Single-cell immune landscape of human atherosclerotic plaques.

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. However, specific characteristics of dysregulated immune cells within atherosclerotic lesions that lead to clinical events such as ischemic stroke or myocardial infarction are poorly understood. Here, using single-cell proteomic and transcriptomic analyses, we uncovered distinct features of both T cells and macrophages in carotid artery plaques of patients with clinically symptomatic disease (recent stroke or transient ischemic attack) compared to asymptomatic disease (no recent stroke). Plaques from symptomatic patients were characterized by a distinct subset of CD4+ T cells and by T cells that were activated and differentiated. Moreover, some T cell subsets in these plaques presented markers of T cell exhaustion. Additionally, macrophages from these plaques contained alternatively activated phenotypes, including subsets associated with plaque vulnerability. In plaques from asymptomatic patients, T cells and macrophages were activated and displayed evidence of interleukin-1ß signaling. The identification of specific features of innate and adaptive immune cells in plaques that are associated with cerebrovascular events may enable the design of more precisely tailored cardiovascular immunotherapies.

Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-ß signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Coronary artery calcification is inversely related to body morphology in patients with significant coronary artery disease: a three-dimensional intravascular ultrasound study.

AIMS: Emerging data have indicated unexpected complexity in the regulation of vascular and bone calcification. In particular, several recent studies have challenged the concept of a universally positive relationship between body morphology [weight, height, body mass index (BMI), body surface area (BSA)] and the extent of vascular calcification. We sought to clarify these discrepancies and investigated the relationship between index lesion coronary artery calcification (CAC) and body morphology in patients undergoing percutaneous coronary intervention (PCI) using three-dimensional intravascular ultrasound (IVUS). METHODS AND RESULTS: We analysed CAC in patients who underwent PCI with pre-intervention IVUS imaging. The main outcome measure was the calcium index (CalcIndex); a three-dimensional IVUS-derived measure of total calcification per obstructive coronary lesion. A total of 346 patients (65.3 ± 10.6 years; 29.5% females) underwent PCI with IVUS-based CAC assessment. CalcIndex was categorized as zero-low (0-0.1399; n = 152) or intermediate-high (0.1400-1.2541; n = 194). All measures of body morphology were lower in patients with intermediate-high CalcIndex (height, P = 0.024; weight, P = 0.008; BMI, P = 0.064; BSA, P = 0.005). In adjusted multivariable models, weight and BSA were independent inverse predictors of intermediate-high CalcIndex [weight: odds ratio (OR) 0.986, P = 0.017; BSA: OR 0.323, P = 0.012] while CalcIndex also trended towards an inverse association with both height (P = 0.068) and BMI (P = 0.064). These independent inverse associations were consistent across multiple clinical subgroups, including stratification by age, race, gender, diabetes, and renal impairment. CONCLUSION: Using three-dimensional IVUS to assess vascular calcification, these data confirm an independent, inverse relationship between body size and index lesion CAC in patients with obstructive coronary artery disease.

Clustering of acute and subacute stent thrombosis related to the introduction of generic clopidogrel.

INTRODUCTION: Generic clopidogrel recently became available in the United States and was rapidly adopted as a cost-effective alternative to the brand name formulation. However, unlike other medications, subtle differences in clopidogrel bioavailability may lead to acute consequences including stent thrombosis (ST). MATERIALS AND METHODS: We studied the incidence of acute and subacute ST during the initial period of generic clopidogrel use (June 18, 2012-September 6, 2012 [80 days]) at a single percutaneous coronary intervention (PCI) center. There were 4 definite ST cases within 30 days of successful PCI in patients receiving generic clopidogrel, which were compared to historic control ST cases from 80 days prior to generic clopidogrel use and for 3 years from June 18, 2009 to June 17, 2012. RESULTS: During generic clopidogrel administration, 1054 PCIs were performed, giving a definite 30-day ST incidence of 0.38% (4 of 1054) among these patients. By comparison, there were 2 episodes of definite 30-day ST during the 80 days immediately preceding generic clopidogrel use (2 of 1114), while 3-year historic data indicated a definite 30-day ST incidence of 0.14% (20 of 14 432), representing a 2.7-fold increase in definite 30-day ST with generic clopidogrel use (P = .076). Exclusion of 3 historic controls with a defined reason for ST (noncompliance, marked thrombocytosis) gave a 3.2-fold increase in 30-day ST with generic clopidogrel (P = .050). An ST-predictive algorithm revealed no difference in the likelihood of ST between patients receiving generic clopidogrel and historic controls. CONCLUSIONS: We observed an unexpected >2-fold increase in ST coincident with generic clopidogrel use. Although we cannot ascribe causality, this observation warrants increased vigilance and close monitoring of patients receiving generic clopidogrel.

