Aortic Atherosclerotic Plaques Research Articles

Enrichment of type 1 innate lymphoid cells in the course of human atherosclerotic plaque development suggests contribution to atherogenesis.

Innate lymphoid cells (ILCs) have been implicated in multiple pathologic conditions, including atherogenesis, as documented in experimental mice studies, however, their role in atherosclerosis in humans remains unexplored. Here, we identify ILCs and their dynamics in early, advanced, and complicated human carotid- and aortic atherosclerotic plaques, using a multiplex immunohistochemical quadruple-staining technique with prototypic transcription factors T-bet, GATA3, or RORgt for identification of the ILC1, ILC2 and ILC3 subsets, respectively, in combination with lineage markers CD3, CD20/ CD79a and CD56 to exclude other lymphoid cell types. ILC subsets were quantified, and to put this in perspective, their numbers were expressed as percentage of the total number of infiltrated lymphoid cells and related to the frequency of conventional T cells, B cells, NK cells, and NKT cells. All ILC subsets were present in every different stage of atherogenesis. ILC1s were the most abundant ILC subset, and their numbers significantly increased in the course of plaque development, but paradoxically, their relative frequency was reduced because of a higher increment of T cells and B cells. The numbers of ILC2s and ILC3s also gradually increased, but this trend did not achieve significance. T cell subsets always significantly outnumbered their ILC counterparts, except for the early lesions where the proportion of ILC1s was markedly higher, albeit not significant. The high abundance of ILC1s in the early stages and further significant enrichment in later stages, suggest they may participate in the initiation and development of atherogenesis, and thus, may represent a novel target to prevent or treat atherosclerosis.

Mussel oil is superior to fish oil in preventing atherosclerosis of ApoE-/- mice.

The present study aimed to explore the preventive effect of mussel oil (MO) on atherosclerosis and the potential mechanism in apolipoprotein E-null (ApoE-/-) mice. ApoE-/- mice were fed with a high-fat and high-cholesterol chow and given corn oil (CO), fish oil (FO), MO, or aspirin (ASP, dissolved in CO) by gavage for 12 weeks. The total n-3 polyunsaturated fatty acids (PUFAs) in MO (51.01%) and FO (46.82%) were comparable (mainly C22:6n-3 and C20:5n-3). Wild-type mice were fed with a normal chow and given equivalent CO as health control (CON). Compared with the CON group, obvious atherosclerotic plaque appeared at aorta and aortic sinus in the CO group. Compared with the CO group, MO but not FO had a significantly smaller atherosclerotic plaque area in the aorta. The aortic atherosclerotic plaque area was comparable in the MO, CON, and ASP groups. The MO group had a significantly smaller atherosclerotic plaque area, lower lipid deposition, lower contents of smooth muscle cell (SMC), and slightly lower contents of macrophage at the aortic sinus than the FO group. Serum concentrations of IL-1β, NF-κB, and VCAM-1 were comparable in the MO and FO groups and were significantly lower than the CO group. Compared with the CO group, the MO group but not FO group had significantly lower aortic protein levels of p65NF-κB, p38MAPK, and VCAM-1. The aortic protein levels of p-p65NF-κB and p-p38MAPK were significantly lower in the MO group than the FO group. In conclusion, MO is more potent than FO in preventing atherosclerosis, and the possible mechanism may be by downregulating p38MAPK/NF-κB signaling pathway, decreasing VCAM-1 and macrophage, and inhibiting proliferation and migration of SMC.

Novel UV-B Phototherapy With a Light-Emitting Diode Device Prevents Atherosclerosis by Augmenting Regulatory T-Cell Responses in Mice.

Ultraviolet B (UV-B) irradiation is an effective treatment for human cutaneous disorders and was shown to reduce experimental atherosclerosis by attenuating immunoinflammatory responses. The aim of this study was to clarify the effect of specific wavelengths of UV-B on atherosclerosis and the underlying mechanisms focusing on immunoinflammatory responses. Based on light-emitting diode technology, we developed novel devices that can emit 282 nm UV-B, which we do not receive from natural sunlight, 301 nm UV-B, and clinically available 312 nm UV-B. We irradiated 6-week-old male atherosclerosis-prone Apoe-/- (apolipoprotein E-deficient) mice with specific wavelengths of UV-B and evaluated atherosclerosis and immunoinflammatory responses by performing histological analysis, flow cytometry, biochemical assays, and liquid chromatography/mass spectrometry-based lipidomics. Irradiation of 282 nm UV-B but not 301 or 312 nm UV-B significantly reduced the development of aortic root atherosclerotic plaques and plaque inflammation. This atheroprotection was associated with specifically augmented immune responses of anti-inflammatory CD4+ Foxp3 (forkhead box P3)+ regulatory T cells in lymphoid tissues, whereas responses of other immune cells were not substantially affected. Analysis of various lipid mediators revealed that 282 nm UV-B markedly increased the ratio of proresolving to proinflammatory lipid mediators in the skin. We demonstrated that 282 nm UV-B irradiation effectively reduces aortic inflammation and the development of atherosclerosis by systemically augmenting regulatory T-cell responses and modulating the balance between proresolving and proinflammatory lipid mediators in the skin. Our findings indicate that a novel 282 nm UV-B phototherapy could be an attractive approach to treat atherosclerosis.

