Study On The Effects Of Apatinib On Vascular Smooth Muscle Cell Phenotypic Switching And Vascular Remodeling

Objectives: To detect the effect of Apatinib on neointimal hyperplasia caused by carotid artery injury using the carotid artery ligation mouse model.To detect the effect of Apatinib on rat primary VSMC proliferation,migration and dedifferentiation induced by PDGF-BB.To clarify the mechanism of Apatinib in vascular remodeling using rat primary VSMC and rat VSMC cell line A7r5.Methods: A total of 18 C57BL/6J mice(WT,8 weeks,25-30 g,male)were randomly divided into 3 groups with 6 mice in each group: sham + vehicle group,injured +vehicle group,and injured + Apatinib group.Mice were injected with Apatinib(10mg/kg)or the same dose of corn oil(vehicle)intraperitoneally one day before the operation.Apatinib or vehicle was given each day after the operation for 2 weeks.After2 weeks,the left carotid arteries were harvested and stained by HE and Masson for the measurement of the intimal as well as media thickness.Intimal thickness,media thickness and the ratio of intima-to-media thickness were used to evaluate the effect of Apatinib on neointimal hyperplasia.VSMCs were isolated from the rat thoracic aorta and cultured in a complete medium.24-hour-starvation in serum-free medium started when the cell confluence was around 80%.Then,VSMCs were pretreated with vehicle or Apatinib at different concentrations(50 n M,100 n M,200 n M)for 4 h,and given the stimulation of PDGF-BB(30 ng/m L)for 24 h.EdU(5-Ethynyl-2’-deoxyuridine)test was used to detect VSMC proliferation;Transwell cell migration test and wound closure test were performed to detect VSMC migration;Immunofluorescence analysis was used to detect contractile phenotype;The protein level of genes associated with cell proliferation,apoptosis,cell migration,cell dedifferentiation and other related processes were detected by Western blot.After 24-hour-starvation,A7r5 was pretreated with vehicle or 200 n M Apatinib for 1 hour,and then stimulated by PDGF-BB for different time(0 min,5 min,15 min,30 min,45 min).The phosphorylated and total protein levels of PDGFR-β as well as related important signaling pathway molecules were detected by Western blot.Results: Compared to the vehicle groups,mice that were performed carotid artery ligation injury and treated with Apatinib produced a reduction in abnormal neointimal area.For in vitro experiment,Apatinib administration inhibited VSMC proliferation,migration and reversed VSMC dedifferentiation with the stimulation of PDGF-BB.In terms of mechanism,with the preincubation of Apatinib,the activations of PDGFR-βand PLC-γ1 induced by PDGF-BB were inhibited in VSMC.With the preincubation of Apatinib,the phosphorylation of PDGFR-β,ERK1/2 and JNK induced by PDGF-BB were also inhibited in rat VSMC cell line A7r5.Conclusions: Apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF-BB in vitro and vascular remodeling in vivo.