GuideLiner mother-and-child guide catheter extension: a simple adjunctive tool in PCI for balloon uncrossable chronic total occlusions.

OBJECTIVES: To investigate the use of the GuideLiner "mother-and-child" guide catheter extension system as a simple solution to facilitate initial device delivery in balloon uncrossable chronic total occlusions (CTOs) undergoing percutaneous coronary intervention (PCI). BACKGROUND: During PCIs for CTO lesions, an important reason for procedural failure is the inability to deliver a balloon or microcatheter across the lesion. METHODS: We retrospectively accessed our interventional registry for 07/01/2010 to 03/21/2012 and extracted data on all CTO lesions involving GuideLiner catheter use. Cine review was performed to identify cases where a guidewire had crossed the CTO and the use of a GuideLiner catheter facilitated initial device delivery. RESULTS: We identified 28 patients that underwent PCI for CTO with a GuideLiner catheter used to assist initial balloon or microcatheter advancement across the culprit lesion. Mean overall CTO length was 26.3 ± 18.1 mm. The GuideLiner catheter was successful in delivering a small balloon to the CTO lesion in 85.7% of cases (24/28). A single CTO PCI resulted in a distal guidewire perforation, but there was no hemodynamic compromise or pericardial effusion and the patient was discharged the next day. Overall procedural success in these selected cases (where a guidewire had already crossed the CTO) was 89.3% (25/28). CONCLUSIONS: The GuideLiner mother-and-child catheter is a simple, safe and efficacious adjunctive device for difficult CTO PCIs where despite standard measures it is not possible to deliver an initial balloon or microcatheter across the occluded segment.

Genome-wide DNA methylation analysis in cohesin mutant human cell lines.

The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS.

Loss of ATRX in chondrocytes has minimal effects on skeletal development.

BACKGROUND: Mutations in the human ATRX gene cause developmental defects, including skeletal deformities and dwarfism. ATRX encodes a chromatin remodeling protein, however the role of ATRX in skeletal development is currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: We induced Atrx deletion in mouse cartilage using the Cre-loxP system, with Cre expression driven by the collagen II (Col2a1) promoter. Growth rate, body size and weight, and long bone length did not differ in Atrx(Col2cre) mice compared to control littermates. Histological analyses of the growth plate did not reveal any differences between control and mutant mice. Expression patterns of Sox9, a transcription factor required for cartilage morphogenesis, and p57, a marker of cell cycle arrest and hypertrophic chondrocyte differentiation, was unaffected. However, loss of ATRX in cartilage led to a delay in the ossification of the hips in some mice. We also observed hindlimb polydactily in one out of 61 mutants. CONCLUSIONS/SIGNIFICANCE: These findings indicate that ATRX is not directly required for development or growth of cartilage in the mouse, suggesting that the short stature in ATR-X patients is caused by defects in cartilage-extrinsic mechanisms.

SMC1A expression and mechanism of pathogenicity in probands with X-Linked Cornelia de Lange syndrome.

Cornelia de Lange Syndrome (CdLS) is a dominantly inherited heterogeneous genetic disorder with multisystem abnormalities. Sixty percent of probands with CdLS have heterozygous mutations in the Nipped-B-like (NIPBL) gene, 5% have mutations in the SMC1A gene, and one proband was found to have a mutation in the SMC3 gene. Cohesin is a multisubunit complex consisting of a SMC1A and SMC3 heterodimer and two non-SMC subunits. SMC1A is located on the human X chromosome and is reported to escape X inactivation. Twenty-nine unrelated CdLS probands with 21 unique SMC1A mutations have been identified including seven males. All mutations identified to date are either missense or small deletions, with all presumably preserving the protein open reading frame. Both wild-type and mutant alleles are expressed. Females quantitatively express twice the amount of SMC1A mRNA compared to males. The transcriptional profiling of 23 selected genes is different in SMC1A mutant probands, controls, and NIPBL mutant probands. These results suggest that mechanistically SMC1A-related CdLS is not due to altered levels of the SMC1A transcript, but rather that the mutant proteins maintain a residual function in males and enact a dominant negative effect in females.