Key Points• Rs12041331 is the first functional CpG-SNP related to platelet function whose regulatory mechanism depends on DNA methylation. • Rs12041331 marks allelespecific methylation at the CpG island encompassing the first untranslated exon during megakaryopoiesis.Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 59-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. (Blood. 2016;128(7):1003-1012

Objective-Thrombospondin-1 (TSP1) is described as a positive regulator of vascular smooth muscle growth in cell culture. However, insight into the in vivo effects of TSP1 on smooth muscle cell (SMC) function is lacking. Methods and Results-We analyzed wild-type (WT) and TSP1-deficient (Tsp1 Ϫ/Ϫ ) mice in a carotid artery ligation model, in which neointimal lesions form without overt mechanical damage to the endothelium. On ligation, the expression of TSP1 increased strongly in the matrix of neointima and adventitia. In the early phase after ligation (day 3 to 7), activation, proliferation, and migration of medial SMCs were delayed and impaired in Tsp1 Ϫ/Ϫ mice, in parallel with defective upregulation of metalloproteinase (MMP)-2 activity. As a result, Tsp1 Ϫ/Ϫ arteries developed smaller neointimal lesions, a thicker media but comparably attenuated patency as in WT arteries, 28 days after ligation. Furthermore, medial and neointimal SMCs in Tsp1 Ϫ/Ϫ mice produced more collagen, more osteopontin, and displayed weaker smooth muscle actin staining than WT SMCs, indicative of a modified SMC phenotype in Tsp1 Ϫ/Ϫ mice. Key Words: carotid artery Ⅲ matricellular proteins Ⅲ neointima Ⅲ smooth muscle cells T hrombospondin-1 (TSP1) is a matricellular protein, with a complex multidomain structure which can interact with a great variety of receptors, ligands, and matrix components (reviewed in 1,2 ). It is highly expressed during development, but present at a low level in adult tissues and upregulated in response to injury. [3][4][5] TSP1 is present in several cell types, including vascular cells, from which it is secreted on cell activation to participate in fine modulation of cell-cell and cell-matrix interactions and regulation of cell function. 6 -8 TSP1 gene-deficient (Tsp1 Ϫ/Ϫ ) mice are viable, and apparently normal, but challenge of these mice in various models has shown that TSP1 inhibits angiogenesis, 9,10 promotes wound healing in an excisional wound healing model, 11 and contributes to pulmonary homeostasis. 12 We have recently shown an in vivo role for TSP1 in platelet and thrombus adhesion to the injured vessel wall. 13 Percutaneous coronary interventions often result in the occurrence of intimal hyperplasia and restenosis. 14 They are triggered by endothelial injury, and involve proliferation and migration of smooth muscle cells (SMCs) from the media into the intima. A role for TSP1 in vascular injury has been suggested by the strong TSP1 upregulation in rat carotid arteries after balloon-angioplasty 15 and at sites of hyperplasia, in hypercholesterolemic lesions, and in the adventitia of diabetic rats. 5,16,17 Although TSP1 is not essential for SMC growth in vitro, the treatment of SMCs with growth factors results in strong TSP1 expression, which further amplifies SMC proliferation. 18 -21 The inhibition of TSP1 by specific antibodies reduced SMC proliferation. 21 TSP1 also acts as a chemoattractant for SMCs via interactions between its carboxyl terminus and CD47, and hence integrins. [22][23][24][25][26] ...

The DxS-induced aggregation of human and murine platelets is the result of activation of PI3K/Akt through direct PEAR1 phosphorylation and parallel Syk-signaling through CLEC-2.

BackgroundPlatelet-endothelial aggregation receptor 1 (PEAR-1) is a transmembrane receptor involved in platelet activation and megakaryopoiesis whose expression is driven by DNA methylation. PEAR1 variants were associated with differential platelet response to activation and cardiovascular outcomes. We aimed at investigating the link between PEAR1 methylation and platelet and leukocyte function markers in a family-based population.ResultsWe measured PEAR1 methylation in 605 Moli-family participants with available blood counts, plasma P-selectin and C-reactive protein, whole blood platelet P-selectin, and platelet-leukocyte mixed conjugate measurements. We performed principal component analysis (PCA) to identify groups of highly correlated CpG sites. We used linear mixed regression models (using age, gender, BMI, smoking, alcohol drinking, being a proband for family recruitment, being a member of myocardial infarction (MI) family as fixed effects, and family as a random effect) to evaluate associations between PEAR1 methylation and phenotypes. PEAR1 methylation Factor2, characterized by the previously identified megakaryocyte-specific CpG sites, was inversely associated with platelet-monocyte conjugates, P-selectin, and WBC counts, while positively associated with the platelet distribution width (PDW) and with leukocyte CD11b and L-selectin. Moreover, PEAR1 Factor2 methylation was negatively associated with INFLAscore, a low-grade inflammation score. The latter was partially mediated by the PEAR1 methylation effect on platelet variables. PEAR1 methylation association with WBC measurements and INFLAscore was confirmed in the independent cohort FLEMENGHO.ConclusionsWe report a significant link between epigenetic signatures in a platelet functional gene and inflammation-dependent platelet function variability measured in two independent cohorts.

BackgroundIn this study we investigated the antiatherogenic effect of dietary enrichment of a combination of extra virgin olive oil (EVO) and seal oil on apolipoprotein E-deficient (apoE-/-).MethodsSix-week-old female and male apoE-/- mice were for 12 weeks fed a lipid rich diet containing 19.5% fat and 1.25% cholesterol without any supplement, with 1% (wt/wt) mixture of extra virgin olive and seal oil (EVO/n-3), or 1% corn oil, respectively.ResultsSupplementation with the combination of EVO/n-3 significantly reduced atherosclerotic lesion formation in the aortic arch, thoracoabdominal, and total aorta of female apoE-/-mice. The effect was less pronounced in male mice and significant reduction was only observed in the thoracoabdominal region of the aorta. There were no differences or changes in dietary intake or body weight gain. However, compared to the other groups, plasma levels of triglycerides were reduced in both female and male mice fed the EVO/n-3 mixture. Male mice on both treatments showed reduced plasma cholesterol compared to the control mice after 12 weeks on diet.ConclusionDietary supplementation of a marine/olive oil combination inhibits atherosclerotic lesion formation in the female apoE-/- mice by antithrombotic, antihypertriglyceridemic, and antioxidant effects.