RESUMO
OBJECTIVE: Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. APPROACH AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.
Assuntos
Aterosclerose/enzimologia , Montagem e Desmontagem da Cromatina , Fator de Transcrição E2F1/metabolismo , Inativação Gênica , Músculo Liso Vascular/enzimologia , Neointima , Telomerase/deficiência , Telomerase/metabolismo , Lesões do Sistema Vascular/enzimologia , Acetilação , Animais , Aterosclerose/genética , Aterosclerose/patologia , Sítios de Ligação , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Artéria Femoral/enzimologia , Artéria Femoral/lesões , Artéria Femoral/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Predisposição Genética para Doença , Histonas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Fenótipo , Fosforilação , Ligação Proteica , Interferência de RNA , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Telomerase/genética , Fatores de Tempo , Transfecção , Remodelação Vascular , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. Furthermore, HDAC inhibition blocked the inhibitory effect of roflumilast on VCAM-1 expression. Both, roflumilast and Epac activation reduced monocyte adhesion to SMCs in vitro. Finally, roflumilast treatment attenuated femoral artery intima-media ratio by more than 50% after 4weeks. In summary, PDE4 inhibition regulates VCAM-1 through a novel Epac-dependent mechanism, which involves regulatory epigenetic components and reduces neointima formation following vascular injury. PDE4 inhibition and Epac activation might represent novel approaches for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis.