Spatial Transcriptomic Approach to Understanding Coronary Atherosclerotic Plaque Stability.

BACKGROUND: Coronary atherosclerotic plaques susceptible to acute coronary syndrome have traditionally been characterized by their surrounding cellular architecture. However, with the advent of intravascular imaging, novel mechanisms of coronary thrombosis have emerged, challenging our contemporary understanding of acute coronary syndrome. These intriguing findings underscore the necessity for a precise molecular definition of plaque stability. Considering this, our study aimed to investigate the vascular microenvironment in patients with stable and unstable plaques using spatial transcriptomics. METHODS: Autopsy-derived coronary arteries were preserved and categorized by plaque stability (n=5 patients per group). We utilized the GeoMx spatial profiling platform and Whole Transcriptome Atlas to link crucial histological morphology markers in coronary lesions with differential gene expression in specific regions of interest, thereby mapping the vascular transcriptome. This innovative approach allowed us to conduct cell morphological and spatially resolved transcriptional profiling of atherosclerotic plaques while preserving crucial intercellular signaling. RESULTS: We observed intriguing spatial and cell-specific transcriptional patterns in stable and unstable atherosclerotic plaques, showcasing regional variations within the intima and media. These regions exhibited differential expression of proinflammatory molecules (eg, IFN-γ [interferon-γ], MHC [major histocompatibility complex] class II, proinflammatory cytokines) and prothrombotic signaling pathways. By using lineage tracing through spatial deconvolution of intimal CD68+ (cluster of differentiation 68) cells, we characterized unique, intraplaque subpopulations originating from endothelial, smooth muscle, and myeloid lineages with distinct regional locations associated with plaque instability. In addition, unique transcriptional signatures were observed in vascular smooth muscle and CD68+ cells among plaques exhibiting coronary calcification. CONCLUSIONS: Our study illuminates distinct cell-specific and regional transcriptional alterations present in unstable plaques. Furthermore, we characterize spatially resolved, in situ evidence supporting cellular transdifferentiation and intraplaque plasticity as significant contributors to plaque instability in human coronary atherosclerosis. Our results provide a powerful resource for the identification of novel mediators of acute coronary syndrome, opening new avenues for preventative and therapeutic treatments.

Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

18F-Fluorodeoxyglucose-Positron Emission Tomography Imaging Detects Response to Therapeutic Intervention and Plaque Vulnerability in a Murine Model of Advanced Atherosclerotic Disease-Brief Report.

OBJECTIVE: This study sought to determine whether 18F-fluorodeoxyglucose-positron emission tomography/computed tomography could be applied to a murine model of advanced atherosclerotic plaque vulnerability to detect response to therapeutic intervention and changes in lesion stability. Approach and Results: To analyze plaques susceptible to rupture, we fed ApoE-/- mice a high-fat diet and induced vulnerable lesions by cast placement over the carotid artery. After 9 weeks of treatment with orthogonal therapeutic agents (including lipid-lowering and proefferocytic therapies), we assessed vascular inflammation and several features of plaque vulnerability by 18F-fluorodeoxyglucose-positron emission tomography/computed tomography and histopathology, respectively. We observed that 18F-fluorodeoxyglucose-positron emission tomography/computed tomography had the capacity to resolve histopathologically proven changes in plaque stability after treatment. Moreover, mean target-to-background ratios correlated with multiple characteristics of lesion instability, including the corrected vulnerability index. CONCLUSIONS: These results suggest that the application of noninvasive 18F-fluorodeoxyglucose-positron emission tomography/computed tomography to a murine model can allow for the identification of vulnerable atherosclerotic plaques and their response to therapeutic intervention. This approach may prove useful as a drug discovery and prioritization method.

Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

De Novo and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects.

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk.

CDKN2B Regulates TGFß Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels.

RATIONALE: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. OBJECTIVE: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. METHODS AND RESULTS: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor ß (TGFß) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFß1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFß activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFß1-induced-1, which is a TGFß-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. CONCLUSIONS: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFß signaling and hypoxic neovessel maturation.

Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including "vascular disease," "disorder of artery," and "occlusion of artery," as well as disease-related cellular functions including "cellular movement" and "cellular growth and proliferation." In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.

Identification and initial functional characterization of a human vascular cell-enriched long noncoding RNA.

OBJECTIVE: Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. APPROACH AND RESULTS: RNA sequencing (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA (Smooth muscle and Endothelial cell-enriched migration/differentiation-associated long NonCoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5' end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. CONCLUSIONS: SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.

Exercise mitigates reductive stress-induced cardiac remodeling in mice.

