Signalling through Src family kinase isoforms is not redundant in models of thrombo-inflammatory vascular disease

Abstract

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad-spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non-redundant fashion in the thrombo-inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE^-/- model of atherosclerosis, we find that SFK signalling regulates platelet-dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet-mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr^-/- /ApoE^-/- or Lyn^-/- /ApoE^-/- animals. SFK signalling is not redundant in thrombo-inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.

Keywords: Src family kinases; atherosclerosis; inflammation; monocytes; platelets.

Figure 1

Dasatinib inhibits platelet activation and recruitment of monocytes on immobilized VWF under flow conditions. A, Percentage platelet coverage from flowing human whole blood (1000^−s) on a matrix of immobilized VWF in the presence of 30 μmol/L ADP ± 20 μmol/L broad spectrum SFK inhibitor, (B) or using murine whole blood (n = 3). C, Representative images of human or (D) murine platelet adhesion to VWF in the presence of 30 μmol/L ADP under flow conditions (1000^−s). E, Total adhesion of Human or (F) murine monocytes in the presence of 30 μmol/L ADP ± 20 μmol/L Dasatinib under flow conditions (100^−s). The level of monocyte and platelet adhesion was assessed by phase contrast microscopy (n = 3). Data are shown as mean ± SD. *P < .05, **P < .01 compared to the ADP positive control using one‐way ANOVA followed by a Dunnett's post‐test

Figure 2

Dasatinib inhibits platelet P‐selectin expression in both human and mouse platelets and prevents the adhesion of platelets and monocytes to TGF‐β1 stimulated endothelium under flow conditions. A, Representative flow cytometry plots of P‐selectin expression in human or (B) murine platelet‐rich plasma (PRP) prepared from freshly drawn blood and stimulated with 30 μmol/L ADP ± 20 μmol/L Dasatinib. C, Mean fluorescent intensity (MFI) of P‐selectin staining on human or (D) murine platelets. E, The effect of Dasatanib on platelet and (F) monocyte recruitment to TGF‐β1 stimulated endothelial cells. Dasatanib (4 μmol/L) was added to blood 15 min prior to perfusion across EC and the level of monocyte and platelet adhesion assessed by phase contrast microscopy (n = 4). Data are mean ± SD. *P < .05, **P < .01, vs ADP only control (C, D) vs blood perfused in the absence of Dasatanib (E, F) using one‐way ANOVA followed by a Dunnett's post‐test

Figure 3

Mice deficient in Fgr or Lyn demonstrate decreased platelet activation, P selectin expression and leucocyte recruitment to VWF under flow conditions. A, Representative images of WT (B) Lyn^−/− or (C) Fgr^−/− platelets activated with 30 μmol/L ADP on a matrix of immobilized VWF in the presence of cells; (D) Average platelet coverage from flow (1000^−s) on a matrix of immobilized VWF in the presence of 30 μmol/L ADP. The level of platelet adhesion was assessed by fluorescent microscopy. Data are mean ± SEM. compared to WT (C57Bl6) ADP positive controls (n = 15). E, Representative flow cytometry plot and (F) MFI ± SD of P‐selectin expression on WT, Lyn^−/− or Fgr^−/− platelets in response to ADP (n = 3). G, Number of WT monocytes adhering to Lyn^−/− or (H) Fgr^−/− platelets under flow conditions (100^−s). The level of monocyte was adhesion assessed by phase contrast microscopy (n = 18). Data are mean ± SEM. Data are shown as mean ± SD. *P < .05, **P < .01 compared to the WT ADP positive control using one‐way ANOVA followed by a Dunnett's post‐test

Figure 4

Decreased plaque burden in the aortas of ApoE^−/− mice on HFD for 6 wk after Fgr and Lyn abolition. A, Plaque burden in the aortas of Apoe^−/− animals or ApoE^−/− mice deficient in Fgr or Lyn assessed using analysis of oil red O staining after 6 wk of HFD (n = 10‐12). B, False colour masks depicting plaque burden in the aortas of Apoe^−/− animals or ApoE^−/− mice deficient in Fgr or Lyn. C, Site specific analysis of plaque burden within the aorta (n = 10‐12). Data are mean ± SEM *P < .05, **P < .01 ApoE ^−/− Fgr ^−/− and ApoE ^−/− Lyn ^−/− mice compared to ApoE ^−/− using one‐way ANOVA followed by a Dunnett (A) or Bonferroni (B) post‐test

Figure 5

Decreased size and number of plaques in the descending aorta of ApoE^−/− mice on HFD for 6 wk after Fgr and Lyn abolition. A, B, ApoE ^−/− mice that were WT or deficient in Fgr or Lyn were placed on a HFD at 6 wk of age for a total of 6 wk. Total number of individual plaques (A) or average plaque size (B) in the descending aorta was analysed (n = 10‐12 per group). *P < .05, ApoE ^−/− Fgr ^−/− and ApoE ^−/− Lyn ^−/− mice compared to ApoE ^−/− using one‐way ANOVA followed by a Dunnett (A) post‐test