Human Atherosclerosis Research Articles

Integrated Single-Cell Analysis Revealed Novel Subpopulations of Foamy Macrophages in Human Atherosclerotic Plaques

Foam cell formation is a hallmark of atherosclerosis, yet the cellular complexity within foam cells in human plaques remains unexplored. Here, we integrate published single-cell RNA-sequencing, spatial transcriptomic, and chromatin accessibility sequencing datasets of human atherosclerotic lesions across eight distinct studies. Through this large-scale integration of patient-derived information, we identified foamy macrophages enriched for genes characteristic of the foamy signature. We further re-clustered the foamy macrophages into five unique subsets with distinct potential functions: (i) pro-foamy macrophages, exhibiting relatively high inflammatory and adhesive properties; (ii) phagocytic foamy macrophages, specialized in efferocytosis; (iii) high-efflux foamy macrophages marked by high NR1H3 expression; (iv) mature foamy macrophages prone to programmed cell death; and (v) synthetic subset. Trajectory analysis elucidated a bifurcated differentiation cell fate from pro-foam macrophages toward either the programmed death (iv) or synthetic (v) phenotype. The existence of these foamy macrophage subsets was validated by immunostaining. Moreover, these foamy macrophage subsets exhibited strong potential ligand–receptor interactions. Finally, we conducted Mendelian randomization analyses to identify a possible causal relationship between key regulatory genes along the programmed death pathway in foamy macrophages and atherosclerotic diseases. This study provides a high-resolution map of foam cell diversity and a set of potential key regulatory genes in atherosclerotic plaques, offering novel insights into the multifaceted pathophysiology underlying human atherosclerosis.

Single cell variant to enhancer to gene map for coronary artery disease.

Although genome wide association studies (GWAS) in large populations have identified hundreds of variants associated with common diseases such as coronary artery disease (CAD), most disease-associated variants lie within non-coding regions of the genome, rendering it difficult to determine the downstream causal gene and cell type. Here, we performed paired single nucleus gene expression and chromatin accessibility profiling from 44 human coronary arteries. To link disease variants to molecular traits, we developed a meta-map of 88 samples and discovered 11,182 single-cell chromatin accessibility quantitative trait loci (caQTLs). Heritability enrichment analysis and disease variant mapping demonstrated that smooth muscle cells (SMCs) harbor the greatest genetic risk for CAD. To capture the continuum of SMC cell states in disease, we used dynamic single cell caQTL modeling for the first time in tissue to uncover QTLs whose effects are modified by cell state and expand our insight into genetic regulation of heterogenous cell populations. Notably, we identified a variant in the COL4A1 / COL4A2 CAD GWAS locus which becomes a caQTL as SMCs de-differentiate by changing a transcription factor binding site for EGR1/2. To unbiasedly prioritize functional candidate genes, we built a genome-wide single cell variant to enhancer to gene (scV2E2G) map for human CAD to link disease variants to causal genes in cell types. Using this approach, we found several hundred genes predicted to be linked to disease variants in different cell types. Next, we performed genome-wide Hi-C in 16 human coronary arteries to build tissue specific maps of chromatin conformation and link disease variants to integrated chromatin hubs and distal target genes. Using this approach, we show that rs4887091 within the ADAMTS7 CAD GWAS locus modulates function of a super chromatin interactome through a change in a CTCF binding site. Finally, we used CRISPR interference to validate a distal gene, AMOTL2 , liked to a CAD GWAS locus. Collectively we provide a disease-agnostic framework to translate human genetic findings to identify pathologic cell states and genes driving disease, producing a comprehensive scV2E2G map with genetic and tissue level convergence for future mechanistic and therapeutic studies.

