Inflammation In Atherosclerosis Research Articles

Classes of Lipid Mediators and Their Effects on Vascular Inflammation in Atherosclerosis.

It is commonly believed that the inactivation of inflammation is mainly due to the decay or cessation of inducers. In reality, in connection with the development of atherosclerosis, spontaneous decay of inducers is not observed. It is now known that lipid mediators originating from polyunsaturated fatty acids (PUFAs), which are important constituents of all cell membranes, can act in the inflamed tissue and bring it to resolution. In fact, PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are precursors to both pro-inflammatory and anti-inflammatory compounds. In this review, we describe the lipid mediators of vascular inflammation and resolution, and their biochemical activity. In addition, we highlight data from the literature that often show a worsening of atherosclerotic disease in subjects deficient in lipid mediators of inflammation resolution, and we also report on the anti-proteasic and anti-thrombotic properties of these same lipid mediators. It should be noted that despite promising data observed in both animal and in vitro studies, contradictory clinical results have been observed for omega-3 PUFAs. Many further studies will be required in order to clarify the observed conflicts, although lifestyle habits such as smoking or other biochemical factors may often influence the normal synthesis of lipid mediators of inflammation resolution.

Association between dietary inflammatory index and atherosclerosis cardiovascular disease in U.S. adults.

To evaluate the association between dietary inflammatory index (DII) and Atherosclerotic cardiovascular disease (ASCVD) among U.S. adults. We collected data from National Health and Nutrition Examination Survey (NHANES) between 1999 and 2018. Adults who reported complete information to diagnose ASCVD and calculate DII were included. We used three models to differentially adjust the covariates, including age, sex, race or ethnicity, education level, smoking status, poverty, insurance, body mass index, hyperlipemia, hypertension, and diabetes. Logistic regression was used to estimate the Odds Ratio (OR) and 95% confidence interval (95% CI) for ASCVD grouped by DII deciles. We additionally conducted spline smoothing with the generalized additive model (GAM) and the log-likelihood ratio to examine the non-linear relationship between DII and ASCVD. If exists, the segmented linear regression will be used to detect the cutoff point. The subgroup analyses were stratified by various atherosclerotic cardiovascular diseases (i.e., CHD, angina, heart attack, and stroke) and sex. A total of 48,733 participants (mean age, 47.13 ± 0.19 years) with 51.91% women were enrolled, of which 5,011 were diagnosed with ASCVD. In the crude model, participants in the five highest deciles (D6, 7, 8, 9, and 10) of DII score had a significantly higher risk of having ASCVD compared to those in the first decile. In the fully adjusted model, those in the tenth decile [OR = 1.47, 95% CI = (1.18,1.84)] of DII had a significantly increased risk of ASCVD compared to the first decile. Notably, when DII is above 3, the ASCVD risk increased by 41% for each one increase in DII [OR = 1.41, 95% CI = (1.15,1.73)]. This relationship was more pronounced in females. Our study revealed a positive and non-linearly association between DII and ASCVD in U.S. adults. This relationship was more pronounced in females. The findings provide a reference for future research and diet recommendations.

Anti-Inflammatory Therapy in Coronary Artery Disease: Where Do We Stand?

Inflammation plays an important role in all stages of atherosclerosis - from endothelial dysfunction, to formation of fatty streaks and atherosclerotic plaque, and its progression to serious complications, such as atherosclerotic plaque rupture. Although dyslipidemia is a key driver of atherosclerosis, pathogenesis of atherosclerosis is now considered interplay between cholesterol and inflammation, with the significant role of the immune system and immune cells. Despite modern therapeutic approaches in primary and secondary cardiovascular prevention, cardiovascular diseases remain the leading cause of mortality worldwide. In order to reduce residual cardiovascular risk, despite the guidelines-guided optimal medical therapy, novel therapeutic strategies are needed for prevention and management of coronary artery disease. One of the innovative and promising approaches in atherosclerotic cardiovascular disease might be inflammation-targeted therapy. Numerous experimental and clinical studies are seeking into metabolic pathways underlying atherosclerosis, in order to find the most suitable pathway and inflammatory marker/s that should be the target for anti-inflammatory therapy. Many anti-inflammatory drugs have been tested, from the well-known broad range anti-inflammatory agents, such as colchicine, allopurinol and methotrexate, to targeted monoclonal antibodies specifically inhibiting a molecule included in inflammatory pathway, such as canakinumab and tocilizumab. To date, there are no approved anti-inflammatory agents specifically indicated for silencing inflammation in patients with coronary artery disease. The most promising results came from the studies which tested colchicine, and studies where the inflammatory-target was NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome/interleukin-1 beta (IL-1 )/interleukin-6 (IL-6)/C-reactive protein (CRP) pathway. A growing body of evidence, along with the ongoing clinical studies, suggest that the anti-inflammatory therapy might become an additional strategy in treating atherosclerotic cardiovascular disease. Herein we present an overview of the role of inflammation in atherosclerosis, the most important inflammatory markers chosen as targets of anti-inflammatory therapy, along with the critical review of the major clinical trials which tested non-targeted and targeted anti-inflammatory drugs in patients with atherosclerotic cardiovascular disease.

