Capstone Project Final (2) (1) (1)

TRANSCRIPTION FACTORS IMPACT CEREBRAL ANEURYSMS

The activity of transcription factors Ets-1 and NF-κB impacts the progression of cerebral

aneurysm rupture

Sallie Ferren and William Heinrich

Florida State College at Jacksonville

Biomedical Degree Capstone

Spring 2015

IDS 4936

DR. Lanh Bloodworth

A course assignment presented to the Department of Biomedical Sciences in partial fulfillment

of the requirements for the Bachelor of Science Degree, Florida State College at Jacksonville

Date

April 24, 2015

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Abstract

Cerebral Aneurysms (CAs) are common cerebrovascular pathologies that typically form

at the bifurcations of cerebral arteries and are a leading cause of stroke death. Studies have

shown that inflammation plays a key role in the development and progression of ruptures. NF-

κB, a chemical mediator stimulating apoptosis and Ets-1 are transcription factors

responsible for the regulation of inflammatory genes during aneurysm formation. The

downstream target Ets-1 in CA development was identified by chromatin immunoprecipitation

analysis and revealed that Ets-1 transactivated MCP-1 in the walls of cerebral aneurysms. Recent

studies have been conducted using a synthesized decoy, oligodeoxynucleotide (ODN), which

synergistically represses the molecular mediators by binding to NF-κB and Ets-1. This

inhibits response showing significant size in reduction and disruption of the internal elastic

lamina. Our study will determine if a decrease in inflammation in VSMC should lead to the

inhibition of CA development. Therefore, if regulation of the expression of Ets-1 and NF-

κB is controlled, then vascular endothelial growth and angiogenesis should be regulated.

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Introduction

General Background

A cerebral aneurysm is a weak or think spot on a blood vessel in the brain that creates a

bulge in the blood vessel and fills with blood (National Institute of Neurological Disorders and

Stroke, 2015) and can occur in anyone, at any age, although CA are more prevalent in adults and

slightly more common in women. It is estimated that 2.3% of the Western population have

intracranial aneurysms, with over 30,000 diagnosed every year. If a cerebral aneurysm develops,

this could eventually lead to pressure on a nerve or surrounding brain tissue. The incidence of

reported ruptured aneurysm is about 10 in every 100,000 persons per year most commonly in

people between ages 30 and 60 years. Possible risk factors for rupture include hypertension,

alcohol abuse, drug abuse (particularly cocaine), and smoking. In addition, the condition and size

of the aneurysm affects the risk of rupture.

An unruptured aneurysm may go unnoticed throughout a person’s lifetime. A burst

aneurysm may be fatal or could lead to hemorrhagic stroke, or vasospasm, which is the leading

cause of disability or death following a burst aneurysm, other problems may occur. Once the

aneurysm has burst, it may bleed into the brain and additional aneurysms may also occur. More

commonly, rupture may cause a subarachnoid hemorrhage, or bleeding into the space between

the skull bone and the brain. It is estimated that approximately forty percent of individuals whose

aneurysm has ruptured do not survive the first twenty-four hours and another twenty five percent

die from complications within six months of the burst. As previously explained, aneurysms can

be fetal or lead to a host of complications, approximately 12% of patients die before they can be

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treated. This paper will discuss how NF-κB and Ets-1 are transcription factors that are

important mediators in the development of cerebral aneurysms and if the expression of NF-

κB and Ets-1 are inhibited, there should be a decrease in the incidence of cerebral

aneurysm formation and rupture. Therefore, determining the molecular cause of CAs is very

important so that further research and more effective diagnosis and treatment methods can be

discovered.

Previous research studies

Research studies in molecular biology using human samples and rats have pointed out

that inflammation is key in the leading development of cerebral aneurysms. These studies have

described the most recent understanding of the inflammation pathways from initiation to rupture

of CAs. Transcription factors such as NF-κB and Ets-1 have proven to lead to

inflammatory chemicals that signal the accumulation of macrophages in vascular smooth muscle

cells in the arteries of humans and rats leading to endothelial dysfunction and pathological

remodeling changes to the vascular walls (Kataoka, 2015).

