Aortic Wall Behavior as a Predictor of Aortic Aneurysm Growth and Rupture
Phase: PHASE2
Conditions: Abdominal Aortic Aneurysm
Sponsor: Rabih A. Chaer

Contrast-Enhanced Ultrasound Microbubble Uptake and 
Abnormal Plasma Biomarkers are Seen in Patients with 
Abdominal Aortic Aneurysms 
Adham N. Abou Ali MD1*, Patrick Cherfan MD1*, Ashraf G. Taha MD1, Michel S. Makaroun MD1, 
Yingze Zhang PhD3, Heather Phelos MPH2, Xucai X. Chen PhD2, Flordeliza S. Villanueva MD2, 
Rabih A. Chaer1 
1. Division of Vascular Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 
2. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 
3. Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh 
Medical Center, Pittsburgh, Pa. 
 
 
 
Presented as an Oral Presentation at the Eastern Vascular Surgery (EVS) 35th Annual meeting 
in Charleston, SC, 2021. 
NCT Number: NCT02022436 
Correspondence 
Rabih A. Chaer, MD 
Professor of Surgery 
University of Pittsburgh Medical Center 
Division of Vascular Surgery 
200 Lothrop St, Suite A1011 
Pittsburgh PA 15213 
Tel: (412) 802-3033 
Fax: (412) 291-1669 
Email: chaerra@upmc.edu 
 
*The two authors Adham N. Abou Ali and Patrick Cherfan contributed equally to this work. 
Document Date: 06/15/2023 
 

SPECIFIC AIMS  
Aging is a major risk factor for abdominal aortic aneurysm (AAA). The incidence of AAA is 
highest in the elderly, and AAA rupture is the 13th leading cause of death among individuals 
aged 65–85 in the United States. AAA rupture is associated with high morbidity and mortality, 
particularly in elderly patients undergoing emergent aortic surgery. Ultrasound surveillance is 
currently recommended to follow progression of small AAA, but it is limited in its ability to 
accurately predict AAA growth or rupture. This is due to the unpredictable clinical behavior of 
AAA. In an autopsy study of 473 non-resected AAA, 40% of AAA between 7 cm and 10 cm had 
not ruptured, while nearly 13% of patients with aneurysms smaller than 5 cm had ruptured. The 
risk of AAA rupture is also higher in women, and tends to occur at a smaller diameter than men, 
raising the possibility of gender disparities in aortic wall vulnerability. These observations 
highlight the limitation of adopting diameter as a predictor of AAA instability, defined as risk for 
growth or rupture.  
Attempts to image the aortic wall in order to predict AAA growth or rupture have been 
inconclusive. Existing imaging modalities include CT, MRI, and intravascular ultrasound, but 
these are limited by low accuracy, radiation exposure, availability, and safety issues. Recently, 
contrast ultrasound (CEUS) using microbubbles has emerged as a method for imaging 
adventitial vasa vasorum (VV) in carotid plaques. We have an active CEUS program and have 
previously correlated VV density with neovascularization in an animal model, and have imaged 
850 patients with carotid CEUS. CEUS can also be used to image AAA, since neovascularization 
and VV are commonly seen on AAA wall histology. Both may be markers of hypoxia and 
weakening of the aortic wall, and can be quantified with CEUS using time–signal intensity curve 
analysis to detect VV density in different regions of interest in the AAA wall.  
Our goal is to non-invasively study the metabolic processes within the aortic wall that are 
thought to explain AAA growth and rupture, and their variation with age and gender. This 
process also affects unstable coronary and carotid disease, where the progression from an 
asymptomatic plaque to rupture and thrombosis, also known as “vulnerable plaque”, is not 

fully explained by the luminal diameter. Similarly, localized hypoxia, neovascularization, and 
inflammation of the aortic wall are thought to contribute to degeneration of AAA, accounting 
for growth and possibly rupture that are otherwise not explained by absolute AAA diameter. In 
addition, pro-inflammatory systemic biomarkers, such as interleukin, tumor necrosis factor-α; 
and matrix-degrading enzymes, are up regulated in patients with AAA suggesting a systemic 
inflammatory process. Non-invasively detecting AAA wall neovascularization using CEUS is a 
novel approach to evaluate aortic wall vulnerability. It could identify regional inflammation, and 
propensity for AAA wall weakening, enlargement or rupture. Our hypothesis is that increased 
neovascularization of the aortic wall correlates with AAA growth and rupture and is more 
pronounced in women. CEUS imaging of AAA, coupled with serum biomarker measurements, 
has the potential to identify the propensity for growth or rupture. The Specific Aims are:  
Aim#1: Determine the relationship between AAA growth rate and VV density. Approach: 
prospective CEUS imaging of a cohort of patients with AAA 4-5.4cm in diameter at 6 month 
intervals for 18 months. Hypothesis: VV density on CEUS is increased in AAA at risk for growth.  
Aim#2: To correlate CEUS findings with serum biomarkers. Approach: longitudinally measure 
AAA specific serum biomarkers with every CEUS. Hypothesis: Levels of pro-inflammatory serum 
biomarkers correlate with VV density and are more elevated in unstable AAA.  
APPROACH  
The study protocol was approved by the Institutional Review Board (IRB) of the University of 
Pittsburgh. The study was funded by the AGS GEMSTAR award from the Society of Vascular 
Surgery (SVS) foundation in addition to the National Institute of Health (NIH) through a R03 
grant.  
Study Design 
The objective of this study was to non-invasively study the metabolic processes within the 
aortic wall that are thought to explain aortic growth. This was a single center prospective 
evaluation of patients with an AAA diameter of at least 4.0  cm. enrolled between December 

