Publications by authors named "Gerard Pasterkamp"

419 Publications

Sex-dependent gene co-expression in the human body.

Sci Rep 2021 Sep 21;11(1):18758. Epub 2021 Sep 21.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, University Utrecht, Heidelberglaan 100, 3584CX, Utrecht, The Netherlands.

Many pathophysiological mechanisms in human health and disease are dependent on sex. Systems biology approaches are successfully used to decipher human disease etiology, yet the effect of sex on gene network biology is mostly unknown. To address this, we used RNA-sequencing data of over 700 individuals spanning 24 tissues from the Genotype-Tissue Expression project to generate a whole-body gene co-expression map and quantified the sex differences per tissue. We found that of the 13,787 genes analyzed in 24 tissues, 29.5% of the gene co-expression is influenced by sex. For example, skeletal muscle was predominantly enriched with genes co-expressed stronger in males, whereas thyroid primarily contained genes co-expressed stronger in females. This was accompanied by consistent sex differences in pathway enrichment, including hypoxia, epithelial-to-mesenchymal transition, and inflammation over the human body. Furthermore, multi-organ analyses revealed consistent sex-dependent gene co-expression over numerous tissues which was accompanied by enrichment of transcription factor binding motifs in the promoters of these genes. Finally, we show that many sex-biased genes are associated with sex-biased diseases, such as autoimmunity and cancer, and more often the target of FDA-approved drugs than non-sexbiased genes. Our study suggests that sex affects biological gene networks by differences in gene co-expression and that attention to the effect of sex on biological responses to medical drugs is warranted.
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http://dx.doi.org/10.1038/s41598-021-98059-9DOI Listing
September 2021

Mast Cell Distribution in Human Carotid Atherosclerotic Plaque Differs Significantly by Histological Segment.

Eur J Vasc Endovasc Surg 2021 Sep 13. Epub 2021 Sep 13.

Department of Vascular Surgery, University Medical Centre Utrecht, Utrecht University, Utrecht, the Netherlands. Electronic address:

Objective: Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated.

Methods: Twenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied.

Results: A statistically significant difference in MCs/mm between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion.

Conclusion: MC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.
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http://dx.doi.org/10.1016/j.ejvs.2021.07.008DOI Listing
September 2021

Pre-Operative Plasma Extracellular Vesicle Proteins are Associated with a High Risk of Long Term Secondary Major Cardiovascular Events in Patients Undergoing Carotid Endarterectomy.

Eur J Vasc Endovasc Surg 2021 Sep 9. Epub 2021 Sep 9.

Department of Vascular Surgery, University Medical Centre Utrecht, Utrecht University, Utrecht, the Netherlands. Electronic address:

Objective: Patients undergoing carotid endarterectomy (CEA) maintain a substantial residual risk of major cardiovascular events (MACE). Improved risk stratification is warranted to select high risk patients qualifying for secondary add on therapy. Plasma extracellular vesicles (EVs) are involved in atherothrombotic processes and their content has been related to the presence and recurrence of cardiovascular events. The association between pre-operative levels of five cardiovascular disease related proteins in plasma EVs and the post-operative risk of MACE was assessed.

Methods: In 864 patients undergoing CEA from 2002 to 2016 included in the Athero-Express biobank, three plasma EV subfractions (low density lipoprotein [LDL], high density lipoprotein [HDL], and tiny extracellular vesicles [TEX]) were isolated from pre-operative blood samples. Using an electrochemiluminescence immunoassay, five proteins were quantified in each EV subfraction: cystatin C, serpin C1, serpin G1, serpin F2, and CD14. The association between EV protein levels and the three year post-operative risk of MACE (any stroke, myocardial infarction, or cardiovascular death) was evaluated using multivariable Cox proportional hazard regression analyses.

Results: During a median follow up of three years (interquartile range 2.2 - 3.0), 137 (16%) patients developed MACE. In the HDL-EV subfraction, increased levels of CD14, cystatin C, serpin F2, and serpin C1 were associated with an increased risk of MACE (adjusted hazard ratios per one standard deviation increase of 1.30, 95% confidence interval [CI] 1.15-1.48; 1.22, 95% CI 1.06-1.42; 1.36, 95% CI 1.16-1.61; and 1.29, 95% CI 1.10-1.51; respectively), independently of cardiovascular risk factors. No significant associations were found for serpin G1. CD14 improved the predictive value of the clinical model encompassing cardiovascular risk factors (net re-classification index = 0.16, 95% CI 0.08-0.21).

Conclusion: EV derived pre-operative plasma levels of cystatin C, serpin C1, CD14, and serpin F2 were independently associated with an increased long term risk of MACE after CEA and are thus markers for residual cardiovascular risk. EV derived CD14 levels could improve the identification of high risk patients who may benefit from secondary preventive add on therapy in order to reduce future risk of MACE.
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http://dx.doi.org/10.1016/j.ejvs.2021.06.039DOI Listing
September 2021

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Nature 2021 09 25;597(7874):92-96. Epub 2021 Aug 25.

