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Gene expression and genetic variation of ERG is associated with inflammation in endothelial cells and risk of coronary artery disease in humans

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Gene expression and genetic variation of ERG is associated with inflammation in endothelial cells and risk of coronary artery disease in humans

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Content Gene Expression and Genetic Variation of ERG is Associated with Inflammation
in Endothelial Cells and Risk of Coronary Artery Disease in Humans
by
Ruowei Zhu
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
August 2020
Copyright 2020 Ruowei Zhu
ii

Acknowledgements
I would like to express my heartfelt gratitude to my mentor, Dr. Hooman Allayee. His
continuous encouragement and guidance kept me moving forward on this path. His passion
towards research and teaching influenced each one of us and motivates me. He is the best teacher
and friend you can ever imagine, and I appreciate the time I spent working with him. This
experience not only impacted me while I was here, but also for the rest of my life.
I also want to especially thank my committee members, Dr. Pragna Patel and Dr. Jian Xu.
Whenever we encountered problems, Dr. Patel was generous with her advice and would always
suggest the best solutions. Her patience and sense of responsibility helped us adjust and move
forward. Similarly, Dr. Xu offered invaluable advice and support for my research project as well.
With her and her lab members’ help, I was able to overcome experimental difficulties and
understand my research project better from a different perspective.
Lastly, I would like to thank my lab members and classmates. Dr. Jaana Hartiala showed me the
rigorous attitude that a well-trained scientist should have towards experimental data. Dr. Yi Han
always made herself available when I needed help and guided me in using state-of-the-art
methods in the field that I would otherwise not have been familiar with. Janet Gukasyan not just
helped me with experimental procedures, but also with adjusting to life in the lab and the United
States. I would also like to thank Zhiheng Cai and James Hilser for their support and
encouragement, as well as my classmates Shulin Liu and Yibo Si who selflessly spared their time
to answer questions that I had in classes. I learned so much from these individuals and
thoroughly enjoyed the time working with them.
iii

Table of Contents
Acknowledgements ................................................................................................... ii
List of Tables ............................................................................................................. v
List of Figures .......................................................................................................... vi
Abbreviations .......................................................................................................... vii
Abstract .................................................................................................................... ix
Introduction ................................................................................................................ 1
Materials and Methods ............................................................................................... 5
Functional Studies in HAECs ......................................................................................... 5
siRNA knockdown of ERG in HAECs ........................................................................... 6
Animal Husbandry and Generation of Erg-deficient Mice ............................................. 6
Gene Expression Analyses in Erg-deficient Mice .......................................................... 7
Human Populations ......................................................................................................... 8
Clinical Definitions in the UK Biobank .......................................................................... 8
Statistical Genetic Analyses in the UK Biobank ............................................................. 9
Meta-analyses with the UK Biobank and the CARDIoGRAM+C4D Consortium ....... 10
Results ......................................................................................................................10
Relationship between ERG Expression, Pro-atherogenic Stimuli, and Inflammatory
Genes in HAECs. .......................................................................................................... 10
In Vivo Evaluation of ERG Deficiency in Endothelial Cells: generation and
characterization of ERG KO mice. ................................................................................ 14
Genetic Variation at the ERG Locus is Associated with Atherosclerosis-related
outcomes in Humans. .................................................................................................... 15
Discussion ................................................................................................................17
iv

References ................................................................................................................22

v

List of Tables
Table 1. Significantly DEGs in aortas of Erg
flox+/Cre+
mice and Erg
flox+/Cre-
control ...................... 14
Table 2. Pathways enriched for DEGs between Erg
flox+/Cre+
mice and Erg
flox+/Cre-
control ............ 15

vi

List of Figures
Figure 1. Development of atherosclerosis in arteries. ..................................................................... 2
Figure 2. Erg isoforms in mice. ...................................................................................................... 4
Figure 3. Relationship between ERG expression, oxPL exposure, and inflammatory gene
expression in HAECs. ................................................................................................................... 13

vii

Abbreviations
ETS-Related Gene (ERG)
Low Density Lipoprotein (LDL)
Smooth Muscle Cell (SMC)
Myocardial Infarction (MI)
E26 transformation-specific (ETS)
Separate Polyadenylation sites (PAS)
Pointed Domain (PNT)
Intercellular Adhesion Molecule 1 (ICAM1)
VE-cadherin/Cadherin 5 (Cdh5)
Human Umbilical Vein Cells (HUVEC)
Interleukin (IL)
Human Aortic Endothelial Cells (HAECs)
Coronary Artery Disease (CAD)
Oxidized Phospholipids (oxPLs)
Robust Multiarray Average (RMA)
small interfering RNA (siRNA)
viii