Mass cytometry analysis reveals altered immune profiles in patients with coronary artery disease.

The importance of inflammation in atherosclerosis is well accepted, but the role of the adaptive immune system is not yet fully understood. To further explore this, we assessed the circulating immune cell profile of patients with coronary artery disease (CAD) to identify discriminatory features by mass cytometry. Mass cytometry was performed on patient samples from the BioHEART-CT study, gated to detect 82 distinct cell subsets. CT coronary angiograms were analysed to categorise patients as having CAD (CAD+) or having normal coronary arteries (CAD-). The discovery cohort included 117 patients (mean age 61 ± 12 years, 49% female); 79 patients (68%) were CAD+. Mass cytometry identified changes in 15 T-cell subsets, with higher numbers of proliferating, highly differentiated and cytotoxic cells and decreases in naïve T cells. Five T-regulatory subsets were related to an age and gender-independent increase in the odds of CAD incidence when expressing CCR2 (OR 1.12), CCR4 (OR 1.08), CD38 and CD45RO (OR 1.13), HLA-DR (OR 1.06) and Ki67 (OR 1.22). Markers of proliferation and differentiation were also increased within B cells, while plasmacytoid dendritic cells were decreased. This combination of changes was assessed using SVM models in discovery and validation cohorts (area under the curve = 0.74 for both), confirming the robust nature of the immune signature detected. We identified differences within immune subpopulations of CAD+ patients which are indicative of a systemic immune response to coronary atherosclerosis. This immune signature needs further study via incorporation into risk scoring tools for the precision diagnosis of CAD.

Metabolic regulation of the proteasome under hypoxia by Poldip2 controls fibrotic signaling in vascular smooth muscle cells

The polymerase delta interacting protein 2 (Poldip2) is a nuclear-encoded mitochondrial protein required for oxidative metabolism. Under hypoxia, Poldip2 expression is repressed by an unknown mechanism. Therefore, low levels of Poldip2 are required to maintain glycolytic metabolism.The Cellular Communication Network Factor 2 (CCN2, Connective tissue growth factor, CTGF) is a profibrogenic molecule highly expressed in cancer and vascular inflammation in advanced atherosclerosis. Because CCN2 is upregulated under hypoxia and is associated with glycolytic metabolism, we hypothesize that Poldip2 downregulation is responsible for the upregulation of profibrotic signaling under hypoxia. Here, we report that Poldip2 is repressed under hypoxia by a mechanism that requires the activation of the enhancer of zeste homolog 2 repressive complex (EZH2) downstream from the Cyclin-Dependent Kinase 2 (CDK2). Importantly, we found that Poldip2 repression is required for CCN2 expression downstream of metabolic inhibition of the ubiquitin-proteasome system (UPS)-dependent stabilization of the serum response factor. Pharmacological or gene expression inhibition of CDK2 under hypoxia reverses Poldip2 downregulation, the inhibition of the UPS, and the expression of CCN2, collagen, and fibronectin. Thus, our findings connect cell cycle regulation and proteasome activity to mitochondrial function and fibrotic responses under hypoxia.

Regulation of Gut Microbiota: Is It Hope for Cure of Colorectal Cancer?

The gut microbiota dysbiosis is linked with inflammatory bowel diseases, hypertension, atherosclerosis, hepatitis, neuropsychiatric disorders, diabetes, obesity, asthma, and development of cancer. Despite the substantial developments in oncology, cancer is still not in the category of curative diseases. However, significant steps are being taken in this direction through the identification of factors that play a role in the stages of complex carcinogenesis. Recent data obtained from human gut microbiota has shown that commensal microorganism species living on the epithelial surfaces of the intestines have an active role in this process and are also associated with personal responses to cancer treatment and toxicity. We now know more about the role of Fusobacterium nucleatum, an important member of the gut microbiota complex, as well as other microorganisms in the formation, treatment, diagnosis, and screening of colorectal cancer. In conclusion, the growing knowledge of microbiota, a key factor in colorectal cancer formation and treatment stages, will be able to change the early detection, treatment, and follow-up of these cancers. In this review, the role of gut microbiota in colorectal carcinogenesis and cancer formation mechanisms, significance in treatment and prognosis, and potential use in cancer prevention and screening have evaluated based on the data obtained from recent studies.

