Inhibition of endothelial to mesenchymal transition in a large animal preclinical arterio-venous fistula model leads to improved remodeling and reduced stenosis.

AIMS: Vein grafts are used for many indications, including bypass graft surgery and arterio-venous fistula (AVF) formation. However, patency following vein grafting or AVF formation is suboptimal for various reasons, including thrombosis, neointimal hyperplasia and adverse remodeling. Recently, endothelial to mesenchymal transition (EndMT) was found to contribute to neointimal hyperplasia in mouse vein grafts. We aimed to evaluate the clinical potential of inhibiting EndMT, and developed the first dedicated preclinical model to study the efficacy of local EndMT inhibition immediately prior to AVF creation. METHODS AND RESULTS: We first undertook pilot studies to optimize the creation of a femoral AVF in pigs and verify that EndMT contributes to neointimal formation. We then developed a method to achieve local in vivo SMAD3 knockdown by dwelling a lentiviral construct containing SMAD3 shRNA in the femoral vein prior to AVF creation. Next, in Phase 1, 6 pigs were randomized to SMAD3 knockdown or control lentivirus to evaluate the effectiveness of SMAD3 knockdown and EndMT inhibition 8 days after AVF creation. In Phase 2, 16 pigs were randomized to SMAD3 knockdown or control lentivirus and were evaluated to assess longer-term effects on AVF diameter, patency and related measures at 30 days after AVF creation.In Phase 1, compared to controls, SMAD3 knockdown achieved a 75% reduction in the proportion of CD31+ endothelial cells co-expressing SMAD3 (p<0.001), and also a significant reduction in the extent of EndMT (p<0.05). In Phase 2, compared to controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56±1.66 versus 4.26±1.71mm, p<0.01) and a reduced degree of stenosis (p<0.01). Consistent with this, neointimal thickness was reduced in the SMAD3 knockdown group (0.88±0.51 versus 0.45±0.19mm, p<0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (p<0.05). CONCLUSIONS: EndMT inhibition in a preclinical AVF model by local SMAD3 knockdown using gene therapy led to reduced neointimal hyperplasia, increased endothelialization and a reduction in the degree of AVF stenosis. This provides important proof-of-concept to pursue this approach as a clinical strategy to improve the patency of AVFs and other vein grafts.

Variants in the Kallikrein Gene Family and Hypermobile Ehlers-Danlos Syndrome.

Hypermobile Ehlers-Danlos syndrome (hEDS) is a common heritable connective tissue disorder that lacks a known genetic etiology. To identify genetic contributions to hEDS, whole exome sequencing was performed on families and a cohort of sporadic hEDS patients. A missense variant in Kallikrein-15 (KLK15 p. Gly226Asp), segregated with disease in two families and genetic burden analyses of 197 sporadic hEDS patients revealed enrichment of variants within the Kallikrein gene family. To validate pathogenicity, the variant identified in familial studies was used to generate knock-in mice. Consistent with our clinical cohort, Klk15 G224D/+ mice displayed structural and functional connective tissue defects within multiple organ systems. These findings support Kallikrein gene variants in the pathogenesis of hEDS and represent an important step towards earlier diagnosis and better clinical outcomes.

Environmentally Not So Friendly: Global Warming, Air Pollution, and Wildfires: JACC Focus Seminar, Part 1.

Environmental stresses are increasingly recognized as significant risk factors for adverse health outcomes. In particular, various forms of pollution and climate change are playing a growing role in promoting noncommunicable diseases, especially cardiovascular disease. Given recent trends, global warming and air pollution are now associated with substantial cardiovascular morbidity and mortality. As a vicious cycle, global warming increases the occurrence, size, and severity of wildfires, which are significant sources of airborne particulate matter. Exposure to wildfire smoke is associated with cardiovascular disease, and these effects are underpinned by mechanisms that include oxidative stress, inflammation, impaired cardiac function, and proatherosclerotic effects in the circulation. In the first part of a 2-part series on pollution and cardiovascular disease, this review provides an overview of the impact of global warming and air pollution, and because of recent events and emerging trends specific attention is paid to air pollution caused by wildfires.

Water, Soil, Noise, and Light Pollution: JACC Focus Seminar, Part 2.

