Platelets: Old Players Revisited284Platelet microvesicles in vascular inflammation285Pharmacological depletion of serotonin promotes atherosclerotic plaque formation in apoE-/- mice286Deletion of junctional adhesion molecule a from platelets increases early stage neointima formation after wire injury in hyperlipidemic mice

# 284 Platelet microvesicles in vascular inflammation {#article-title-2}Background: Microvesicles are gathering increasing attention as mediators of cell communication and as integral effectors of disease. Platelets present a major source of microvesicles and release these microvesicles either spontaneously or upon activation. Platelet microvesicles (PMVs) retain many features of their parent cells and have been shown to exert modulatory effects on vascular and immune cells.Purpose: We hypothesize that PMVs interact with vascular smooth muscle cells (SMCs) and modulate their function in the context of vascular remodeling.Methods: PMVs were isolated from aging human platelet concentrates by serial centrifugation steps. PMVs were quantified and characterized by flow cytometry using annexin A5/phosphatidylserine and antibodies against CD41a/GPIIb. Size calibrated micro beads were used to quantify the absolute amount of PMVs/mL. Cell migration experiments were performed using a boyden chemotaxis chamber. Platelet receptors implicated in PMV-SMC interaction were identified by blocking antibodies. Proliferation of SMCs was measured by the BrdU-cell proliferation kit. Adhesion of monocytic cells to SMCs was determined by a flow adhesion assay. Relative quantification of gene expression was determined by real time and quantitative PCR.Results: In the presence of PMVs, SMCs show increased migration. Under resting conditions, the PMV binding to SMCs was specifically abrogated by the integrin αIIbβ3 inhibitor (integrilin) indicating an integrin-dependent mechanism of interaction. A proliferative effect on SMCs was measured after 48 hours after incubation with PMVs and this proliferation relied on interactions via integrin αMβ2, CD40 and P-selectin. The firm adhesion of monocytic cells to PMVs stimulated SMCs under flow conditions was significantly increased compared to untreated, resting SMCs. The adhesion mainly depended on the integrin αIIbβ3 and P-selectin but also CD40 and fractalkine. PMVs decreased gene expression of contractile proteins, i.e. αSMA and calponin.Conclusion: Isolated PMVs have shown to exert an immunomodulatory activity on various cell types. The present data indicate a role of PMVs in inducing a phenotypic switch towards a synthetic inflammatory SMC phenotype, thus contributing to vascular atherogenesis, in particular vascular remodeling.# 285 Pharmacological depletion of serotonin promotes atherosclerotic plaque formation in apoE-/- mice {#article-title-3}Cardiovascular disease, like myocardial infarction and stroke, is the major cause of death in western countries, which mainly arise from atherosclerosis. This chronic disorder is characterized by inflammation of the vessel wall driving lesion formation. These so called plaques contain macrophages, T cells and other immune cells as well as an accumulation of lipids. In the early phase an activation of the endothelium induces adhesion molecule expression and secretion of pro-inflammatory cytokines and chemokines leading to the recruitment of leukocytes. Platelets also contribute to the leukocyte recruitment by interacting with the endothelium and secretion of granule content including serotonin. Platelet serotonin was recently shown to promote selectin-dependent leukocyte interaction and recruitment to post-capillary venules in a mouse model of acute inflammation. Clinically available serotonin reuptake inhibitors (SERT) used as anti-depressive drugs might be a useful strategy to impair peripheral serotonin storage in platelets and thus reduce serotonin-mediated endothelium activation in cardiovascular disease patients. We therefore hypothesized that treatment with serotonin transporter inhibitor fluoxetine (FLX) inhibits atherosclerotic plaque formation by limiting leukocyte adhesion.We fed apolipoprotein E-deficient (ApoE-/-) mice for 4 or 16 weeks with high-cholesterol diet (HCD) and treated them with FLX via the drinking water. We analyzed atherosclerotic lesion formation and composition via oil-red-O lipid staining and immunohistology of aortic root cross-sections. Leukocyte subsets in blood, bone marrow, spleen and the abdominal aorta were assessed by flow cytometry. Surprisingly, pharmacological serotonin depletion resulted in significantly increased plaque size after 4 weeks HCD (ctrl, 44592 ± 5920 µm2 vs fluoxetine, 68749 ± 6118 µm2; n=9-10; P=0.01), in part due to an increased macrophage infiltration (ctrl, 10965 ± 1931 µm2 vs FLX, 22424 ± 4906 n=14-15; P=0.058). After 16 weeks HCD feeding, we no longer observed a difference in lesion size. The enhanced arterial leukocyte recruitment was not due to enhanced myelopoiesis or leukocyte mobilization from bone marrow or spleen, but rather mediated by increased adhesion of myeloid cells to aortic lesions as evidenced by intravital microscopy of carotid arteries. The live imaging revealed increased numbers of adhering CD11b-stained myeloid cells in carotids of fluoxetine-treated mice compared to control mice. Further investigations of early and advanced plaque composition, adhesion molecule expression and role of endothelial versus leukocyte-dependent effects of serotonin-mediated activation are ongoing to explain the unexpected pro-atherogenic effect of pharmacological SERT inhibition in early plaque formation. Our findings might have important clinical implications in particular for cardiovascular risk patients treated with SERT inhibitors for depression.# 286 Deletion of junctional adhesion molecule a from platelets increases early stage neointima formation after wire injury in hyperlipidemic mice {#article-title-4}Objective: Platelets play an important role in the pathogenesis of vascular remodeling after injury. Junctional Adhesion Molecule A (JAM-A) was recently described to regulate platelet activation. Specific deletion of JAM-A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet-derived JAM-A to neointima formation after vascular injury.Approach and Results: Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe–/–) background underwent wire-induced injury of the common carotid artery. Ex vivo imaging by 2-photon microscopy revealed increased platelet coverage at the site of injury in trJAM-A–deficient mice, hour after wire injury (Figure 1A,B). Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM-A–deficient platelets than to control platelets (Figure 1C). Up to 4 weeks after wire-injury, intimal neoplasia and neointimal cellular content were analyzed. Neointimal lesion area was increased in trJAM-A–/– apoe–/– mice (Figure 1D,E) and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM-A+/+ apoe–/– littermates 2 weeks (Figure 1F,G), but not 4 weeks after injury. Re-endothelialization was decreased in trJAM-A–/– apoe–/– mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks.Conclusions: A platelet gain-of-function by deletion of JAM-A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.![Graphic][1] Figure 1 [1]: /embed/inline-graphic-1.gif