Abstract 4120821: Electrochemical Impedance Spectroscopy Unmasks High-Risk Atherosclerotic Features in Human Coronary Artery Disease

Background: Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. Methods: In this single-center, prospective study, n=26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N=61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Results: Necrotic cores were identified in n=19 vessels (median area 1.53 mm 2 ) with median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm 2 vs <1.75 mm 2 (19.8±4.4 kΩ vs 7.2±1.0 kΩ, P =0.019), fibrous cap thickness ≤65 μm vs >65 μm (19.1±3.5 kΩ vs 6.5±0.9 kΩ, P =0.004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) vs <20 macrophages per HPF (19.8±4.1 kΩ vs 10.2±0.9 kΩ, P=0.002). Impedance identified necrotic core area ≥1.75 mm 2 , fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716–1.000) ( P =0.013), 0.852 (0.646—1.000) ( P =0.025), and 0.835 (0.577—1.000) ( P =0.028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573—0.962) ( P =0.035). Conclusions: EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.

Abstract 4132752: The immune checkpoint landscape in human atherosclerosis: influence of lipid lowering and type 2 diabetes

Immune checkpoint inhibitor (ICI) therapies substantially increase the risk of atherosclerotic cardiovascular events in cancer survivors. Here, we mapped the expression of immune checkpoints (ICs) within human atherosclerotic plaques, unraveling an intricate local network of immune cell-cell interactions in the context of relevant comorbidities like dyslipidemia and diabetes. Our findings identified a population of mature, regulatory CCR7 + FSCN1 + dendritic cells as pivotal orchestrators of IC-mediated signaling within human and murine plaques, a function akin to their role in tumor environments. In clinical cohorts, type 2 diabetes diminished CCR7 + FSCN1 + dendritic cells within atherosclerotic plaques and dampened interactions steered by PD-1 or CTLA4 consistently across immune cell subpopulations. This effect was also present and further decreased by lipid lowering in the circulation, where CD86–CTLA4 was among the most prominent IC pairs signaling between regulatory T cells and myeloid populations including CCR7 + FSCN1 + dendritic cells. These findings underline the vulnerability of atherosclerotic plaques to cancer immunotherapies and underscore the critical impact of cardiometabolic disease and lipid-lowering strategies on the atherosclerotic IC landscape, offering a framework toward the reshaping of clinical approaches.

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies.

Early coronary artery atherosclerosis in humans is associated with attenuated coronary retention of klotho

Abstract Background Vascular aging is a key feature of cardiovascular disease. α-klotho (klotho) is a protein inversely associated with aging and may contribute to vascular repair. Lower levels of systemic klotho have been identified in humans with features of early and advanced coronary atherosclerosis. It remains unknown if circulating klotho is present within human coronary circulation and whether cardiovascular klotho retention correlates with atherosclerosis. Purpose The current study aimed to assess the presence of klotho within coronary circulation compared to systemic circulation and hypothesized that net coronary klotho retention is attenuated in patients with early age-related features of coronary artery disease (CAD). Methods A total of 42 patients that underwent clinically indicated coronary angiogram for chest pain that had no obstructive CAD underwent intravascular ultrasound (IVUS) assessment were retrospectively examined. Virtual histology assessed coronary plaque characteristics. Blood samples simultaneously collected from the proximal aorta and coronary sinus were used for klotho detection using enzyme-linked immunosorbent assay. The net gradient of klotho across cardiovascular circulation was calculated by multiplying the arteriovenous klotho gradient by basal coronary blood flow. A net positive gradient indicated less cardiovascular klotho retention; a net negative gradient indicated greater cardiovascular klotho retention. Results Overall, participants were age 54±10 years old and 76% were female. There were overall higher levels of klotho within the coronary sinus (median 0.49 ng/mL [0.15; 1.69]) compared to systemic circulation (median 0.30 ng/mL [0.16; 1.14]). Participant groups were determined by those with positive (n = 20) or negative (n = 22) cardiovascular retention of klotho. Both groups were similar in age (56 ±2 vs. 51 ±2 years old, p = 0.080), gender (72% vs. 80% female, p = 0.580), and had similar occurrences of traditional cardiovascular comorbidities. Those with less cardiovascular klotho retention in the coronary vascular bed had significantly greater degree of non-obstructive coronary artery plaque (Gensini score 3.5 vs. 0, p < 0.001) and had higher percent plaque burden (46% [35; 53] vs. 34% [28; 46], p = 0.044) than those with greater cardiovascular klotho retention. Conclusions Patients with early coronary atherosclerosis may be characterized by attenuated retention of klotho within coronary circulation. Moreover, humans appear to have higher circulating klotho in coronary circulation than in systemic circulation. The current study may support a role for mechanism or potential therapeutic target for klotho in coronary atherosclerosis in humans.