Pathophysiology and Etiology

In a broad, general spectrum of the pathophysiology of cerebral aneurysms, CAs may be

more prevalent in people with certain genetic diseases. The embryological develop of connective

tissue is linked to mesenchyme and may lead to CAs in these people. Moreover, high blood

pressure, infection in the arterial wall, tumors in the head and neck, and atherosclerosis has also

been linked to cause cerebral aneurysms (National Institute of Neurological Disorders and

Stroke, 2015).

This paper will include an analysis that will be specific on molecular and cellular causes

of cerebral aneurysm, based around NF-κB and Ets-1 general transcription factors that

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regulate inflammation in vascular smooth muscle cells. We will explain the Proto-oncogene E26

transformation-specific-1 (Ets-1) not only plays a crucial role in reciprocal regulation of

inflammatory and anti-inflammatory responses but also in the regulation of a wide variety of

biological processes such as cellular growth (Grenningloh, Kang, & Ho, 2005). The Ets

transcription family is involved in direct gene expression and binds to specific promoters and

enhancers, allowing assembly to occur of components for transcription. The Ets domain is a

helix-turn-helix binding protein responsible for the recognition of a specific sequence called 5′-

GGA (A/T)-3′ (Sharrocks, A., Brown, A., Ling, Y., & Yates, P., 1997). In addition to the role as

a binding protein, protein-protein interactions also occur and have been identified as either a

transcriptional activator or repressor and are regulated by signal transduction pathways. An

important function of Ets domain transcription factor is to regulate hematopoiesis, the production

of red blood cells, in adults.

MCP-1, or monocyte chemoattractant protein-1, a member of the C-C chemokine

subfamily, was originally identified for its potent chemotactic activity toward monocytes

(Feinberg, 2004). Monocyte chemoattractant protein-1 (MCP-1) led to the enlargement of

VSMC and development of cerebral aneurysms through the recruitment of monocytes or

macrophages in the walls of CA. There is a secretion of proteinases such as metalloproteinase

(MMP)-2, -9 and cysteine cathepsins, causing degeneration of the walls surrounding the cerebral

aneurysm (Aoki et al., 2010). Levels of MMP were found to be higher in patients with ruptured

CAs versus patients with unruptured CAs (Chalouhi, 2012).

In the inflammatory process, macrophages have three major functions, antigen

presentation, followed by phagocytosis, and then production of various chemical mediators of

inflammation including cytokines and growth factors. This is important in the development of

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cerebral aneurysms as macrophages play a key role in the synthesis due to the Ets-1 transcription

factor. Expression of Ets genes is associated with mesenchymal-epithelial interactions and

changes in extracellular matrix proteins that contribute to tissue remodeling and integrity

(Maroulakou & Bowe, 2000).

A higher prevalence of CAs in women is thought to be due to decrease in estrogen level after

menopause. Estrogen protects several components within the artery wall and inhibits the activity

of TNF- that protects against inflammation of the blood vessels, which, can lead to aneurysms

(Francis, Tu, Qian, & Avolio, 2013). In humans, Ets-1 is expressed in high levels in proliferating

vascular endothelial cells of an embryo and in the blood vessels of an adult during angiogenesis,

or the formation of blood vessels from existing vasculature (Maroulakou & Bowe, 2000).

Treatments

When an unruptured aneurysm is detected, the decision to treat can be complicated

because usually only 1-2% eventually ruptures but the mortality rate within the first month after

a rupture is about 50% (Francis et al., 2013). Treatment sometimes involves complications,

therefore it is mainly recommended for larger or oddly shaped unruptured CAs or if they are

causing symptoms in the patient (NINDS, 2015). Morbidity and mortality are associated with

CA despite clinical advances in diagnosis and therapy. Currently there are only invasive

treatment modalities such as microsurgical clipping and endovascular coiling. Endovascular

coiling has showed a better clinical outcome when compared to microsurgical clipping especially

when using biologically active coated coils (Hudson, Hoyne, & Hasan, 2013). Liquid embolic

materials, such as Onyx HD500 have also been used as treatment but are mostly effective at only

treating smaller CAs. All of the aforementioned interventions have procedural complication rates

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and, thus, illustrate the need for noninvasive alternatives and a clearer understanding of the

pathophysiology of CAs (Hudson et al., 2013).

Future Treatments

An independent study performed by Hasan and colleagues for Mayo Clinic on subjects

enrolled in the International Study of Unruptured Intracranial Aneurysms (ISUIA) found that

patients who used aspirin 3x weekly to daily had an occurrence rate (OR) for hemorrhage 60%

less when compared to the reference group who did not use aspirin or used it ‘< once a month.