Institute of Medicine, University of Gothenburg, Göteborg, Sweden.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs) and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.
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http://dx.doi.org/10.1038/s41586-021-03818-3DOI Listing
September 2021

Mildly Increased Renin Expression in the Absence of Kidney Injury in the Murine Transverse Aortic Constriction Model.

Front Pharmacol 2021 15;12:614656. Epub 2021 Jun 15.

Laboratory for Experimental Cardiology, University Medical Center Utrecht, Utrecht, Netherlands.

Cardiorenal syndrome type 2 is characterized by kidney failure as a consequence of heart failure that affects >50% of heart failure patients. Murine transverse aortic constriction (TAC) is a heart failure model, where pressure overload is induced on the heart without any systemic hypertension or its consequences. Whether renal function is altered in this model is debated, and if so, at which time post-TAC renal dysfunction starts to contribute to worsening of cardiac function. We therefore studied the effects of progressive heart failure development on kidney function in the absence of chronically elevated systemic blood pressure and renal perfusion pressure. C57BL/6J mice (N = 129) were exposed to TAC using a minimally invasive technique and followed from 3 to 70 days post-TAC. Cardiac function was determined with 3D ultrasound and showed a gradual decrease in stroke volume over time. Renal renin expression and plasma renin concentration increased with progressive heart failure, suggesting hypoperfusion of the kidney. In addition, plasma urea concentration, a surrogate marker for renal dysfunction, was increased post-TAC. However, no structural abnormalities in the kidney, nor albuminuria were present at any time-point post-TAC. Progressive heart failure is associated with increased renin expression, but only mildly affected renal function without inducing structural injury. In combination, these data suggest that heart failure alone does not contribute to kidney dysfunction in mice.
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http://dx.doi.org/10.3389/fphar.2021.614656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239225PMC
June 2021

Plasma Testosterone Levels and Atherosclerotic Plaque Gene Expression in Men With Advanced Atherosclerosis.

Front Cardiovasc Med 2021 14;8:693351. Epub 2021 Jun 14.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

Low plasma testosterone levels have been shown to predict worse outcome in men with severe atherosclerotic disease. We hypothesized that a low plasma testosterone level affects disease risk through changes in gene expression in atherosclerotic plaques. Therefore, we studied plasma testosterone levels in relation to gene expression levels in atherosclerotic plaque tissue of men with advanced atherosclerotic disease. Plasma testosterone levels were measured in 203 men undergoing carotid endarterectomy. The corresponding atherosclerotic plaque tissue was used for RNA sequencing. First, we assessed how often the androgen receptor gene was expressed in the plaque. Second, correlations between plasma testosterone levels and pre-selected testosterone-sensitive genes were assessed. Finally, differences within the RNA expression profile of the plaque as a whole, characterized into gene regulatory networks and at individual gene level were assessed in relation to testosterone levels. Testosterone plasma levels were low with a median of 11.6 nmol/L (IQR: 8.6-13.8). RNA-seq of the plaque resulted in reliable expression data for 18,850 genes to be analyzed. Within the RNA seq data, the androgen-receptor gene was expressed in 189 out of 203 (93%) atherosclerotic plaques of men undergoing carotid endarterectomy. The androgen receptor gene expression was not associated with testosterone plasma levels. There were no significant differences in gene expression of atherosclerotic plaques between different endogenous testosterone levels. This remained true for known testosterone-sensitive genes, the complete transcriptomic profile, male-specific gene co-expression modules as well as for individual genes. In men with severe atherosclerotic disease the androgen receptor is highly expressed in plaque tissue. However, plasma testosterone levels were neither associated with pre-selected testosterone sensitive genes, gene expression profiles nor gene regulatory networks in late-stage atherosclerotic plaques. The effect of testosterone on gene expression of the late-stage atherosclerotic plaque appears limited, suggesting that alternate mechanisms explain its effect on clinical outcomes.
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http://dx.doi.org/10.3389/fcvm.2021.693351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236711PMC
June 2021

Scientists on the Spot: Re-defining atherosclerosis through biobanks.

Cardiovasc Res 2021 Jul;117(8):e99-e100

Laboratory of Experimental Cardiology and Clinical Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1093/cvr/cvab090DOI Listing
July 2021

Persistent Symptoms and Health Needs of Women and Men With Non-Obstructed Coronary Arteries in the Years Following Coronary Angiography.