Vitamin K2 and the Vitamin-K-Cycle-Enzyme VKORC1L1 improve endothelial regeneration by inhibiting endothelial-mesenchymal transition, inflammatory cell activation and ferroptosis

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while dietary vitamin K intake was proposed to reduce incidence of coronary artery disease, but the mechanisms remain unresolved. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of Vitamin K2 and the VKOR-enzymes in endothelial cell inflammation. Methods and Results In human coronary artery endothelial cells (HCAEC), Vitamin K2 improves endothelial regeneration by increasing proliferation and cell viability and by reducing both apoptosis and ferroptosis (relative viability: RSL3 0.11 ± 0.01 vs 0.32 ± 0.05 RSL3+K2-1µM vs. 0.44 ± 0.11 RSL3+K2-10µM, p<0.001). Moreover, Vitamin K2-supplementation inhibits EndMT by reducing expression of mesenchymal markers (Calponin, SM22; Fig.1). In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress and ER-Stress promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Likewise, siRNA-mediated VKORC1L1 knockdown induces an inflammatory gene signature with upregulation of NFkB-signalling and endoplasmic reticulum stress (NF-κB: 1.95-fold ± 0.13, GRP78: 1.32-fold ± 0.04 vs. control, p<0.01). Furthermore, VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01). In contrast, VKORC1-knockdown does not impair endothelial regeneration and function (Fig.2). Vitamin K2 reduces vascular inflammation and endoplasmic reticulum stress in the case of VKORC1-, but not VKORC1L1-knockdown. In addition, inducing EndMT resulted in a reduction of VKORC1L1, but not of VKORC1, while VKORC1L1-knockdown aggravates EndMT. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Prediction of cardiovascular outcomes by subtle changes in the peripheral immune cell landscape - insights from the LURIC single cell RNA-sequencing study (LuRNA)

Abstract Background and Aims Atherosclerosis and its clinical sequelae, myocardial infarction and stroke, represent the main causes of death worldwide. Although preclinical evidence has suggested the existence of an inflammatory response driving disease, the extend of cellular alterations in human atherosclerosis remains enigmatic. Here, we employ single cell RNA-sequencing (scRNA-seq) on peripheral blood mononucleated cells (PBMCs) in a well-defined cardiovascular risk cohort from the "Ludwigshafen Risk and Cardiovascular Health (LURIC) trial" to define changes in the immune cell landscape in atherosclerotic patients. Methods and Results Of 3317 patients enrolled in the LURIC trial between 1997 and 2000, we selected individuals with stable coronary-artery disease (CAD) (n=31) and healthy patients (no CAD) (n=16) by propensity score matching, adjusted for CRP, NTproBNP, Troponin T, LDL-C, and Cystatin-C, in a case-control design. scRNA-seq was performed by droplet sequencing on PBMCs stored in liquid nitrogen. Individual data sets were integrated and changes between healthy individuals and patients with CAD were evaluated on numeric and transcriptional level. Single cell transcriptomes were annotated using an established CITE-seq reference atlas. While bulk RNA-sequencing of PBMC preparations revealed only minor changes between both conditions, we identified several leukocyte populations on a single cell level with specific transcriptomes that were differentially regulated between both conditions. Interestingly, we found that several subsets of Natural Killer (NK) cells, T cells with an enrichment of proliferation-associated pathways, and a population resembling hematopoietic stem cells were more frequent in patients with CAD, while naïve phenotypes of B and T cells were overrepresented in the absence of CAD. Consistently, most T and B cell populations in patients with CAD were enriched in pathways associated with cell activation, immune receptor signalling, and differentiation. In addition, we detected restricted repertoires of T cell receptor sequences in several adaptive immune cell subtypes from patients with CAD, suggestive of increased cellular clonality and antigen-specificity. Finally, in a comparison of cellular frequencies with cardiovascular outcomes over more than 15 years, several immune cell subsets and cell-type specific transcriptional programs predicted cardiovascular death in this case-control scenario in multi-variate adjusted regression analysis. Notably, addition of scRNA-seq derived parameters was superior to traditional risk prediction with clinical characteristics and soluble biomarkers alone. Conclusion Employing scRNAseq, we demonstrate that atherosclerosis is associated with a profound change in the circulating immune cell landscape even in the absence of measurable differences in inflammatory biomarkers. These findings propose the usage of scRNAseq for the discovery of outcome-relevant cellular biomarkers in the future.