UltraPleX (UPX)
Differential Expressed Genes (DEGs)
National Health Service (NHS)
International Classification of Diseases version-10 (ICD10)
Single Nucleotide Polymorphisms (SNPs)
Minor Allele Frequencies (MAF)
Odds Ratios (ORs)
Standard Errors (SEs)
Nitric Oxide Synthase 3 (NOS3)
Vascular Cell Adhesion Molecule (VCAM)
Selectin P (SELP)
Selectin E (SELE)
Linkage Disequilibrium (LD)
expression Quantitative Trait Locus (eQTL)
Genotype-Tissue Expression (GTEx)

ix

Abstract
Atherosclerosis is the primary cause of heart disease and stroke, resulting in significant
morbidity and mortality. It is an inflammatory disease characterized initially by the accumulation
of lipids in coronary arteries and other large vessels. Dysfunction of endothelial cells is an
important contributor to the pathogenesis of this complex process. ETS-related Gene (ERG) is a
transcription factor highly expressed in endothelial cells and a major genetic determinant of
endothelial cell homeostasis. For example, ERG upregulates genes that maintain endothelial cell
junction integrity and downregulates pro-inflammatory genes. Thus, ERG could be directly
involved in atherogenic processes, but evidence for this notion in the literature, particularly with
respect to in vivo studies, has been limited. In this study, we provide functional in vitro and
genetic evidence in humans showing that ERG could potentially influence the development of
atherosclerosis through inflammatory mechanisms at the level of the vessel wall. An in vivo
study of gene expression in aortas from endothelial cell-specific Erg-deficient mice also
demonstrated that lack of this homeostatic factor in endothelial cells results in dysregulation of
pathways related to cardiac function and heart failure. Collectively, our data suggests that ERG‘s
atheroprotective properties could be related to regulation of inflammatory genes in endothelium
in humans, while the underlying mechanisms for the effects of Erg deficiency in endothelial cells
of mice will require further investigation.
1

Introduction
Atherosclerosis is the leading cause of heart disease and stroke, and accounts for a significant
fraction of morbidity and mortality in developed countries. Multiple environmental and genetic
risk factors contribute to atherosclerosis, including unhealthy diet, lack of physical activities,
smoking, obesity, aging, diabetes mellitus and family history (Singh et al. 2002). Atherosclerosis
is an inflammatory disease in which atheromatous plaques accumulate in the inner layers of large
arteries. Formation of these plaques is initiated by the trapping of low-density lipoprotein-
cholesterol (LDL) at lesion sites. Oxidation of LDL along with the cytokines produced in this
microenvironment activate endothelial cells and promote adhesion and transmigration of
monocytes into the artery wall. Inside the intima, monocytes proliferate and differentiate into
macrophages, which are subsequently transformed into foam cells as they engulf the oxidized
LDL formed in the subendothelial space. In the center of more advanced plaques, the apoptosis
of foam cells forms a lipid-rich “necrotic core” capped by migrated smooth muscle cells (SMCs)
and synthesized extracellular matrix. The accumulation of plaques leads to narrowing of the
arterial lumen, but in more advanced plaques, the fibrous caps could be ruptured. The release of
intraplaque contents, including collagen and lipids, into the lumen of the artery initiates platelet
aggregation and coagulation that culminate in thrombotic events, such as myocardial infarction
(MI) or stroke (Rafieian-Kopaei et al. 2014 and Lusis. 2000) (see Fig. 1).
2

Figure 1. Development of atherosclerosis in arteries. (Adapted from Rafieian-Kopaei et al.
2014)

Among the various types of vascular cells that actively participate in the atherogenic process are
endothelial cells. For example, endothelial cells regulate and maintain vascular tone by releasing
various substances regulating dilation and constriction, such as nitric oxide, prostacyclin,
angiotensin II, and endothelin (Drexler. 1998). However, during the inflammatory cascade
associated with atherogenesis, endothelial cells become activated and begin to express multiple
proinflammatory cytokines and adhesion molecules, which further exacerbates lesion
progression (Yang et al. 2016). Thus, endothelial cell dysfunction could be an early indicator of
atherosclerosis.
3

Nearly all active enhancers and promoters discovered in endothelial cells thus far harbor several
E26 transformation-specific (ETS) binding sites (De Val et al. 2008). These observations suggest
that ETS transcription factors may play an important role in regulating endothelial cell
homeostasis and gene expression (Randi et al. 2009). ETS transcription factor was first
discovered in E26 avian leukemia retrovirus as a viral oncogene (Leprince et al. 1983) and 27
members of this gene family are now recognized to exist in humans (Gutierrez-Hartmann et al.
2007). All ETS factors share a conservative domain characterized by a winged helix-turn-helix
structure that binds to the 5’-GGA(A/T)-3 core DNA sequence (Meadows et al. 2011).
Furthermore, ETS transcription factors can function as activators or repressors of gene
expression, or both, depending on the cellular context (Sharrocks 2001, Lelièvre et al, 2001 and
Yuan et al. 2009). First described in 1987 by Reddy et al. (Reddy et al. 1987), ETS-related gene
(ERG) is one important member of the ETS transcription factor family. For example, ERG is
involved in a wide range of biological process, including endothelial homeostasis (McLaughlin
et al. 2001), angiogenesis (Birdsey et al. 2012), vascular integrity (Birdsey et al. 2008 and Han et
al. 2015), hematopoiesis (Loughran et al. 2008), bone and cartilage development (Iwamoto et al.
2000), and multiple cancers (Shing et al. 2003, Shimizu et al. 1993, and Adamo et al 2016). In
humans, ERG maps to chromosome 21q.22, which is syntenic to mouse chromosome 16
(Owczarek et al. 2004). Considering the various known alternative promoters, alternative
splicing sites and separate polyadenylation sites (PAS) of ETS in humans, approximately 30
different ERG isoforms can be transcribed but only a few can be translated into protein
(Zammarchi et al. 2013). In mice, seven Erg isoforms are known, with four sharing the
translational start site located in exon 3. By comparison, the translational start site for the other
three Erg isoforms are in exon 4 (see Fig. 2). Mice with a homozygous deletion of exon 3 are
4