Front Cardiovasc Med 2021 3;8:670843. Epub 2021 May 3.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

The prognosis of women and men with persistent anginal complaints and non-obstructed coronary arteries is impaired as compared with asymptomatic women and men. The increased healthcare burden in the hospital due to repeated coronary angiography in these women and men has been documented, yet little is known about the percentage of women and men who remain symptomatic and under care of the general practitioner in the years following a coronary angiographic outcome of non-obstructed coronary arteries. From the Utrecht Coronary Biobank study, including individuals who underwent a coronary angiography from 2011 to 2015 ( = 2,546, 27% women), we selected women and men with non-obstructed coronary arteries ( = 687, 39% women). This population was linked to the Julius General Practitioners Network (JGPN); a database with routine care data of general practitioners. For every individual with non-obstructed coronary arteries, we selected an asymptomatic non-referred age-, sex-, and general practitioner-matched individual from the JGPN. We compared the healthcare consumption of men and women with non-obstructed coronary arteries to these matched individuals. The McNemar's test was used for pairwise comparison, and sex differences were assessed using stratified analyses. The prevalence of non-obstructed coronary arteries was higher in women as compared with men (39 vs. 23%). During a median follow-up of 7 years [IQR 6.4-8.0], 89% of the individuals with non-obstructed coronary arteries (91% women and 87% men) visited their general practitioner for one or more cardiovascular consultations. This was compared to 34% of the matched individuals (89 vs. 34%, < 0.001). The consultations were most often for angina (equivalents) (57 vs. 11%, < 0.001) and heart failure (10 vs. 2%, = 0.015). In addition, they more often consulted the general practitioner for psychosocial complaints (31 vs. 15%, = 0.005). Findings were similar for women and men. A coronary angiographic outcome of non-obstructed coronary arteries is more common in women than in men. In the years following the coronary angiography, the majority of the population remains symptomatic. Both women and men with non-obstructed coronary arteries had higher health needs for angina, heart failure, and psychosocial complaints than matched asymptomatic individuals.
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http://dx.doi.org/10.3389/fcvm.2021.670843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126611PMC
May 2021

Common Variants Associated With Expression Contribute to Carotid Plaque Vulnerability, but Not to Cardiovascular Disease in Humans.

Front Cardiovasc Med 2021 20;8:658915. Epub 2021 Apr 20.

Central Diagnostics Laboratory, University Medical Center Utrecht, University of Utrecht, Utrecht, Netherlands.

Oncostatin M (OSM) signaling is implicated in atherosclerosis, however the mechanism remains unclear. We investigated the impact of common genetic variants in and its receptors, and , on overall plaque vulnerability, plaque phenotype, intraplaque and expression, coronary artery calcification burden and cardiovascular disease susceptibility. We queried Genotype-Tissue Expression data and found that rs13168867 (C allele) was associated with decreased expression and that rs10491509 (A allele) was associated with increased expression in arterial tissues. No variant was significantly associated with expression. We associated these two variants with plaque characteristics from 1,443 genotyped carotid endarterectomy patients in the Athero-Express Biobank Study. After correction for multiple testing, rs13168867 was significantly associated with an increased overall plaque vulnerability (β = 0.118 ± s.e. = 0.040, = 3.00 × 10, C allele). Looking at individual plaque characteristics, rs13168867 showed strongest associations with intraplaque fat (β = 0.248 ± s.e. = 0.088, = 4.66 × 10, C allele) and collagen content (β = -0.259 ± s.e. = 0.095, = 6.22 × 10, C allele), but these associations were not significant after correction for multiple testing. rs13168867 was not associated with intraplaque expression. Neither was intraplaque expression associated with plaque vulnerability and no known eQTLs were associated with coronary artery calcification burden, or cardiovascular disease susceptibility. No associations were found for rs10491509 in the locus. Our study suggests that rs1316887 in the OSMR locus is associated with increased plaque vulnerability, but not with coronary calcification or cardiovascular disease risk. It remains unclear through which precise biological mechanisms OSM signaling exerts its effects on plaque morphology. However, the OSM-OSMR/LIFR pathway is unlikely to be causally involved in lifetime cardiovascular disease susceptibility.
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http://dx.doi.org/10.3389/fcvm.2021.658915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093786PMC
April 2021

Exploring the causal inference of shear stress associated DNA methylation in carotid plaque on cardiovascular risk.

Atherosclerosis 2021 05 6;325:30-37. Epub 2021 Apr 6.

Central Diagnostics Laboratory, Division Laboratories, Pharmacy, and Biomedical Genetics, University Medical Center Utrecht, University of Utrecht, Utrecht, the Netherlands. Electronic address:

Background And Aims: Atherosclerosis is a lipid-driven inflammatory disease presumably initiated by endothelial activation. Low vascular shear stress is known for its ability to activate endothelial cells. Differential DNA methylation (DNAm) is a relatively unexplored player in atherosclerotic disease development and endothelial dysfunction. Previous studies showed that the expression of 11 genes was associated with differential DNAm due to low shear stress in murine endothelial cells. We hypothesized a causal relationship between DNAm of shear stress associated genes in human carotid plaque and increased risk of cardiovascular disease.

Methods: Using Mendelian randomisation (MR) analysis, we explored the potential causal role of DNAm of shear stress associated genes on cardiovascular disease risk. We used data from the Athero-Expression Biobank Study for the discovery of methylation quantitative trait loci (mQTLs) in 442 advanced carotid plaques. Next, we performed MR analysis using these mQTLs and publicly available GWAS summary statistics of coronary artery disease (CAD) and ischemic stroke (IS).

