Wild-type p53-induced phosphatase 1 promotes vascular smooth muscle cell proliferation and neointima hyperplasia after vascular injury via p-adenosine 5′-monophosphate-activated protein kinase/mammalian target of rapamycin complex 1 pathway

Abstract

Objectives:Vascular smooth muscle cell (VSMC) proliferation is a crucial cause of vascular neointima hyperplasia and restenosis, thus limiting the long-term efficacy of percutaneous vascular intervention. We explored the role of wild-type p53-induced phosphatase 1 (Wip1), a potent regulator of tumorigenesis and atherosclerosis, in VSMC proliferation and neointima hyperplasia.Methods and results:Animal model of vascular restenosis was established in wild type C57BL/6J and VSMC-specific Tuberous Sclerosis 1 (TSC1)-knockdown mice by wire injury. We observed increased protein levels of Wip1, phospho (p)-S6 Ribosomal Protein (S6)Ser235/236, p-4EBP1Thr37/46 but decreased p-adenosine 5′-monophosphate-activated protein kinase (AMPK)αThr172 both in carotid artery at day 28 after injury and in VSMCs after 48 h of platelet derived growth factor-BB (PDGF-BB) treatment. By using hematoxylin-eosin staining, Ki-67 immunohistochemical staining, cell counting kit-8 assay and Ki-67 immunofluorescence staining, we found Wip1 antagonist GSK2830371 (GSK) or mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin both obviously reversed the neointima formation and VSMC proliferation induced by wire injury and PDGF-BB, respectively. GSK also reversed the increase in mRNA level of Collagen I after wire injury. However, GSK had no obvious effects on VSMC migration induced by PDGF-BB. Simultaneously, TSC1 knockdown as well as AMPK inhibition by Compound C abolished the vascular protective and anti-proliferative effects of Wip1 inhibition. Additionally, suppression of AMPK also reversed the declined mTORC1 activity by GSK.Conclusion:Wip1 promotes VSMC proliferation and neointima hyperplasia after wire injury via affecting AMPK/mTORC1 pathway.

NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

“…The mice (8-10 weeks old) were housed as following described: 12-hour light/dark cycle, 22-25°C, free access to food and water and periodic air changes. The injury of left common carotid artery was conducted as our previous study described [46]. Briefly, WT and Nf2 -/mice (8-10 weeks old) were firstly anesthetized with pentobarbital (40 mg/kg, intraperitoneal).…”

“…Samples were then incubated with primary antibody at 4°C overnight, and immunocomplexes were precipitated after 1 hour of incubation with sepharose A/G beads (Santa Cruz, Delaware, CA). Western blot analysis was performed as described previously [46]. Extraction of artery and VSMC were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China).…”

“…Total RNA was extracted by using Trizol agent (Life Technologies, Carlsbad, CA) as previously described [46]. Briefly, RNA (1 μg) was reverse-transcribed into cDNA through a Bestar qPCR RT Kit (DBI Bioscience, Ludwigshafen, RP).…”

“…Histological analyses of carotid arteries were conducted by standard hematoxylin and eosin (H&E) and Ki-67 AGING staining as previously described [46]. For H&E staining, the artery sections (4-5 μm) were stained with hematoxylin and eosin.…”

“…VSMC was obtained from thoracic aortas of 8-to 10week-old WT and Nf2 -/mice by enzyme digestion method as previously described [46]. Briefly, VSMC was digested with 0.25% trypsin (Beyotime Institute of Biotechnology) and cultured in complete medium [Dulbecco's modified Eagle's medium (DMEM; HyClone, Carlsbad, CA) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA), streptomycin (100 mg/mL) and penicillin (100 units/mL)] at 37°C in humidified 5% CO2 atmosphere.…”