viable and fertile without distinctive phenotype, whereas ablation of exon 4 in homozygous
form, even only in endothelial cells, results in embryonic lethality with vascular defects and
compromised development of the cardiovascular system (Birdsey et al. 2015 and Vijayaraj et al.
2012). Thus, exon 4 of Erg encodes an important functional component of the protein that is
essential for survival.

Figure 2. Erg isoforms in mice. Exon 3 encodes the translational start site for Erg isoforms 1-4.
Exon 4 encodes the translational start site for Erg isoforms 5-7. The exonic locations of the
pointed domain (PNT), variable, and Ets domains are indicated. (Adapted from Vijayaraj et al.
2012)

ERG is abundantly expressed in resting endothelial cells (Yuan et al. 2009) and regulates genes
that define endothelial lineages, such as endoglin, von Willebrand Factor, intercellular adhesion
molecule 1 (ICAM1), and VE-cadherin/Cadherin 5 (Cdh5) (Birdsey, Graeme M., et al. 2012). Its
expression is markedly down-regulated after exposure to inflammatory cytokines, such as tumor
necrosis factor alpha (McLaughlin et al. 1999), and is absent in the endothelium that overlays
active shoulders of human atherosclerotic plaques (Sperone et al. 2011). Furthermore,
5

knockdown of ERG in human umbilical vein cells (HUVECs) by RNA interference significantly
increases expression of pro-inflammatory genes, such as interleukin (IL) 8 (Yuan et al. 2009). By
comparison, overexpression of ERG in Xenopus laevis embryos invokes ectopic erythrocyte and
endothelial cell accumulation (Baltzinger et al. 1999). These data collectively suggest that ERG
plays a protective role in the development of vascular inflammation. However, to our knowledge,
a clear association between ERG and atherosclerosis in mice or humans has yet to be directly
demonstrated.
In the present study, we investigated the relationship between ERG, pro-inflammatory stimuli,
and atherosclerosis-related genes in human aortic endothelial cells (HAECs) in vitro. An Erg-
deficient mouse model was also used to examine the role of Erg in atherosclerosis-related gene
expression in the aorta. Finally, we carried out a population-based analysis to determine the
relevance of genetic variation at the ERG locus to risk of atherosclerosis-related outcomes, such
as coronary artery disease (CAD) and MI.
Materials and Methods
Functional Studies in HAECs
Collection and isolation of HAECs have been described previously (Romanoski et al. 2010 and
Romanoski et al. 2011). Briefly, HAECs were isolated from aortic explants of 149 heart
transplant donors of anonymous origin through the UCLA transplant program. Cells were grown
to 90% confluence and incubated for 4 hrs in M-199 (ThermoFisher Scientific, Waltham, MA,
MT10–060-CV) and 1% FBS with or without 40mg/mL oxidized phospholipids (oxPLs). RNA
was hybridized to Affymetrix HT-HU133A microarrays with a standard protocol described
previously (Romanoski et al. 2010). Intensity values were normalized with the robust multiarray
average (RMA) (Bolstad, et al. 2003 and Irizarry et al. 2003) normalization method in R 2.5.0
6

with the justRMA function of the Affymetrix package of Bioconductor. Transcript expression
values were averaged between duplicate arrays per condition and donor. Ancestry of each subject
was determined with principal components analysis of whole-genome genotype data and using
publicly available data from the 1,000 Genomes Project. Differences in normalized log2 ERG
mRNA levels between oxPL exposed and unexposed groups in all subjects, within each ancestry
group, or separately in males and females were assessed by paired t-tests. Correlation
coefficients and P-values for the relationship between mRNA levels of ERG and selected
atherosclerosis-related inflammatory genes with and without oxPL exposure were derived from
linear regression analyses that included all 149 donors and the inclusion of ancestry and sex as
covariates.

siRNA knockdown of ERG in HAECs
The effect of knocking down ERG in HAECs on global gene expression was carried out as
described previously (Hogan, et al. 2017). HAECs from three donors independent of the 149
subjects described above were transfected with 1nM small interfering RNA (siRNA)
oligonucleotides in Opti-MEM (ThermoFisher Scientific) with Lipofectamine 2000
(ThermoFisher Scientific) in serum-free media for 4 hrs and the cells were then grown in full
growth media for 48 hrs. Expression levels of candidate inflammatory genes after transfection
with siRNA against ERG or a control scrambled siRNA were extracted from RNAseq-based data
and evaluated for differences using paired t-tests.

