Fig 2: CKLF1 promotes monocyte adhesion and HASMC migration via VCAM-1. (A) CKLF1 promotes U937 cell adhesion to HASMCs, and this adhesion effects can be inhibited by VCAM-1 inhibitor (K-7174). Scale bar = 200 µm. (B) CKLF1 could promote HASMC migration and HASMC migration can be inhibited by VCAM-1 inhibitor (K-7174). Scale bar = 200 µm. The migration assay was performed in Transwell inserts with a pore of 8 µm. Five areas were randomly selected and the cell numbers of adhesion or migration in each area were calculated for the statistical analysis. Data are shown as the mean ± SEM. Differences between two groups were analyzed by Student's t-test (n = 3 independent experiments, ***P < 0.001). K-7174 represents VCAM-1 inhibitor (10 µm). Dimethylsulfoxide (final concentration 0.1%).

Fig 3: CKLF1 regulates the expression of VCAM-1 in the rat carotid artery and HASMCs. (A) Immunohistochemical staining showed that CKLF1 overexpression upregulated the expression of VCAM-1, whereas knocking down of CKLF1 downregulated the expression of VCAM-1 in rats. Scale bar = 100 µm. Square areas are presented at 40× magnification. Data are shown as the mean ± SEM. Differences between two groups were analyzed by Student's t-test (n = 5 independent experiments, *P < 0.05, **P < 0.01). (B) Western blotting analysis indicated that CKLF1 overexpression upregulated the expression of VCAM-1, whereas knockdown of CKLF1 downregulated the expression of VCAM-1 in rats (n = 3 independent experiments). (C) The mRNA and protein level of VCAM-1 increased after Ad-CKLF1 transfection in HASMCs. For immunohistochemical staining, density values of positive areas were calculated for the statistical analysis. Data are shown as the mean ± SEM. Differences between two groups were analyzed by Student's t-test (n = 3 independent experiments, *P < 0.05).

Fig 4: Immunofluorescence staining of rat carotid artery. Upregulation of CKLF1 is tightly associated with VSMC accumulation and thereby NIH in the rat carotid artery after balloon injury. Histological analysis of adenovirus‐transfected carotid arteries was carried out using anti‐SMA and anti‐VCAM‐1 antibodies and cell nuclei were stained with DAPI. The arteries were harvested 2 weeks after balloon injury (n = 5 independent experiments). Representative photomicrographs are shown. Scale bar = 25 μm.

Fig 5: Both CKLF1 and VCAM‐1 are highly expressed in human carotid plaque. (A) Immunohistochemical staining indicated that both CKLF1 and VCAM‐1 expression in human carotid plaque was higher compared to that in the control. Scale bar = 100 μm. Square areas are presented at 40× magnification. (B) Western blotting analysis showed that the levels of CKLF1 and VCAM‐1 in human carotid plaque were obviously increased compared to the control group. (C) Immunofluorescence staining suggested the level of VCAM‐1 was significantly upregulated in human carotid plaque compared to the control group. Smooth muscle cells were co‐localized with specific antibody to α‐SMA and the cell nuclei was stained with DAPI. Scale bar = 25 μm. Density values of positive areas in each slide were used for the statistical analysis. Healthy human radial artery tissue served as the control. Data are shown as the mean ± SEM. Differences between two groups were analyzed by Student's t‐test (n = 3 independent experiments, *P < 0.05, ***P < 0.001).