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis.

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.

The role of T cells in vascular aging, hypertension, and atherosclerosis.

Vascular dysfunction has emerged as a significant risk factor for the development of cardio- and cerebrovascular diseases (CVDs), which are currently the leading cause of morbidity and mortality worldwide. T lymphocytes (T cells) have been shown to be important modulators of vascular function in primary aging and CVDs, likely by producing inflammatory cytokines and reactive oxygen species that influence vasoprotective molecules. This review summarizes the role of T cells on vascular function in aging, hypertension, and atherosclerosis in animals and humans, and discusses potential T-cell targeted therapeutics to prevent, delay, or reverse vascular dysfunction.

Reduced circulating CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesterolaemic mice and humans

AimsThe association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis.Methods and resultsWild type (WT), PCSK9−/−, and LDLR−/− C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR−/− and PCSK9−/− mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9−/− mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR−/− mice showing high levels while PCSK9−/− were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR−/− mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63+ EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63+ EVs were significantly depleted.ConclusionsThis research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.

Cellular communication network factor 2 regulates smooth muscle cell transdifferentiation and lipid accumulation in atherosclerosis.

Accruing evidence illustrates an emerging paradigm of dynamic vascular smooth muscle cell (SMC) transdifferentiation during atherosclerosis progression. However, the molecular regulators that govern SMC phenotype diversification remain poorly defined. This study aims to elucidate the functional role and underlying mechanisms of cellular communication network factor 2 (CCN2), a matricellular protein, in regulating SMC plasticity in the context of atherosclerosis. In both human and murine atherosclerosis, an up-regulation of CCN2 is observed in transdifferentiated SMCs. Using an inducible murine SMC CCN2 deletion model, we demonstrate that SMC-specific CCN2 knockout mice are hypersusceptible to atherosclerosis development as evidenced by a profound increase in lipid-rich plaques along the entire aorta. Single-cell RNA sequencing studies reveal that SMC deficiency of CCN2 positively regulates machinery involved in endoplasmic reticulum stress, endocytosis, and lipid accumulation in transdifferentiated macrophage-like SMCs during the progression of atherosclerosis, findings recapitulated in CCN2-deficient human aortic SMCs. Our studies illuminate an unanticipated protective role of SMC-CCN2 against atherosclerosis. Disruption of vascular wall homeostasis resulting from vascular SMC CCN2 deficiency predisposes mice to atherosclerosis development and progression.

ADAMTS7 Promotes Smooth Muscle Foam Cell Expansion in Atherosclerosis.