This data suggest that a low dose of acetylsalicylic acid may attenuate inflammation in CA and

histologically showed a decrease in macrophage and pro-inflammatory molecule expression

(Hudson et al., 2013).

According to Hudson et al., 2013 diagnostic imaging has been used at a cellular level to

identify macrophages as a biomarker for inflammation. Human patients were injected with

ferumoxytol and then imaged with T2-MRI to show the macrophages localizing in the wall of

cerebral aneurysms. Ferumoxytol is an ultra-small super-paramagnetic iron oxide, until recently

used as an iron replacement but now as an intravenous contrast for Magnetic Resonance Imaging

(MRI) to demonstrate signal enhancement or loss (Bashir, Bhatti, Marin, & Nelson, 2014). This

will be crucial in diagnosing and preventing CA ruptures.

The activated DNA binding form of transcription factor NF-κB with MCP-1 and

Vascular Cell Adhesion Molecule-1 (VCAM-1) showed a molecular basis which caused

activation of macrophages during CA inflammation (Hudson et al., 2013). A recent study by

Aoki et al, 2010 showed an overexpression of NF-κB in the CA wall of rats and created

mice that were NF-κB deficient and saw a reduction in macrophage recruitment, MCP-1,

and VCAM-1 mRNA, and overall size.

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In another experiment, Aoki et al, 2010 performed a synthesized decoy

oligodeoxynucleotide (ODN) that was used to bind and inhibit NF-κB. They found a

significant reduction in CAs when decoy ODN was administered after one week of induction.

When used simultaneously for both NF-κB and Ets-1, they produce a synergistic effect

that represses molecular mediators of inflammation while promoting collagen biosynthesis

(Hudson et al., 2013).

Another area of interest for possible treatment of CAs is statins due to their inhibitory

action on NF-κB and beneficial results for vascular disease. One study by Aoki et al.,

2010 found that in rats, after administrating an oral dose one month after CA induction, there

was a decrease in expression of inflammatory mediators MMP-1 and MMP-9. Another study

found that lower doses attenuated CA progression but that higher doses promoted CA growth

and rupture. Furthermore, a study done by an independent hospital based control group found

that there was actually an inverse relationship between statins and CAs (Hudson et al., 2013).

Materials and Methods

To determine if there will be a decrease in the incidence of cerebral aneurysm formation

and rupture by controlling the expression of NF-κB and Ets-1, an article by Aoki et al.,

2010 was reviewed. Aoki et al., 2012 extended their previous findings by examining the

regressive effect of decoy ODN, which simultaneously inhibit NF-κB and Ets-1 in the

development of CAs. They performed several experiments on 7 week-old rats as described

below, using numerous techniques at Kyoto University School of Medicine and Osaka

University School of Medicine.

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CAs were introduced into rat cerebral walls, after CA induction, the animals were perfused

transcardially with 4% paraformaldehyde and the anterior cerebral artery/olfactory artery

(ACA/OA) bifurcation was subjected to fluorescence immunohistochemistry among other tests

using several antibodies. The number of SMA (smooth muscle -actin), CD68 or CD31 were

identified and calculated for Ets-1 double positive cells.

To detect specific proteins, a Western Blotting technique was used, the ACA/OA

bifurcation was homogenized and dissolved into samples. Electrophoresis was ran, the

membranes were then incubated with primary antibodies and then incubated again with

secondary antibodies. A quantitative (real-time) Polymerase Chain Reaction (qPCR) was also

used to analyze RNA from the whole circle of Willis (anastomotic system of arteries at the base

of the brain that provides communication between blood supply of forebrain and hindbrain) then

converted into cDNA from dead rats to amplify and simultaneously detect MCP-1 expression.

For quantification, a second-derivative was used for cross-point determination.

Next, nuclear protein was extracted from a whole circle of Willis using electrophoretic

mobility shift assay (EMSA) with a biotin 3 oligonucleotides containing c-Ets-binding

consensus sequence and then followed by an anti-Ets-1 super-shift assay. Then, Chromatin

Immunoprecipitation (CHIP) was carried out after homogenization, cell lysis, and sonication

using anti-Ets-1 antibodies. Prior to this, a whole circle of Willis, with or without CA induction

was dissected and cross-linked by formaldehyde, then PCR was then carried out using primers or

rat MCP-1 and -9.