2011 and December 2016, in the outpatient vascular clinic. Patients were followed up until 
October 2019.  
Aneurysm diameter size was determined on duplex ultrasound. Patients were excluded if they 
had contraindications to the administration of Definity contrast, such as pulmonary 
hypertension, unstable cardiopulmonary conditions, known right-to-left cardiac shunting and 
pregnancy. Patients underwent CEUS and serum PIB testing at enrollment and every 6 months 
up for 18 months or until eventual repair. PIB testing was also done one year after AAA repair..  
CEUS imaging protocol 
Patients were positioned on their back in the non-fasting state. An intravenous line was placed 
by the research staff at the time of the office visit. The abdominal aorta was imaged initially 
without contrast administration. Following non-contrasted duplex imaging, CEUS was 
performed via intravenous administration of the ultrasound contrast agent, Definity® 
(Lantheus). 1ml of the contrast agent was diluted in 9ml of normal saline to achieve a 
concentration of approximately 10 microliters/kg. For each injection sequence, 0.5 mL contrast 
was injected over 2-3 sec during simultaneous burst replenishment real time imaging. Two 
injection sequences were typically performed. Each CEUS evaluation took approximately 20-30 
minute to complete. Patient’s heart rate, blood pressure and breathing were monitored during 
the examination and afterwards in the waiting area. The total monitoring time ranged between 
30-90 minutes. Microbubble replenishment was analyzed using ImageJ (NIH) via manually 
drawn regions of the aortic wall and intraluminal thrombus (ILT) (Figure 1).  
Ultrasound evaluation was performed using the GE US system (GE Healthcare, Chicago, IL, USA) 
with the standard curved array abdominal transducer. The contrast settings on the ultrasound 
system were set to a low mechanical index of 0.07 and a dynamic range of 50 with the focal 
zone placed underneath the posterior aortic wall.  
Microbubble uptake was determined by the ultrasound technologist and the location was 
recorded on a study sheet. Uptake was then separately recorded by two of the investigators 
(RC, PC). Care was taken to correlate with color flow duplex ultrasound to avoid lumbar and 
mesenteric vessels as well as artifact. The visualization of the microbubbles within the 

abdominal aortic aneurysm wall was defined as contrast uptake. This was determined by 
consensus agreement between the ultrasound sonographer and two investigators (The PI, RC 
and PC) for every single patient.  
Uptake was quantified via video intensity analysis in a region of interest in the aortic adventitia. 
Background-subtracted peak video intensity, which has been shown to correlate with VV 
density, was derived and normalized to peak intensity in the lumen (Figure 2).9  
Aneurysm growth, rupture and repair were recorded during the follow up period. Matched 
controls with no aneurysm disease underwent one-time CEUS and PIB testing using biomarker 
panels (Meso scale discovery) and Bioplex 100 analyzer (Bio-rad).  
Biomarker testing 
Blood samples were withdrawn from patients at each visit and one year following AAA repair. 
Blood was drawn in the Vascular Surgery clinic by the research staff. Blood samples were 
banked and used for the measurement of biomarkers. This is the list of plasma inflammatory 
biomarkers that were tested: Osteoprotegerin (OPGN), Galectin 3, Cystatin C, Neutrophil 
gelatinase-associated lipocalin (NGAL), Uromodulin (UMOD), C-reactive protein (CRP), 
Macrophage migration inhibitory  factor (MIF), Intercellular adhesion molecule (ICAM1), 
Interferon γ (IFN-γ), Interleukin 10 (IL10), Interleukin 6 (IL6), Interleukin 8 (IL8), Tumor necrosis 
factor-a (TNF-a), Serum amyloid A (SAA), Vascular cell-adhesion molecule (VCAM1).  
Statistical analysis plan: Sample size: No previous literature has reported the mean and 
standard deviation of AAA VV density measured in vivo by CEUS. Statistical analysis: Data will be 
summarized as mean and SD for continuous variables after testing for normality. 
Categorical data will be summarized with frequency and percentage. Correlation coefficients 
will be determined for the correlation of VV and microbubble density measured by CEUS, as 
well as serum biomarkers with AAA growth. We will use linear regression to test the 
significance of such higher VV and microbubble density by CEUS as predictor of AAA growth 
(Aim1). We will include serum biomarkers in the modeling process. Adjustment for any 

demographics differences will be also attempted including age, gender and race. A p-
value<0.05 will determine significance.  
Anticipated findings and alternative approaches: We anticipate that this clinical research study 
will identify biomarker and CEUS differences in AAA adventitial VV density based on age, 
gender and AAA stability or growth. Our findings will support a larger clinical trial and future 
investigations of treatment paradigms of AAA based on biologic risk of growth or rupture 
instead of absolute diameter. If CEUS and biomarker differences are not detected during the 
study period in otherwise stable AAA, longer follow up with repeated imaging and biomarker 
testing at 6month intervals will be sought. If our hypothesis is not confirmed (i.e. differences in 
AAA wall neovascularization do not exist), imaging of AAA wall inflammation will be considered 
when available to human subjects using microbubbles targeted to either activated leucocytes 
or endothelial cell adhesion molecules that are upregulated in inflammation and mediate 
leukocyte recruitment and adhesion. If CEUS is not accurate given the depth of penetration of 
ultrasound, alternative imaging modalities such as IVUS or FDG-PET CT scanning will be 
explored. We anticipate that our results will identify active angiogenesis and ischemia of the 
AAA wall and biomarker predictors of growth, which may improve real time monitoring of AAA 
progression and response to systemic or targeted microbubble anti-angiogenesis therapies.