Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes

Abstract

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. cultured cells, TERT is induced in both activated macrophages and proliferating SMC. 14,17 In SMC, the transient increase in TERT abundance in response to growth factor stimulation serves a mitogenic role because inhibition of TERT by oligonucleotide or pharmacological targeting inhibits SMC proliferation. 15,[17][18][19][20] Furthermore, acute telomerase downregulation using TERT antisense RNA decreases proliferation of SMC isolated from genetically hypertensive rats. 21 Although recent genetic experiments established that the mitogenic activity of TERT requires neither telomere extension nor the catalytic activity, 5-8 the molecular mechanisms by which TERT supports cellular proliferation remain elusive. Conclusions-TheseIn this study, we used a genetic model of TERT deficiency to investigate the transcriptional mechanisms underlying the mitogenic activity of TERT during proliferative vascular remodeling. Our studies reveal that TERT deletion prevents neointima formation and SMC proliferation by inducing a silenced chromatin environment at S phase gene promoters. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. Results TERT Is Expressed in Replicating Neointimal Smooth Muscle CellsTERT protein expression was first analyzed in neointimal SMC of human coronary artery atherosclerotic lesions from hearts explanted during cardiac transplantation. Although TERT expression is considered negligible in normal arteries, 13,14 we confirmed abundant TERT expression in the SMCrich neoint...

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“…The localization of E2F1 protein in remodeled arteries corresponds to sites of SMC proliferation in the vessel wall. These results agree with previous studies showing that E2F1 and its targets regulate SMC proliferation and vessel remodeling (41,42). In addition, E2F1 is expressed in the endothelium.…”

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“…For example, in vitro recombinant expression of TERT can prolong the survival time of vascular smooth muscle cells (VSMC) in atherosclerotic plaques, while inhibition of TERT can terminate the proliferation of VSMC, leading to the reduction of the regeneration of damaged vascular intima. 44 In addition, activation of telomeric repeat binding factor 2 (TRF2), namely telomeric protective protein, can inhibit the aging process of vascular endothelium, enhance the repair ability of damaged DNA, protect vascular intima and increase plaque stability. 45 As expected, MLT, which has been demonstrated to improve telomerase activity as stated before, was also found to have the ability to inhibit senescence as it greatly reduced SA-β-gal level and protein expression of p16 and p21, as well as increasing protein expression of CDK2 and cyclinD1 in injured endothelial cells and tissues.…”

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“…For instance, high expression levels of telomerase reverse transcriptase in mice induce atherosclerosis-like smooth muscle cell morphology, whereas deletion of TERT decreases neointima formation through epigenetic regulation of proliferative gene expression. 98 Aortic valve interstitial cells from Notch1 heterozygous deficient mice become fully activated myofibroblasts leading to enhanced dystrophic calcification. 99 Genetic ablation of CaV3.2 channels enhances the arterial myogenic response through modulation of the RyR-BKCa (ryodine receptors-large conductance Ca 2+ -activated K + channels) axis.…”