In a decoy ODNs treatment, Ets was synthesized along with scrambled decoy ODNs to

serve as a control. Ets or the scrambled decoy ODNs was then injected into the cisterna magna

every two weeks under general anesthesia. After 1 month, the ACA/OA bifurcation was stripped

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and observed using Elastica van Gieson (EvG) staining and macrophage accumulation was

assessed. Lastly, Immunohistochemistry was conducted with human CA samples retrieved from

neck clippings of un-ruptured CAs; the middle cerebral artery from an autopsy was used as a

control. Samples were fixed and then double stained to determine if Ets-1 was expressed in

human CA walls.

Results

Aoki et al., 2010 results showed in the immunohistochemistry test that Ets-1 was

expressed in the CA walls 1 month after induction but was barely present in the control cerebral

arterial walls. The result for the western blotting showed an increase in Ets-1 expression 1 month

after CA induction and was reduced from 1 to 3 months. The results for the EMSA produced a

band specific to the Ets family; this was later eliminated due to competition with

oligonucleotides. In the CHIP assay performed on the MCP-1 promoter with anti-Ets-1

polyclonal antibody showed MCP-1 binding site at -102 an increase from 1 month to 3 months,

conversely the -832 binding site for Ets showed a decrease from 1 month to 3 months and no

band was detected for the MMP-9 promoter. The ets decoy ODN-treated group compared to the

scrambled decoy ODN-treated group was significantly different, Ets decoy ODN inhibited DNA

binding activity. The qPCR analysis showed the MCP-1 expression increased in the scrambled

decoy ODN while the ets decoy ODN was significantly inhibited. This preserved the MCP-1

expression in endothelial cells after the ets decoy ODN treatment while inhibiting macrophage

infiltration in CA walls. In the immunohistochemistry for human samples, Ets-1 was found

generously in CA walls along with SMA and MCP-1 but rarely in the middle cerebral artery of

the control group.

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Feinberg et al., 2004 explains that several lines of evidence suggest a critical role for

MCP-1 in vascular disease. Studies have also demonstrated a reduction in atherosclerotic lesion

formation in mice deficient in MCP-1 or its chemokine receptor (CCR2) in atherosclerotic-prone

mice as explained. The information supported by Feinberg et al., 2004 also suggests

macrophage-specific overexpression of MCP-1 resulted in the increased development of vascular

lesion size and infiltration of macrophages in atherosclerosis-prone mice. In several animal

models, blockade of MCP-1 or deficiency of CCR2 decreased neointimal hyperplasia after

arterial injury. A link was discovered between macrophages and lymphocytes, which infiltrate

the aneurysm wall leading to the presence of the development of a cerebral aneurysm (Jayaraman

et al., 2008).

Another study conducted by Chyatte, Bruno, & Desai, 1999 discovered there was an

increased level of macrophages, T-lymphocytes, and cell adhesion molecule-1 in aneurysm

vascular tissue, but rarely in the control tissue. Feinberg et al., 2004 stated that in a previous

research that cytokines are a major chemical mediator in inflammation by activation of MCP-1

that occurs primarily at the level of transcription. This supports that Ets-1 inhibition from

binding may inhibit inflammation in the brain and therefore, less cerebral aneurysms will

development.

Studies also demonstrate that MCP-1 is a downstream gene of Ets-1 during CA

development. This study used Ets decoy ODNs which inhibited MCP-1 expression and shown a

significant difference in the media. MCP-1 was expressed at a high level in epithelial cells

especially at the early phase of CA formation. The Ets decoy ODN treatment did not abolish

MCP-1 expression in endothelial cells. Therefore, this suggests that different role between NF-

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κB and Ets-1 in transcriptional regulation of MCP-1 in CA walls. Ets decoy ODN

treatment resulted in suppression of CA enlargement without affecting systemic blood pressure.

According to Chalouhi et al., 2012 one study histologically compared 42 ruptured CAs

with 24 unruptured CAs and confirmed the infiltration of macrophages in the vessel walls

associated with aneurysm ruptures, apoptosis, and VSMC proliferation. While another

histological study compared 44 ruptured and 27 unruptured and found significantly more

endothelial damage, structural changes, and inflammatory cell invasion in the ruptured CAs.