Macrophage-targeted ultrasound nanobubbles for highly efficient sonodynamic therapy of atherosclerotic plaques by modulating M1-to-M2 polarization

Background and aimsSonodynamic therapy (SDT) is a new approach for the treatment of atherosclerosis (AS), yet the poor targeting ability of sonosensitizers limits its therapeutic efficacy. Herein, we reported a plaque-targeted nanoplatform modified with macrophage type A scavenger receptor (SR-A)-targeted peptide (designated as SR-A-Ce6NB) to augment the efficacy of low-intensity pulsed ultrasound (LIPUS)-mediated SDT of atherosclerotic plaque. MethodsSR-A-Ce6NB was fabricated by thin hydration method and biotin-avidin system, and its physicochemical properties, biocompatibility and plaque-targeting ability were investigated. RAW 264.7 cells were used for in vitro experimental studies. Male 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 16 weeks to induce aortic atherosclerotic plaques. Plaque-bearing mice were randomly allocated into five groups (n = 6): control group, Ce6 + LIPUS group, Ce6NB + LIPUS group, SR-A-Ce6NB + LIPUS group and atorvastatin group. After treatment in each group, the aortic artery was harvested for Oil red O, H&E, Masson's trichrome staining, immunohistochemical and immunofluorescent staining. ResultsSR-A-Ce6NB with high stability and excellent biocompatibility was successfully fabricated. SR-A-Ce6NB could actively target activated macrophages and selectively accumulate in the plaque. SR-A-Ce6NB could be triggered by LIPUS and had a more potent sonodynamic effect than free Ce6 to potentiate SDT. SR-A-Ce6NB-mediated SDT enhanced the anti-atherogenic effect via modulating M1-to-M2 macrophage polarization and had an earlier onset of action on plaque than the statin-mediated effect. No apparent side effect was observed after intravenous SR-A-Ce6NB injection and LIPUS exposure. ConclusionsMacrophage-targeted nanoplatform SR-A-Ce6NB-mediated SDT provides a safe, effective and preferable anti-atherogenic therapy by mediating M1-to-M2 macrophage polarization.

Association between aortic thrombi detected using non-obstructive general angioscopy and atrial fibrillation.

Atrial fibrillation (AF) is an independent risk factor for stroke and systemic embolism. Cardiogenic and aortogenic emboli are causes of stroke or systemic embolism. Non-obstructive general angioscopy (NOGA) can be used to diagnose aortic intimal findings, including thrombi and atherosclerotic plaques, but little is known about NOGA-derived aortic intimal findings in patients with AF. This study focused on aortic intimal findings in patients with AF and evaluated the association between AF and aortic thrombi detected using NOGA. We enrolled 283 consecutive patients with coronary artery disease who underwent NOGA of the aorta between January 2017 and August 2022. Aortic intimal findings were screened using NOGA after coronary arteriography. The patients were divided into two groups according to their AF history (AF, n = 50 and non-AF, n = 233). Patients in the AF group were older than those in the non-AF group. Sex, body mass index, and coronary risk factors were not significantly different between the two groups. In the NOGA findings, the presence of intense yellow plaques and ruptured plaques was not significantly different between the two groups. Aortic thrombi were more frequent in the AF group than in the non-AF group (92.0 vs. 71.6%, p < 0.001). Multivariate logistic regression found that AF was independently associated with aortic thrombi (odds ratio 3.87 [95% CI 1.28-11.6], p = 0.016). The presence of aortic thrombi observed using NOGA was associated with AF in patients with coronary artery disease. The roles of aortic thrombi as well as cardiogenic embolism may require clarification.

Mechanisms and Therapeutic Strategies to Reverse TET2 Mutant Clonal Hematopoiesis and the Risk of MDS, AML, and Atherosclerotic Cardiovascular Disease