Animal Husbandry and Generation of Erg-deficient Mice
7

Erg-deficient mice were generated previously (Ohta et al. 2015) and engineered to carry loxP
sites flanking exon 6 (floxed). Breeding of Erg-floxed mice to transgenic mice expressing Cre in
only a tissue of interest would result in the elimination of all Erg isoforms in a tissue-specific
manner. Therefore, we used this strategy to generate endothelial cell-specific Erg knockout mice.
To ablate Erg in endothelial cells, floxed Erg mice (Erg
flox+/flox+
) were bred to hemizygous Cre
transgenic animals in whom Cre expression was driven by the endothelial cell-specific Cdh5
promoter. By crossing homozygous Erg floxed mice with Cdh5-Cre mice, 12 Erg
flox+/Cre+
littermates and 12 Erg
flox+/Cre-
littermates as wildtype controls were generated. Mice were housed
in sterilized microisolator cages (n=4-5/cage) at 25ºC and maintained on a 12 hrs dark/12 hrs
light cycle with access to food (Purina chow diet #5053) and water ad libitum. All animal studies
were approved by the USC Keck School of Medicine Institutional Animal Care and Use
Committee and conducted in accordance with the Department of Animal Resources’ guidelines.

Gene Expression Analyses in Erg-deficient Mice
Mice were euthanized at 10 weeks of age and the aorta was carefully dissected out under a light
microscope. Total RNA was extracted using RNAeasy kits (Qiagen) and assessed for purity
using the ratio of light absorbance at 260nm and 280nm. Whole transcriptome analysis was
carried out using QIASeq UltraPleX (UPX) 3’ Transcriptome system (Qiagen) and raw counts
obtained were uploaded to iDEP.91. Among 30,633 genes in 24 samples, 8,107 genes passed a
quality control filter of having a minimum of 10 counts per million in at least 3 libraries.
Differentially expressed genes (DEGs) were analyzed by limma-voom, followed by DEG2
analysis embedded in iDEP.91. GO Biological Processes were used for enrichment analyses of
DEGs.
8

Human Populations
To assess whether genetic variation at the ERG locus influenced risk of atherosclerosis-related
outcomes in humans, we leveraged phenotype and genetic data available in the UK Biobank as
well as summary statistics from the CARDIoGRAM+C4D Consortium. Briefly, the UK Biobank
recruited 503,325 individuals between 2006-2010 who were 40-69 years of age and were
registered with a general practitioner of the UK National Health Service (NHS) (Bycroft et al.
2018). All study participants provided informed consent and the study was approved by the
North West Multi-centre Research Ethics Committee. Individual cohorts in the
CARDIoGRAM+C4D Consortium have been previously described in detail (Nikpay et al. 2015).
MI and CAD status in CARDIoGRAM+C4D was based on individual study-specific parameters
and resulted in the definition of ~44,000 and 60,801 cases, respectively, and 123,504 controls.
All subjects gave written consent for participation in genetic studies, and the protocol of each
study was approved by the corresponding local research ethics committee or institutional review
board. The present genetic analyses were approved by the Institutional Review Boards of the
Cleveland Clinic and USC Keck School of Medicine.

Clinical Definitions in the UK Biobank
MI cases were defined as positive for International Classification of Diseases version-10
(ICD10) codes I21, I22, I23, I25.2, which included MI, and complications following acute MI.
Doctor-diagnosed and self-reported MI were also included in the definition of MI. CAD cases
were defined as subjects positive for MI according to the criteria described above, as well as
positive for other ICD-10 codes I24.0, I24.8, I24.9, I25.0, I25.1, I25.4, I25.8, and I25.9, which
9

included ischemic heart diseases. Additional criteria to define CAD included Office of
Population Censuses and Surveys Classification of Interventions and Procedures, version 4
(OPCS-4) codes K40-K46, K49, K50 and K75, covering replacement, transluminal balloon
angioplasty, other therapeutic transluminal operations on coronary artery and percutaneous
transluminal balloon angioplasty and insertion of stent into coronary artery. To avoid any
misclassification of cases, we also excluded 59 samples who were only positive for ICD10 codes
I24.1 (Dressler's syndrome), I25.3 (aneurysm of heart), I25.5 (ischaemic cardiomyopathy), or
I25.6 (silent myocardial ischaemia). This overall strategy in the UK Biobank led to the definition
of 17,505 MI cases, 33,085 all-inclusive CAD cases, and 454,212 controls who were negative for
all of the clinical designations used to define CAD and MI.