The relationship between neutrophil-to-lymphocyte ratio and cerebral collateral circulation in patients with symptomatic severe intracranial artery stenosis or occlusion

BackgroundThe neutrophil/lymphocyte ratio (NLR) has been considered a prognostic indicator for determining the systemic inflammatory response and atherosclerosis. We aimed to determine the relationship between NLR and the development of cerebral collateral circulation in patients with symptomatic severe stenosis or occlusion of intracranial arteries. MethodsAll patients underwent digital subtraction angiography (DSA) within 14 days of admission and were divided into a group with good collateral circulation (77 patients) and a group with poor collateral circulation (86 patients) according to the DSA collateral compensation grading method. Apo B, total cholesterol, LDL, and Neutrophil count in the poor side branch group were significantly higher than in the good side branch group. Multifactorial analysis showed that high NLR levels were a valid predictor of poor collateral circulation in patients with symptomatic severe intracranial artery stenosis or occlusion. Spearman correlation analysis showed that the size of the collateral branch score was negatively correlated with NLR (r = −0.509, P < 0.001) and cholesterol content (r = −0.249, P = 0.002). NLR predicted poor collateral circulation with an AUC of 0.620 (sensitivity 66.7 %, specificity 61.3 %, 95 % CI = 0.517–0.723,P < 0.05). ConclusionWe demonstrate a correlation between NLR levels and the development of collateral circulation in the brain in patients with symptomatic severe stenosis or occlusion of the intracranial arteries.

The Role of Transcription Factors in Coronary Artery Disease and Myocardial Infarction.

Coronary artery disease (CAD) and its main complication, myocardial infarction (MI), is a complex disease caused by environmental and genetic factors and their interaction. Family-based linkage analysis and genome-wide association studies have indicated many of genetic variations related to CAD and MI in recent years. Some are in the coding sequence, which mediates the coding protein, while others are in the non-coding region, which affects the expression of adjacent genes and forms differential gene expression. These variants and differential expressions will have varying degrees of impact on the development of the cardiovascular system and normal heart electrical activity function, subsequently leading to CAD and MI. Among these affected genes, some Transcription Factors (TFs), as important means of transcriptional regulation, have a key role in the pathogenesis of coronary artery disease and myocardial infarction. The GATAs binding protein 2 (GATA2) enhances monocyte adhesion and promoted vessel wall permeabilization through vascular EC adhesion molecule 1 (VCAM-1) upregulation, further revealing its atherosclerosis-promoting role. Myocyte enhancer factor 2 (MEF2) has a role in fostering many functions of the atherosclerotic endothelium and is a potential therapeutic target for atherosclerosis, thrombosis, and inflammation. Nuclear factor-kappa B (NF-κB) is an important promoter of vascular endothelial growth factor (VEGF)-driven angiogenesis, and its pathway has a key role in atherosclerosis-related complications such as angiogenesis, inflammation, apoptosis, and immune effects. Activating transcription factor 3 (ATF3) may be a novel prognostic biomarker and therapeutic target for atherosclerosis. The important role of signal transducer and activator of transcription 3 (STAT3) (especially in mitochondria) in endothelial cells (EC) dysfunction, inflammation, macrophage polarization and immunity in atherosclerosis.

PET/MR imaging of inflammation in atherosclerosis.

Myocardial infarction, stroke, mental disorders, neurodegenerative processes, autoimmune diseases, cancer and the human immunodeficiency virus impact the haematopoietic system, which through immunity and inflammation may aggravate pre-existing atherosclerosis. The interplay between the haematopoietic system and its modulation of atherosclerosis has been studied by imaging the cardiovascular system and the activation of haematopoietic organs via scanners integrating positron emission tomography and resonance imaging (PET/MRI). In this Perspective, we review the applicability of integrated whole-body PET/MRI for the study of immune-mediated phenomena associated with haematopoietic activity and cardiovascular disease, and discuss the translational opportunities and challenges of the technology.

