This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 w g/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca 2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 w M)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.

Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model. Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1^−/− mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1^−/− splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1^−/− spleen displayed anomalous T_reg and T_H cell differentiation. NEAT1^−/− bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1^−/− BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1^−/− bone marrow and blood. NEAT1^−/− mice displayed aortic wall CD68⁺ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1^−/− mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.

We have shown that the G protein–coupled receptor (GPCR) agonists, thrombin and Factor Xa, stimulate smooth muscle cell (SMC) proliferation through transactivation of the EGF receptor (EGFR) or the FGF receptor (FGFR), both of which are tyrosine kinase receptors. In the present study, we investigated whether platelet-derived growth factor (PDGF), a tyrosine kinase receptor agonist, might transactivate another tyrosine kinase receptor to induce SMC proliferation. Because heparin inhibits PDGF-mediated proliferation in human SMCs, we investigated whether the heparin-binding growth factor basic fibroblast growth factor (bFGF) and one of its receptors, FGFR-1, play a role in the response of human arterial SMCs to PDGF-BB. PDGF-BB induced the release of bFGF and sustained phosphorylation of FGFR-1 (30 minutes to 6 hours). A bFGF-neutralizing antibody inhibited PDGF-BB–mediated phosphorylation of FGFR-1, DNA synthesis, and cell proliferation. In the presence of bFGF antibody, PDGF-BB–induced early activation of ERK (0 to 60 minutes) was not affected, whereas late ERK activation (2 to 4 hours) was reduced. When FGFR-1 expression was suppressed using small interfering RNA (siRNA), ERK activation was reduced at late, but not early, time points after PDGF-BB stimulation. Addition of bFGF antibody to cells treated with siRNA to FGFR-1 had no further effect on ERK activation. Our results provide support for a novel mechanism by which PDGF-BB induces the release of bFGF and activation of FGFR-1 followed by the sustained activation of ERK and proliferation of human SMCs.