary endothelial dysfunction and increased levels of inflammatory markers. Methods: Next generation sequencing was used to detect mutations among patients who had coronary endothelial dysfunction and control group (normal coronary endothelial function). Endothelial dysfunction was defined by ≥ 20% decrease in coronary artery diameter or ≤50% increase in blood flow in response to acetylcholine injection compared to baseline. Plasma cytokine levels of Interleukin (IL)-6 and IL-8 were also assessed. Results: Clonal hematopoiesis relevant gene mutations were found in 1 out of 64 patients with normal endothelial function (1.5%) and 11 out of 119 cases in the endothelial dysfunction group (9.2%) (P = 0.04) with ASXL1 (Additional sex combs-like 1) being the most frequent gene (4.2%) mutated. Cytokine analysis demonstrated that mutations in ASXL1, DNMT3A (DNA methyltransferase 3A) and TET2 (Ten-eleven-translocation-2) in the endothelial dysfunction group were associated with increased levels of IL-6 and IL-8 (P = 0.001, P = 0.003; respectively). Conclusions: The current study supports a potential role for CHIP in mechanisms of coronary artery disease starting at early stage of atherosclerosis. Furthermore, enhanced expression levels of IL-6 and IL-8 seem to be related to mutations in DNMT3A, ASXL1 and TET2 genes, more than other gene mutations relevant CHIP.

Abstract 16284: Patient Derived Endothelial Progenitor Cells as a Model to Unravel New Mechanisms of Coronary Artery Disease

Introduction: Endothelial colony forming cells (ECFCs) are progenitor cells that can differentiate into mature endothelial cells. Ability to culture these cells from peripheral blood mononuclear cells (PBMCs) make them a suitable tool for investigation into individual susceptibility to coronary artery disease (CAD). We hypothesise that patient-derived ECFCs provide a model to study endothelial cell signalling in patients with CAD. Here we aim to identify biasing factors in spontaneous growth of ECFCs, and confirm whether they retain an “imprint” of patient disease burden. Methods: ECFCs were purified from patient samples in the BioHEART-CT biobank. Mitochondrial ROS assays (MitoSOX) were performed and protein lysate was collected to determine expression of redox signalling proteins by immunoblotting. CT coronary angiograms were analysed using Gensini scores to determine the burden of disease. Results: Spontaneous growth of ECFCs occurred in 178 of the 828 patients (21.50%). Spontaneous growth was lower in those with obesity (BMI ≥ 30: 15.7%, n = 30/191 vs BMI &lt; 30: 23.2%, n = 148/637, p = 0.03) and higher in hypertensive patients (25.4% n = 81/319 vs normotensive 19.1% n = 97/509, p = 0.03), although growth was not affected by presence of CAD (21.0% n = 59/281 vs CAD- 21.8% n = 119/547, p = 0.8). ECFCs expressed endothelial markers such as CD31 and eNOS, validating their differentiation into endothelial cells. However, ECFCs of patients with CAD expressed higher NADPH oxidase-2 protein level (1.9±0.4 fold-change vs CAD-, n = 13, p = 0.02) and were associated with higher mitochondrial superoxide production (Pearson correlation= 0.66, n = 18, p = 0.003). Conclusion: Spontaneous growth is affected by obesity and hypertension however the impact of this is reasonably small. The biochemical characteristics identified in these cells correlate with the patient’s disease state indicating that they are a helpful tool to further investigate individual susceptibility to CAD.

NFAT activating protein with ITAM motif 1 (NFAM1) is upregulated on circulating monocytes in coronary artery disease and potentially correlated with monocyte chemotaxis