Innate Immune Memory in Monocytes and Macrophages: The Potential Therapeutic Strategies for Atherosclerosis.

Atherosclerosis is a complex metabolic disease characterized by the dysfunction of lipid metabolism and chronic inflammation in the intimal space of the vessel. As the most abundant innate immune cells, monocyte-derived macrophages play a pivotal role in the inflammatory response, cholesterol metabolism, and foam cell formation. In recent decades, it has been demonstrated that monocytes and macrophages can establish innate immune memory (also termed trained immunity) via endogenous and exogenous atherogenic stimuli and exhibit a long-lasting proinflammatory phenotype. The important cellular metabolism processes, including glycolysis, oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, fatty acid synthesis, and cholesterol synthesis, are reprogrammed. Trained monocytes/macrophages with innate immune memory can be persistently hyperactivated and can undergo extensive epigenetic rewiring, which contributes to the pathophysiological development of atherosclerosis via increased proinflammatory cytokine production and lipid accumulation. Here, we provide an overview of the regulation of cellular metabolic processes and epigenetic modifications of innate immune memory in monocytes/macrophages as well as the potential endogenous and exogenous stimulations involved in the progression of atherosclerosis that have been reported recently. These elucidations might be beneficial for further understanding innate immune memory and the development of therapeutic strategies for inflammatory diseases and atherosclerosis.

Differential Expression of Inflammarafts in Macrophage Foam Cells and in Nonfoamy Macrophages in Atherosclerotic Lesions-Brief Report.

Reprogramming of monocytes and macrophage manifests in hyperinflammatory responses and chronification of inflammation in atherosclerosis. Recent studies focused on epigenetic, transcriptional, and metabolic alterations that characterize trained immunity. However, the underlying effector mechanisms driving the hyperinflammatory response of reprogrammed macrophages remain unclear. We hypothesized that the plasma membrane of atherosclerotic lesion macrophages undergoes reprogramming to maintain inflammarafts, enlarged lipid rafts (LR) serving as a platform for assembly of inflammatory receptor complexes. Single-cell suspensions from the aortae of Western diet-fed Ldlr-/- mice were gated for BODIPY-high foamy and BODIPY-low nonfoamy F4/80 macrophages by flow cytometry. Inflammarafts were characterized by increased levels of LR, TLR4 (toll-like receptor-4) localization to LR, TLR4 dimers, and the proximity between TLR2, TLR1, and CD36. In a cellular model of trained immunity, LR, TLR4 dimers, and the inflammatory response were measured in bone marrow-derived macrophages subjected to a 24-hour treatment with LPS (lipopolysaccharide) or OxLDL (oxidized low-density lipoprotein), followed by a 6-day wash-out period. Nonfoamy macrophages, which constituted ≈40% of macrophages in atherosclerotic lesions, expressed significantly higher levels of LR and TLR4 dimers, as well as proximity ligation signals for TLR4-LR, TLR2-CD36, and TLR2-TLR1 complexes, compared with foamy macrophages. These inflammaraft measures associated, to a different degree, with plasma cholesterol and inflammatory cytokines, as well as the size of the atherosclerotic lesions and necrotic cores. The bone marrow-derived macrophages trained with LPS simulated nonfoamy atherosclerotic lesion macrophages and continued to express inflammarafts and inflammatory genes for 6 days after LPS removal and displayed a hyperinflammatory response to Pam3CSK4, a TLR2/TLR1 agonist. OxLDL-exposed, lipid-laden macrophages did not express inflammarafts. Our data support the hypothesis that persistent inflammarafts in nonfoamy macrophages in atherosclerotic lesions serve as effectors of macrophage reprogramming into a hyperinflammatory phenotype.

Non-tumoral uptake of 68Ga-FAPI-04 PET: A retrospective study.