Inactivating mutations of the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). These mutations in hematopoietic stem and progenitor cells (HSPCs) impart a clonal advantage with increased self-renewal. CHIP is associated with an increased risk of hematological malignancies and, surprisingly, with atherosclerotic cardiovascular disease, supported by studies in mice and humans. The heightened cardiovascular risk in CHIP patients may result from a pro-inflammatory slant of the mutant HSPC progeny, particularly monocytes and macrophages, independent from the risk imparted by elevated LDL cholesterol levels. Targeting molecules like IL-1β may provide a strategy to lower the risk of cardiovascular morbidity in patients with CHIP. However, as an alternative strategy, administration of drugs that selectively suppress the growth of mutant CHIP clones in the bone marrow (BM) might reduce the risk of both i) progression to MDS or hematologic malignancy and of ii) atherosclerotic cardiovascular disease. This study focused on identifying drugs that reverse the inflammatory and atherosclerotic properties of TET2-mutant macrophages. Our previous investigations demonstrated that the nuclear export inhibitor eltanexor selectively kills Tet2-mutant HSPCs in zebrafish embryos and reduces the number of mutant colonies in murine colony formation assays. A murine competitive repopulation model of CHIP tested the in vivo efficacy of eltanexor. We found that eltanexor selectively reduces Tet2-mutant circulating monocytes while having no effect on overall total white blood cell counts. Moreover, a second trial using Ldlr-/- atherosclerosis-prone mice consuming a high-cholesterol diet, showed that treatment with eltanexor selectively reduces the amount of aortic atherosclerotic plaque formation as well as spleen weight in mice injected with Tet2+/- (mutant) BM cells, whereas eltanexor had no significant effect in mice injected with Tet2+/+ (wild-type) BM cells. Single-cell CITE-seq analysis of Ldlr-/- mice injected with a mix of Tet2+/- and Tet2+/+ BM cells showed that eltanexor selectively reduces the percentage of Tet2+/- HSPCs. Additionally, CITE-seq analysis revealed a significant reduction in pro-inflammatory macrophages in the arterial wall after eltanexor treatment, along with a decrease in IL-1β expression by these cells. As TET2 functions in mechanistically regulating gene regulation through epigenetic DNA methylation, we employed ChIP-seq and CUT&amp;RUN-Seq technologies to investigate the mechanisms underlying enhancer dysregulation caused by loss of Tet2. Enhancer profiling of bone marrow-derived macrophages showed that the negative regulator of the macrophage inflammatory response, ATF3, is associated with a super-enhancer. Notably, the ATF3 super-enhancer emerged as the largest super-enhancer in wild-type macrophages but was reduced in Tet2-mutant macrophages. Our investigation also revealed that ATF3 binds with IL-1β at H3K27ac modified regions, and this binding was significantly diminished in Tet2-mutant macrophages. Remarkably, treatment with eltanexor restored the binding of ATF3 to IL1β, providing a mechanism to explain the anti-inflammatory effect of eltanexor concordant with our in vivo results. In summary, this study proposes eltanexor treatment as a novel therapeutic approach to specifically target TET2-mutant cells in individuals with CHIP and sheds light on the underlying mechanisms of the pro-inflammatory slant of TET2-mutant macrophages. These findings offer potential new avenues for precise therapies in humans with CHIP and associated cardiovascular risks from atherosclerosis.

Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway

BackgroundCircZBTB46 has been identified as being associated with the risk of coronary artery disease (CAD) and has the potential to be a diagnostic biomarker for CAD. However, the specific function and detailed mechanism of circZBTB46 in CAD are still unknown.MethodsThe expression levels and properties of circRNAs were examined using qRT‒PCR, RNA FISH, and subcellular localization analysis. ApoE−/− mice fed a high-fat diet were used to establish an atherosclerosis model. HE, Masson, and Oil Red O staining were used to analyze the morphological features of the plaque. CCK-8, Transwell, and wound healing assays, and flow cytometric analysis were used to evaluate cell proliferation, migration, and apoptosis. RNA pull-down, silver staining, mass spectrometry analysis, and RNA-binding protein immunoprecipitation (RIP) were performed to identify the interacting proteins of circZBTB46.ResultsCircZBTB46 is highly conserved and is significantly upregulated in atherosclerotic lesions. Functional studies revealed that knockdown of circZBTB46 significantly decreased the atherosclerotic plaque area, attenuating the progression of atherosclerosis. In addition, silencing circZBTB46 inhibited cell proliferation and migration and induced apoptosis. Mechanistically, circZBTB46 physically interacted with hnRNPA2B1 and suppressed its degradation, thereby regulating cell functions and the formation of aortic atherosclerotic plaques. Additionally, circZBTB46 was identified as a functional mediator of PTEN-dependent regulation of the AKT/mTOR signaling pathway and thus affected cell proliferation and migration and induced apoptosis.ConclusionOur study provides the first direct evidence that circZBTB46 functions as an important regulatory molecule for CAD progression by interacting with hnRNPA2B1 and regulating the PTEN/AKT/mTOR pathway.