Various forms of pollution carry a substantial burden with respect to increasing the risk of causing and exacerbating noncommunicable diseases, especially cardiovascular disease. The first part of this 2-part series on pollution and cardiovascular disease provided an overview of the impact of global warming and air pollution. This second paper provides an overview of the impact of water, soil, noise, and light pollution on the cardiovascular system. This review discusses the biological mechanisms underlying these effects and potential environmental biometrics of exposure. What is clear from both these pollution papers is that significant efforts and redoubled urgency are needed to reduce the sources of pollution in our environment, to incorporate environmental risk factors into medical education, to provide resources for research, and, ultimately, to protect those who are particularly vulnerable and susceptible.

The protocol for an observational Australian cohort study of CADASIL: The AusCADASIL study.

Introduction: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a rare genetic condition with a broad phenotypic presentation. This study aims to establish the first Australian cohort of individuals affected by CADASIL (AusCADASIL) and examine its clinical features and longitudinal course, and to investigate neuroimaging and blood biomarkers to assist in early diagnosis and identify disease progression. Methods: Participants will be recruited from six study centres across Australia for an observational study of CADASIL. We aim to recruit 150 participants with diagnosed CADASIL, family history of CADASIL or suspected CADASIL symptoms, and 150 cognitively normal NOTCH3 negative individuals as controls. Participants will complete: 1) online questionnaires on medical and family history, mental health, and wellbeing; 2) neuropsychological evaluation; 3) neurological examination and brain MRI; 4) ocular examination and 5) blood sample donation. Participants will have annual follow-up for 4 years to assess their progression and will be asked to invite a study partner to corroborate their self-reported cognitive and functional abilities.Primary outcomes include cognitive function and neuroimaging abnormalities. Secondary outcomes include investigation of genetics and blood and ocular biomarkers. Data from the cohort will contribute to an international consortium, and cohort participants will be invited to access future treatment/health intervention trials. Discussion: AusCADASIL will be the first study of an Australian cohort of individuals with CADASIL. The study will identify common pathogenic variants in this cohort, and characterise the pattern of clinical presentation and longitudinal progression, including imaging features, blood and ocular biomarkers and cognitive profile.

Macrotroponin in the COVID-19 Era: An Under-Recognised Cause of Persistent Troponin Elevation.

Troponin is an important diagnostic tool, however, as the assay sensitivity and frequency of testing has increased in the COVID-19 era, a new cohort of patients with persistently elevated troponin has emerged. Interfering antibodies should be considered in patients with persistent and stable troponin elevation, where there is no ongoing cause.

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

The Tricuspid Valve: A Review of Pathology, Imaging, and Current Treatment Options: A Scientific Statement From the American Heart Association.

Tricuspid valve disease is an often underrecognized clinical problem that is associated with significant morbidity and mortality. Unfortunately, patients will often present late in their disease course with severe right-sided heart failure, pulmonary hypertension, and life-limiting symptoms that have few durable treatment options. Traditionally, the only treatment for tricuspid valve disease has been medical therapy or surgery; however, there have been increasing interest and success with the use of transcatheter tricuspid valve therapies over the past several years to treat patients with previously limited therapeutic options. The tricuspid valve is complex anatomically, lying adjacent to important anatomic structures such as the right coronary artery and the atrioventricular node, and is the passageway for permanent pacemaker leads into the right ventricle. In addition, the mechanism of tricuspid pathology varies widely between patients, which can be due to primary, secondary, or a combination of causes, meaning that it is not possible for 1 type of device to be suitable for treatment of all cases of tricuspid valve disease. To best visualize the pathology, several modalities of advanced cardiac imaging are often required, including transthoracic echocardiography, transesophageal echocardiography, cardiac computed tomography, and cardiac magnetic resonance imaging, to best visualize the pathology. This detailed imaging provides important information for choosing the ideal transcatheter treatment options for patients with tricuspid valve disease, taking into account the need for the lifetime management of the patient. This review highlights the important background, anatomic considerations, therapeutic options, and future directions with regard to treatment of tricuspid valve disease.

Heterogeneous Effects of Continuous Positive Airway Pressure in Non-Sleepy Obstructive Sleep Apnea on Cardiovascular Disease Outcomes: Post Hoc Machine Learning Analysis of the ISAACC Trial (ECSACT Study).

