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Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN , the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient’s condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Objective: Reported associations between kidney risk variants (G1 and G2) in APOL1 (apolipoprotein L1), encoding APOL1, and cardiovascular disease have been conflicting. We sought to explore associations of APOL1 risk variants with cause of sudden death using the CVPath Sudden Death Autopsy Registry. Approach and Results: APOL1 haplotypes and causes of sudden death, as determined through autopsy and histopathology, were obtained for 764 Black subjects. Genotyping revealed APOL1 risk alleles in 452 of 764 (59%) subjects with 347 (77%) subjects carrying one risk allele and 105 (23%) subjects harboring 2 risk alleles. APOL1 risk allele carrier status was associated with a significantly increased risk of coronary thrombosis due to plaque rupture, versus noncarriers (odds ratio for rupture, 1.655 [95% CI, 1.079–2.539]; P =0.021). Histological examinations showed coronary plaques in carriers of 2 APOL1 risk alleles had larger necrotic cores compared with noncarriers (necrotic core area/total plaque area: 46.79%±6.47% versus 20.57%±5.11%; P =0.0343 in ruptured plaques, and 41.48%±7.49% versus 18.93%±3.97%; P =0.0342 in nonruptured plaques), and immunohistochemical and immunofluorescent staining revealed APOL1-positive areas localized primarily to the necrotic core. Conclusions: APOL1 risk alleles were independently associated with an increased risk of thrombotic coronary death due to plaque rupture. Our results suggest that carriers of both 1 and 2 APOL1 risk alleles have greater accumulation of APOL1 protein within culprit plaques and greater necrotic core sizes than noncarriers. These findings suggest that APOL1 plays a role in determining plaque stability.

Introduction: In-stent restenosis (ISR) has been one of the biggest limitations to the success of percutaneous coronary intervention for the treatment of coronary artery disease (CAD). The introduc...

To co-register conventional computed tomography angiography (CTA), with ex vivo micro-computed tomography (microCT) and histology of popliteal atherosclerotic plaques. Improving the non-invasive imaging capabilities may be valuable to advance patient care with peripheral arterial obstructive disease towards lesion and individual based treatment.In this prospective observational study, 12 popliteal arteries from 11 symptomatic patients who had undergone transfemoral amputations for chronic limb threatening ischaemia and who had pre-operative CTA, were analysed ex vivo by microCT and histology. A total of 353 histological cross sections were co-registered with microCT and CTA, and classified as: lipid rich (LP, n = 26), fibrous (FP, n = 80), or calcific (CP, n = 247) plaques. CTA and microCT plaque density was calculated in 791 regions of interest as Hounsfield units (HU).CTA and microCT could identify plaque components that were confirmed by histology such as fibrous tissue (FP), lipid pool/core (LP), and calcification (CP). MicroCT densities were 77.8 HU for FP (IQR 52.8, 129.5 HU), -28.4 HU for LP (IQR -87.1, 13.2 HU), and 3826.0 HU for CP (IQR 2989.0, 4501.0 HU). CTA densities of the three components of the plaque were: 78.0 HU for FP (IQR 59.5, 119.8 HU), 32.5 HU for LP (IQR 15.0, 42 HU), and 641.5 HU for CP (IQR 425.8, 1135 HU). The differences were statistically significant between the HU densitometric characteristics among the three groups (p .0001) for both imaging modalities. Overall, microCT performed better diagnostically than conventional CTA for the three types of plaques: areas under the receiving operator characteristics curve were greater for microCT than CTA for FP (0.97 vs. 0.90), for LP (0.88 vs. 0.67), and for CP (0.97 vs. 0.90).CTA and microCT can be used to identify histological atherosclerotic plaque components, with better diagnostic performance for microCT. This study demonstrates the feasibility of using microCT to assess plaque morphology lesions in a manner that approaches histology thus becoming a useful tool for ex vivo assessment of atherosclerosis and towards lesion based treatment.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. Methods and Results: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

