Circulating Exosomal circRNAs Contribute to Potential Diagnostic Value of Large Artery Atherosclerotic Stroke

Large artery atherosclerotic (LAA) stroke is closely associated with atherosclerosis, characterized by the accumulation of immune cells. Early recognition of LAA stroke is crucial. Circulating exosomal circRNAs profiling represents a promising, noninvasive approach for the detection of LAA stroke. Exosomal circRNA sequencing was used to identify differentially expressed circRNAs between LAA stroke and normal controls. From a further validation stage, the results were validated using RT-qPCR. We then built logistic regression models of exosomal circRNAs based on a large replication stage, and receiver operating characteristic (ROC) curves were constructed to assess the diagnostic efficacy. Using exosomal circRNA sequencing, large sample validation, and diagnostic model construction revealed that exosomal circ_0043837 and circ_ 0001801were independent predictive factors for LAA stroke, and had better diagnostic efficacy than plasma circRNAs. In the atherosclerotic group (AS), we developed a nomogram for clinical use that integrated the two-circRNA-based risk factors to predict which patients might have the risk of plaque rupture. Circulating exosomal circRNAs profiling identifies novel predictive biomarkers for the LAA stroke and plaque rupture, with superior diagnostic value than plasma circRNAs. It might facilitate the prevention and better management of this disease.

Immune Mechanisms of Plaque Instability

Inflammation crucially drives atherosclerosis from disease initiation to the emergence of clinical complications. Targeting pivotal inflammatory pathways without compromising the host defense could compliment therapy with lipid-lowering agents, anti-hypertensive treatment, and lifestyle interventions to address the substantial residual cardiovascular risk that remains beyond classical risk factor control. Detailed understanding of the intricate immune mechanisms that propel plaque instability and disruption is indispensable for the development of novel therapeutic concepts. In this review, we provide an overview on the role of key immune cells in plaque inception and progression, and discuss recently identified maladaptive immune phenomena that contribute to plaque destabilization, including epigenetically programmed trained immunity in myeloid cells, pathogenic conversion of autoreactive regulatory T-cells and expansion of altered leukocytes due to clonal hematopoiesis. From a more global perspective, the article discusses how systemic crises such as acute mental stress or infection abruptly raise plaque vulnerability and summarizes recent advances in understanding the increased cardiovascular risk associated with COVID-19 disease. Stepping outside the box, we highlight the role of gut dysbiosis in atherosclerosis progression and plaque vulnerability. The emerging differential role of the immune system in plaque rupture and plaque erosion as well as the limitations of animal models in studying plaque disruption are reviewed.

CD74 in Apoptotic Macrophages Is Associated with Inflammation, Plaque Progression and Clinical Manifestations in Human Atherosclerotic Lesions

The aim of this study was to investigate whether CD74 levels in atherosclerotic lesions are associated with inflammation, apoptosis, plaque severity, and clinical symptoms among patients with carotid atherosclerosis. We further studied whether CD74 expression is associated with apoptosis in macrophages induced by 7ketocholesterol (7keto). Sixty-one carotid samples (39 males and 22 females) were immunostained with macrophages, smooth muscle cells, CD74, ferritin, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), and thrombin receptors. Double immunocytochemistry of CD74 and caspase 3 or CD74 and Annexin V was performed on THP-1 macrophages exposed to 7keto. In human carotid plaques, CD74 expression is lesion-dependently increased and is associated with necrotic core formation and plaque rupture, clinical symptoms, macrophage apoptosis, ferritin, and thrombin receptors. CD74 levels were inversely correlated to high-density lipoproteins and statin treatment, and positively correlated to triglycerides. In THP-1 macrophages, 7keto induced a significant increase in levels of CD74, ferritin, and apoptotic cell death. This study suggests that CD74 in apoptotic macrophages is linked to inflammation and thrombosis in progression of human atherosclerotic plaques, lipid metabolism, and clinical manifestation in atherosclerosis. Surface CD74 in apoptotic macrophages and ferritin production induced by oxidized lipids may contribute to inflammation and plaque vulnerability in atherosclerosis.

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Identification of Carotid Artery Microstructure and Plaque Rupture Using C-Arm Cone-Beam CT: A Case Report

C-arm cone-beam computed tomography (CBCT) offers a high imaging resolution with a wide range of contrast to visualize vessels, soft tissue, and bone. We report the usefulness of CBCT in observing neovascularization, microcalcification, and plaque rupture. A 56-year-old man presented with vertigo and complain of an unsteady gait for 5 months. Catheter angiography demonstrated right severe carotid stenosis with irregular filling defect, which on high-resolution MRI showed vessel wall enhancement. The CBCT showed high density structures and linear contrast enhancement from the vascular lumen to the plaque, related to microstructure and plaque rupture. Carotid endarterectomy was performed, and histopathology confirmed that the high-density areas represented neovascularization and microcalcification, with linear enhancement representing plaque rupture. This is the first report showing that microcalcifications and plaque rupture can be identified by CBCT. Thus, CBCT can be used as a promising supplement to current imaging modalities to evaluate plaque components more accurately.