Statistical Genetic Analyses in the UK Biobank
Quality control of samples and DNA variants and imputation were performed by the Wellcome
Trust Centre for Human Genetics (Bycroft et al. 2018). To evaluate the ERG locus, we selected
an 800 kb interval on chromosome 21 that encompassed the gene and any potentially flanking
regulatory elements. Prior to analysis, single nucleotide polymorphisms (SNPs) were filtering on
having INFO scores >0.8 (directly from the UK Biobank) and with minor allele frequencies
(MAF) >0.5% in the 487,379 individuals with imputed genotype data. Association testing was
performed with BOLT-LMM V2.3.2 using a standard (infinitesimal) mixed model to correct for
structure due to relatedness, ancestral heterogeneity, with adjustment for age, sex, the first 20
principal components, and genotyping array (Loh et al. 2015). Since BOLT-LMM relies on
linear models even for qualitative traits, SNP effect size estimates on the quantitative scale were
10

transformed to obtain odds ratios (ORs) and standard errors (SEs) using the following formula: β
or SE/(μ ∗ (1 - μ)), where μ = case fraction (Loh et al. 2015)

Meta-analyses with the UK Biobank and the CARDIoGRAM+C4D Consortium
Publicly available summary statistics for association of SNPs in the 800 kb ERG interval with
CAD and MI from the CARDIoGRAM+C4D Consortium were combined with our results in the
UK Biobank. We carried out a fixed-effects meta-analysis with ~2,800 SNPs common to both
datasets assuming an additive model, as implemented in GWAMA (Marchini. 2010). In total,
~639,000 and ~655,000 subjects were included in the meta-analyses that tested association of the
ERG locus with MI and CAD, respectively. Based on testing ~2,800 SNPs in the 800 kb interval,
significance was set at a Bonferroni-corrected threshold of P=1.8x10
-5
(0.05/2800).
Results
Relationship between ERG Expression, Pro-atherogenic Stimuli, and Inflammatory Genes
in HAECs.
Prior evidence from the literature has highlighted the importance of the endothelium in CAD
(Gimbrone et al. 2016). Based on this notion, we carried out functional studies with the ERG
locus since this gene had previously been implicated in multiple processes related to endothelial
homeostasis. We first used a well-characterized HAEC dataset to examine ERG expression in
response to oxPL and in relation to several pro-atherosclerotic inflammatory genes. ERG mRNA
levels were significantly and consistently downregulated in HAECs after incubation with oxPL
in subjects of various ethnicities (Fig. 3a). When the data were stratified by sex, downregulation
of ERG was observed to be similar in both men and women (Fig. 3b). We next examined the
11

relationship between ERG mRNA levels and genes known to be involved in atherogenesis. As
shown in Fig. 3c, ERG expression was inversely correlated with pro-inflammatory genes, such as
IL6 and IL8, and positively correlated with athero-protective genes, such as nitric oxide synthase
3 (NOS3), under both control conditions and after oxPL treatment (Fig. 3c). Interestingly, the
significance of the correlations of ERG mRNA levels with those of IL6 and IL8 after oxPL
exposure were higher by several orders of magnitude than under control conditions (Fig. 3c).
Direct perturbation of ERG through siRNA knockdown experiments in an independent set of
HAEC donors validated the inverse relationship between expression of ERG and the same
inflammatory genes (Fig. 3d). However, regardless of oxPL exposure, there were weak or no
significant correlations between ERG and expression of vascular adhesion molecule genes (i.e.
ICAM1, vascular cell adhesion molecule (VCAM1), selectin p (SELP), selectin E (SELE)) that
encode proteins involved in facilitating trans-endothelial migration of monocytes into the intima
of the artery wall (Fig. 3e). Taken together, these data provide functional evidence that ERG
could potentially influence the pathogenesis of CAD through inflammatory mechanisms in
endothelial cells.
12

a.
b.

13

c. d.

e.

Figure 3. Relationship between ERG expression, oxPL exposure, and inflammatory gene
expression in HAECs. ERG mRNA levels in HAECs from 149 donors is downregulated after
exposure to oxPLs compared to control exposure regardless of ethnicity a or sex b. c. ERG
expression is significantly correlated with mRNA levels of atherosclerosis related inflammatory
genes (NOS3, IL8 and IL6) in HAECs with or without exposure to oxPLs. d. Relationship
between ERG and NOS3, IL8 and IL6 was validated through siRNA-mediated ERG knockdown
in 3 independent HAECs. e. There were very weak or no correlations between ERG expression
and adhesion molecule genes, such as ICAM1, VCAM1, SELP, and SELE, in HAECs regardless
of oxPL exposure. (Data (Fig. 3a, 3c, 3d, 3e) kindly shared by Romanoski’s lab. Fig. 3b made
by J. Gukasyan and R. W. Zhu based on their gene expression data)
SELE
r=0.09; P=0.69
ox-PL
Control SELP
r=0.12; P=0.32
SELP
r=0.16; P=0.17
SELE
r=0.09; P=0.68
Males
Females
VCAM1
r=0.24; P=0.32
VCAM1
r=0.05; P=0.87
ICAM1
r=0.19; P=0.03
ERG
ICAM1
r=0.23; P=0.01
ERG
14