The endoplasmic reticulum (ER) regulates protein folding and maintains proteostasis in cells. We observed that the ER transcriptome is impaired during chronic reductive stress (RS) in cardiomyocytes. Here, we hypothesized that a prolonged moderate treadmill exercise mitigates the RS-induced ER dysfunction and cardiac remodeling in cardiac-specific constitutively active Nrf2 mice (CaNrf2-TG). RNA sequencing showed notable alterations in the ER transcriptome of TG hearts at 4, 12, and 24 weeks (16, 28, and 35 genes, respectively). Notably, the downregulation of ER genes was significant at 12 weeks, and further pronounced at 24 weeks, at which the cardiac pathology is evident. We also observed increased levels of ubiquitinated proteins in CaNrf2-TG hearts across all ages, along with VCP, a marker of ERAD function, at 24 weeks. These findings indicate that constitutive Nrf2 activation and RS impair protein-folding activity and augments ERAD function over time. Exercise intervention for 20 weeks (beginning at 6 weeks of age), reduced cardiomyocyte hypertrophy (from 448 µm2 to 280 µm2) in TG mice, through adaptive remodeling, and preserved the cardiac function. However, while exercise did not influence antioxidants or ER stress protein levels, it significantly improved ERAD function and autophagy flux (LC-I to LC-II) in the TG-EXE hearts. Collectively, our findings underscore the prophylactic potential of exercise in mitigating RS-associated pathology, highlighting its essential role in maintaining cellular proteostasis through ER-independent mechanisms.

Leiomodin 1, a new serum response factor-dependent target gene expressed preferentially in differentiated smooth muscle cells.

Smooth muscle cell (SMC) differentiation is defined largely by a number of cell-restricted genes governed directly by the serum response factor (SRF)/myocardin (MYOCD) transcriptional switch. Here, we describe a new SRF/MYOCD-dependent, SMC-restricted gene known as Leiomodin 1 (Lmod1). Conventional and quantitative RT-PCRs indicate that Lmod1 mRNA expression is enriched in SMC-containing tissues of the mouse, whereas its two paralogs, Lmod2 and Lmod3, exhibit abundant expression in skeletal and cardiac muscle with very low levels in SMC-containing tissues. Western blotting and immunostaining of various adult and embryonic mouse tissues further confirm SMC-specific expression of the LMOD1 protein. Comparative genomic analysis of the human LMOD1 and LMOD2 genes with their respective mouse and rat orthologs shows high conservation between the three exons and several noncoding sequences, including the immediate 5' promoter region. Two conserved CArG boxes are present in both the LMOD1 and LMOD2 promoter regions, although LMOD1 displays much higher promoter activity and is more responsive to SRF/MYOCD stimulation. Gel shift assays demonstrate clear binding between SRF and the two CArG boxes in human LMOD1. Although the CArG boxes in LMOD1 and LMOD2 are similar, only LMOD1 displays SRF or MYOCD-dependent activation. Transgenic mouse studies reveal wild type LMOD1 promoter activity in cardiac and vascular SMC. Such activity is abolished upon mutation of both CArG boxes. Collectively, these data demonstrate that Lmod1 is a new SMC-restricted SRF/MYOCD target gene.

Prediction of In-Hospital Mortality in Patients With Traumatic Brain Injury Using the Rotterdam and Marshall CT Scores: A Retrospective Study From Western India.

Objective Head trauma of any severity, including concussions and skull fractures, can cause a traumatic brain injury (TBI). Prognostication plays a vital role in the scenario of urgency put forth by TBI. The application of CT-based scoring systems developed by the Rotterdam CT score and Marshall classification system appears to be appropriate for the early and precise prediction of clinical outcomes in TBI patients. The present study was designed to determine the predictive value of the Rotterdam CT score and Marshall classification system for in-hospital mortality in patients with TBI. Methods All adult patients (≥ 18 years) with acute traumatic brain injury presented over a period from February 2019 to November 2022 were included. Only those patients who had undergone a plain CT scan of the brain during the initial presentation at the emergency department (ED) were considered. Patients who presented with penetrating brain injury as well as those who died on arrival or who died prior to the initial CT scan of the brain were excluded. A total of 127 patients were included in the final data analysis. Based on initial CT-scan findings, the Rotterdam CT score and Marshall classification system were calculated in order to predict in-hospital mortality. Results The study was dominated by male patients (85.8%) as compared to female patients (14.2%). The overall mortality rate was 32.3% (n = 41). The mortality rate among males and females was 30.3% (33/109) and 44.4% (8/18), respectively. As per the Glasgow Coma Scale (GCS) classification, the severity of the injury was mild in 12.6% of the study subjects, moderate in 22%, and severe in 65.4%. The mortality rate among the patients with mild severity was 12.5% (2/16), while it was 28.6% in moderate (8/28) and 37.3% (31/83) in the severe category group. The best cut-off point of the Rotterdam score for predicting mortality was >4 (as per the Youden Index), which had a sensitivity and specificity of 60.98% and 90.70%, respectively, while the cut-off point of the Marshall CT classification for predicting mortality was >3 (as per the Youden Index), which had a sensitivity of 82.93% and a specificity of 75.58%. There was only a minor difference in the area under the curve (AUC) value of the receiver operating characteristic curve (ROC) curve between the Rotterdam CT score (0.827) and the Marshall classification system (0.833). Conclusion The Rotterdam and Marshall CT scores have demonstrated significant independent prognostic value and may serve as a useful initial evaluation tool for risk stratification of in-hospital mortality among patients with TBI.