Results: We discovered 9 mQTLs in plaque in the promoters of shear stress associated genes. We found no significant effect of shear stress gene promoter methylation and increased risk of CAD and IS.

Conclusions: Differential methylation of shear stress associated genes in advanced atherosclerotic plaques in unlikely to increase cardiovascular risk in human.
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http://dx.doi.org/10.1016/j.atherosclerosis.2021.03.043DOI Listing
May 2021

Hunt for the (Multi)-Marker Grail in the Diverse Landscape of Atherosclerosis.

Arterioscler Thromb Vasc Biol 2021 05 8;41(5):1789-1791. Epub 2021 Apr 8.

Central Diagnostic Laboratory, University Medical Center Utrecht, the Netherlands.

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http://dx.doi.org/10.1161/ATVBAHA.121.316167DOI Listing
May 2021

Common Genetic Variation in MC4R Does Not Affect Atherosclerotic Plaque Phenotypes and Cardiovascular Disease Outcomes.

J Clin Med 2021 Mar 1;10(5). Epub 2021 Mar 1.

Department Medicine, Division Endocrinology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

We analyzed the effects of the common BMI-increasing melanocortin 4 receptor (MC4R) rs17782313-C allele with a minor allele frequency of 0.22-0.25 on (1) cardiovascular disease outcomes in two large population-based cohorts (Copenhagen City Heart Study and Copenhagen General Population Study, = 106,018; and UK Biobank, = 357,426) and additionally in an elderly population at risk for cardiovascular disease ( = 5241), and on (2) atherosclerotic plaque phenotypes in samples of patients who underwent endarterectomy ( = 1439). Using regression models, we additionally analyzed whether potential associations were modified by sex or explained by changes in body mass index. We confirmed the BMI-increasing effects of +0.22 kg/m per additional copy of the C allele ( < 0.001). However, we found no evidence for an association of common MC4R genetic variation with coronary artery disease (HR 1.03; 95% CI 0.99, 1.07), ischemic vascular disease (HR 1.00; 95% CI 0.98, 1.03), myocardial infarction (HR 1.01; 95% CI 0.94, 1.08 and 1.02; 0.98, 1.07) or stroke (HR 0.93; 95% CI 0.85, 1.01), nor with any atherosclerotic plaque phenotype. Thus, common MC4R genetic variation, despite increasing BMI, does not affect cardiovascular disease risk in the general population or in populations at risk for cardiovascular disease.
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http://dx.doi.org/10.3390/jcm10050932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957774PMC
March 2021

Sclerostin Downregulation Globally by Naturally Occurring Genetic Variants, or Locally in Atherosclerotic Plaques, Does Not Associate With Cardiovascular Events in Humans.

J Bone Miner Res 2021 07 30;36(7):1326-1339. Epub 2021 Mar 30.

University Medical Centre Utrecht, Utrecht, The Netherlands.

Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.6 ± 10.4 years). Sclerostin staining was absent in most plaques (67%), and when detected, it was of reduced intensity compared with normal aorta and was located in deeper regions of the plaque/wall but was not observed in areas considered relevant to plaque stability (fibrous cap and endothelium). Additionally, genetic variants associated with lifelong reduced sclerostin expression were explored for associations with phenotypes including those related to bone physiology and CV risk factors/events in a population-based phenomewide association study (PheWAS). Natural genetic modulation of sclerostin by variants with a significant positive effect on bone physiology showed no association with lifetime risk of MI or stroke. These data do not support a causal association between the presence of sclerostin, or its inhibition, in the vasculature and increased risk of serious cardiovascular events. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360163PMC
July 2021

Prosaposin mediates inflammation in atherosclerosis.

Sci Transl Med 2021 03;13(584)

Department of Internal Medicine and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient ( ) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified , a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of bone marrow to low-density lipoprotein receptor knockout ( ) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.
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http://dx.doi.org/10.1126/scitranslmed.abe1433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209679PMC
March 2021

Preclinical Feasibility and Patency Analyses of a New Distal Coronary Connector: The ELANA Heart Bypass.

Innovations (Phila) 2021 Mar-Apr;16(2):163-168. Epub 2021 Mar 7.

8124 Department of Cardiothoracic Surgery, University Medical Center Utrecht, The Netherlands.

Objective: This preclinical study determines the feasibility and 6-month patency rates of a new distal coronary connector, the Excimer Laser Assisted Nonocclusive Anastomosis (ELANA) Heart Bypass.

Methods: Twenty Dutch Landrace pigs received either a hand-sewn ( = 8) or an ELANA ( = 12) left internal thoracic artery to left anterior descending artery anastomosis, using off-pump coronary artery bypass grafting. Six-month patency rates were demonstrated by coronary angiography and histological evaluation. Throughout, procedural details and complication rates were collected.