Human genetic studies have repeatedly associated ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury and that the proatherogenicity of ADAMTS7, a secreted protein, is due to its catalytic activity. However, the cell-specific mechanisms governing ADAMTS7 proatherogenicity remain unclear. To determine which vascular cell types express ADAMTS7, we interrogated single-cell RNA sequencing of human carotid atherosclerosis and found ADAMTS7 expression in smooth muscle cells (SMCs), endothelial cells (ECs), and fibroblasts. We subsequently created SMC- and EC-specific Adamts7 conditional knockout and transgenic mice. Conditional knockout of Adamts7 in either cell type is insufficient to reduce atherosclerosis, whereas transgenic induction in either cell type increases atherosclerosis. In SMC transgenic mice, this increase coincides with an expansion of lipid-laden SMC foam cells and decreased fibrous cap formation. RNA-sequencing in SMCs revealed an upregulation of lipid uptake genes typically assigned to macrophages. Subsequent experiments demonstrated that ADAMTS7 increases SMC oxLDL uptake through increased CD36 levels. Furthermore, Cd36 expression is increased due to increased levels of PU.1, a transcription factor typically associated with myeloid fate determination. In summary, Adamts7 expression in either SMCs or ECs promotes SMC foam cell formation and atherosclerosis. In SMCs, ADAMTS7 promotes oxLDL uptake via increased PU.1 and Cd36 expression, thereby increasing SMC foam cell formation and atherosclerosis.

Understanding the Complex Interplay of Epigenetic Factors in Atherosclerosis: A Review

This review focuses on the epigenetic factors that play a vital role in the formation of plaques and lead to atherosclerosis and related diseases in humans during current times. Apart from the epigenetics there are plenty more factors that influence the atherosclerotic risk in the older population of the current era. Lack of physical activities has become one of the most commonly found traits that causes obesity in people especially the middles aged adults. People working 9 - 5 in an office, or an IT firm tend spend idle time with their computer for 4 - 5 consecutive hours with barely changing their position. Lack of exercise tends to affect not only the physical health but also stress a person mentally. Chronic inflammatory diseases like atherosclerosis have a major role in the morbidity and mortality from cardiovascular disease worldwide. Atherosclerotic plaques are created by the pathophysiological process of lipids, inflammatory cells and fibrous components gradually accumulating within artery walls. Endothelial dysfunction signals the start of atherosclerosis and allows low-density lipoproteins (LDL) to enter the subendothelial region. Oxidative stress-induced changes in retained low-density lipoprotein (LDL) set off an inflammatory cascade that draws T cells and monocytes to the site of the lesion. The interaction of these immune cells with the local vascular cells encourages the production of foam cells, which fills the plaque’s centre with lipids. Smooth muscle cells multiply and help produce fibrous caps as the lesion ages, which affects the stability of the plaque. Studies shows how the brain responds to physical labour and how differently the effectively the enzymes and hormones are secreted, and their actions are vastly guided based on the physical stress an individual undergoes. Among the most prominent causes of atherosclerosis epigenetics has been focussed majorly in this review, where we focus on the underlying genetic factors that play a vital role in atherosclerosis and related cardiovascular diseases and also the genetic approach to a treatment is also elaborated. A number of miRNAs are involved in the cause/cure for the atherosclerosis, the following tables give an outlook regarding the same. This review aims to focus on the causes, risk factors, pathophysiology and the treatment and therapies that are currently available all the way concentrating on the genetic approach in the above-mentioned perspective. HIGHLIGHTS The review revolves around the modifiable and non-modifiable risk factors leading to atherosclerosis. Key risk factors and epigenetics causes of atherosclerosis and atherosclerosis related Cardio vascular Diseases (CVD). The article explains in detail regarding the pathophysiology of atherosclerosis. The progression and stages of atherosclerosis. GRAPHICAL ABSTRACT

Effect of phospholipid transfer protein on plasma sphingosine-1-phosphate

Plasma phospholipid transfer protein (PLTP) is a risk factor for cardiovascular diseases. Sphingosine-1-phosphate (S1P), carried by high-density lipoprotein (HDL), is a potent lipid mediator and is also associated with cardiovascular diseases. We found that germline Pltp gene knockout (KO) mice have decreased circulating S1P without influencing apoM, a major S1P carrier on HDL. We then hypothesized that, like apoM, PLTP is another S1P carrier. We established inducible Pltp-KO, Apom-KO, and Pltp/Apom double KO mice and measured plasma lipoprotein and S1P levels under different diets. We found that PLTP deficiency, and the double deficiency have a similar effect on HDL reduction. Importantly, we found that all mice have about 50% reduction in plasma S1P levels, compared to WT mice, and PLTP deficiency significantly reduces apoM levels (about 40%), while apoM deficiency has no effect on PLTP activity, indicating that PLTP depletion reduces S1P through HDL reduction. To further evaluate this HDL reduction-mediated effect, we overexpressed PLTP which also caused a reduction of HDL. We found that the overexpression reduces S1P and apoM as well as apoA-I, a major apolipoprotein on HDL. Furthermore, we found that albumin (another reported S1P carrier) deficiency in mice has no effect on plasma S1P. We also found that the influence of PLTP on HDL may not require its direct binding to the particle. In conclusion, PLTP is not a direct S1P carrier. PLTP depletion or overexpression in adulthood dramatically reduces plasma S1P through HDL reduction. ApoM, but not albumin, deficiency reduces plasma S1P levels.