NR1D1 Deletion Induces Rupture-Prone Vulnerable Plaques by Regulating Macrophage Pyroptosis via the NF-κB/NLRP3 Inflammasome Pathway

Vulnerable plaque rupture is the main trigger of most acute cardiovascular events. But the underlying mechanisms responsible for the transition from stable to vulnerable plaque remain largely unknown. Nuclear receptor subfamily 1 group D member 1 (NR1D1), also known as REV-ERB α, is a nuclear receptor that has shown the protective role in cardiovascular system. However, the effect of NR1D1 on vulnerable plaque rupture and its underlying mechanisms are still unclear. By generating the rupture-prone vulnerable plaque model in hypercholesterolemic ApoE−/− mice and NR1D1−/−ApoE−/− mice, we demonstrated that NR1D1 deficiency significantly augmented plaque vulnerability/rupture, with higher incidence of intraplaque hemorrhage (78.26% vs. 47.82%, P = 0.0325 ) and spontaneous plaque rupture with intraluminal thrombus formation (65.21% vs. 39.13%, P = 0.1392 ). In vivo experiments indicated that NR1D1 exerted a protective role in the vasculature. Mechanically, NR1D1 deficiency aggravates macrophage infiltration, inflammation, and oxidative stress. Compared with the ApoE−/− mice, NR1D1−/−ApoE−/− mice exhibited a significantly higher expression level of pyroptosis-related genes in macrophages within the plaque. Further investigation based on mice bone marrow-derived macrophages (BMDMs) confirmed that NR1D1 exerted a protective effect by inhibiting macrophage pyroptosis in a NLRP3-inflammasome-dependent manner. Besides, pharmacological activation of NR1D1 by SR9009, a specific NR1D1 agonist, prevented plaque vulnerability/rupture. In general, our findings provide further evidences that NR1D1 plays a protective role in the vasculature, regulates inflammation and oxidative stress, and stabilizes rupture-prone vulnerable plaques.

Biomarkers of Inflammation and Oxidative stress in the Prediction and Management of Acute Coronary Syndrome

The assessment of patients presenting with chest pain or symptoms indicative of cardiac ischemia remains a diagnostic challenge. Many types of research have focused on the search for ideal biological markers for the rapid detection of cardiac cell injuries. Markers of inflammation and oxidative stress are the way forward. At present, the biomarker most widely used for diagnosing acute coronary syndrome is cardiac troponin though it has some limitations. Apart from cardiac troponin, several other biomarkers, especially inflammation and oxidative stress markers in acute coronary syndrome, have been investigated. However, most of them still require validation in further studies. As markers of inflammation and oxidative stress address a particular aspect of the pathophysiology of acute coronary syndrome, these biomarkers may provide unique information to the managing clinician separate from that of markers of myocyte necrosis. Serum markers of inflammation and oxidative stress appear before cardiac necrosis markers and are valuable targets for early and timely diagnosis of an acute cardiac event. Using these markers in combination with biomarkers of plaque formation, unstable plaque development, plaque rupture, thrombosis, and myocardial necrosis (multimarker approach) could increase their diagnostic and prognostic value.

363 Old but fashioned: IVUS and chromaflo guidance for definition of thrombosis mechanism

Aims Plaque rupture and plaque erosion are the main causes of coronary thrombosis. While the first one involves fibrous cap disruption, the second one is caused by loss of endothelial continuity. In selected cases with evidence of plaque erosion, antithrombotic therapy without stenting has been suggested as a possible option. OCT is considered the gold standard for definition of thrombosis mechanism and has recently been included in algorithms for evaluation and management of patients with ACS. Also, high definition IVUS was compared with OCT in defining plaque erosion showing promising results. However, the cost and the large amount of contrast medium needed for OCT performance make these diagnostic tools of scarce applicability in daily practice. Methods and results We herein describe the case of a young man acceding to the Cath Lab with the diagnosis of NSTEMI. After baseline angiography and IVUS confirmed presence of Thrombus (Figure 1A and B), thromboaspiration was successfully performed (Figure 1D). The definition of thrombosis mechanism, revealing plaque rupture, was then performed with IVUS and ChromaFlo devices (Figure 1C and E). Also, IVUS was used to optimize stent implantation. Conclusions Although requiring further confirmations, we believe that in selected cases IVUS and ChromaFlo could provide a more applicable first-line diagnostic tool to define thrombosis mechanism. 363 Figure 1Baseline angiographic and IVUS evaluation confirming presence of coronary thrombus (A, B). After successful performance of thromboaspiration (D), plaque rupture was revealed by IVUS and ChromaFlo (C).