Deletion of Elovl5 leads to dyslipidemia and atherosclerosis in LDLR-deficient mice

Atherosclerosis is a chronic inflammatory disease for which hepatic steatosis and atherogenic dyslipidemia are significant risk factors. We investigated the effects of endogenously generated very-long-chain polyunsaturated fatty acids (VL-PUFAs) on dyslipidemia and atherosclerosis development using mice that lack ELOVL5, a PUFA elongase that is required for the synthesis of arachidonic acid, EPA, and DHA from the essential fatty acids linoleic and linolenic acids, and the LDL receptor (LDLR). Elovl5−/−;Ldlr−/− mice manifest increased liver triglyceride and cholesterol concentrations due to the activation of sterol regulatory element binding protein-1, a transcription factor that activates enzymes required for de novo lipogenesis. Plasma levels of triglycerides and cholesterol in VLDL, IDL, and LDL were markedly elevated in Elovl5−/−;Ldlr−/− mice fed a chow and the mice exhibited marked aortic atherosclerotic plaques. Bone marrow-derived monocytes from wild-type (WT) and Elovl5−/− mice were polarized to M1 and M2 macrophages, and the effects of ELOVL5 on inflammatory activity were determined. There were no differences in most of the markers tested for M1 and M2 polarized cells between WT and Elovl5−/− cells, except for a slight increase in PGE2 secretion in Elovl5−/− cells, likely due to elevated Cox-2 expression. These results suggest that the deletion of Elovl5 leads to hepatic steatosis and dyslipidemia, which are the major factors in severe atherosclerosis in Elovl5−/−;Ldlr−/− mice.

Abstract 12048: Aortic Vulnerable Plaques Including Cholesterol Crystals: Non-Obstructive General Angioscopy and Pathological Study

Introduction: Severe atherosclerosis of the aorta is known to be a risk of embolism to peripheral organs such as cerebral infarction and acute lower limb ischemia. Non-obstructive general angioscopy (NOGA) has high spatial and temporal resolution, and it is possible to dynamically observe various atherosclerosis in the aorta. Cholesterol crystals (CCs) which could be observed by NOGA scattered into the blood from ruptured plaques and caused inflammation. However, which NOGA-derived aortic plaques are high risk has not fully investigated yet. Aims: The purpose of our study was to evaluate aortic vulnerable plaques based on their frequency of scattering CCs. Methods: We investigated 52 consecutive patients with coronary artery disease who underwent NOGA between September 2021 and May 2023. Blood specimens were taken from a total of 118 NOGA-derived atheromatous plaques in the infrarenal abdominal aorta. Then, presence of CCs at each plaque was evaluated with polarized light microscopy using the filter paper rinse method. Results: The mean age of 52 patients in this study was 67.4 years, 82.7% were male, 36.5% had diabetes, 76.9% had dyslipidemia, and 82.7% had hypertension. CCs were detected in 28.8% (n=34/118) of the samples. The frequency of detection of CCs in aortic plaques was 52.5% for Puff Chandelier Rupture (PCR), 15.4% for Puff Rupture (PR), 4.5% for yellow plaques (YP), 0% for thrombi, and 0% for ulcer and fissure. P &amp;lt; 0.0001). Conclusions: Among aortic atherosclerotic plaques, many CCs were detected from PCR. PCR may be a vulnerable plaque that has a high impact on peripheral organs.

Can the Abdominal Aortic Atherosclerotic Plaque Index Predict Functional Outcomes after Robot-Assisted Partial Nephrectomy?

This study aims to evaluate the abdominal aortic atherosclerotic plaque index (API)'s predictive role in patients with pre-operatively or post-operatively developed chronic kidney disease (CKD) treated with robot-assisted partial nephrectomy (RAPN) for renal cell carcinoma (RCC). One hundred and eighty-three patients (134 with no pre- and post-operative CKD (no CKD) and 49 with persistent or post-operative CKD development (post-op CKD)) who underwent RAPN between January 2019 and January 2022 were deemed eligible for the analysis. The API was calculated using dedicated software by assessing the ratio between the CT scan atherosclerotic plaque volume and the abdominal aortic volume. The ROC regression model demonstrated the influence of API on CKD development, with an increasing effect according to its value (coefficient 0.13; 95% CI 0.04-0.23; p = 0.006). The Model 1 multivariable analysis of the predictors of post-op CKD found that the following are independently associated with post-op CKD: Charlson Comorbidity Index (OR 1.31; p = 0.01), last follow-up (FU) Δ%eGFR (OR 0.95; p < 0.01), and API ≥ 10 (OR 25.4; p = 0.01). Model 2 showed API ≥ 10 as the only factor associated with CKD development (OR 25.2; p = 0.04). The median follow-up was 22 months. Our results demonstrate API to be a strong predictor of post-operative CKD, allowing the surgeon to tailor the best treatment for each patient, especially in those who might be at higher risk of CKD.

Wnt signaling pathway and sclerostin in the development of atherosclerosis and vascular calcification.