Electrochemical impedance spectroscopy unmasks high-risk atherosclerotic features in human coronary artery disease.

Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. In this single-center, prospective study, n = 26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N = 61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Necrotic cores were identified in n = 19 vessels (median area 1.53 mm2) with a median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm2 versus <1.75 mm2 (19.8 ± 4.4 kΩ vs. 7.2 ± 1.0 kΩ, p = .019), fibrous cap thickness ≤65 μm versus >65 μm (19.1 ± 3.5 kΩ vs. 6.5 ± 0.9 kΩ, p = .004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) versus <20 macrophages per HPF (19.8 ± 4.1 kΩ vs. 10.2 ± 0.9 kΩ, p = .002). Impedance identified necrotic core area ≥1.75 mm2, fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716-1.000) (p = .013), 0.852 (0.646-1.000) (p = .025), and 0.835 (0.577-1.000) (p = .028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573-0.962) (p = .035). EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.

&lt;i&gt;CDKN2B-AS1&lt;/i&gt; polymorphism rs1333049 is associated with advanced carotid artery atherosclerosis in a Slovenian population

Several studies have reported an association between the 9p21 region in the human genome and atherosclerosis. The rs1333049 polymorphism is a single nucleotide polymorphism (SNP) in CDKN2B-AS1, located in the 9p21 region. The aim of our study was to investigate the association between the rs1333049 polymorphism and advanced carotid atherosclerosis, as well as its effect on CDKN2B expression in endarterectomy sequesters. In our case-control study, we included 881 participants, divided into two groups. The case group comprised 308 participants with advanced atherosclerosis of the internal or common carotid artery (stenosis &gt; 75 %) who underwent a revascularization procedure. The control group included 573 participants without hemodynamically significant carotid atherosclerosis. We analyzed the rs1333049 polymorphism using the StepOne real-time polymerase chain reaction (PCR) and TaqMan SNP genotyping assay. We found a statistically significant association according to the co-dominant (P=0.014, OR=3.29, 95% CI: 1.32-8.91, and P=0.015, OR=2.50, 95% CI: 1.22-5.37) and dominant (P=0.006, OR=2.74, 95% CI: 1.36-5.71) models. We performed immunohistochemical analysis of CDKN2B expression on 26 endarterectomy sequesters. The C allele of rs1333049 was associated with a lower numerical area density of CDKN2B-positive cells in atherosclerotic plaques. In conclusion, the C allele of the rs1333049 SNP is associated with an increased risk of developing advanced carotid atherosclerosis and lower CDKN2B expression in the plaques.

Atherosclerosis on a Chip: A 3-Dimensional Microfluidic Model of Early Arterial Events in Human Plaques.