Rationale: Randomized controlled trials of continuous positive airway pressure (CPAP) therapy for cardiovascular disease (CVD) prevention among patients with obstructive sleep apnea (OSA) have been largely neutral. However, given that OSA is a heterogeneous disease, there may be unidentified subgroups demonstrating differential treatment effects. Objectives: We sought to apply a novel data-drive approach to identify nonsleepy OSA subgroups with heterogeneous effects of CPAP on CVD outcomes within the Impact of Sleep Apnea Syndrome in the Evolution of Acute Coronary Syndrome (ISAACC) study. Methods: Participants were randomly partitioned into two datasets. One for training (70%) our machine-learning model and a second (30%) for validation of significant findings. Model-based recursive partitioning was applied to identify subgroups with heterogeneous treatment effects. Survival analysis was conducted to compare treatment (CPAP vs. usual care [UC]) outcomes within subgroups. Results: A total of 1,224 nonsleepy OSA participants were included. Of 55 features entered into our model, only two appeared in the final model (i.e., average OSA event duration and hypercholesterolemia). Among participants at or below the model-derived average event duration threshold (19.5 s), CPAP was protective for a composite of CVD events (training hazard ratio [HR], 0.46; P = 0.002). For those with longer event duration (>19.5 s), an additional split occurred by hypercholesterolemia status. Among participants with longer event duration and hypercholesterolemia, CPAP resulted in more CVD events compared with UC (training HR, 2.24; P = 0.011). The point estimate for this harmful signal was also replicated in the testing dataset (HR, 1.83; P = 0.118). Conclusions: We discovered subgroups of nonsleepy OSA participants within the ISAACC study with heterogeneous effects of CPAP. Among the training dataset, those with longer OSA event duration and hypercholesterolemia had nearly 2.5 times more CVD events with CPAP compared with UC, whereas those with shorter OSA event duration had roughly half the rate of CVD events if randomized to CPAP.

Endothelial to mesenchymal transition: at the axis of cardiovascular health and disease.

Endothelial cells (ECs) line the luminal surface of blood vessels and play a major role in vascular (patho)-physiology by acting as a barrier, sensing circulating factors and intrinsic/extrinsic signals. ECs have the capacity to undergo endothelial-to-mesenchymal transition (EndMT), a complex differentiation process with key roles both during embryonic development and in adulthood. EndMT can contribute to EC activation and dysfunctional alterations associated with maladaptive tissue responses in human disease. During EndMT, ECs progressively undergo changes leading to expression of mesenchymal markers while repressing EC lineage-specific traits. This phenotypic and functional switch is considered to largely exist in a continuum, being characterized by a gradation of transitioning stages. In this report, we discuss process plasticity and potential reversibility and the hypothesis that different EndMT-derived cell populations may play a different role in disease progression or resolution. In addition, we review advancements in the EndMT field, current technical challenges, as well as therapeutic options and opportunities in the context of cardiovascular biology.

Computational protocol to identify shared transcriptional risks and mutually beneficial compounds between diseases.

The accumulation of omics and biobank resources allows for a genome-wide understanding of the shared pathologic mechanisms between diseases and for strategies to identify drugs that could be repurposed as novel treatments. Here, we present a computational protocol, implemented as a Snakemake workflow, to identify shared transcriptional processes and screen compounds that could result in mutual benefit. This protocol also includes a description of a pharmacovigilance study designed to validate the effect of compounds using electronic health records. For complete details on the use and execution of this protocol, please refer to Gao et al.1 and Baylis et al.2.

Hybrid Magnetic Resonance Positron Emission Tomography Is Associated With Cardiac-Related Outcomes in Cardiac Sarcoidosis.