Fibroblast activation protein (FAP)-targeting radiopharmaceutical based on the FAP-specific inhibitor (FAPI) is considered as a potential alternative agent to FDG for tumor-specific imaging. However, FAP is also expressed in normal adult tissues. The aim of this study was to explore the image features of non-tumoral regions with high uptake of 68Ga-FAPI-04 in positron emission tomography (PET) imaging and to reveal the physiological mechanisms of these regions. A total of 137 patients who underwent whole-body 68Ga-FAPI-04 PET/MR (n=46) or PET/CT (n=91) were included in this retrospective study. Three experienced nuclear medicine physicians determined the non-tumoral regions according to other imaging modalities (CT, MRI, 18F-FDG PET, or ultrasound), clinical information, or pathological results. The regions of interest (ROIs) were drawn manually, and the maximum standardized uptake value (SUVmax) was measured. A total of 392 non-tumoral uptake regions were included in this study. The included physiological regions were uterus (n=38), submandibular gland (n=118), nipple (n=37), gingiva (n=65), and esophagus (n=31). The incidence of 68Ga-FAPI-04 uptake in physiological regions was independent of age, the tracer uptakes in the gingiva and esophagus were more common in male patients (p=0.006, 0.009), while that in the nipple was more common in female patients (p < 0.001). The included benign regions were inflammatory lymph node (n =10), pneumonia (n=13), atherosclerosis (n=10), pancreatitis (n=18), osteosclerosis (n=45), and surgical scar (n=7). No significant difference was observed in SUVmax between physiological and benign regions. A number of organs exhibit physiological uptakes of 68Ga-FAPI-04. Our study showed that regions with high 68Ga-FAPI-04 uptake did not necessarily represent malignancy. Being familiar with physiological and typical benign 68Ga-FAPI-04 uptake regions can be helpful for physicians to interpret images and to make an accurate diagnosis.

Emerging vascular cell-specific roles for mineralocorticoid receptor: implications for understanding sex differences in cardiovascular disease.

As growing evidence implicates extrarenal mineralocorticoid receptor (MR) in cardiovascular disease (CVD), recent studies have defined both cell- and sex-specific roles. MR is expressed in vascular smooth muscle (SMC) and endothelial cells (ECs). This review integrates published data from the past 5 years to identify novel roles for vascular MR in CVD, with a focus on understanding sex differences. Four areas are reviewed in which there is recently expanded understanding of the cell type- or sex-specific role of MR in 1) obesity-induced microvascular endothelial dysfunction, 2) vascular inflammation in atherosclerosis, 3) pulmonary hypertension, and 4) chronic kidney disease (CKD)-related CVD. The review focuses on preclinical data on each topic describing new mechanistic paradigms, cell type-specific mechanisms, sexual dimorphism if addressed, and clinical implications are then considered. New data support that MR drives vascular dysfunction induced by cardiovascular risk factors via sexually dimorphic mechanisms. In females, EC-MR contributes to obesity-induced endothelial dysfunction by regulating epithelial sodium channel expression and by inhibiting estrogen-induced nitric oxide production. In males with hyperlipidemia, EC-MR promotes large vessel inflammation by genomic regulation of leukocyte adhesion molecules, which is inhibited by the estrogen receptor. In pulmonary hypertension models, MRs in EC and SMC contribute to distinct components of disease pathologies including pulmonary vessel remodeling and RV dysfunction. Despite a female predominance in pulmonary hypertension, sex-specific roles for MR have not been explored. Vascular MR has also been directly implicated in CKD-related vascular dysfunction, independent of blood pressure. Despite these advances, sex differences in MR function remain understudied.

USP14-mediated NLRC5 upregulation inhibits endothelial cell activation and inflammation in atherosclerosis

Atherosclerosis, a chronic inflammatory condition that leads to a variety of life-threatening cardiovascular diseases, is a worldwide public health concern. Endothelial cells (ECs), which line the inside of blood vessels, play an important role in atherogenic initiation. Endothelial activation and inflammation are indispensable for the early stage of atherosclerosis. Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme that regulates the stability and activity of target proteins, has been identified as a potential therapeutic target for many inflammatory diseases. However, the role of USP14 on ECs is undefined. In this study, we found that USP14 is downregulated in either atherosclerosis patient specimens or oxidized low-density lipoprotein (ox-LDL)-stimulated ECs as compared to the control group. Overexpression of USP14 in ECs restrains ox-LDL-stimulated nuclear transcription factor kappa B (NF-κB) activation and subsequent adhesion molecule production. USP14 inhibits endothelium proinflammatory activation by suppressing the degradation of the negative regulator of NF-κB signaling, nod-like receptor family caspase recruitment domain family domain containing 5 (NLRC5). Finally, our in vivo experiments confirmed that USP14 adenovirus injection in apolipoprotein E deficient (ApoE−/−) mice fed with a western diet reduced the atherosclerotic lesion size, inhibited macrophage accumulation in the intima, and restricted the progression of atherosclerosis. Our results reveal that USP14 may represent a new therapeutic target for atherosclerosis.