Realistic reconstruction of the in vivo human atherosclerotic environment requires the coculture of different cell types arranged in atherosclerotic vessel-like structures with exposure to flow and circulating cells, presenting challenges for disease modeling. This study aimed to develop a 3-dimensional tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells, human umbilical cord vein endothelial cells, and foam cells to recreate a complex human atherosclerotic vessel in vitro to study the effects of flow and circulating immune cells. We developed a coculture protocol utilizing BFP (blue fluorescent protein)-labeled human aortic smooth muscle cells, GFP (green fluorescent protein)-labeled human umbilical cord vein endothelial cells, and THP-1 macrophage-derived, Dil-labeled oxidized LDL (low-density lipoprotein) foam cells within a fibrinogen/collagen I-based 3-dimensional ECM (extracellular matrix). Perfusion experiments were conducted for 24 hours on both atherosclerotic vessels and healthy vessels (BFP-labeled human aortic smooth muscle cells and GFP-labeled human umbilical cord vein endothelial cells without foam cells). Additionally, perfusion with circulating THP-1 monocytes was performed to observe cell extravasation and recruitment. The resulting vessels displayed early lesion morphology, with a layered composition including an endothelium and media, and foam cells accumulating in the subendothelial space. The layered wall composition of both atherosclerotic and healthy vessels remained stable under perfusion. Circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment to the foam cell core. The qPCR analysis indicated increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation of vascular smooth muscle cell migration in response to flow and the plaque microenvironment, compared with control vessels. The human 3-dimensional atherosclerosis model demonstrated stability under perfusion and allowed for the observation of immune cell behavior, providing a valuable tool for the atherosclerosis research field.

Epsins oversee smooth muscle cell reprograming by influencing master regulators KLF4 and OCT4.

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA-seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

Assessing the causal relationship between metabolic biomarkers and coronary artery disease by Mendelian randomization studies

The development of coronary artery disease (CAD) is significantly affected by impaired endocrine and metabolic status. Under this circumstance, improved prevention and treatment of CAD may result from knowing the connection between metabolites and CAD. This study aims to delve into the causal relationship between human metabolic biomarkers and CAD by using two-sample Mendelian randomization (MR). Utilizing two-sample bidirectional MR analysis, we assessed the correlation between 1400 blood metabolites and CAD, and the metabolites data from the CLSA, encompassing 8299 participants. Metabolite analysis identified 1091 plasma metabolites and 309 ratios as instrumental variables. To evaluate the causal link between metabolites and CAD, we analyzed three datasets: ebi-a-GCST005195 (547,261 European & East Asian samples), bbj-a-159 (29,319 East Asian CAD cases & 183,134 East Asian controls), and ebi-a-GCST005194 (296,525 European & East Asian samples). To estimate causal links, we utilized the IVW method. To conduct sensitivity analysis, we used MR-Egger, Weighted Median, and MR-PRESSO. Additionally, we employed MR-Egger interception and Cochran’s Q statistic to assess potential heterogeneity and pleiotropy. What’s more, replication and reverse analyses were performed to verify the reliability of the results and the causal order between metabolites and disease. Furthermore, we conducted a pathway analysis to identify potential metabolic pathways. 59 blood metabolites and 27 metabolite ratios nominally associated with CAD (P < 0.05) were identified by IVW analysis method. A total of four known blood metabolites, namely beta-hydroxyisovaleroylcarnitine (OR 1.06, 95% CI 1.027–1.094, FDR 0.07), 1-palmitoyl-2-arachidonoyl (OR 1.07, 95% CI 1.029–1.110, FDR 0.09), 1-stearoyl-2- docosahexaenoyl (OR 1.07, 95% CI 1.034–1.113, FDR 0.07) and Linoleoyl-arachidonoyl-glycerol, (OR 1.07, 95% CI 1.036–1.105, FDR 0.05), and two metabolite ratios, namely spermidine to N-acetylputrescine ratio (OR 0.94, 95% CI 0.903–0.972, FDR 0.09) and benzoate to linoleoyl-arachidonoyl-glycerol ratio (OR 0.87, 95% CI 0.879–0.962, FDR 0.07), were confirmed as having a significant causal relationship with CAD, after correcting for the FDR method (p < 0. 1). A causal relationship was found to be established between beta -hydroxyisovalerylcarnitine and CAD with the validation in other two datasets. Moreover, multiple metabolic pathways were discovered to be associated with CAD. Our study supports the hypothesis that metabolites have an impact on CAD by demonstrating a causal relationship between human metabolites and CAD. This study is important for new strategies for the prevention and treatment of CAD.