BACKGROUND: Imaging with late gadolinium enhancement (LGE) magnetic resonance (MR) and 18F-fluorodeoxyglucose (18F-FDG) PET allows complementary assessment of myocardial injury and disease activity and has shown promise for improved characterization of active cardiac sarcoidosis (CS) based on the combined positive imaging outcome, MR(+)PET(+). OBJECTIVES: This study aims to evaluate qualitative and quantitative assessments of hybrid MR/PET imaging in CS and to evaluate its association with cardiac-related outcomes. METHODS: A total of 148 patients with suspected CS underwent hybrid MR/PET imaging. Patients were classified based on the presence/absence of LGE (MR+/MR-), presence/absence of 18F-FDG (PET+/PET-), and pattern of 18F-FDG uptake (focal/diffuse) into the following categories: MR(+)PET(+)FOCAL, MR(+)PET(+)DIFFUSE, MR(+)PET(-), MR(-)PET(+)FOCAL, MR(-)PET(+)DIFFUSE, MR(-)PET(-). Further analysis classified MR positivity based on %LGE exceeding 5.7% as MR(+/-)5.7%. Quantitative values of standard uptake value, target-to-background ratio, target-to-normal-myocardium ratio (TNMRmax), and T2 were measured. The primary clinical endpoint was met by the occurrence of cardiac arrest, ventricular tachycardia, or secondary prevention implantable cardioverter-defibrillator (ICD) before the end of the study. The secondary endpoint was met by any of the primary endpoint criteria plus heart failure or heart block. MR/PET imaging results were compared between those meeting or not meeting the clinical endpoints. RESULTS: Patients designated MR(+)5.7%PET(+)FOCAL had increased odds of meeting the primary clinical endpoint compared to those with all other imaging classifications (unadjusted OR: 9.2 [95% CI: 3.0-28.7]; P = 0.0001), which was higher than the odds based on MR or PET alone. TNMRmax achieved an area under the receiver-operating characteristic curve of 0.90 for separating MR(+)PET(+)FOCAL from non-MR(+)PET(+)FOCAL, and 0.77 for separating those reaching the clinical endpoint from those not reaching the clinical endpoint. CONCLUSIONS: Hybrid MR/PET image-based classification of CS was statistically associated with clinical outcomes in CS. TNMRmax had modest sensitivity and specificity for quantifying the imaging-based classification MR(+)PET(+)FOCAL and was associated with outcomes. Use of combined MR and PET image-based classification may have use in prognostication and treatment management in CS.

Polygenic Risk in Families With Spontaneous Coronary Artery Dissection.

Importance: Spontaneous coronary artery dissection (SCAD) is a poorly understood cause of acute coronary syndrome that predominantly affects women. Evidence to date suggests a complex genetic architecture, while a family history is reported for a minority of cases. Objective: To determine the contribution of rare and common genetic variants to SCAD risk in familial cases, the latter via the comparison of a polygenic risk score (PRS) with those with sporadic SCAD and healthy controls. Design, Setting, and Participants: This genetic association study analyzed families with SCAD, individuals with sporadic SCAD, and healthy controls. Genotyping was undertaken for all participants. Participants were recruited between 2017 and 2021. A PRS for SCAD was calculated for all participants. The presence of rare variants in genes associated with connective tissue disorders (CTD) was also assessed. Individuals with SCAD were recruited via social media or from a single medical center. A previously published control database of older healthy individuals was used. Data were analyzed from January 2022 to October 2023. Exposures: PRS for SCAD comprised of 7 single-nucleotide variants. Main Outcomes and Measures: Disease status (familial SCAD, sporadic SCAD, or healthy control) associated with PRS. Results: A total of 13 families with SCAD (27 affected and 12 unaffected individuals), 173 individuals with sporadic SCAD, and 1127 healthy controls were included. A total of 188 individuals with SCAD (94.0%) were female, including 25 of 27 with familial SCAD and 163 of 173 with sporadic SCAD; of 12 unaffected individuals from families with SCAD, 6 (50%) were female; and of 1127 healthy controls, 672 (59.6%) were female. Compared with healthy controls, the odds of being an affected family member or having sporadic SCAD was significantly associated with a SCAD PRS (where the odds ratio [OR] represents an increase in odds per 1-SD increase in PRS) (affected family member: OR, 2.14; 95% CI, 1.78-2.50; adjusted P = 1.96 × 10-4; sporadic SCAD: OR, 1.63; 95% CI, 1.37-1.89; adjusted P = 5.69 × 10-4). This association was not seen for unaffected family members (OR, 1.03; 95% CI, 0.46-1.61; adjusted P = .91) compared with controls. Further, those with familial SCAD were overrepresented in the top quintile of the control PRS distribution (OR, 3.70; 95% CI, 2.93-4.47; adjusted P = .001); those with sporadic SCAD showed a similar pattern (OR, 2.51; 95% CI, 1.98-3.04; adjusted P = .001). Affected individuals within a family did not share any rare deleterious variants in CTD-associated genes. Conclusions and Relevance: Extreme aggregation of common genetic risk appears to play a significant role in familial clustering of SCAD as well as in sporadic case predisposition, although further study is required.

Multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci.

Genome-wide association studies (GWASs) have identified hundreds of risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWASs, and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotypes to identify quantitative trait loci for expression (eQTLs) and splicing (sQTLs) in coronary arteries from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary artery; 19% exhibited cell-type-specific expression. Colocalization revealed subgroups of eGenes unique to CAD and blood pressure GWAS. Fine-mapping highlighted additional eGenes, including TBX20 and IL5. We also identified sQTLs for 1,690 genes, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing to accurately identify disease-relevant isoform expression. Our work provides a patient-derived coronary artery eQTL resource and exemplifies the need for diverse study populations and multifaceted approaches to characterize gene regulation in disease processes.