Matr3 reshapes m6A modification complex to alleviate macrophage inflammation during atherosclerosis

Atherosclerosis, characterized as the chronic inflammation of the arterial wall, is one of the leading causes of coronary artery disease (CAD), and macrophages are found to play essential roles in the initiation and progression of inflammation in atherosclerosis. N6-methyladenosine (m6A) modification, as the most abundant epi-transcriptomic modification in mRNA, is found to mediate the atherogenic inflammatory cascades in vascular endothelium. The detailed molecular mechanism of m6A methylation regulating inflammatory response during atherosclerosis is still not fully known. In this study, we find oxidized low-density lipoprotein (oxLDL) stimulation increases methyltransferases Mettl3 and Mettl14 expressions in macrophages, whereas the total m6A modification level in macrophages decreases under oxLDL stimulation. Matrin-3 (Matr3), an RNA binding protein, is identified to play a suppressive role on oxLDL-mediated macrophage inflammatory responses through inhibiting activation of pro-inflammatory signaling, mitogen-activated protein kinase (Mapk) by m6A-mediated mRNA decay via regulating the formation of Mettl3-Mettl14 complex. Moreover, we find that Matr3 expression decreases in the oxLDL-stimulated macrophages, and the peripheral blood-derived monocytes from patients with CAD, and overexpression of Matr3 significantly alleviates atherosclerosis development in vivo. Our study for the first time clarifies the role of Matr3 on macrophage inflammatory responses during atherosclerotic development, and supplies deep understanding on the relationship of m6A modification and inflammatory responses in atherosclerosis.

Abstract 12879: Endothelial Cu Exporter Atp7a Deficiency Promotes Cu-Dependent Endothelial-to-Mesenchymal Transition and Atherosclerosis via Orchestrating Glycolysis and Ros Signaling

Introduction: Oxidative stress, inflammation and metabolic reprogramming contribute to endothelial cell (EC) dysfunction and atherosclerosis. Copper (Cu), an essential micro-nutrient, promotes atherosclerosis via unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu exporter ATP7A. However, the role of ATP7A and its molecular linkage with inflammation, oxidative stress and metabolism in EC dysfunction and atherosclerosis are entirely unknown. Results: Here we found a dramatic reduction of ATP7A expression in human and mouse atherosclerotic aorta. Surprisingly, ATP7A knockdown (KD) in human aortic ECs using shRNA changed the cell morphology from EC to mesenchymal types. This was confirmed by RNAseq/qPCR showing reduced EC markers and increased mesenchymal markers, indicating endothelial to mesenchymal transition (EndMT). Mechanistically, loss of ATP7A in ECs increased [Cu]i and PFKFB3-dependent glycolysis (1.8- fold by ECAR in Seahorse assay). Overexpression of PFKFB3 or constitutively active PFK2, which enhances glycolysis, or direct Cu treatment induced EndMT. In parallel, ATP7A KD in ECs increased mitochondrial (mito)ROS (9.8 fold)-histone demethylase JMJD2B, which induced inflammatory EndMT inducer TGFβ2-EndMT transcription factor Snail (2 fold) axis. Of note, ATP7A KD-induced glycolysis as well as mitoROS- TGFβ2-Snail axis involved in EndMT were inhibited by Cu chelator TTM. In vivo, Cu transport dysfunctional ATP7A mutant/ApoE -/- mice showed enhanced atherosclerosis and CD31 + /αSMA + cells representing EndMT in aorta compared to control ApoE -/- mice, which was further confirmed by EC-specific ATP7A -/- /mTmG reporter mice. Conclusion: Our study demonstrates that ATP7A dysfunction in ECs drives EndMT by orchestrating mitoROS-JMJD2B-TGFβ as well as PFKFB3-glycolysis axis in a Cu dependent manner, leading to Snail induction to accelerates atherosclerosis. Thus, ATP7A is a potential therapeutic target for treatment of inflammatory vascular disease.

