Elsevier

Life Sciences

Volume 244, 1 March 2020, 117342
Life Sciences

BRCA1 protects cardiac microvascular endothelial cells against irradiation by regulating p21-mediated cell cycle arrest

https://doi.org/10.1016/j.lfs.2020.117342Get rights and content

Highlights

  • BRCA1 plays a protective role in radiation-induced heart disease.

  • Endothelial cell BRCA1-/- knockout mice were more prone to severe Radiation-related heart disease (RIHD).

  • BRCA1 in cardiac endothelial cells plays a role in the response to radiation by regulating p21-cell cycle arrest.

Abstract

Aims

Microvascular endothelial cell dysfunction is a leading cause of radiation-induced heart disease (RIHD). BRCA1 plays an important role in DNA damage repair. The study aims to explore the effect of BRCA1 in endothelial cells involved in RIHD.

Materials and methods

BRCA1 and p21 expression were detected in human umbilical vein endothelial cells (HUVECs) and in mouse heart tissue after irradiation exposure. The effects of BRCA1 on cell proliferation, cell cycle and radiosensitivity were determined in HUVECs with overexpression and knockdown of BRCA1. A mouse model of RIHD was established. Heart damage was detected in C57BL/6J mice and endothelial cell specific knockout BRCA1 mice (EC-BRCA1/).

Key findings

BRCA1 and p21 expression was significantly increased both in vitro and vivo response to irradiation. BRCA1 overexpression in endothelial cells enhanced cell growth and G1/S phase arrest, and the opposite results were observed in BRCA1 knockdown endothelial cells. BRCA1 downregulated endothelial cell cycle-related genes cyclin A, cyclin D1, cyclin E and p-Rb through increasing p21 expression, and HUVECs with BRCA1 gene knockdown were more sensitive to radiation. In vivo, a decrease in cardiac microvascular density, as well as cardiomyocyte hypoxia and apoptosis were observed in a time-dependent manner. EC-BRCA1/ mice were more prone to severe RIHD than EC-BRCA1+/ mice after 16Gy radiation exposure due to endothelial dysfunction caused by loss of BRCA1, and p21 was declined in EC-BRCA1/ mice heart.

Significance

These findings indicate that BRCA1 plays a protective role in RIHD by regulating endothelial cell cycle arrest mediated by p21 signal.

Graphical abstract

Graphical abstract of the findings.

BRCA1 plays a protective role in radiation-induced heart disease by regulating vessel endothelial cell cycle arrest mediated by p21 signaling pathway. Abbreviation: CMECs, cardiac microvascular endothelial cells; RIHD, radiation-induced heart disease.

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Introduction

Radiation-induced heart disease (RIHD) has become one of the major late side effects of thoracic radiotherapy with the increasing number of long-term cancer survivors [1]. Accumulated epidemiological studies have shown that a relative increase in the risk of RIHD was observed in cancer survivors who had received thoracic irradiation decades ago, such as in patients with (especially left-sided) breast cancer [2], Hodgkin's lymphoma (relative risks (RR) 2-7-fold) [3,4], childhood cancers [5] and other thoracic malignant tumors [6]. As a late effect, the symptoms of RIHD usually manifest several decades later. The pathologies of RIHD are chronic progressive processes. Previous studies have reported that endothelial cell damage, especially in microvascular cells, may play a vital role in RIHD [[7], [8], [9]]. Cardiac microvascular endothelial cells are more sensitive to radiation than other types of cells in heart tissue [10]. The DNA damage pathway and DNA repair pathway in normal tissue cells, including cell cycle-related pathways, would be activated after ionizing radiation [11,12]. However, the molecular mechanism underlying RIHD has not been fully defined.

BRCA1 is a tumor suppressor gene and plays an important role in DNA damage repair [13]. In addition, BRCA1 is able to directly mediate transcriptional activation of p21 [14]or form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC) [15] in cancer cells. Furthermore, BRCA1 in complex with CDK, cyclin A and cyclin D participates in modulating the cell cycle [16]. Recently, some studies have illustrated that BRCA1 can also regulate cardiac endothelial cells. Praphulla C. Shukla and colleagues reported that BRCA1 in cardiomyocytes played an important role in protecting myocardial infarction patients against cardiac remodeling, poor ventricular function and higher mortality via DNA double-strand break repair and inactivation of p53-mediated pro-apoptotic signaling [17]. Accumulated studies found that p21 is a crucial determinant of DNA repair [18,19].

Until now, the role of BRCA1 in protecting endothelial cells from DNA damage has rarely been explored, especially in RIHD. Therefore, we proposed a hypothesis that BRCA1 protects radiation-induced heart damage by blocking the endothelial cell cycle via the activation of p21. In this study, we demonstrated the underlying mechanism and designed a radiation-induced heart damage model in C57BL/6 mice to confirm this observation both in vitro and in vivo. BRCA1 protected endothelial cells against irradiation by p21-mediated cell cycle arrest.

Section snippets

Cell culture and irradiation

HUVECs were obtained from the Key Laboratory of Molecular Medical in Jiangxi and cultured in endothelial growth medium (Gibco, Grand Island, USA) enriched with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO2 humidified atmosphere. Cells were irradiated with 2–20 Gy of X-rays (6 MeV, SSD = 100 cm) using a Varian linear accelerator (Varian Clinical 23EX, USA) at a dose rate of 600 cGy/min.

Transfection and RNA interference

The plasmids pSin-EF2-BRCA1 and pCDEF-BRC1-Flag were purchased from Ribobio

BRCA1 and p21 expression are up-regulated in vascular endothelial cells both in vitro and vivo after radiation exposure

To examine the mechanism and prove our hypothesis, we investigated the effect of the BRCA1 on the cellular level using HUVECs. BRCA1 and p21 protein expression levels were detected via Western blotting after the cells received 20 Gy X-ray at different times and ionizing radiation. We found that BRCA1 and p21 protein expression in HUVECs increased in a dose- and time-dependent manner after radiation exposure (Fig. 1A–D). In vivo, BRCA1 and p21 expression levels in mouse cardiac vascular

Discussion

Here, we demonstrated the protective role BRCA1 in RIHD by establishing an irradiation-induced heart damage model in C57BL/6 mice (Fig. 7). In this study, we first demonstrated that BRCA1 plays a role in the response of cardiac endothelial cells to radiation by regulating p21-cell cycle arrest and crisis in HUVEC and ultimately leading to endothelial cell apoptosis. In the wild mouse RIHD model, we confirmed that that the pathological process of RIHD presented as microvessel density decline,

Author contributions

Z-M Z, in charge of design of the work, performed cell and animal experiment, evaluated experimental data, and drafted the manuscript. H-Y D, L X, X-L Z performed part of cell and animal experiment. P-Z performed mouse radiation. H-Y D, X-L and J-C helped to analyze the data. L H and A-W L, the Corresponding author, was in charge of guidance of the design and analysis the whole research.

Declaration of competing interest

The authors report no conflict of interest.

Acknowledgements

This work was supported by the National Nature Science Foundation [grant number 81560509, 81760566 and 81960571], Key Research and Development Project of Jiangxi province [grant number 20171ACB20034, 20181ACG70011 and r20192ACB70013], Science and Technology Innovation Platform of Jiangxi Province [grant number 20171BCD40022 and 20192BCBL23023]. We thank all the other members in the Zhejiang key laboratory of radiation oncology for supporting our work. Thank Caifeng Xie, Associate Professor of

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