Background and aimsCirculating monocytes have been proven to be critical mediators in the propagation and progression of atherosclerosis and myocardial infarction. The present study was designed to characterise a new transmembrane protein—NFAT activating protein with ITAM motif 1 (NFAM1)—on monocytes and uncover the potential effects and underlying mechanisms in coronary artery disease. MethodsMonocytes from a population of four controls, five stable coronary artery disease patients and five acute coronary syndrome patients were isolated for RNA sequencing. A potential monocyte biomarker molecule was discovered and then validated with a group of 79 controls, 70 stable coronary artery disease patients and 183 acute coronary syndrome patients. A stable cell line was generated as an in vitro model to determine chemotaxis migration and chemokine receptor expression. ResultsNFAM1 was identified through RNA sequencing analysis. The validation results confirmed that NFAM1 expression on monocytes was significantly increased by coronary artery disease status. A higher expression level of NFAM1 on classical and intermediate monocytes was observed compared with that on nonclassical monocytes. As shown in the in vitro cell model, knockdown of NFAM1 significantly attenuated chemotactic migration of monocytes by downregulating chemokine receptor expression and the p38 MAPK signalling pathway. Multivariable regression analysis of a group of 16 individuals suggested that NFAM1 was positively correlated with CCR2 expression. ConclusionsThe present study reported for the first time that distinctive alterations of NFAM1 expression on monocytes may correlate with atherosclerosis pathobiology and serve as a potential monocyte biomarker and therapeutic target for coronary artery disease.

Molecular mechanisms of coronary disease revealed using quantitative trait loci for TCF21 binding, chromatin accessibility, and chromosomal looping

BackgroundTo investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we create a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMCs).ResultsWe use a pooling approach with HCASMC lines to map regulatory variants that mediate binding of the CAD-associated transcription factor TCF21 with ChIPseq studies (bQTLs), variants that regulate chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with Hi-C methods (clQTLs). We examine the overlap of these QTLs and their relationship to smooth muscle-specific genes and transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlate to lead SNPs where they show allelic effects. By utilizing genome editing, we verify that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease-associated TGFB1-SMAD3 pathway to the CAD-associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping.ConclusionsTogether, these studies represent the most thorough mapping of multiple QTL types in a highly disease-relevant primary cultured cell type and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

Pericardial adipose tissue is an independent risk factor of coronary artery disease and is associated with risk factors of coronary artery disease

ObjectivePericardial adipose tissue volume (PATV) is related to the mechanism of coronary artery disease (CAD), but its association with CAD risk factors is not clear. This study aimed to investigate the relationships between PATV and its associated factors.MethodsA total of 682 inpatients were consecutively enrolled in this study. Patients were divided into the high PATV group (PATV ≥174.5 cm3; n = 506) and low PATV group (PATV < 174.5 cm3; n = 176). Multiple linear regression analysis was conducted to evaluate the related factors of PATV. Multivariable logistic regression was used to analyze the risk factors of CAD.ResultsLeft ventricular fat volume, right ventricular fat volume, body mass index, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were significant and independent risk factors of enlargement of PATV. Increased PATV was identified as an independent risk factor of CAD, and increased pulse pressure was also independently and positively correlated with CAD.ConclusionsPATV is significantly correlated with the classic risk factors of CAD. Pulse pressure is also correlated with PATV. PATV is an independent risk factor of CAD, and pericardial adipose tissue may alternatively be used in non-invasive diagnostic examination of CAD.

A novel lncRNA-miRNA-mRNA triple network identifies lncRNA TWF1 as an important regulator of miRNA and gene expression in coronary artery disease

BackgroundLong non-coding RNAs (lncRNAs) are involved in numerous physiological functions. Yet, their mechanisms in coronary artery disease (CAD) are not well understood.MethodsThe expression profile of genes associated to CAD was reannotated into the lncRNA-mRNA biphasic profile. The target microRNA data were used to design a global CAD triple network. Thereafter, we conducted a functional enrichment analysis and clustering using the triple network from the level of topology analyses. The expression of four non-coding RNAs (ncRNAs) was measured by qRT-PCR and the risk of CAD was calculated by nomogram. The prognostic value of three ncRNAs was evaluated using receiver operating characteristic (ROC) curve.ResultsA CAD lncRNA-miRNA-mRNA network was constructed which included 15 mRNAs, 3 miRNAs, 19 edges and one lncRNA. Nomogram showed that four ncRNAs were the risk of CAD. After RT-PCR validation in four ncRNAs between CAD and non-CAD samples, only three ncRNAs had significant meaning for further analysis. ROC curve showed that TWF1 presented an area under curve (AUC) of 0.862, the AUC of hsa -miR-142-3p was 0.856 and hsa -miR126-5p was 0.822. After the pairwise comparison, we found that TWF1 had significant statistical significance (PTWF1–142 < 0.05 and PTWF1–126 < 0.01). The results of functional enrichment analysis of interacting gene and microRNA showed that the shared lncRNA TWF1 may be a new factor for CAD.ConclusionsThis investigation on the regulatory networks of lncRNA-miRNA-mRNA in CAD suggests that a novel lncRNA, lncRNA TWF1 is a risk factor for CAD, and expands our understanding into the mechanisms involved in the pathogenesis of CAD.