Atherosclerosis is a complex process involving endothelial dysfunction, vascular inflammation, vascular smooth muscle cell (VSMC) proliferation, angiogenesis, and calcification. One of the pathomechanisms of atherosclerosis is the upregulation of Wnt signaling. This study aimed to summarize the current knowledge regarding the role of Wnt signaling and sclerostin in atherosclerosis, vascular calcification, aneurysms, and mortality based on the PubMed database. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendation and identified 160 papers that were included in this systematic review. The published data highlight that the upregulation of Wnt components facilitates the initiation and progression of atherosclerosis, arterial remodeling, VSMCs proliferation and phenotypic transition to the osteoblastic lineage in the arterial wall. This results in protein secretion, cell migration, calcification, fibrosis and aneurysm formation. The transformation of VSMCs into osteoblast-like cells that is observed in atherosclerosis results in sclerostin expression inhibiting the Wnt pathway. Furthermore, it was shown that sclerostin, expressed in atherosclerotic plaques, inhibits aneurysm formation in a mouse model. However, in humans, while the antisclerostin antibody romosozumab inhibits bone resorption, biochemical parameters of endothelial activation and inflammation are not affected, and the incidence of aneurysms is not increased. It was suggested that detecting sclerostin in the calcified aortic atherosclerotic plaques reflects a defense mechanism against Wnt activation and inhibition of atherosclerosis, although this has only been shown in animal models. Moreover, an increased number of vascular cells converted to osteogenic phenotypes results in increased plasma sclerostin concentrations. Therefore, plasma sclerostin derived from bone limits its importance as a global marker of vascular calcification.

Allogeneic Adipose-Derived Mesenchymal Stem Cell Transplantation Alleviates Atherosclerotic Plaque by Inhibiting Ox-LDL Uptake, Inflammatory Reaction and Endothelial Damage in Rabbits.

Atherosclerosis (AS) is a chronic inflammatory disease of arteries fueled by lipids. It is a major cause of cardiovascular morbidity and mortality. Mesenchymal stem cells have been used for the treatment of atherosclerotic lesions. Adipose-derived stem cells (ADSCs) have been shown to regulate the activation state of macrophages and exhibit anti-inflammatory capabilities. However, the effect of allogeneic ADSCs in the treatment of AS have not been investigated. In this study, the early treatment effect and preliminary mechanism analysis of allogeneic rabbit ADSCs intravenous transplantation were investigated in a high-fat diet rabbit model. The polarization mechanism of rabbit ADSCs on the macrophage was further analyzed in vitro. Compared with the model group, blood lipid levels declined, the plaque area, oxidized low-density lipoprotein (ox-LDL) uptake, scavenger receptor A1 and cluster of differentiation (CD) 36 levels were all significantly reduced, and the accumulation of inflammatory M1 macrophages, apoptosis, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression were decreased. The endothelial cells (CD31), M2 macrophages, IL-10 and the transforming growth factor (TGF)-β levels increased. In vitro, ADSCs can promote the M1 macrophage phenotypic switch toward the M2 macrophage through their secreted exosomes, and the main mechanism includes increasing arginase 1 expression and IL-10 secretion, declining inducible nitric oxide synthase (iNOS) expression and TNF-α secretion, and activating the STAT6 pathway. Therefore, allogeneic rabbit ADSC transplantation can transmigrate to the aortic atherosclerotic plaques and show a good effect in lowering blood lipids and alleviating atherosclerotic plaque in the early stage of AS by inhibiting ox-LDL uptake, inflammatory response, and endothelial damage.

Hyperuricemia promotes the progression of atherosclerosis by activating endothelial cell pyroptosis via the ROS/NLRP3 pathway.

Hyperuricemia closely correlates with the development of atherosclerosis, but little is known of the mechanism by which atherosclerosis progression occurs in hyperuricemia. Atherosclerosis appears to involve pyroptosis, an emerging mechanism of proinflammatory regulated cell death. This study tested the hypothesis that pyroptosis underlies the relationship between hyperuricemia and atherosclerosis, using ApoE-/- mice (a model of atherosclerosis), human umbilical vein endothelial cells (HUVECs), and human atherosclerotic arterial samples. We found that hyperuricemia can aggravate the aortic atherosclerotic plaque-load in ApoE-/- mice and promote endothelial cell pyroptosis. Additionally, hyperuricemia can increase the levels of serum inflammatory factors (including IL-1β and IL-18). Exposure to lipopolysaccharideplus a high concentration of soluble uric acid (≥12 mg/dL) induced cell pyroptosis in HUVECs, as evidenced by increased expression of pyroptosis-related proteins and elevated release of lactate dehydrogenase (a marker of tissue damage). Further, MCC950, a selective nucleotide-binding oligomerization domain (NOD)-like receptor 3(NLRP3) inflammasome inhibitor, and N-acetyl- l-cysteine, an antioxidant, attenuated HUVEC pyroptosis by inhibiting activation of the NLRP3 inflammasome and production of intracellular reactive oxygen species (ROS). Finally, we detected significantly higher expression of pyroptosis-associated proteins in carotid specimens from patients with hyperuricemia. Collectively, our findings suggest that hyperuricemia can aggravate endothelial cell pyroptosis in aortic atherosclerotic plaques, promoting the development of atherosclerosis. Additionally, a high concentration of soluble uric acid can trigger the activation stage of the NLRP3 inflammasome, mediating endothelial cell pyroptosis, and this process is regulated by the cellular ROS level.

