Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor‐β

Figures

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  Figure 1. Effect of apatinib on intimal thickness induced by wire ligation injury for 14 days in vivo. A, C57BL/6 mice were treated with vehicle or apatinib for 14 days since the first day of being performed sham‐surgery injury or wire ligation injury. After 14 days of vehicle or apatinib administration, carotid arteries were harvested, fixed and embedded in paraffin. H&E‐stained and elastic Masson trichrome‐stained sections of all groups were shown. The images shown are representative of those obtained from 6 independent experiments. Arrowheads indicate elastic lamina. B, Measurement of mean intimal thickness. Data are represented as means ± SEM (n = 6 for each experimental group). **P < .01 versus the sham + vehicle group. ##P < .01 versus the injured + vehicle group. C, Quantification of intima/media ratio. Data are represented as means ± SEM (n = 6 for each experimental group). **P < .01 versus the sham + vehicle group. ##P < .01 versus the injured + vehicle group

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  Figure 2. Effect of apatinib on VSMC proliferation induced by PDGF‐BB. A, VSMCs were stained with EdU (red) and DAPI (blue). B, Quantification of the proliferative cells. Data are represented as mean ± SEM (n = 5). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group

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  Figure 3. Effect of apatinib on the expression of cell proliferation and apoptosis marker genes. A, The protein level of PCNA, Cyclin D1, P27 and P21 were determined by Western blots. B, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group. C, The protein level of caspase3 (cleaved), Bcl‐2 and Bax was determined by Western blots. D, Quantification of Bcl‐2/ Bax relative density using the Western blots. Data are represented as mean ± SEM (n = 3)

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  Figure 4. Effect of apatinib on VSMC migration induced by PDGF‐BB. A, Migrated VSMCs were photographed after being stimulated with PDGF‐BB (30 ng/mL) for 12 hours in the presence or absence of apatinib treatment. B, Quantification of migrated cells. Data are represented as mean ± SEM (n = 5). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group. C, Cell monolayers were scratched and treated with vehicle or indicated concentrations of apatinib in the presence of PDGF‐BB (30 ng/mL) for 24 hours. D, Quantification of the area of wound closure (%). Data are represented as mean ± SEM (n = 4). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group. E, The protein levels of MMP2 and MMP9 were detected by Western blots. F, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). *P < .05, **P < .01 versus the vehicle group. #P < .05, ##P < .01 versus the PDGF‐BB + vehicle group

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  Figure 5. Effect of apatinib on VSMC dedifferentiation induced by PDGF‐BB in vitro. A, The protein levels of SMα‐actin, SM22α were determined by Western blots. B, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). *P < .05 versus the vehicle group. #P < .05, ##P < .01 versus the PDGF‐BB + vehicle group. C, Cells were stained with antibodies SMα‐actin (green); nuclei were stained with DAPI (blue). Each group was photographed within the same exposure time. D, Quantification of the SMα‐actin positive cells. Data are represented as mean ± SEM (n = 4). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group

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  Figure 6. Effect of apatinib on VSMC dedifferentiation induced by PDGF‐BB in vivo. A, SMα‐actin staining in the injured or sham‐surgery carotid vessel walls. Cells were stained with antibodies SMα‐actin (green); nuclei were stained with DAPI (blue). Each group was photographed within the same exposure time

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  Figure 7. Effect of apatinib on the phosphorylation of PDGFR‐β in VSMCs and cell line A7r5. A, The protein levels of phospho‐PDGFR‐β, PDGFR‐β in VSMCs were determined using Western blot. B, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). *P < .05 versus the vehicle group. #P < .05, ##P < .01 versus the PDGF‐BB + vehicle group. C, The protein levels of phospho‐PDGFR‐β, PDGFR‐β in cell line A7r5 were determined using Western blots. D, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). *P < .05, **P < .01 versus the vehicle control. E, The protein levels of phospho‐PLC‐γ1, PLC‐γ1 in VSMCs were determined using Western blots. F, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). **P < .01 versus the vehicle group. ##P < .01 versus the PDGF‐BB + vehicle group

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  Figure 8. Effect of apatinib on the MAPK signalling pathway in cell line A7r5. A, The protein levels of phospho‐ERK1/2, ERK1/2, phospho‐p38, p38, phospho‐JNK and JNK in cell line A7r5 were determined using Western blots. B, Quantification of the Western blots. Data are represented as mean ± SEM (n = 3). *P < .05 versus the vehicle control