Transgenic Expression of Nrf2 Induces a Pro-Reductive Stress and Adaptive Cardiac Remodeling in the Mouse.

Nuclear factor, erythroid 2 like 2 (Nfe2l2 or Nrf2), is a transcription factor that protects cells by maintaining a homeostatic redox state during stress. The constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Transgenic and non-transgenic (NTG) mice at 7−8 months of age were used to analyze the myocardial transcriptome, structure, and function. Next generation sequencing (NGS) for RNA profiling and qPCR-based validation of the NGS data, myocardial redox levels, and imaging (echocardiography) were performed. Transcriptomic analysis revealed that out of 14,665 identified mRNAs, 680 were differently expressed (DEG) in TG hearts. Of 680 DEGs, 429 were upregulated and 251 were downregulated significantly (FC > 2.0, p < 0.05). Gene set enrichment analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of the glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 µM/mg protein in mNrf2-TG and CaNrf2-TG vs. NTG). Genes involved in fetal development, hypertrophy, cytoskeletal rearrangement, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG. Non-invasive echocardiography analysis revealed an increase in systolic function (ejection fraction) in mNrf2-TG, suggesting an adaptation, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e., pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

Marijuana-induced acute myocardial infarction in a young adult male.

A 27-year-old man presented to the emergency department of a tertiary care centre with complaints of acute onset breathing difficulty and retrosternal chest discomfort of 6 hours' duration. On primary survey, he was dyspnoeic with a room air saturation of 85% and a blood pressure of 80/50 mm Hg. A bedside ultrasound revealed an ejection fraction of around 40%, with hypokinesia of interventricular septum, left ventricular apex and anterior wall with bilateral multiple B lines in all lung zones. A 12 lead ECG showed ST segment elevation in leads V2-V6. He sustained a cardiac arrest. Return of spontaneous circulation was achieved following high-quality cardiopulmonary resuscitation. After successful resuscitation, the patient underwent primary percutaneous coronary intervention and recovered fully from the event. On further evaluation, an acute binge of marijuana smoking prior to the onset of symptoms was identified as the cause of the acute coronary syndrome.

Measuring the impact of a single dose of ChAdOx1 nCoV-19 (recombinant) coronavirus vaccine on hospital stay, ICU requirement, and mortality outcome in a tertiary care centre.

OBJECTIVE: To comparatively evaluate ICU requirement, length of stay, and mortality between single-dose vaccinated and non-vaccinated hospitalized COVID-19 patients. DESIGN: A retrospective observational study was carried out in a tertiary care hospital in western Indian, from April 1 to June 30, 2021. RESULTS: Of the 569 patients who fulfilled the eligibility criteria and were enrolled in the study, 137 (24.08%) patients had received a single dose of ChAdOx1 nCoV-19 vaccine, while 432 (75.92%) patients had not received any form of vaccination. The overall length of stay in hospital was similar for both groups; however, a significant difference was seen in length of stay in the ward and in the ICU. Vaccinated patients were admitted to the ward for 6.21 ± 3.204 days, while non-vaccinated patients were admitted for 5.56 ± 4.55 days (p < 0.001). The mean length of ICU stay for the 21 vaccinated patients requiring intensive care was 4.47 ± 2.3 days, while that for the 145 non-vaccinated patients was 6.29 ± 2.19 days (p < 0.001). Mortality was observed in four patients in the vaccinated group and in 95 patients in the non-vaccinated group. CONCLUSION: A single dose of ChAdOx1 nCoV-19 vaccine was associated with a significantly lower severity of SARS-CoV-2 infection compared with no vaccination.

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

AIMS: Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. METHODS AND RESULTS: Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CONCLUSION: CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.