In Vivo Evaluation of ERG Deficiency in Endothelial Cells: generation and characterization
of ERG KO mice.
We first characterized Erg
flox+/Cre+
knockout and Erg
flox+/Cre-
littermates control mice with respect
to Erg expression. Although Erg expression was decreased in Erg
flox+/Cre+
mice compared to
controls (Erg
flox+/Cre-
), the P-value did not achieve statistical significance (data not shown). We
also did not observe any significant changes in expression of candidate inflammatory (IL6, IL8,
NOS3) and adhesion molecule (ICAM1, VCAM1, SELP, SELE) genes. We next carried out an
unbiased analysis to identify genes differentially expressed between Erg
flox+/Cre+
knockout mice
and Erg
flox+/Cre-
controls. As determined by limma-voom, Nppa, Myh6, and Tnni3 were among
the most significantly DEGs in aortas of Erg
flox+/Cre+
mice and control mice (Table 1). Notably,
these genes were found to be upregulated in Erg
flox+/Cre+
knockout mice compared to control
littermates. Based on the DEG2 analysis, we also observed that genes in pathways related to
regulation of blood circulation and blood pressure, along with cardiac muscle contraction and
adaptation were also strongly upregulated in Erg
flox+/Cre+
mice, suggesting the lack of Erg in the
vascular wall may be associated with deterioration of heart function (Table 2).

Table 1. Significantly DEGs in aortas of Erg
flox+/Cre+
mice and Erg
flox+/Cre-
control (data
generated by J. Gukasyan, N. Woodward and R.W. Zhu)

log2 Fold Change adj.Pval Symbol Chr Type
2.51 1.78E-04 Nppa 4.00E+02 Protein Coding
2.21 3.56E-04 Myh6 14C3 Protein Coding
1.73 4.55E-02 Tnni3 7A1 Protein Coding

15

Table 2. Pathways enriched for DEGs between Erg
flox+/Cre+
mice and Erg
flox+/Cre-
control.
(data generated by J. Gukasyan, N. Woodward and R.W. Zhu)

Direction adj.Pval nGenes Pathways
Up regulated 1.40E-05 3 Regulation of blood circulation

1.40E-05 3 Regulation of blood pressure

1.40E-05 3 Cardiac muscle contraction

2.90E-05 2 Cardiac muscle adaptation

Genetic Variation at the ERG Locus is Associated with Atherosclerosis-related outcomes in
Humans.
We next used a genetics approach to determine whether variants at the ERG locus influenced risk
of atherosclerosis-related outcomes. We first tested SNPs in an 800 kb interval encompassing
ERG for association with CAD and MI in the UK Biobank (see Methods for details), followed by
a meta-analysis with summary statistics for the same interval from the CARDIoGRAM+C4D
Consortium. Based on sample sizes of ~655,000 and ~639,000 cases and controls for CAD and
MI, respectively, in the meta-analysis, several SNPs in high and low linkage disequilibrium (LD)
with each other around the promoter and upstream of ERG were associated with CAD (Fig. 4a).
The lead SNP (rs2836633) in this interval, indicated by the purple diamond, yielded a P-value of
4.16e-6 and would be considered significant at a Bonferroni-corrected threshold P-value of 1.8e-
5 for testing 2,830 SNPs (P=0.05/2830). We next used expression quantitative trait locus (eQTL)
data from Genotype-Tissue Expression (GTEx) project to determine whether these association
signals implicated ERG as the causal gene in this interval. Although there were no eQTLs with
the lead CAD SNP for ERG or other genes in vascular tissues available in GTEx, there was a cis
eQTL for ERG in mucosa of the esophagus with a modest P-value of 1.17e-2 (Fig. 4b).
16

Importantly, mRNA levels of ERG were lower among carriers of the G allele, which was
associated with increased risk of CAD (OR=1.03 (1.02-1.04).
We next carried out the equivalent association analysis for MI, which also identified the same
lead SNP for CAD as being associated with MI (P=4.54e-6). However, another variant
(rs2836620) in low LD with the lead CAD SNP yielded a slightly more significant association
with MI (P =3.87e-6) (Fig. 4c). The lead SNP for MI also yielded a more significant cis eQTL
for ERG in esophagus (Fig. 4d). Similar to the lead CAD SNP, the allele (C) of rs2836620 that
increased risk of MI (OR=1.04 (1.02-1.06) was also associated with decreased ERG expression.
However, there were no cis eQTLs for ERG with either the lead CAD or MI SNP under control
or oxPL conditions in the HAEC dataset described above (data not shown). Nonetheless, these
observations are consistent with the notion that ERG has cardioprotective properties that may be
related to the suppression of inflammatory pathways.
17

