Deletion of junctional adhesion molecule A from platelets increases early-stage neointima formation after wire injury in hyperlipidemic mice

Abstract

Platelets play an important role in the pathogenesis of vascular remodelling 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. 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 two-photon microscopy revealed increased platelet coverage at the site of injury in trJAM-A-deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM-A-deficient platelets than to control platelets. Inhibition of α_M β₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM-A^-/- apoe^-/- mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM-A^+/+ apoe^-/- littermates 2 weeks, 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. 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.

Keywords: junctional adhesion molecule; leucocyte; neointima; platelet.

Figure 1

trJAM‐A deficiency increases platelet aggregation. Aggregation of platelets from JAM‐A^+/+ and JAM‐A^−/− mice in response to collagen (3 μg/ml) is shown as representative tracings (A) and quantified as area under the curve (AU*min) (B). Activation of JAM‐A^+/+ and JAM‐A^−/− platelets after stimulation with thrombin at indicated concentrations as measured by CD62P surface exposure by flow cytometry. (C) Representative histograms before (solid grey) and after (black line) stimulation with 0.5 nM of thrombin. (D) Median fluorescence intensity of CD62P surface exposure after thrombin stimulation. Ctrl is isotype control and 10 nM of thrombin. P values were calculated by anova with Tukey's post‐test (n = 8–15).

Figure 2

trJAM‐A deficiency increases platelet adhesion after vascular injury. Three‐dimensional (3D) luminal view of the vessel wall recorded with two‐photon laser scanning microscopy (A) shows platelets (CD41⁺, red) adherent to endothelium (CD31⁺, green) at the border of the arterial injury (1 hr after denudation). Platelet coverage (μm³) was quantified at sites of complete denudation (B). P values were calculated by unpaired two‐tailed t‐test without Welch correction (B, n = 5). Scale bar: 50 μm.

Figure 3

trJAM‐A deficiency promotes flow‐resistant monocytic cell adhesion. Transient tethering and stable interactions of RAW264.7 cells with platelets from JAM‐A^+/+ apoe^−/− and JAM‐A^−/−apoe^−/− mice immobilized on APTES‐coated glass slides under flow conditions, without or with thrombin activation (IIa, 1 nM) (A). Stable interactions of RAW264.7 cells with platelets from JAM‐A^+/+ and JAM‐A^−/− mice immobilized on collagen‐coated glass slides under flow conditions, without or with thrombin activation (IIa, 0.5 nM) in the presence of indicated inhibitors (B, C). Representative micrographs of RAW264.7 cells adherent to JAM‐A^+/+ and JAM‐A^−/− platelets after treatment with isotype control or anti‐GPIbα antibodies (D). Cell adhesion expressed as percentage of initially adherent cells under incrementally increased shear stress (dyne/cm²) on immobilized platelets after thrombin activation (IIa, 0.5 nM) (E). Adherent cells (%) on thrombin‐activated platelets at a shear stress of 20 dyne/cm² in the presence of blocking anti‐CD11b antibodies (F). P values were calculated by anova with Tukey's or Bonferroni's post‐test (B, C, F n = 3–5). Scale bar: 100 μm.

Figure 4

trJAM‐A deficiency increases neointima formation and plaque macrophage content. Neointima 2 weeks after wire injury was visualized by EVG staining (A) and quantification (B) of neointimal area (μm²). Immunofluorescence of neointimal macrophages (C, MAC2⁺, green) and quantification (D). P values were calculated by unpaired two‐tailed t‐test with (B, n = 9–10) or without (D, n = 6) Welch correction. Scale bars: 200 μm (A) and 100 μm (C).

Figure 5

Cellular composition of neointima from trJAM‐A^+/+ apoe^−/− and trJAM‐A^−/− apoe^−/− mice, 2 weeks after wire injury. Immunofluorescence of SMC (A, α‐Actin⁺, red) and proliferating cells (A, Ki67⁺, green) and their quantification (B, SMC, left panel and Ki67⁺ SMC, right panel). Proliferating SMC were marked with arrows (A, α‐Actin⁺, Ki67⁺ and DAPI ⁺). Endothelium was visualized by staining of VWF (green) (C), and length of VWF ⁺ lining was expressed as percentage of luminal circumference (D). P values were calculated by Mann–Whitney U‐test (B, n = 6 and D, n = 4–5). Neointima areas were demarcated with dashed lines. Nuclei were stained with DAPI (blue). Scale bars: 100 μm.