Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-β-induced endothelial-mesenchymal transition and cardiac fibrosis

Abstract

Cardiac fibrosis (CF), a process characterized by potentiated proliferation of cardiac fibroblasts and excessive secretion and deposition of extracellular matrix (ECM) from the cells, contributes strongly to the pathogenesis of a series of cardiovascular (CV) diseases, including AMI, heart failure and atrial fibrillation. Endothelial-mesenchymal transition (EndMT), one of the sources of transformed cardiac fibroblasts, has been reported as a key factor involved in CF. However, the molecular basis of EndMT has not been thoroughly elucidated to date. At the posttranscriptional level, of the three epigenetic regulators, writer and eraser are reported to be involved in EndMT, but the role of reader in the process is still unknown. In this study, we aimed to explore the role of Bromodomain-containing protein 4 (BRD4), an acetyl-lysine reader protein, in EndMT-induced CF and related mechanisms. We found that BRD4 was upregulated in endothelial cells (ECs) in the pressure-overload mouse heart and that its functional inhibitor JQ1 potently attenuated the TAC-induced CF and preserved cardiac function. In umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs), bothJQ1 and shRNA-mediated silencing of BRD4 blocked TGF-β-induced EC migration, EndMT and ECM synthesis and preserved the EC sprouting behavior, possibly through the downregulation of a group of transcription factors specific for EndMT (Snail, Twist and Slug), the Smads pathway and TGF-β receptor I. In the absence of TGF-β stimulation, ectopic expression of BRD4 alone could facilitate EndMT, accelerate migration and increase the synthesis of ECM. In vivo, JQ1 also attenuated TAC-induced EndMT and CF, which was consistent with JQ1's intracellular mechanisms of action. Our results showed that BRD4 plays a critical role in EndMT-induced CF and that targeting BRD4 might be a novel therapeutic option for CF.

Introduction

Heart failure (HF) refers to a syndrome caused by a variety of heart diseases leading to cardiac insufficiency [1]. It is now recognized that the basic mechanism leading to pathogenesis of HF is ventricular remodeling, notably extensive cardiomyocyte loss and excessive cardiac fibrosis (CF) [2]. However, no specific therapies to cure or even reverse CF are currently available. New therapeutic targets and treatment options are urgently required.

Many studies have shown that myofibroblasts are the cause of CF [3]. There has been extensive investigation aimed at the precise identification of the cellular origin of myofibroblasts [4,5]. These studies have shown that myofibroblasts can originate from various sources, including activation and proliferation of quiescent resident tissue fibroblasts [6], migration and tissue accumulation of bone marrow-derived CD34⁺ fibrocytes [7], or from the phenotypic transition of epithelial cells, a process known as epithelial mesenchymal transition (EMT) [8]. More recent studies have demonstrated that another source of myofibroblasts is endothelial cells (ECs) that have acquired a mesenchymal phenotype through a process known as endothelial-mesenchymal transition (EndMT) [9]. During the process of EndMT, ECs become delaminated and detach from the endothelial layer, change their morphological characteristics to become elongated and fusiform, lose their specific molecular markers, such as CD31/PECAM-1, von-Willebrand Factor (vWF), and VE-cadherin, and initiate the expression of mesenchymal cell products, including α-SMA, vimentin and fibroblasts-specific protein 1 (FSP1). Eventually, ECs acquire invasive and migratory phenotypes which, in turn, promote the development of fibrotic diseases [10].

The mechanisms by which EndMT leads to the development of fibrotic diseases are still incompletely understood. Previous research focused on the impact of changes in gene expression on EndMT [11,12]. However, cell fate is determined to a great extent by posttranslational modifications, and such mechanisms in the process of EndMT have not been fully explored.

BRD4 (Bromodomain Containing Protein 4) is a member of the BET (Bromodomain and Extraterminal motif) protein family, which also includes BRD2, BRD3 and Bromodomain testis-specific protein (BRDt) [13]. As the “readers” of lysine acetylation, BRD4 is responsible in transducing the signal carried by acetylated lysine residues and translating it into various normal or abnormal phenotypes [14]. It has previously been reported that disrupting the interaction of BRD4 with diacetylated Twist suppresses EMT in basal-like breast cancer cells [15]. The process of EndMT shares many features with the extensively described process of EMT, through which epithelial cells gain expression of mesenchymal proteins and ECM proteins, such as CTGF and collagen type I and III [16]. One study reported that BRD4 functions as a critical coactivator of pathological gene transactivation during cardiomyocyte hypertrophy in cardiac remodeling [17,18], but the therapeutic relevance of these observations remains unknown. In particular, the function of BRD4 in EC differentiation through EndMT warrants further study.

In this study, we aimed to elucidate a potential role for BRD4 in the induction of EndMT in ECs. Our studies demonstrated that BRD4 can induce EndMT through EndMT-related transcription factors and the Smads signaling pathway. Therefore, our results indicate that BRD4 is a critical regulator of EndMT, that BRD4-regulated EndMT plays an important role in EndMT-mediate CF, and that targeting BRD4 might be a potential therapeutic option for CF and HF.