Results: The ELANA Heart Bypass demonstrated 0% mortality and complication rates during follow-up. It was demonstrated feasible, with comparable perioperative flow measurements (ELANA vs hand-sewn, median [min to max], 24 [14 to 28] vs 17 [12 to 31] mL/min; = 0.601) and fast construction times (3 [3 to 7] vs 31 [26 to 37] min; < 0.001). Yet, an extra hemostatic stitch was needed in 25% of the ELANA versus 12.5% of the hand-sewn anastomoses. The 6-month patency rate of the ELANA Heart Bypass was 83.3% versus 100% in hand-sewn anastomoses. The 2 occluded ELANA-anastomoses were defined model-based errors.

Conclusions: The ELANA Heart Bypass facilitates a sutureless distal coronary anastomosis. A design change is suggested to improve hemostasis and will be evaluated in future translational studies. This new technique is a potential alternative to hand-sewn anastomoses in (minimally invasive) coronary surgery.
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http://dx.doi.org/10.1177/1556984521991519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108114PMC
March 2021

Sex-Stratified Gene Regulatory Networks Reveal Female Key Driver Genes of Atherosclerosis Involved in Smooth Muscle Cell Phenotype Switching.

Circulation 2021 Feb 27;143(7):713-726. Epub 2021 Jan 27.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands (R.J.G.H., M.M., H.M.d.R.).

Background: Although sex differences in coronary artery disease are widely accepted with women developing more stable atherosclerosis than men, the underlying pathobiology of such differences remains largely unknown. In coronary artery disease, recent integrative systems biological studies have inferred gene regulatory networks (GRNs). Within these GRNs, key driver genes have shown great promise but have thus far been unidentified in women.

Methods: We generated sex-specific GRNs of the atherosclerotic arterial wall in 160 women and age-matched men in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). We integrated the female GRNs with single-cell RNA-sequencing data of the human atherosclerotic plaque and single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic smooth muscle cell (SMC) lineage-tracing mice.

Results: By comparing sex-specific GRNs, we observed clear sex differences in network activity within the atherosclerotic tissues. Genes more active in women were associated with mesenchymal cells and endothelial cells, whereas genes more active in men were associated with the immune system. We determined that key drivers of GRNs active in female coronary artery disease were predominantly found in (SMCs by single-cell sequencing of the human atherosclerotic plaques, and higher expressed in female plaque SMCs, as well. To study the functions of these female SMC key drivers in atherosclerosis, we examined single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic SMC lineage-tracing mice. The female key drivers were found to be expressed by phenotypically modulated SMCs and affected by Klf4, suggesting that sex differences in atherosclerosis involve phenotypic switching of plaque SMCs.

Conclusions: Our systems approach provides novel insights into molecular mechanisms that underlie sex differences in atherosclerosis. To discover sex-specific therapeutic targets for atherosclerosis, an increased emphasis on sex-stratified approaches in the analysis of multi-omics data sets is warranted.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930467PMC
February 2021

The prognostic value of automated coronary calcium derived by a deep learning approach on non-ECG gated CT images from Rb-PET/CT myocardial perfusion imaging.

Int J Cardiol 2021 04 4;329:9-15. Epub 2021 Jan 4.

Department of Vascular Surgery, University Medical Centre Utrecht, the Netherlands; Netherlands Heart Institute, Utrecht, the Netherlands.

Background: Assessment of both coronary artery calcium(CAC) scores and myocardial perfusion imaging(MPI) in patients suspected of coronary artery disease(CAD) provides incremental prognostic information. We used an automated method to determine CAC scores on low-dose attenuation correction CT(LDACT) images gathered during MPI in one single assessment. The prognostic value of this automated CAC score is unknown, we therefore investigated the association of this automated CAC scores and major adverse cardiovascular events(MACE) in a large chest-pain cohort.

Method: We analyzed 747 symptomatic patients referred for RubidiumPET/CT, without a history of coronary revascularization. Ischemia was defined as a summed difference score≥2. We used a validated deep learning(DL) method to determine CAC scores. For survival analysis CAC scores were dichotomized as low(<400) and high(≥400). MACE was defined as all cause death, late revascularization (>90 days after scanning) or nonfatal myocardial infarction. Cox proportional hazard analysis were performed to identify predictors of MACE.

Results: During 4 years follow-up, 115 MACEs were observed. High CAC scores showed higher cumulative event rates, irrespective of ischemia (nonischemic: 25.8% vs 11.9% and ischemic: 57.6% vs 23.4%, P-values <0.001). Multivariable cox regression revealed both high CAC scores (HR 2.19 95%CI 1.43-3.35) and ischemia (HR 2.56 95%CI 1.71-3.35) as independent predictors of MACE. Addition of automated CAC scores showed a net reclassification improvement of 0.13(0.022-0.245).

Conclusion: Automatically derived CAC scores determined during a single imaging session are independently associated with MACE. This validated DL method could improve risk stratification and subsequently lead to more personalized treatment in patients suspected of CAD.
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http://dx.doi.org/10.1016/j.ijcard.2020.12.079DOI Listing
April 2021

Cardiovascular imaging of women and men visiting the outpatient clinic with chest pain or discomfort: design and rationale of the ARGUS Study.