Thoracic aortic atherosclerosis in patients with a bicuspid aortic valve; a case–control study

IntroductionBicuspid aortic valve (BAV) patients have an increased risk to develop thoracic aortic complications. Little is known about the prevalence and severity of atherosclerosis in the BAV ascending aortic wall. This study evaluates and compares the prevalence of thoracic aortic atherosclerosis in BAV and tricuspid aortic valve (TAV) patients.MethodsAtherosclerosis was objectified using three diagnostic modalities in two separate BAV patient cohorts (with and without an aortic dilatation). Within the first group, atherosclerosis was graded histopathologically according to the modified AHA classification scheme proposed by Virmani et al. In the second group, the calcific load of the ascending aorta and coronary arteries, coronary angiographies and cardiovascular risk factors were studied. Patients were selected from a surgical database (treated between 2006–2020), resulting in a total of 128 inclusions.ResultsHistopathology showed atherosclerotic lesions to be more prevalent and severe in all TAV as compared to all BAV patients (OR 1.49 (95%CI 1.14 – 1.94); p = 0.003). Computed tomography showed no significant differences in ascending aortic wall calcification between all BAV and all TAV patients, although a tendency of lower calcific load in favor of BAV was seen. Coronary calcification was higher in all TAV as compared to all BAV (OR 1.30 (95%CI 1.06 – 1.61); p = 0.014).ConclusionAscending aortic atherosclerotic plaques were histologically more pronounced in TAV as compared to the BAV patients, while CT scans revealed equal amounts of calcific depositions within the ascending aortic wall. This study confirms less atherosclerosis in the ascending aortic wall and coronary arteries of BAV patients as compared to TAV patients. These results were not affected by the presence of a thoracic aortic aneurysm.

TREM2 promotes cholesterol uptake and foam cell formation in atherosclerosis.

Disordered lipid accumulation in the arterial wall is a hallmark of atherosclerosis. Previous studies found that the expression of triggering receptor expressed on myeloid cells 2 (TREM2), a transmembrane receptor of the immunoglobulin family, is increased in mouse atherosclerotic aortic plaques. However, it remains unknown whether TREM2 plays a role in atherosclerosis. Here we investigated the role of TREM2 in atherosclerosis using ApoE knockout (ApoE-/-) mouse models, primary vascular smooth muscle cells (SMCs), and bone marrow-derived macrophages (BMDMs). In ApoE-/- mice, the density of TREM2-positive foam cells in aortic plaques increased in a time-dependent manner after the mice were fed a high-fat diet (HFD). Compared with ApoE-/- mice, the Trem2-/-/ApoE-/- double-knockout mice showed significantly reduced atherosclerotic lesion size, foam cell number, and lipid burden degree in plaques after HFD feeding. Overexpression of TREM2 in cultured vascular SMCs and macrophages exacerbates lipid influx and foam cell formation by upregulating the expression of the scavenger receptor CD36. Mechanistically, TREM2 inhibits the phosphorylation of p38 mitogen-activated protein kinase and peroxisome proliferator activated-receptor gamma (PPARγ), thereby increasing PPARγ nuclear transcriptional activity and subsequently promoting the transcription of CD36. Our results indicate that TREM2 exacerbates atherosclerosis development by promoting SMC- and macrophage-derived foam cell formation by regulating scavenger receptor CD36 expression. Thus, TREM2 may act as a novel therapeutic target for the treatment of atherosclerosis.