Discussion

Ets-1 is involved in the regulation of vascular inflammation and remodeling in various

vascular diseases, but has been considered in the development of cerebral aneurysms (Aoki et

al., 2010). The main experiment by Aoki et al., 2010 used fluorescence immunohistochemistry

with different animal antibodies, Western Blotting, qPCR, and (EMSA) with a biotin 3

oligonucleotides containing c-Ets-binding consensus sequence and then followed by an anti-Ets-

1 super-shift assay.

Aoki et al., 2010 demonstrated in their research on Ets-1 that immunochemistry showed

that Ets-1 expression in media of CA walls at one month after the induction of a CA, while Ets-1

was barely expressed in the control arterial walls used. Treatment with Ets decoy

oligodeoxynucleotides resulted in the prevention of CA development, upregulation of MCP-1

expression and increase in macrophage accumulation in CA walls

The study showed that Ets-1 regulates expression of vascular endothelial growth factor,

regulating angiogenesis. Also, Ets-1 is highly expressed in VSMC derived from human

atherosclerotic plaques and in VSMC in the rat carotid artery after balloon injury (Aoki et al.,

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2010). Deficient mice showed a marked reduction in vascular remodeling in response to a

systemic administration of angiotensin II. With decreased vascular remodeling, this could lead to

an increase risk of developing cerebral aneurysms based on that the vascular smooth muscle cells

in the area become weaker (Aoki et al., 2010). Results also demonstrated in this study that

transactivation of Ets-1 induces the expression of platelet derived growth factor, thus promoting

VSMC proliferation. An increased expression and activation of Ets-1 in VSMCs in CA walls

strongly suggest the contribution of Ets-1 to vascular inflammation and remodeling occurring in

CA walls. This team hypothesized that pro-inflammatory cell types are the prime source of TNF-

alpha that initiate damage to endothelium, Smooth Muscle Cells (SMC) and Internal Elastic

Lamina (IEL). This supports that inflammation is a source of the development of cerebral

aneurysms, though a link between the Ets-1 transcription factors was not found in this study.

This study does support that inflammation is a primary cause of cerebral aneurysm development

however inflammation is triggered by stimulating the binding of Ets-1 to a sequence of DNA,

triggering the process of synthesizing various chemical mediators, like macrophages.

Ets-1 was expressed in the CA wall, although a small amount of Ets-1 was expressed in

the adjacent arterial wall. Ets-1 expression was reduced from 1 to 3 of CA induction. In western

blotting, Ets-1 expression was significantly upregulated one month after CA induction.

With the correlation of findings in this review between Ets-1 and its’ role in inflammation, it

can be determined that if Ets-1 is regulated, then inflammation can be controlled, leading to

decrease in the formation of CAs.

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Conclusion

In review of this analysis, an investigation of the molecular causes of the formation of

cerebral aneurysms was completed. NF-κB and Ets-1 is considered prime transcription

factors mediating transcription and therefore, protein synthesis and/or other mechanisms to

signal inflammation response. This leads to the accumulation of inflammatory chemicals,

including macrophages within SMVC of the arteries within the brain. Results have shown that

there is probable cause that these chemical mediators lead to inflammation in the muscle with

macrophages involvement and therefore, a cerebral aneurysm. They have also shown that by

inhibiting these transcription factors, which play a crucial role in CA development, we can

inhibit their expression and therefore control and possibly prevent CAs (Aoki et al., 2007). More

conclusive studies need to be completed to determine if other means or variables will control

cerebral aneurysms as well. Therefore, with the medical advances used today and with the

studies that show promising results for CAs, such as treatment with Ets decoy ODN; soon there

should be an alternative noninvasive medical treatment for management and preventions of CAs.

Future Perspective

Further studies should be completed using Ets decoy ODN to inhibit the development and

progression of cerebral aneurysms in rat and human vascular smooth muscle cells. A larger

sample size will further indicate accurate results to depict the usefulness of this approach.

Furthermore, different species of animal should be examined in addition to rats to instill that

inhibition of Ets transcription factor through the use of Ets ODN decoys actually

inhibits/decreases the development of cerebral aneurysms. Also, a better understanding of the

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exact mechanisms in the inflammation processes leading to CA ruptures will aid in the

development of more noninvasive treatments and possibly prevention.

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Capstone Project Final (2) (1) (1)

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