The proliferation of lymphocytes after interfered OX40-OX40L interaction.

<p>*<i>p</i><0.05 compared to control.</p

The proliferation of lymphocytes at 24 h, 48 h and 72 h after stimulated OX40-OX40L interaction by anti-OX40 (10, 20, 40 µg/ml).

<p>***<i>p</i><0.001 compared to respective control.</p

The proliferation of lymphocytes at 24 h, 48 h and 72 h after inhibition of OX40-OX40L interaction by anti-OX40L mAb (20, 80, 100 and 120 µg/ml).

<p>***<i>p</i><0.001 compared to control.</p

R2 were CD4⁺ lymphocytes (A).

<p>Th1 cells were labeled with IFNγ CD4⁺ lymphocytes. Th2 cells were labeled with IL-4 CD4⁺ lymphocytes. The proportion of Th1 and Th2 cells in the lymphocytes isolated from spleen of ApoE<b>^−/−</b> mice(B). When lymphocytes were stimulated with anti-OX40, Th1 cells were significantly increased in lymphocytes(C).</p

The levels of IL-2, IFN-γ and IL-4 induced by OX40-OX40L interaction in lymphocytes.

<p>*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ns <i>p</i>>0.05, respectively.</p

HE-stained cross sections of carotid 6 weeks in ApoE^−/− mice injecting with anti-OX40(100 µg/ml) and anti-OX40L(100 µg/ml) 2 times a week.

<p>Representative of plaques in control(C) and treated with anti-OX40(A) and anti-OX40L(B) stained with HE.</p

Platelet PI3Kγ mediates activated platelet-induced leukocyte recruitment after partial ligation.

<p>Representative images of PMN⁺ neutrophils (3 d) and Mac2⁺ monocyte/macrophages (21 d) immunofluorescence staining for left common carotid arteries from WT mice treated with PBS, WT platelets, or PI3Kγ^-/- platelets after partial ligation, as well as their quantitative analysis (A, B) (n = 5 per group). Scale bars: 50 μm. Data are expressed as mean ± SEM. * <i>P</i><0.05 versus vehicle, # <i>P</i><0.05 versus WT platelet-infused mouse.</p

Platelet PI3Kγ Contributes to Carotid Intima-Media Thickening under Severely Reduced Flow Conditions

<div><p>Studies have begun to focus on the emerging function of platelets as immune and inflammatory cells that initiate and accelerate vascular inflammation. Phosphoinositide 3-kinase gamma (PI3Kγ) is critically involved in a number of inflammatory and autoimmune diseases. This study aims to investigate the contribution of platelet PI3Kγ to vascular remodeling under flow severely reduced conditions. Mouse partial left carotid artery ligation with adoptive transfer of activated, washed wild-type or PI3Kγ^-/- platelets was used as the model. Intima-media area, leukocyte recruitment, and proinflammatory mediator expression were assessed. In vitro PI3Kγ^-/- platelets were used to verify the effect of PI3Kγ on platelet activation, interaction with leukocytes, and endothelial cells. Mice injected with activated platelets showed a significant increase in intima-media thickening, recruitment of neutrophils (at 3 d) and macrophages (at 21 d), and intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor alpha, and interleukin-6 expression (at 3 d) in the flow-reduced area. These effects were abrogated by platelet PI3Kγ deficiency. Circulating platelet-leukocyte aggregates were reduced in PI3Kγ^-/- mice after partial ligation. In vivo data confirmed that PI3Kγ mediated Adenine di-Phosphate -induced platelet activation through the Akt and p38 MAP kinase signaling pathways. Moreover, platelet PI3Kγ deficiency reduced platelet-leukocyte aggregation and platelet-endothelial cell (EC) interaction. These findings indicate that platelet PI3Kγ contributes to platelet-mediated vascular inflammation and carotid intima-media thickening after flow severely reduced. Platelet PI3Kγ may be a new target in the treatment of vascular diseases.</p></div

Platelet PI3Kγ mediates ADP-induced platelet activation through Akt- and p38 MAPK-dependent mechanisms.

<p>(A, B) Western blot analysis of phosphorylated p-p38 and p-Akt in platelets stimulated with ADP (20 μmol/L), with total p38 and Akt protein as internal controls. Average densitometric values normalized against those of internal control from three independent experiments are shown in the bar graph. Density from WT resting platelet sample was set as 1.0. Data are expressed as mean ± SEM. (C) Representative flow cytometric analyses of P-selectin, CD147, and CD154 expressions on WT platelet surface pretreated with Akti-1/2 (5 μmol/L) or SB203580 (10 μmol/L) for 5min, followed by stimulation with or without ADP (20 μmol/L) for 15 min, as well as the statistical data analysis from three separate experiments.</p

Platelet PI3Kγ deficiency impaired the platelet-leukocyte aggregates in vivo and in vitro.

<p>(A) Flow cytometry of leukocyte subpopulations, R1: CD45⁺ leukocyte, R2: neutrophils R3: monocytes. (A) Representative flow cytometric analyses of platelet-neutrophil aggregates (% total CD45+ Ly6G+ CD11b+ cells) and platelet-monocyte aggregates (% total CD45⁺ Ly6G^- CD11b⁺ cells) in WT or PI3Kγ^-/- mice before (0 h), 2 h, and 4 h after partial ligation in vivo, (B) as well as the statistical data analysis. (C) Representative flow cytometric analyses of platelet-neutrophil aggregates (% total CD45+ Ly6G+ CD11b+ cells) and platelet-monocyte aggregates (% total CD45⁺ Ly6G^- CD11b⁺ cells) in vitro, as well as the statistical analysis (n = 5 per group). Data represent mean ± SEM. * <i>P</i><0.05 versus PI3Kγ^-/- group.</p