Rosuvastatin acts on the lymphatic system to improve atherosclerosis

Objective: To investigate whether rosuvastatin acts on lymphatic system and influences lymphatic system-mediated reverse cholesterol transport to play an anti-atherosclerosis role. Methods: Forty-eight apolipoprotein E-/- mice fed a high fat diet were used to construct the atherosclerosis model. They were randomly divided into 4 groups with 12 rats in each group. They were treated with rosuvastatin, vascular endothelial growth factor-C (VEGF-C) and rosuvastatin+VEGF-C inhibitors as experimental group, and no intervention measures were given in control group. After 8 weeks, aortic plaque area, high density lipoprotein cholesterol (HDL-C) content in lymph fluid, the function of popliteal lymphatic drainage of peripheral Evans blue, and the ability of lymphatic system to transport peripheral cell membrane red fluorescent probes to label high-density lipoprotein (HDL) were detected. Subsequently, the effects of rosuvastatin on proliferation, migration and tubular function of lymphoendothelial cells and the expression of scavenger receptor class B type 1 (SR-B1) on lymphoendothelial cells at different concentrations were detected. Results: Compared with the control group, Rosuvastatin and VEGF-C could reduce the area of aortic atherosclerotic plaque (P<0.05). In addition to rosuvastatin plus VEGF-C inhibitor, the intra-aortic plaque area increased (P<0.05). Compared with the control group, Rosuvastatin could increase the content of HDL-C in lymphatic fluid (P<0.05), enhance the drainage function of lymphatic vessels, and enhance the capacity of HDL in the transport tissue fluid of lymphatic system. Compared with the control group, VEGF-C increased the content of HDL-C in mouse lymph fluid (P<0.01), enhanced the drainage function of popliteal lymphatic canal, and enhanced the ability of lymphatic system to transport HDL. With the addition of VEGF-C inhibitor on the basis of rosuvastatin, the content of HDL-C in lymph fluid was reduced, the drainage of popliteal lymphatic canal was interrupted, and the ability of lymphatic system to transport HDL was reduced. Western blotting showed that rosuvastatin increased the protein expression of SR-B1. Conclusion: Rosuvastatin can promote the proliferation, migration and tube formation of lymphatic endothelial cells. At the same time, SR-B1 expression on lymphatic endothelial cells is promoted, thus enhancing the lymphatic system mediated cholesterol reversal transport and playing the role of anti-atherosclerosis.

Abstract 13392: 3D TEE Aids in Assessment of Embolic Stroke Due to Aortic Atherosclerotic Plaque

Case Presentation: A 69-year-old right-handed man presented with new left-sided weakness and recurrent ischemic strokes. His had history of coronary artery disease, diabetes mellitus, hypertension, dyslipidemia, chronic kidney disease stage 3, obstructive sleep apnea and obesity. Plasma low-density lipoprotein was 127 mg/dL. Brain MRI showed diffusion restriction in the left caudate head, left corona radiata, left anterior lentiform nucleus, and left periventricular white matter from acute to subacute infarction. CT angiogram showed diffuse atheromatous disease in the anterior and posterior circulation, bilateral mild cervical internal carotid artery stenosis, and calcifications in the aortic arch ( Figure 1 ). Transthoracic echocardiogram (TTE) showed mild generalized left ventricular hypokinesis (ejection fraction 45%) without thrombus. Transesophageal echocardiogram (TEE) showed complex mobile atherosclerotic plaques in the aortic arch ( Figure 2 ) after which Apixaban &amp; Clopidogrel were started and statins increased. At 1-year follow-up there was no recurrent stroke. Discussion: This case highlights the role of 3D TEE in the detection of complex aortic arch atherosclerotic plaque with mobile components as the etiology of recurrent embolic stroke. 2D TTE &amp; TEE and CT chest were unable to show mobile components in the aortic arch plaque. Many patients identified as having “cryptogenic stroke” after 2D TTE &amp; TEE may in fact have mobile aortic arch plaques which can only be delineated by 3D TEE, thus reducing the need for further workup and expediting appropriate treatment.

Role of CD40 ligand-mediated endothelial cell-monocyte interaction at atherosclerosis predilection sites

BackgroundMonocyte recruitment into the vessel wall at atherosclerosis predilection sites is essential for lesion development in the early phase of atherosclerosis. Platelets interacting with ultra-large von Willebrand Factor (ULVWF) multimers deposited after CD40 receptor ligation on the endothelial surface form adhesive bridges and facilitate monocyte diapedesis. We hypothesise that enhanced endothelial CD40 expression at arterial bifurcations is responsible for monocyte recruitment and that its absence reduces susceptibility to atherosclerosis. MethodsY-shaped channel slides covered with endothelial cells (HUVEC) and isolated perfused carotid artery bifurcations from different mouse lines were used for adhesion studies with isolated fluorescent dye-labelled platelets and monocytes. Monocyte adherence was quantified via fluorescence imaging. Oil Red O staining visualised aortic atherosclerotic plaques, and mRNA expression was determined by qRT-PCR. ResultsIn response to soluble CD40 ligand (sCD40L) stimulated ULVWF release, the number of monocytes bound distal to the bifurcation of the Y-slide was 1.8-fold greater than without stimulation. The number of adherent monocytes in sCD40L-treated carotid artery bifurcations was 6 to 12.3-fold greater in ApoE knockout mice as compared to bifurcations derived from CD40/ApoE-deficient or control mice. CD40 mRNA expression was 2-fold higher in carotid artery bifurcations of ApoE knockout mice as compared to the proximal unbranched segment. Introduction of the CD40 knockout into the ApoE-/- background reduced the atherosclerosis burden along the entire aorta of these mice by 60 %. ConclusionsOur data demonstrate the importance of endothelial CD40 expression at atherosclerosis predilection sites for endothelial cell-platelet-monocyte interaction in the early phase of atherosclerosis.

