Direct Targeting of the mTOR (Mammalian Target of Rapamycin) Kinase Improves Endothelial Permeability in Drug-Eluting Stents-Brief Report.

Objective- Drug-eluting stents eluting canonical mTOR (mammalian target of rapamycin) inhibitors are widely used to treat coronary artery disease but accelerate the development of atherosclerosis within the stent (neoatherosclerosis)-a leading cause of late stent failure. We recently showed that canonical mTOR inhibitors bind FKBP12.6 (12.6-kDa FK506-binding protein 12), displace it from calcium release channels, resulting in activation of PKCα (protein kinase Cα) and dissociation of p-120-catenin (p120) from VE-CAD (vascular endothelial cadherin; promoting endothelial barrier dysfunction [EBD]). However, the relevance of these findings to drug-eluting stents remains unknown. Newer generation direct mTOR kinase inhibitors do not bind FKBP12.6 and offer the potential of improving endothelial barrier function while maintaining antirestenotic efficacy, but their actual effects are unknown. We examined the effects of 2 different pharmacological targeting strategies-canonical mTOR inhibitor everolimus and mTOR kinase inhibitors Torin-2-on EBD after stenting. Approach and Results- Using the rabbit model of stenting and a combination of Evans blue dye, confocal and scanning electron microscopy studies, everolimus-eluting stents resulted in long-term EBD compared with bare metal stents. EBD was mitigated by using stents that eluted mTOR kinase inhibitors (Torin-2-eluting stent). At 60 days after stent placement, everolimus-eluting stents demonstrated large areas of Evans blue dye staining and evidence of p120 VE-CAD dissociation consistent with EBD. These findings were absent in bare metal stents and significantly attenuated in Torin-2-eluting stent. As proof of concept of the role of EBD in neoatherosclerosis, 100 days after stenting, animals were fed an enriched cholesterol diet for an additional 30 days. Everolimus-eluting stents demonstrated significantly more macrophage infiltration (consistent with neoatherosclerosis) compared with both bare metal stents and Torin-2-eluting stent. Conclusions- Our results pinpoint interactions between FKBP12.6 and canonical mTOR inhibitors as a major cause of vascular permeability and neoatherosclerosis, which can be overcome by using mTOR kinase inhibitors. Our study suggests further refinement of molecular targeting of the mTOR complex may be a promising strategy (Graphic Abstract).

Genetic Variants Associated With Unexplained Sudden Cardiac Death in Adult White and African American Individuals.

Importance: Unexplained sudden cardiac death (SCD) describes SCD with no cause identified. Genetic testing helps to diagnose inherited cardiac diseases in unexplained SCD; however, the associations between pathogenic or likely pathogenic (P/LP) variants of inherited cardiomyopathies (CMs) and arrhythmia syndromes and the risk of unexplained SCD in both White and African American adults living the United States has never been systematically examined. Objective: To investigate cases of unexplained SCD to determine the frequency of P/LP genetic variants of inherited CMs and arrhythmia syndromes. Design, Setting, and Participants: This genetic association study included 683 African American and White adults who died of unexplained SCD and were included in an autopsy registry. Overall, 413 individuals had DNA of acceptable quality for genetic sequencing. Data were collected from January 1995 to December 2015. A total of 30 CM genes and 38 arrhythmia genes were sequenced, and variants in these genes, curated as P/LP, were examined to study their frequency. Data analysis was performed from June 2018 to March 2021. Main Outcomes and Measures: The frequency of P/LP variants for CM or arrhythmia in individuals with unexplained SCD. Results: The median (interquartile range) age at death of the 413 included individuals was 41 (29-48) years, 259 (62.7%) were men, and 208 (50.4%) were African American adults. A total of 76 patients (18.4%) with unexplained SCD carried variants considered P/LP for CM and arrhythmia genes. In total, 52 patients (12.6%) had 49 P/LP variants for CM, 22 (5.3%) carried 23 P/LP variants for arrhythmia, and 2 (0.5%) had P/LP variants for both CM and arrhythmia. Overall, 41 P/LP variants for hypertrophic CM were found in 45 patients (10.9%), 9 P/LP variants for dilated CM were found in 11 patients (2.7%), and 10 P/LP variants for long QT syndrome were found in 11 patients (2.7%). No significant difference was found in clinical and heart characteristics between individuals with or without P/LP variants. African American and White patients were equally likely to harbor P/LP variants. Conclusions and Relevance: In this large genetic association study of community cases of unexplained SCD, nearly 20% of patients carried P/LP variants, suggesting that genetics may contribute to a significant number of cases of unexplained SCD. Our findings regarding both the association of unexplained SCD with CM genes and race-specific genetic variants suggest new avenues of study for this poorly understood entity.

Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries.

Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, which increases red cell membrane-derived free cholesterol in plaque content and inflammatory cell recruitment. Evans Blue Dye (EBD) assay is widely used as a standard assay for vasculature permeability. However, the method has not been established in fresh human coronary artery autopsy samples to evaluate intraplaque microvessel permeability and angiogenesis. In this protocol, we describe a method to evaluate human coronary samples for microvascular permeability, including procedures to perfuse coronary arteries, collection of artery samples for histological analysis and immunostaining as well as the use of appropriate methodology to analyze the images. An optional procedure is also provided for the use of FITC-dextran in mouse model to evaluate vascular permeability. These Evans Blue Dye procedures may be useful in providing functional measure of the endothelium integrity and permeability in both human samples and animal models in various pathological conditions.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.