BMJ Open 2020 12 15;10(12):e040712. Epub 2020 Dec 15.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, the Netherlands

Introduction: Chest pain or discomfort affects 20%-40% of the general population over the course of their life and may be a symptom of myocardial ischaemia. For the diagnosis of obstructive macrovascular coronary artery disease (CAD), algorithms have been developed; however, these do not exclude microvascular angina. This may lead to false reassurance of symptomatic patients, mainly women, with functionally significant, yet non-obstructive coronary vascular disease. Therefore, this study aims to estimate the prevalence of both macrovascular and microvascular coronary vascular disease in women and men presenting with chest pain or discomfort, and to subsequently develop a decision-support tool to aid cardiologists in referral to cardiovascular imaging for both macrovascular and microvascular CAD evaluation.

Methods And Analysis: Women and men with chest pain or discomfort, aged 45 years and older, without a history of cardiovascular disease, who are referred to an outpatient cardiology clinic by their general practitioner are eligible for inclusion. Coronary CT angiography is used for anatomical imaging. Additionally, myocardial perfusion imaging by adenosine stress cardiac MRI is performed to detect functionally significant coronary vascular disease. Electronic health record data, collected during regular cardiac work-up, including medical history, cardiovascular risk factors, physical examination, echocardiography, (exercise) ECG and blood samples for standard cardiovascular biomarkers and research purposes, are obtained. Participants will be classified as positive or negative for coronary vascular disease based on all available data by expert panel consensus (a cardiovascular radiologist and two cardiologists). After completion of the clinical study, all collected data will be used to develop a decision support tool using predictive modelling and machine-learning techniques.

Ethics And Dissemination: The study protocol was approved by the Institutional Review Board of the University Medical Center Utrecht. Results will be disseminated through national and international conferences and in peer-reviewed journals in cardiovascular disease.

Trial Registration Number: Trialregister.nl Registry NL8702.
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http://dx.doi.org/10.1136/bmjopen-2020-040712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745322PMC
December 2020

Preemptive Stenting of the Vulnerable Plaque: Fixing a Dogma?

J Am Coll Cardiol 2020 11;76(20):2302-2304

Cardiology Department, Utrecht University Medical Center, Utrecht, the Netherlands.

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http://dx.doi.org/10.1016/j.jacc.2020.09.600DOI Listing
November 2020

Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

Circ Res 2020 12 12;127(12):1552-1565. Epub 2020 Oct 12.

Center for Public Health Genomics (R.A., A.A., J.H., L.C., J.Y.S., D.L., E.F., R.A.-W., N.A., A.W.M., S.O.-G., C.L.M., M.C.), University of Virginia, Charlottesville.

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 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC 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 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718324PMC
December 2020

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics.

Circ Res 2020 11 28;127(11):1437-1455. Epub 2020 Sep 28.

Laboratory of Clinical Chemistry and Haematology, University Medical Center, Heidelberglaan 100, Utrecht, the Netherlands (L.S., A.B., F.W.A., S.W.v.d.L., M.M., G.P.).

Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed.

Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis.

Methods And Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 and CD8 T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells.

Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641189PMC
November 2020

The TAXINOMISIS Project: A multidisciplinary approach for the development of a new risk stratification model for patients with asymptomatic carotid artery stenosis.

Eur J Clin Invest 2020 Dec 2;50(12):e13411. Epub 2020 Oct 2.

Department of Materials Science and Engineering, Unit of Medical Technology and Intelligent Information Systems, University of Ioannina, Ioannina, Greece.

Introduction: Asymptomatic carotid artery stenosis (ACAS) may cause future stroke and therefore patients with ACAS require best medical treatment. Patients at high risk for stroke may opt for additional revascularization (either surgery or stenting) but the future stroke risk should outweigh the risk for peri/post-operative stroke/death. Current risk stratification for patients with ACAS is largely based on outdated randomized-controlled trials that lack the integration of improved medical therapies and risk factor control. Furthermore, recent circulating and imaging biomarkers for stroke have never been included in a risk stratification model. The TAXINOMISIS Project aims to develop a new risk stratification model for cerebrovascular complications in patients with ACAS and this will be tested through a prospective observational multicentre clinical trial performed in six major European vascular surgery centres.

Methods And Analysis: The risk stratification model will compromise clinical, circulating, plaque and imaging biomarkers. The prospective multicentre observational study will include 300 patients with 50%-99% ACAS. The primary endpoint is the three-year incidence of cerebrovascular complications. Biomarkers will be retrieved from plasma samples, brain MRI, carotid MRA and duplex ultrasound. The TAXINOMISIS Project will serve as a platform for the development of new computer tools that assess plaque progression based on radiology images and a lab-on-chip with genetic variants that could predict medication response in individual patients.