Abstract 14719: Erk5 S496 Phosphorylation, but Not Erk5 Kinase Activation, Promotes Senescence-Associated Cell Growth (sacg) and Inflammation of Myeloid Cells and Atherosclerosis via Upregulating Sumoylation at a Novel Site (k518) on Nrf2 and Aryl Hydrocarbon Receptor

Senescence induced by various stresses reprograms cancer cells to acquire senescence-associated (SA) cell growth (SACG), which allows them to escape senescence-induced cell cycle arrest with enhanced growth potential. ERK5 is a dual kinase-transcription factor, containing an N-terminal kinase domain and transactivation domains in the C-terminal half. Recent data have raised questions regarding the functional role of ERK5 kinase inhibitors in cancer and inflammatory diseases. We aimed to investigate how ERK5 reprograms myeloid cells (MC) to the SA secretory phenotype (SASP) and SACG, consequently leading to atherosclerosis (AthS). AthS was inhibited in ERK5 S496A knock-in (KI) mice. Single-cell analysis of p53 and Ki67 with imaging mass cytometry revealed three groups of MCs with different p53 and Ki67 expression patterns in the plaque. One group showed that p53 and Ki67 were co-expressed lineally, suggesting that this group of cells escaped from the growth-suppression effects of p53, which is also known as a senescence marker. The % of ERK5 S496A KI cells in this group was significantly lower than in wild-type (WT). Also, we found an increase of SA β-gal (SAβ-gal) + Ki67 + and 53BP1 + Ki67 + cells in bone narrow-derived macrophage (BMDMs) isolated from hypercholesterolemia (H-chol) WT mice, suggesting H-chol-induced MC SACG. MC SACG were significantly inhibited in ERK5 S496A KI mice, supporting the role of ERK5 S496 phosphorylation on SACG. RNA sequencing analysis identified the ERK5 S496 phosphorylation-dependent AHR induction by H-chol, and AHR depletion significantly inhibited SACG. We also discovered a critical effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity and antagonized oxidized LDL (oxLDL)-induced SASP. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) inhibited oxLDL-induced ERK5 S496 phosphorylation, suggesting that ERK5 S496 phosphorylation was involved at least in part of the effects of these inhibitors. We have discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibits NRF2 activity by upregulating NRF2 K518 SUMOylation and AHR induction, subsequently inducing SACG and AthS.

Citrullination of histoneH3 in neutrophil via CXCL1 enhances neutrophil adhesion to femoral artery of LDLR−/− mice fed HFD

Abstract Background Vascular inflammation plays an important role in the development of atherosclerosis. Previously we have shown that a high-fat diet (HFD) increased neutrophil adhesion to the vascular intima in wild-type (wt) mice (Osaka M. Sci Rep. 2016). However, the involvement of neutrophils in atherosclerosis-related vascular inflammation is not well known. Purpose This study examined that neutrophil extracellular trap (NETs) or the hypercitrullination of histone H3 in neutrophils enhances neutrophil adhesion to atheroprone-arteries in LDL receptor null (LDLR−/−) mice. Methods We observed leukocyte adhesion in the femoral artery of LDLR−/− mice fed normal chow (NC) or HFD, and determined leukocyte subtype that adhered on vascular endothelium under neutrophil or monocyte depletion using intravital microscopy. Importantly, neutrophil adhesion was examined under the administration of TDFA which inhibits NETs and citrullination of histone H3, in LDLR−/− mice fed HFD. Furthermore, immunohistochemistry for citrullinated histone H3 in peripheral neutrophils of mice was examined. Comprehensive cytokine/chemokine analysis for a plasma of mice was performed to determine the factors citrullinating histone H3 in LDLR−/− mice. Moreover, these mice were treated with a novel specific PPARα agonist, to reduce the elevation of plasma triglyceride levels. Results Leukocyte adhesion in LDLR−/− mice fed HFD significantly increased compared to NC. More interestingly, it significantly enhanced compared to wt mice fed HFD. Furthermore, neutrophil depletion rather than monocyte depletion diminished leukocyte adhesion, suggesting that the leukocyte subtype that adhered in LDLR−/− mice fed HFD was neutrophil. Neutrophil adhesion in these mice significantly was reduced by the administration of TDFA, suggesting a pivotal role for histone H3 citrullination in neutrophil adhesion. Moreover, citrullination of histone H3 in neutrophils from LDLR−/− mice fed HFD but not from those without HFD was significantly enhanced. In addition, comprehensive cytokine/chemokine analysis revealed an increase of CXCL1 in plasma of LDLR−/− mice fed HFD. CXCL1 enhanced neutrophil adhesion to HUVECs, and the adhesion significantly decreased by the treatment of TDFA to neutrophil in vitro non-static adhesion assay. These results showed that CXCL1 enhanced neutrophil adhesion in LDLR−/− mice fed HFD through citrullination. Furthermore, when these mice were treated with PPARα agonist, observed histone citrullination, as well as neutrophil adhesion, was significantly reduced. Conclusion These results suggest that HFD induced histone citrullination in neutrophils in LDLR−/− mice and PPARα agonist plays a role during hypertriglyceridemia-mediated vascular inflammation in atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research(C)