Biomaterials containing extracellular matrix molecules as biomimetic next-generation vascular grafts.

The performance of synthetic biomaterial vascular grafts for the bypass of stenotic and dysfunctional blood vessels remains an intractable challenge in small-diameter applications. The functionalization of biomaterials with extracellular matrix (ECM) molecules is a promising approach because these molecules can regulate multiple biological processes in vascular tissues. In this review, we critically examine emerging approaches to ECM-containing vascular graft biomaterials and explore opportunities for future research and development toward clinical use.

Strain Assessment in Aortic Stenosis: Pathophysiology and Clinical Utility.

Contemporary echocardiographic criteria for grading aortic stenosis severity have remained relatively unchanged, despite significant advances in noninvasive imaging techniques over the last 2 decades. More recently, attention has shifted to the ventricular response to aortic stenosis and how this might be quantified. Global longitudinal strain, semiautomatically calculated from standard two-dimensional echocardiographic images, has been the focus of extensive research. Global longitudinal strain is a sensitive marker of subtle hypertrophy-related impairment in left ventricular function and has shown promise as a relatively robust prognostic marker, both independently and when added to severity classification systems. Herein we review the pathophysiological basis underpinning the potential utility of global longitudinal strain in the assessment of aortic stenosis, as well as its potential role in quantifying myocardial recovery and prognostic discrimination following aortic valve replacement.

Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence.

Senescent cells are a major contributor to age-dependent cardiovascular tissue dysfunction, but knowledge of their in vivo cell markers and tissue context is lacking. To reveal tissue-relevant senescence biology, we integrate the transcriptomes of 10 experimental senescence cell models with a 224 multi-tissue gene co-expression network based on RNA-seq data of seven tissues biopsies from ∼600 coronary artery disease (CAD) patients. We identify 56 senescence-associated modules, many enriched in CAD GWAS genes and correlated with cardiometabolic traits-which supports universality of senescence gene programs across tissues and in CAD. Cross-tissue network analyses reveal 86 candidate senescence-associated secretory phenotype (SASP) factors, including COL6A3. Experimental knockdown of COL6A3 induces transcriptional changes that overlap the majority of the experimental senescence models, with cell-cycle arrest linked to modulation of DREAM complex-targeted genes. We provide a transcriptomic resource for cellular senescence and identify candidate biomarkers, SASP factors, and potential drivers of senescence in human tissues.

Integrative single-cell meta-analysis reveals disease-relevant vascular cell states and markers in human atherosclerosis.

Coronary artery disease (CAD) is characterized by atherosclerotic plaque formation in the arterial wall. CAD progression involves complex interactions and phenotypic plasticity among vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but human cell phenotypes remain controversial. Here, we perform an integrated meta-analysis of 22 scRNA-seq libraries to generate a comprehensive map of human atherosclerosis with 118,578 cells. Besides characterizing granular cell-type diversity and communication, we leverage this atlas to provide insights into smooth muscle cell (SMC) modulation. We integrate genome-wide association study data and uncover a critical role for modulated SMC phenotypes in CAD, myocardial infarction, and coronary calcification. Finally, we identify fibromyocyte/fibrochondrogenic SMC markers (LTBP1 and CRTAC1) as proxies of atherosclerosis progression and validate these through omics and spatial imaging analyses. Altogether, we create a unified atlas of human atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

Molecular biomarkers for vascular cognitive impairment and dementia.

As disease-specific interventions for dementia are being developed, the ability to identify the underlying pathology and dementia subtypes is increasingly important. Vascular cognitive impairment and dementia (VCID) is the second most common cause of dementia after Alzheimer disease, but progress in identifying molecular biomarkers for accurate diagnosis of VCID has been relatively limited. In this Review, we examine the roles of large and small vessel disease in VCID, considering the underlying pathophysiological processes that lead to vascular brain injury, including atherosclerosis, arteriolosclerosis, ischaemic injury, haemorrhage, hypoperfusion, endothelial dysfunction, blood-brain barrier breakdown, inflammation, oxidative stress, hypoxia, and neuronal and glial degeneration. We consider the key molecules in these processes, including proteins and peptides, metabolites, lipids and circulating RNA, and consider their potential as molecular biomarkers alone and in combination. We also discuss the challenges in translating the promise of these biomarkers into clinical application.