RFX1 downregulation contributes to TLR4 overexpression in CD14+ monocytes via epigenetic mechanisms in coronary artery disease

BackgroundToll-like receptor 4 (TLR4) expression is increased in activated monocytes, which play a critical role in the pathogenesis of coronary artery disease (CAD). However, the mechanism remains unclear. Regulatory factor X1 (RFX1) is a critical transcription factor regulating epigenetic modifications. In this study, we investigated whether RFX1 and epigenetic modifications mediated by RFX1 contributed to the overexpression of TLR4 in activated monocytes.ResultsCompared with those of the controls, the mRNA and protein expression of RFX1 were downregulated and the mRNA expression of TLR4 was upregulated in CD14+ monocytes obtained from CAD patients and CD14+ monocytes obtained from healthy controls treated with low-density lipoprotein (LDL). The mRNA expression of RFX1 was negatively correlated with the mRNA expression of TLR4 in CD14+ monocytes. RFX1 knockdown led to the overexpression of TLR4 and the activation of CD14+ monocytes. In contrast, the overexpression of RFX1 inhibited TLR4 expression and the activation of CD14+ monocytes stimulated with LDL. Moreover, TLR4 was identified as a target gene of RFX1. The results indicated that RFX1 downregulation contributed to the decreased DNA methylation and histone H3 lysine 9 trimethylation and the increased H3 and H4 acetylation in the TLR4 promoter via the lack of recruitments of DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone-lysine N-methyltransferase SUV39H1 (SUV39H1), which were observed in CD14+ monocytes of CAD patients.ConclusionsOur results show that RFX1 expression deficiency leads to the overexpression of TLR4 and the activation of CD14+ monocytes in CAD patients by regulating DNA methylation and histone modifications, which highlights the vital role of RFX1 in the pathogenesis of CAD.

DYSLIPIDEMIA AS RISK FACTOR OF ATHEROSCLEROSIS IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Introduction: Atherosclerosis is one of the most common co-morbidities observed in chronic obstructive pulmonary disease. A better understanding of mechanisms of atherosclerosis in patients with chronic obstructive pulmonary disease is needed to improve clinical outcomes. The aim: to evaluate the plasma levels of lipid parameters, atherogenic indices, systemic inflammatory markers and to assess their relationship with the severity of chronic obstructive pulmonary disease. Materials and methods: A total of 72 subjects diagnosed with chronic obstructive pulmonary disease and 41 healthy controls, the same gender and age categories, with ≥ 10 pack years smoking history, were followed-up of about 5.8 years. Blood tests with determination of lipid profiles, atherogenic indices and systemic inflammatory markers were conducted in remaining patients who fulfilled inclusion criteria of the study. Results: Triglyceride, atherogenic index of plasma, cardiogenic risk ratio and atherogenic coefficient values were significantly higher, but high-density lipoprotein cholesterol- significantly lower in patients with chronic obstructive pulmonary disease than in controls. Lipid profiles were similar in lower-risk (stage A and B) and higher-risk (stage C and D) patients with chronic obstructive pulmonary disease. The analysis showed that atherogenic indices and serum high sensitive C-reactive protein were inversely correlated with forced expiratory volume in 1 sec, especially in higher-risk patients with chronic obstructive pulmonary disease (r = - 0.61 p < 0.05; r = - 0.57 p < 0.05; r = - 0.54 p < 0.05 and r = - 0.49 p < 0.05 respectively). Conclusions: Atherogenic indices and serum high sensitive C-reactive protein can be considered as useful biochemical markers to predict an early stage of atherosclerosis especially in higher-risk patients with chronic obstructive pulmonary disease.