The effects of colchicine in patients with diabetes mellitus and chronic coronary artery disease: a post-hoc analysis of the LoDoCo2-trial

Abstract Background Atherosclerosis is an inflammatory disease and is accelerated by diabetes mellitus (DM). Patients with chronic coronary artery disease (CAD) who also have DM are at high risk of recurrent cardiovascular events. The role of inflammation in atherosclerosis is well established, whereas the role of inflammation on incident and progression of DM has been hypothesized. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in particular, may play an important role in the onset and progression of T2DM. The anti-inflammatory drug colchicine attenuates the NLRP3-inflammasome. The Low-Dose Colchicine 2 (LoDoCo2) trial showed that colchicine reduces cardiovascular risk in patients with chronic CAD. Purpose The purpose of this study was to assess the effects of colchicine in patients with chronic CAD and DM on cardiovascular events as well as the effect of colchicine on the development of new-onset DM. Methods The LoDoCo2 trial randomized 5522 to placebo or colchicine, with a median follow-up of 28.6 months (interquartile range 20.5–44.4). The primary endpoint was a composite of cardiovascular death, spontaneous myocardial infarction, ischaemic stroke, or ischaemia-driven revascularization. Secondary outcomes consisted of the aforementioned events, separately. Cox proportional hazards models were used to investigate univariable associations between DM status for all endpoints in the placebo group. The interactions between treatment group and DM status were evaluated with the addition of treatment and the treatment-by-DM variable interaction. Results In total, 1007 participants (18.2%) had DM at baseline. The hazard ratio for the primary endpoint was 0.87 (95% CI, 0.61–1.25) in those with DM and 0.64 (95% CI, 0.51–0.80) in those without DM (p for interaction&amp;gt;0.05). Treatment effects of colchicine were consistent over all secondary endpoints (p for interaction&amp;gt;0.05). The incidence of new-onset DM was 1.5% (34/2270) in the colchicine group and 2.2% (49/2245) in the placebo group (p=0.10). Participants with DM were at higher risk for all endpoints. The primary composite end point in the placebo group occurred in 13.0% (67/515) patients with DM and in 8.8% (197/2245) of the patients without DM (unadjusted hazard ratio 1.54 [95% CI 1.16–2.03, p&amp;lt;0.01]) compared to the group without DM. DM was also strongly associated with the occurrence of all secondary end points. Conclusion This study shows that the beneficial effects of colchicine on cardiovascular endpoints are consistent regardless of DM status. The data indicate that larger trials are needed to assess whether colchicine reduces the incidence of new-onset DM. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): National Health Medical Research Council of Australia and the Netherlands Organization for Health Research and Development

Identification of a PRDM1-regulated T cell network to regulate T cell driving plaque inflammation in human and mouse atherosclerosis

Abstract T cells have a prominent role in the pathogenesis of atherosclerosis, although their exact function remains elusive. Here, we pursued a network-driven approach to identify T cell-associated gene programs driving the transition from low- to high-risk human plaques. In this study 43 human carotid arterial plaques were collected and stratified based on absence (low-risk) or presence (high-risk) of intraplaque haemorrhage (IPH). Lesion RNA was subjected to microarray gene expression analysis and analysed by Weighted Gene Co-expression Network Analysis (WGCNA). We identified a co-expressed gene cluster displaying a strong T cell signalling signature in high- versus low-risk plaque, which was tightly connected to subnetworks of angiogenesis and interferon-signalling. WGCNA-based Bayesian network inference, cell-type deconvolution and single-cell gene expression revealed that this T cell-associated gene program was likely linked to effector-memory cytotoxic CD8+ T cells, underpinning the central role of T cells in plaque destabilization. Gene regulatory analysis identified cytotoxic T cell-related transcription factors, like PRDM1, regulating this plaque T cell gene program. Moreover, we demonstrated in LDL receptor knockout mice with T cell-specific Prdm1 deficiency, that lack of Prdm1 in T cells resulted in larger, more advanced plaques. In conclusion, our study reveals a PRDM1-regulated T cell footprint in high- versus low-risk human atherosclerotic lesions and murine atherosclerotic plaque development, thereby identifying this network as a potential target for intervention in adverse T cell responses. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): The European Research Area Network on Cardiovascular Diseases (ERA-CVD and Dutch Heart Foundation)