BackgroundChronic innate and adaptive immune activation may contribute to high prevalence of cardiovascular disease in persons living with HIV (PLWH).MethodsWe assessed coronary plaques from deceased PLWH (n=6) and HIV-negative (n=6) persons matched by age and gender. Formalin-fixed, paraffin-embedded 5μm thick sections were processed using Movat, hematoxylin and eosin, immunohistochemical and immunofluorescence stains. Immune cell populations were measured using surface antibodies, and immune-related protein expression from macrophage rich, T-cell rich and perivascular adipose tissue regions using GeoMx® digital spatial profiling.ResultsCoronary plaques from PLWH and HIV-negative persons had similar plaque area and percent stenosis. Percent CD163+ cells as measured by immunohistochemical staining was significantly higher in PLWH, median 0.29% (IQR 0.11-0.90) vs. 0.01% (IQR 0.0013-0.11) in HIV-negative plaque, p = 0.02 (Figure 1A). Other surface markers of innate cells (CD68 +, p=0.18), adaptive immune cells (CD3+, p=0.39; CD4+, p=0.09; CD8+, p=0.18) and immune trafficking markers (CX3CR1+, p=0.09) within the coronary plaque trended higher in HIV-positive plaques but did not reach statistical significance. GeoMx® digital spatial profiling showed higher differential protein expression of CD163 (scavenger receptor for hemoglobin-haptoglobin complex), stimulator of interferon gamma (STING, a cytosolic DNA sensor), CD25 and granzyme-B in the HIV-positive compared to HIV-negative, pConclusionsIncreased inflammation within the coronary plaques of PLWH is characterized by more innate and adaptive immune cells. Higher STING expression in PLWH suggests that immune response to viral antigens within the plaque might be a driver above other stimulants. STING inhibitors are available and could be investigated as a future therapeutic target in PWH if these results are replicated with a larger number of plaques.Graphical AbstractHighlightsImmunohistochemical and fluorescent stains combined with GeoMx® digital spatial profiling allowed for deep characterization of immune cells within intact coronary plaques and perivascular adipose tissueCoronary plaques from HIV-positive persons had higher proportion of CD163+ immune cells compared to HIV-negative personsDifferential protein expression of immune-rich regions of interest within intact 5μm sections of coronary plaques revealed higher levels of stimulator of interferon gamma (STING) in HIV-positive persons

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet–fed Ldlr(—/—) mice into diabetic Ager(—/—) (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow–derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

Mammalian target of rapamycin (mTOR) inhibitors have been applied to vascular coronary devices to avoid neointimal growth and have become the predominant pharmacological agents used to prevent restenosis. mTOR inhibitors can affect not only proliferating vascular smooth muscle cells but also endothelial cells and therefore can result in delayed healing of the vessel including endothelialization. Emerging evidence suggests accelerated atherosclerosis due to the downstream negative effects on endothelial barrier functional recovery. The development of neoatherosclerosis within the neointima of drug-eluting stents can result in late thrombotic events. This type of problematic healing response may open the way for specific mTOR kinase inhibitors, such as ATP-competitive mTOR inhibitors. These inhibitors demonstrate a better healing profile than traditional limus-based drug-eluting stent and their clinical efficacy remains unknown.

The data presented here are related to the research article, entitled Genetic linkage of oxidative stress with cardiometabolic traits in an intercross derived from hyperlipidemic mouse strains, published in Atherosclerosis 2019 Dec 3;293:1–10 (D. Fuller, A.T. Grainger, A. Manichaikul, W. Shi). The supporting materials include original genotypic and phenotypic data obtained from 266 female F2 mice derived from an intercross between C57BL/6 (B6) and BALB/cJ (BALB) Apoe−/- mice. F2 mice were fed 12 weeks of Western diet, starting at 6 weeks of age. Plasma levels of HDL, LDL cholesterol, triglycerides, glucose and malondialdehyde (MDA) and atherosclerosis in the aortic root and the left carotid artery were measured. 127 microsatellite markers across the entire genome were genotyped. The data is provided in the format ready for QTL analysis with J/qtl and MapManager QTX. Keywords: Genetic linkage, Oxidative stress, Cardiometabolic trait, Atherosclerosis, Mouse

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.

Purpose To compare the long-term vascular healing responses of healthy swine iliofemoral arteries treated with a polymer-free paclitaxel-eluting stent (Z-PES, Zilver PTX) or a fluoropolymer-based paclitaxel-eluting stent (FP-PES, Eluvia). Materials and Methods Bilateral iliofemoral arteries in 20 swine were treated with a Z-PES (n = 16) or a FP-PES (n = 24) and were examined histologically at 1, 3, 6, and 12 months. Results Morphometric analysis revealed larger external and internal elastic lamina, stent expansion, and lumen area in the FP-PES than in the Z-PES at all timepoints. Luminal narrowing was similar in the 2 groups at 1 month; however, greater stenosis was observed in the Z-PES group at 3 months, with significant regression thereafter, resulting in equivalent stenosis at 6 and 12 months. Greater drug effect and less complete vessel healing were found in the FP-PES group at all timepoints, including greater numbers of malapposed struts with excessive fibrin deposition at 1 and 3 months, than in the Z-PES group. Three of 12 FP-PESs from the 6- and 12-month cohorts also showed circumferential medial disruption with peri-strut inflammation, whereas no abnormal findings were observed in contralateral Z-PESs. Conclusions Prolonged paclitaxel release with the presence of a permanent polymer may contribute to the differential vascular responses seen for the Z-PES and FP-PES groups, including medial layer disruption and aneurysmal vessel degeneration that was sometimes observed in the FP-PES group. These distinct features should be confirmed by pathology and in vivo imaging of human superficial femoral arteries to determine their clinical significance.