Neutrophils as major drivers of increased atherosclerosis in a murine model of chronic colitis

Abstract Background Recent findings showed a higher risk of premature atherosclerosis and cardiovascular events in patients with inflammatory bowel disease (IBD) especially during acute flare of the chronic disease. The underlying mechanisms remain to be defined since traditional risk factors such as hypercholesterinemia are not present in these patients. Purpose The present study aimed to unravel the underlying pathomechanisms of enhanced atherogenesis and -progression in a murine model of colitis. Methods Chronic colitis was induced in male Apolipoprotein E-deficient mice (Apoe−/−) using dextran sodium sulphate (DSS) in drinking water for 2,3 or 5 cycles, while the control group received regular drinking water. Each cycle consisted of 6 days DSS application and two weeks of recovery. Aortic atherosclerotic plaque burden was determined by en face Oil Red O staining and immune cell subsets were analysed by flow cytometry in the circulation, the bone marrow, and the aorta. Neutrophil depletion was performed via i.p injection of a Ly6G-depleting antibody or respective isotype control. Bone marrow-derived neutrophils were further analysed by transcriptome analysis. Results Similar to IBD patients, mice with chronic colitis exhibited an increased aortic plaque burden after 15 weeks of treatment despite the absence of classical risk factors. Over time, both circulating and aortic neutrophils showed an oscillatory detection pattern with the first significant increase after the 2nd DSS administration whereby the second increase after the 3rd DSS cycle was even stronger. Also, pro-inflammatory cytokines were elevated in the plasma and specifically G-CSF showed the same oscillatory pattern with increased plasma level already after the 2nd DSS administration and an even stronger increase after the 3rd thereby pointing towards alterations in bone marrow hematopoiesis. In line, flow cytometric analyses confirmed a greater rise of hematopoietic stem and even myeloid progenitor cells compared with the 2nd DSS application in IBD mice after 3rd treatment. scRNA-Seq analysis of progenitor cells revealed changes in cell differentiation towards neutrophils and upregulation of proinflammatory genes in isolated neutrophils of DSS-treated mice. These neutrophils showed also a more adhesive phenotype revealed by increased mRNA expression of Glg1 and Selplg. Accordingly, also Sele mRNA was increased in the aorta. The reduction of circulating neutrophils by an anti-Ly6G antibody during the acute phases of colitis reduced the aortic plaque burden compared to isotype treaded mice. Conclusion The current findings suggest detrimental effects of chronic colitis on atherogenesis and -progression in Apoe−/− mice via increased differentiation of myeloid cells into neutrophils and the promotion of a more adhesive and proinflammatory phenotype. These modified neutrophils may act as initiators of atherogenesis by promoting the invasion of immune cells into the aortic wall. Funding Acknowledgement Type of funding sources: None.

A dual Keap1 and p47phox inhibitor Ginsenoside Rb1 ameliorates high glucose/ox-LDL-induced endothelial cell injury and atherosclerosis.

Oxidative stress is a vital contributor to the development and progression of diabetes-accelerated atherosclerosis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a well-known molecule that participates in cellular defense against oxidative stress. Utilizing luciferase reporter assay from 379 natural products, we reported here that Ginsenoside Rb1 played a dual role in inhibiting Kelch-like ECH-associated protein 1 (Keap1) and p47phox luciferase reporter activities. In endothelial cells (ECs), Rb1 pretreatment enhanced cell viability, reduced oxidative stress, inflammation, endothelial-mesenchymal transition (EndMT), and apoptosis, as well as ameliorated mitochondrial quality following oxidized low-density lipoprotein (ox-LDL) plus high glucose (HG) challenge. Rb1 directly bound to Keap1 and promoted its ubiquitination and proteasomal degradation dependent on lysine residues (K108, K323, and K551) by recruiting the E3 ligase synovial apoptosis inhibitor 1 (SYVN1), leading to Nrf2 dissociation from Keap1, Nrf2 nuclear translocation, Nrf2/PGC-1α complex formation. We further identified that Rb1 could bind to p47phox and reduce its phosphorylation and membrane translocation, thereby disrupting the assembly of the NOX2 complex. Importantly, Rb1-mediated preservation of cytoplasmic p47phox stabilized and contributed to Nrf2 activation. Additionally, we revealed that Rb1 reduced aortic atherosclerotic plaque formation along with reductions in oxidative stress and inflammatory response in streptozotocin (STZ)-induced ApoE−/− mice, but not in ApoE−/− mice with deficiency of Nrf2 and PGC-1α. Collectively, we demonstrated that Rb1, which directly targeted Keap1 and p47phox in ECs, may be an attractive candidate for the treatment of atherosclerosis in diabetes.