Role of polypyrimidine tract-binding protein 1/yin yang 2 signaling in regulating vascular smooth muscle cell proliferation and neointima hyperplasia.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2.

Plin5 inhibits proliferation and migration of vascular smooth muscle cell through interacting with PGC-1α following vascular injury.

Abnormal proliferation and migration of vascular smooth muscle cell (VSMC) is a hallmark of vascular neointima hyperplasia. Perilipin 5 (Plin5), a regulator of lipid metabolism, is also confirmed to be involved in vascular disorders, such as microvascular endothelial dysfunction and atherosclerosis. To investigate the regulation and function of plin5 in the phenotypic alteration of VSMC, -an animal model of vascular intima hyperplasia was established in C57BL/6 J and Plin5 knockdown (Plin5±) mice by wire injure. Immunohistochemical staining was used to analyze neointima hyperplasia in artery. Ki-67, dihydroethidium immunofluorescence staining and wound healing assay were used to measure proliferation, reactive oxygen species (ROS) generation and migration of VSMC, respectively. Plin5 was downregulated in artery subjected to vascular injury and in VSMC subjected to platelet-derived growth factor (PDGF)-BB. Plin5 knockdown led to accelerated neointima hyperplasia, excessive proliferation and migration of VSMC after injury. In vitro, we observed increased ROS content in VSMC isolated from Plin5± mice. Antioxidative N-acetylcysteine (NAC) inhibited VSMC proliferation and migration induced by PDGF-BB or plin5 knockdown. More importantly, plin5-peroxlsome proliferator-activated receptor-γ coactivator (PGC)-1α interaction was also attenuated in VSMC after knockdown of plin5. Overexpression of PGC-1α suppressed PDGF-BB-induced ROS generation, proliferation, and migration in VSMC isolated from Plin5± mice. These data suggest that plin5 serves as a potent regulator of VSMC proliferation, migration, and neointima hyperplasia by interacting with PGC-1α and affecting ROS generation.

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

Neurofibromin 2 (NF2), a potent tumor suppressor, is reported to inhibit proliferation in several cell types. The role of NF2 in neointima hyperplasia after vascular injury is unknown. We explored the role of NF2 in proliferation, migration of vascular smooth muscle cell (VSMC) and neointima hyperplasia after vascular injury. NF2 phosphorylation was elevated in VSMC subjected to platelet-derived growth factor (PDGF)-BB and in artery subjected to vascular injury. Mice deficient for Nf2 in VSMC showed enhanced neointima hyperplasia after injury, increased proliferation and migration of VSMC after PDGF-BB treatment. Mechanistically, we observed increased nuclear p-NF2, declined p-Yes-Associated Protein (YAP), nuclear translocation of YAP after PDGF-BB treatment or injury. NF2 knockdown or YAP overexpression showed similar phenotype in VSMC proliferation, migration and neointima hyperplasia. YAP inhibition abolished the above effects mediated by NF2 knockdown. Finally, NF2 knockdown further promoted YAP-TEA Domain Transcription Factor 1 (TEAD1) interaction after PDGF-BB treatment. Inhibition of TEAD1 blocked PDGF-BB-induced VSMC proliferation and migration, which were not reversed by either NF2 knockdown or YAP overexpression. In conclusion, NF2 knockdown promotes VSMC proliferation, migration and neointima hyperplasia after vascular injury via inducing YAP-TEAD1 interaction.

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.

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), p-4EBP1 but decreased p-adenosine 5'-monophosphate-activated protein kinase (AMPK)α 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.