Figure 4. Association and functional evaluation of genetic variation at ERG locus with CAD
and MI. a. Regional plot of an 800 kb region at chromosome 21 encompassing ERG for
association with CAD. The lead CAD SNP (rs2836633) is indicated by the purple diamond
(P=4.16e-6, OR=1.03, 95% CI 1.02-1.04). b. cis eQTL from the GTEx Project for rs2836633
and ERG expression in esophagusmucosa (P=1.17e-2). c. Regional plot of the same 800 kb
interval encompassing ERG for association with MI. The lead CAD SNP (rs2836633) is
indicated by the purple diamond (P =4.54e-6) whereas the lead MI SNP (rs2936620; P=3.87e-6,
OR=1.04, 95% CI 1.02-1.06) is indicated by the light blue dot directly to the left of rs2836633.
d. cis eQTL from GTEx Project for ERG with the lead MI SNP in esophagus-mucosa (P=1.60e-
9). (GWAS data generated by Q. Jia and Y. Han. Regional plots and eQTL generated by R. W.
Zhu)
Discussion
A large body of data from prior studies indicate that disruption of normal endothelial function
through mechanisms such as oxidative stress, pro-inflammatory pathways involving cytokines
18

and infectious agents, environmental toxins, and hemodynamic forces, promotes the
development of atherosclerosis (Gimbrone et al. 2016). We now provide functional and genetic
evidence with respect to ERG that further supports this concept. For example, under
experimental conditions with natural variation in mRNA levels, ERG expression in HAECs was
not only downregulated in response to oxPL exposure but inversely correlated with
proinflammatory genes and positively correlated with athero-protective genes. Importantly, these
observations were independently validated by siRNA-mediated perturbation of ERG expression
in HAECs. Furthermore, our results are also consistent with previous studies in which
overexpression of ERG in HUVECs repressed expression of not only IL8 (Yuan et al. 2009) but
several other important inflammatory genes involved in atherosclerosis (Sperone et al. 2011).
Taken together, these data lend support for the notion that ERG represents a novel causal
candidate gene for CAD and that this transcription factor may functionally influence
development of atherosclerosis in the endothelium through mechanisms related to inflammation
and vascular homeostasis.
The genetic evaluation of the ERG locus on chromosome 21q22.2 revealed several variants that
were significantly associated with risk of CAD and MI. In this regard, two recent large-scale
genetics studies identified another nearby variant at the ERG locus (rs117870289) as being
associated with pulse pressure and diastolic blood pressure (Warren et al. 2017 and Giri et al.
2019), suggesting that genetic variation of ERG influences risk atherosclerosis-related outcomes
through its effects on blood pressure regulation. Interestingly, the lead SNPs we identified for MI
(rs2836620) and CAD (rs2836633) are not in LD with the blood pressure variant and only in
weak LD with each other (r
2
=0.31). These latter observations suggest that the association signals
for MI and CAD are independent. rs2836620 and rs2836633 are located ~20 kb and ~34 kb
19

upstream, respectively, of the promoter of several longer isoforms of ERG, with rs2836633
localizing to a putative regulatory region within a CCCTC-binding factor (CTCF). However, it is
not known whether this putative CTCF site is functional and whether rs2836633 disrupts its
regulatory effects, which will require additional studies to determine. However, both variants did
yield directionally consistent eQTLs for ERG in esophageal tissue based on data from GTEx,
suggesting these SNPs are functional with respect to gene expression. By comparison, cis eQTLs
with rs2836620 and rs2836633 were not observed in HAECs under control or oxPL exposure
conditions. In this regard, complete ablation of Erg expression in mice, whether globally or even
only in endothelial cells, resulted in embryonic lethality (Vijayaraj et al. 2012 and Han et al.
2015). These observations suggest that ERG expression is tightly regulated in vivo, particularly
in endothelial cells. This may explain, at least in part, why our lead SNPs for CAD or MI at the
ERG locus were not associated with ERG mRNA levels in HAECs since the 149 subjects
available in the dataset for this cell type may not have provided sufficient power for detecting a
cis eQTL for ERG in HAECs.
Based on our functional and genetic studies, and those from the literature, it is reasonable to
assume that increased expression of ERG would protect against atherosclerosis and risk of CAD
and MI. While a mouse model for Erg overexpression has yet to be developed, this hypothesis is
supported by studies of a natural occurring genetics experiment in humans. For example, ERG is
located within the critical interval for Down syndrome, which is caused by three copies of
chromosome 21 (Owczarek et al. 2004). Aside from various levels of intellectual disability, such
patients have approximately 20-fold higher risk of childhood leukaemia, which is thought to be
due, at least in part, to increased expression of ERG and its cell growth-promoting properties in
bone marrow-derived cells (Tsuzuki et al. 2011 and de Smith et al. 2019). By the same token,
20

