Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Pharmacology and Nutritional Sciences

First Advisor

Dr. Dennis C. Bruemmer

Abstract

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and the limiting factor for the enzyme activity. The expression of TERT and telomerase activity is increased in atherosclerotic plaques. However, the role of TERT dysregulation during atherosclerosis formation remains unknown.

The work herein first identified a multi-tiered regulation of TERT expression in smooth muscle cells (SMC) through histone deacetylase (HDAC) inhibition. HDAC inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Furthermore, during vascular remodeling in vivo, TERT protein expression in the neointima is prevented by HDAC inhibition. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition. TERT is highly expressed in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, neointima formation was reduced 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 which could not be reversed by ectopic overexpression of E2F1. Chromatin immunoprecipitation and accessibility assays revealed that TERT was recruited to E2F1 target sites to increase chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a mitogenic effect of TERT on SMC growth and neointima formation through epigenetic regulation of proliferative gene expression. Furthermore, TERT expression is induced in activated macrophages during experimental and human atherosclerosis formation. To investigate the role for TERT in lesional macrophages and the subsequent effect on atherosclerosis formation, TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wild-type controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, gene ontology (GO) enrichment analysis of silenced inflammatory genes indicated that TERT positively regulates signal transducer and activator of transcription (STAT) cascade, which was confirmed by the decreased tyrosine phosphorylation of STAT3 protein resulting from TERT deletion. These findings indicate genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway in activated macrophages.

In conclusion, the dysregulation of TERT expression within atherosclerotic plaques plays a causative role for vascular remodeling, including injury-induced neointima formation and hypercholesterolemia-induced atherosclerosis, through inducing SMC proliferation and a pro-inflammatory phenotype in infiltrating macrophages. These findings unveil a mechanism of TERT exacerbating the pathological vascular remodeling, which may provide a novel therapeutic target to combating vascular diseases.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.158

Available for download on Saturday, May 04, 2019

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