Conclusion: Results from the TAXINOMISIS study could potentially improve future risk stratification in patients with ACAS to assist personalized evidence-based treatment decision-making.
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http://dx.doi.org/10.1111/eci.13411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757200PMC
December 2020

Elevated Lp(a) (Lipoprotein[a]) Levels Increase Risk of 30-Day Major Adverse Cardiovascular Events in Patients Following Carotid Endarterectomy.

Stroke 2020 10 3;51(10):2972-2982. Epub 2020 Sep 3.

Department of Experimental Vascular Medicine (K.E.D., J.G.S., J.K.), Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, the Netherlands.

Background And Purpose: General population studies have shown that elevated Lp(a) (lipoprotein[a]) levels are an emerging risk factor for cardiovascular disease and subsequent cardiovascular events. The role of Lp(a) for the risk of secondary MACE in patients undergoing carotid endarterectomy (CEA) is unknown. Our objective is to assess the association of elevated Lp(a) levels with the risk of secondary MACE in patients undergoing CEA.

Methods: Lp(a) concentrations were determined in preoperative blood samples of 944 consecutive patients with CEA included in the Athero-Express Biobank Study. During 3-year follow-up, major adverse cardiovascular events (MACE), consisting of myocardial infarction, stroke, and cardiovascular death, were documented.

Results: After 3 years follow-up, Kaplan-Meier cumulative event rates for MACE were 15.4% in patients with high Lp(a) levels (>137 nmol/L; >80th cohort percentile) and 10.2% in patients with low Lp(a) levels (≤137 nmol/L; ≤80th cohort percentile; log-rank test: =0.047). Cox regression analyses adjusted for conventional cardiovascular risk factors revealed a significant association between high Lp(a) levels and 3-year MACE with an adjusted hazard ratio of 1.69 (95% CI, 1.07-2.66). One-third of MACE occurred within 30 days after CEA, with an adjusted hazard ratio for the 30-day risk of MACE of 2.05 (95% CI, 1.01-4.17). Kaplan-Meier curves from time point 30 days to 3 years onward revealed no significant association between high Lp(a) levels and MACE. Lp(a) levels were not associated with histological carotid plaque characteristics.

Conclusions: High Lp(a) levels (>137 nmol/L; >80th cohort percentile) are associated with an increased risk of 30-day MACE after CEA. This identifies elevated Lp(a) levels as a new potential risk factor for secondary cardiovascular events in patients after carotid surgery. Future studies are required to investigate whether Lp(a) levels might be useful in guiding treatment algorithms for carotid intervention.
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http://dx.doi.org/10.1161/STROKEAHA.120.030616DOI Listing
October 2020

The age- and sex-specific composition of atherosclerotic plaques in vascular surgery patients.

Atherosclerosis 2020 10 29;310:1-10. Epub 2020 Jul 29.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, University of Utrecht, Utrecht, the Netherlands. Electronic address:

Background And Aims: The sex- and age-related differences in the composition of iliofemoral atherosclerotic plaques are largely unknown. Therefore, the aim of the current study is to gain insight into plaque composition across strata of age and sex in a large cohort of vascular surgery patients.

Methods: Peripheral atherosclerotic plaques of patients who underwent iliofemoral endarterectomy (n = 790) were harvested between 2002 and 2014. The plaques were semi-quantitatively analyzed for the presence of lipid cores, calcifications, plaque hemorrhages (PH), collagen, macrophage and smooth muscle cell (SMC) content, and quantitatively for microvessel density. Patients were stratified by age tertiles and sex.

Results: Ageing was independently associated with rupture-prone iliofemoral plaque characteristics, such as higher prevalence of plaque calcifications (OR 1.52 (95%CI:1.03-2.24) p = 0.035) and PH (OR 1.46 (95%CI:1.01-2.09) p = 0.042), and lower prevalence of collagen (OR 0.52 (95%CI:0.31-0.86) p = 0.012) and SMCs (OR 0.59 (95%CI:0.39-0.90) p = 0.015). Sex-stratified data showed that men had a higher prevalence of lipid cores (OR 1.62 (95%CI:1.06-2.45) p = 0.025) and PH (OR 1.62 (95%CI:1.16-2.54) p = 0.004) compared to women. These sex-differences attenuated with increasing age, with women showing an age-related increase in calcifications (p = 0.002), PH (p = 0.015) and decrease in macrophages (p = 0.005). In contrast, men only showed a decrease in collagen (p = 0.043).

Conclusions: Atherosclerotic iliofemoral plaques derived from men display more rupture-prone characteristics compared to women. Yet, this difference is attenuated with an increase in age, with older women having more rupture-prone characteristics compared to younger women.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.07.016DOI Listing
October 2020

Intrinsic transcriptomic sex differences in human endothelial cells at birth and in adults are associated with coronary artery disease targets.