risk of atherosclerosis would presumably be comparatively lower in Down syndrome patients,
given the presence of three copies of ERG, than in normal subjects. The first observations
supporting this concept were reported over 30 years ago in small numbers of subjects in whom
post-mortem examinations revealed very little to no coronary atherosclerosis (Rodger et al.1977
and Ylä-Herttuala et al. 1989). These initial findings have since been replicated by other groups
as well. In particular, a large Australian study with ~20,000 subjects demonstrated a remarkable
65-70% reduced risk of CAD and MI in male Down syndrome patients compared to age-matched
controls (Sobey et al. 2015). In another, adults with Down syndrome had significantly lower
carotid atherosclerosis compared that in age-, sex-, and ethnicity-matched adults (Draheim et al.
2010). Thus, increased expression of ERG in humans, either under naturally occurring conditions
or as the result of an having an extra copy of chromosome 21, is associated with decreased
atherosclerosis and related adverse clinical outcomes.
With respect to the in vivo mouse study, heterozygous deficiency of Erg (Erg
flox+/Cre+
) in
endothelial cells did not alter expression of inflammatory cytokines that were correlated with
ERG expression in HAECs. However, we note that decreased expression of Erg was not
observed in the Erg
flox+/Cre+
vs. control mice. One possible explanation for this observation could
be because we profiled the transcriptome of whole aorta. However, endothelial cells represent
only a small fraction of the cellular composition of the aorta since they only present as a single
monolayer along the artery wall. Thus, any gene expression differences that are specific to
endothelial cells may not have been detected without a more focused study such as single cell
RNAseq analysis. Alternatively, only one Erg allele was ablated in the Erg
flox+/Cre+
mice and it is
possible that the unmodified allele was upregulated to compensate for the 50% reduced Erg
expression. Despite these potential confounders, Erg
flox+/Cre+
mice demonstrated a surprising
21

dysregulation of genes and pathways related to cardiac function. For example, the most
differentially expressed genes (Nppa and Myh6) in Erg
flox+/Cre+
mice were canonical gene
expression markers for cardiomyocytes. Overexpression of Nppa (Song et al. 2015) has been
implicated in hypertrophic cardiomyopathy and heart failure, while Myh6 plays vital roles in
cardiac contraction (England et al. 2013). These observations suggest that Erg
flox+/Cre+
mice may
exhibit alterations in blood pressure, consistent with the genetic associations observed with the
ERG locus in humans (Warren et al. 2017 and Giri et al. 2019), and possible susceptibility to
development of heart failure. However, evidence for these hypotheses will require a thorough in
vivo assessment of vascular and cardiac function.
There are several limitations in our present study. First, we examined the expression profile of
the whole aorta while the Erg knockout only took place in the endothelium. Second, the
manipulation of Erg in mice only affected cardiac functional genes while there were no
significant changes in our candidate inflammatory genes. Since mice are resistant to developing
atherosclerosis by nature, the mice should have received an appropriate atherogenic stimulus.
Third, the Qiagen UPX3’ Transcriptome system is a low input system that uses ultralow amounts
of RNA and is based on 3’ RNA sequencing. However, while 3’ RNA sequencing has a better
ability to detect short transcripts, whole-transcript RNA-seq is better able to detect more
differentially expressed genes (Ma et al. 2019). In future studies, Erg-deficient mice could be
stressed with western diet or through knockout of the LDL receptor gene. Endothelial cells could
then be isolated from the aorta and used for single-cell RNAseq analysis to validate other
atherosclerosis-related genes identified in HAECs from Erg-deficient mice.
22

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Abstract (if available)

Abstract Atherosclerosis is the primary cause of heart disease and stroke, resulting in significant morbidity and mortality. It is an inflammatory disease characterized initially by the accumulation of lipids in coronary arteries and other large vessels. Dysfunction of endothelial cells is an important contributor to the pathogenesis of this complex process. ETS-related Gene (ERG) is a transcription factor highly expressed in endothelial cells and a major genetic determinant of endothelial cell homeostasis. For example, ERG upregulates genes that maintain endothelial cell junction integrity and downregulates pro-inflammatory genes. Thus, ERG could be directly involved in atherogenic processes, but evidence for this notion in the literature, particularly with respect to in vivo studies, has been limited. In this study, we provide functional in vitro and genetic evidence in humans showing that ERG could potentially influence the development of atherosclerosis through inflammatory mechanisms at the level of the vessel wall. An in vivo study of gene expression in aortas from endothelial cell-specific Erg-deficient mice also demonstrated that lack of this homeostatic factor in endothelial cells results in dysregulation of pathways related to cardiac function and heart failure. Collectively, our data suggests that ERG‘s atheroprotective properties could be related to regulation of inflammatory genes in endothelium in humans, while the underlying mechanisms for the effects of Erg deficiency in endothelial cells of mice will require further investigation.

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Asset Metadata

Creator Zhu, Ruowei (author)

Core Title Gene expression and genetic variation of ERG is associated with inflammation in endothelial cells and risk of coronary artery disease in humans

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 01/29/2021

Defense Date 05/13/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag atherosclerosis,endothelium,ERG,Inflammation,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Allayee, Hooman (committee chair), Patel, Pragna (committee member), Xu, Jian (committee member)

Creator Email ruoweiz@usc.edu,ruowzhu@gmail.com

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