BackgroundUnexplained-sudden cardiac death (SCD) describes SCD with no cause identified after a comprehensive autopsy and toxicologic examination. Genetic testing helps to diagnose inherited cardiac diseases in unexplained-SCD, however, the relationship between pathogenic or likely pathogenic (P/LP) variants of inherited cardiomyopathies and primary electrical disorders (PED) and risk of unexplained-SCD in adults living the United States has never been systematically examined.MethodsWe performed sequencing of 29 cardiomyopathy and 39 arrhythmia genes in 413 African-Americans and Caucasians (≥18 years-old) who died of unexplained-SCD (median age; 41 years-old, 37% females, 50% African-Americans) and whose hearts were found to have no gross pathological finding upon referral to our institution for pathologic consultation from the State of Maryland Medical Examiner. We examined P/LP variants in these genes to study the association between presence of these variants and unexplained-SCD.Results143/413 (34.6%) subjects had variants considered P/LP for cardiomyopathy and/or PED (i.e. Brugada Syndrome (BrS), long QT syndrome (LQTS), and arrhythmogenic right ventricular dysplasia (ARVD)). In total, 102 (24.7%) subjects harbored 86 P/LP variants for cardiomyopathies and 60 (14.5%) subjects carried 76 P/LP variants for PED. The following pathogenic variants were identified: 68 P/LP variants for hypertrophic cardiomyopathy (HCM) in 79/413 (19.1%) subjects, 18 P/LP variants for dilated cardiomyopathy (DCM) in 22/413 subjects (5.3%), and 76 P/LP variants in 60/413 (14.5%) for PED. There were greater than 121.0- and 138.5-fold median enrichments (431.4- and 200.0-fold cumulative enrichments) in these cardiomyopathy and arrhythmia variants in victims of unexplained SCD versus the general population, respectively. Among these P/LP positive carriers, combinations of conditions were found, including 14/413 (2.4%) having both HCM and PED variants, and 5/413 (1.2%) with DCM and PED variants. African Americans (AA) and Caucasians were equally likely to harbor P/LP variants (32.7% versus 36.6%, p=0.5), but AA had a higher frequent variants of unknown significance.ConclusionsThis study represents the largest examination reported on the association between cardiomyopathy and arrhythmia P/LP genetic variants and unexplained-SCD in adults with no gross abnormality on rigorous pathological examination. Nearly one-third of those with unexplained-SCD were carriers of P/LP variants. Our findings with respect to both the association of unexplained SCD with cardiomyopathy genes and race-specific genetic variants suggest new avenues of study for this poorly understood entity.

The presence of macrophages within the plaque is a defining hallmark of atherosclerosis. Macrophages are exposed to various microenvironments such as oxidized lipids and cytokines which effect their phenotypic differentiation and activation. Classically, macrophages have been divided into two groups: M1 and M2 macrophages induced by T-helper 1 and T-helper 2 cytokines, respectively. However, for a decade, greater phenotypic heterogeneity and plasticity of these cells have since been reported in various models. In addition to M1 and M2 macrophage phenotypes, the concept of additional macrophage phenotypes such as M (Hb), Mox, and M4 has emerged. Understanding the mechanisms and functions of distinct phenotype of macrophages can lead to determination of their potential role in atherosclerotic plaque pathogenesis. However, there are still many unresolved controversies regarding their phenotype and function with respect to atherosclerosis. Here, we summarize and focus on the differential subtypes of macrophages in atherosclerotic plaques and their differing functional roles based upon microenvironments such as lipid, intraplaque hemorrhage, and plaque regression.

Summary Persons with HIV are at increased risk for diabetes mellitus compared with individuals without HIV. Adipose tissue is an important regulator of glucose and lipid metabolism, and adipose tissue T cells modulate local inflammatory responses and, by extension, adipocyte function. Persons with HIV and diabetes have a high proportion of CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells in adipose tissue, a subset of which are cytomegalovirus specific, whereas individuals with diabetes but without HIV have predominantly CD69+ CD4+ T cells. Adipose tissue CD69+ and C-G-C+ CD4+ T cell subsets demonstrate higher receptor clonality compared with the same cells in blood, potentially reflecting antigen-driven expansion, but C-G-C+ CD4+ T cells have a more inflammatory and cytotoxic RNA transcriptome. Future studies will explore whether viral antigens have a role in recruitment and proliferation of pro-inflammatory C-G-C+ CD4+ T cells in adipose tissue of persons with HIV., Graphical Abstract, Highlights Adipose tissue memory CD4+ T cells are frequently CD69+ in persons with diabetes Persons with HIV and diabetes have more CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells Adipose tissue C-G-C+ CD4+ T cells and CD69+ CD4+ T cells are clonally expanded C-G-C+ CD4+ T cells are often CMV specific and have more inflammatory transcriptomes, Wanjalla et al. demonstrate that adipose tissue in persons with HIV and diabetes includes a large proportion of clonally expanded, inflammatory, and frequently CMV-specific CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells. These cells may contribute to the altered adipocyte function and elevated risk of metabolic disease observed among persons with HIV.