Deletion of the FHL2 gene attenuates intima‐media thickening in a partially ligated carotid artery ligated mouse model

Figure 1. Lack of FHL2 retards neointima lesion formation. A, The workflow of animal study. Representative photomicrographs showing cross‐sectional areas (at 0.8‐1 mm from bifurcation) of the ligated carotid artery of wild‐type (FHL2+/+, C57Bl/6) and FHL2‐deficient (FHL2−/−) mice isolated 28 d after ligation (N = 10). Scale bars: 500 μm, (B) haematoxylin and eosin stain and (C) Verhoeff‐Van Gieson Stain images of cross‐sections from carotid arteries harvested 28 d after carotid ligation in FHL2+/+ and FHL2−/− mice. D, The quantitative results of intima and (E) medial area were measured by the ImageJ software (NIH Image software). Bars represent average ± SEM of intimal and medial area. (*P < .01)

Figure 2. Expression of FHL2 in arterial SMCs after ligation. Neointimal formation was induced by ligation of left carotid artery of FHL2+/+ and FHL2−/− mice. Immunofluorescence analysis (28 d after ligation) showed co‐localization of FHL2 with αSMA+ cells (n = 10). The common carotid arteries from mice were fixed with 4% formaldehyde and cut into 7 μm frozen sections. Immunostaining for FHL2 (green), α‐SMA (red) and Hoechst (blue)

Figure 3. Expression of ERK and AKT of the artery of the mice, and the secretion level of PDGF after carotid ligation. The vascular injury was induced by ligation of the carotid artery in mice. Arteries were collected 2 wk after ligation. A, The ratio of ERK and AKT phosphorylation was analysed using Western blotting. B, Serum was collected from wild‐type mice in different time‐points after carotid artery ligation. The level of PDGF secretion was measured using ELISA. C, The comparison of PDGF level in FHL2+/+ and FHL2−/− mice. The results were expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01 vs before ligation) (D) HASMCs were treated with PDGF for 24 h. Knockdown of FHL2 decreased the level of PDGF receptor phosphorylation. E, HASMCs were treated with a series of cytokines for 24 h. PDGF significantly increased the level of FHL2. FHL2 protein expression up‐regulated in a dose and time‐dependent manners (F, G) after PDGF treatment. These results are expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01 vs unstimulated cells)

Figure 4. Effect of FHL2 on the signalling mediated the phosphorylation of MEK, ERK and p38. FHL2 deficiency modulated cell proliferation ability and the MAPK signalling pathway. A, FHL2 siRNA inhibited the FHL2 protein expression in HASMCs after 24 h transfection. B, Cell proliferation of transfected HASMCs evaluated by a Cell Counting Kit‐8. HASMCs were transfected with the control or FHL2 siRNA. After 24 h, cells were stimulated with PDGF for 15 min, MEK/ERK (C) and p38 (D) phosphorylation were analysed using Western blotting. These results are expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01)

Figure 5. FHL2 deficiency induced G1 cell cycle arrest in HASMCs. A, HASMCs were transfected with FHL2 siRNA and serum starved for 24 h. Cells were stimulated with PDGF for 24 h, and the cell cycle distribution was analysed using flow cytometry and expressed as a percentage of cells in the S phase. B, HASMCs transfected FHL2 siRNA and serum starved for 24 h. Cells were stimulated with PDGF for the indicated time, and Western blot analysis for cell cycle‐regulatory proteins was performed. These results are expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01)

Figure 6. Effects of FHL2 deficiency in migration of HASMCs. FHL2 deficiency modulated HASMCs migration and cytoskeleton conformational changes. A, Effect of FHL2 deficiency on the migration of HASMCs. Cells were transfected with FHL2 siRNA for 24 h and placed onto the upper side of a transwell towards PDGF (10 ng/mL PDGF located in the lower chamber). The histogram showed the percentage of migrating cells. B, The effect of FHL2 deficiency on the formation of lamellipodia was induced by PDGF in HASMCs. After 15 min of PDGF treatment, HASMCs were stained with F‐actin and DAPI. These results are expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01)

Figure 7. FHL2 deficiency modulated Rac1 activation and related signalling pathway. A, Cell extracts were harvested after 24 h transfection and proceeded the Rac1 pull‐down assay. FHL2 siRNA down‐regulated the levels of activated Rac1. B, Cells were transfected with FHL2 siRNA for 24 h and calculated the ratio of AKT phosphorylation after PDGF treatment using Western blotting. C, Cells were stimulated in PDGF for 15 min after pre‐treated with Akt inhibitor (LY294002) and ERK inhibitor (PD98059) for 1 h. Western blot showed the expression of Akt and ERK phosphorylation. D, PDGF treated transfected cells for 15 min. Co‐immunoprecipitation revealed the interaction of FHL2 and AKT. E, Proposed scheme of HASMCs antiproliferation and antimigration by FHL2 through Rac1‐AKT and MAPK pathways. FHL2 directly controlled AKT phosphorylation. FHL2 deficiency led to Rac1 inactivation and cell cycle arrest. FHL2 plays a pivotal role in the AKT and MAPK signalling pathway in neointimal formation. These results are expressed as the mean ± SEM of five separate experiments run in triplicate. (*P < .01)