Sci Rep 2020 07 23;10(1):12367. Epub 2020 Jul 23.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Sex differences in endothelial cell (EC) biology may reflect intrinsic differences driven by chromosomes or sex steroid exposure and gender differences accumulated over life. We analysed EC gene expression data from boy-girl twins at birth and in non-twin adults to detect sex differences at different stages of life, and show that 14-25% of the EC transcriptome is sex-biased. By combining data from both stages of life, we identified sex differences that are present at birth and maintained throughout life, and those that are acquired over life. Promisingly, we found that genes that present with an acquired sex difference in ECs are more likely to be targets of sex steroids. Annotating both gene sets with data from multiple genome-wide association studies (GWAS) revealed that genes with an intrinsic sex difference in ECs are enriched for coronary artery disease GWAS hits. This study underscores the need for treating sex as a biological variable.
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http://dx.doi.org/10.1038/s41598-020-69451-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378217PMC
July 2020

Plasma extracellular vesicle proteins are associated with stress-induced myocardial ischemia in women presenting with chest pain.

Sci Rep 2020 07 23;10(1):12257. Epub 2020 Jul 23.

Department of Cardiology, St. Antonius Hospital, Nieuwegein, the Netherlands.

Diagnosing stable ischemic heart disease (IHD) is challenging, especially in females. Currently, no blood test is available. Plasma extracellular vesicles (EV) are emerging as potential biomarker source. We therefore aimed to identify stress induced ischemia due to stable IHD with plasma extracellular vesicle protein levels in chest pain patients. We analyzed 450 patients suspected for stable IHD who were referred for Rb PET/CT in the outpatient clinic. Blood samples were collected before PET/CT and plasma EVs were isolated in 3 plasma subfractions named: TEX, HDL, LDL. In total 6 proteins were quantified in each of these subfractions using immuno-bead assays. CD14 and CystatinC protein levels were independent significant predictors of stress-induced ischemia in the LDL and the HDL subfraction and SerpinC1 and SerpinG1 protein levels in the HDL fraction. Subgroup-analysis on sex revealed that these associations were completely attributed to the associations in women. None of the significant EV proteins remained significant in men. Plasma EV proteins levels are associated with the presence of stable IHD in females presenting with chest pain. This finding, if confirmed in larger cohort studies could be a crucial step in improving diagnostic assessment of women with suspected IHD.
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http://dx.doi.org/10.1038/s41598-020-69297-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378184PMC
July 2020

Sex differences in flow cytometry-based platelet reactivity in stable outpatients suspected of myocardial ischemia.

Res Pract Thromb Haemost 2020 Jul 15;4(5):879-885. Epub 2020 May 15.

Department of Cardiology University Medical Center Utrecht Utrecht The Netherlands.

Background: Antiplatelet therapy is the mainstay of secondary prevention of cardiovascular events. Studies suggest that women do not obtain equal therapeutic benefit from antiplatelet therapy compared with men. The link between sex differences in platelet biology and response to antiplatelet therapies is unclear. We therefore investigated the role of sex differences in platelet reactivity in a cohort of outpatients with chest pain, in response to treatment with antiplatelet agents.

Methods: Platelet reactivity was measured in 382 randomly selected patients participating in the Myocardial Ischemia Detection by Circulating Biomarkers (MYOMARKER) study, an observational cohort study of outpatients suspected of myocardial ischemia. In all patients, blood was collected during diagnostic workup, and platelet reactivity was assessed with a flow cytometry-based platelet activation test that quantifies both platelet degranulation (P-selectin expression) and platelet aggregation (fibrinogen binding to integrin αIIbβ3) in whole blood.

Results: Platelet reactivity was higher in women compared with men when activated with protease activating receptor 1-activating peptide SFLLRN (PAR1-AP) and adenosine 5'-phosphate (ADP), independent of age, basal activation status, estimated glomerular filtration rate < 60, platelet count, statin use, the use of P2Y12 inhibitors, or the use of aspirin. P2Y12 inhibitor use strongly reduced fibrinogen binding after stimulation with PAR1-AP, but only slightly reduced platelet P-selectin expression. Calculation of the relative inhibition in P2Y12 users indicated 62% inhibition of the response toward ADP. Stratified analysis showed that women (n = 14) using P2Y12 inhibitors showed less inhibition of fibrinogen binding after PAR1-AP stimulation than men (n = 38) using P2Y12 inhibitors.

Conclusions: These findings call for further study of differential effects of P2Y12 inhibitors in women with suspected myocardial ischemia.
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http://dx.doi.org/10.1002/rth2.12344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354392PMC
July 2020

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

Circulation 2020 Nov 17;142(21):2045-2059. Epub 2020 Jul 17.

Robert M. Berne Cardiovascular Research Center (G.F.A., K.M.O, S.K., A.N., C.M.W., S.S., C.A.H., R.H., R.A.B., C.A.M., E.R.Z., G.K.O.), University of Virginia, Charlottesville.

Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability.

Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis.

Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11, Lgals3 population with a chondrocyte-like gene signature that was markedly reduced with SMC- knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization.

Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682794PMC
November 2020
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