Thoracic Aortic Aneurysm Frequency and Dissection are Associated with Fibrillin-1 Fragment Concentrations in Circulation

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1 Thoracic Aortic Aneurysm Frequency and Dissection are Associated with Fibrillin-1 Fragment Concentrations in Circulation Lynn M. Marshall 1,2, Eric J. Carlson 5, Jean O Malley 2, Caryn K. Snyder 1, Noe L. Charbonneau 5, Susan J. Hayflick 3, Joseph S. Coselli 6, Scott A. LeMaire 6, Lynn Y. Sakai 4,5 1 Departments of Orthopedics and Rehabilitation; 2 Public Health and Preventive Medicine; 3 Medical and Molecular Genetics, and; 4 Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR; 5 Portland Shriners Hospital for Children, Portland, OR, and; 6 Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine; and the Department of Cardiovascular Surgery, the Texas Heart Institute at St. Luke s Episcopal Hospital, Houston, TX. Downloaded from by guest on April 27, 2018 L.M.M., E.J.C., S.A.L. and L.Y.S. contributed equally to this manuscript. Running title: Plasma Fibrillin-1 in Thoracic Aortic Aneurysms Subject codes: [8] Epidemiology [35] CV surgery: aortic and vascular disease [97] Other vascular biology [98] Other research Address correspondence to: Dr. Lynn Y. Sakai Shriners Hospital for Children 3101 SW Sam Jackson Park Road Portland OR Tel: Fax: lys@shcc.org In August 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.8 days. DOI: /CIRCRESAHA

2 ABSTRACT Rationale: Mutations in fibrillin-1 are associated with thoracic aortic aneurysm (TAA) in Marfan syndrome. Genome-wide association studies also implicate fibrillin-1 in sporadic TAA. Fragmentation of the aortic elastic lamellae is characteristic of TAA. Objective: Immunoassays were generated to test whether circulating fragments of fibrillin-1, or other microfibril fragments, are associated with TAA and dissection. Methods and Results: Plasma samples were obtained from 1265 patients with aortic aneurysm or dissection and 125 control subjects. Concentrations of fibrillin-1, fibrillin-2, and fibulin-4 were measured with novel immunoassays. One hundred and seventy-four patients (13%) had aneurysms with only abdominal aortic involvement (AAA), and 1091 (86%) had TAA. Of those with TAA, 300 patients (27%) had chronic dissection and 109 (10%) had acute or subacute dissection. Associations of fragment concentrations with TAA (vs. AAA) or with dissection (vs. no dissection) were estimated with odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, and smoking. Compared to controls, significantly higher percentages of aneurysm patients had detectable levels of fibrillin fragments. TAA was significantly more common (than AAA) in the highest compared with lowest quartile of fibrillin-1 concentration (OR=2.9; 95% CI, ). Relative to TAA without dissection, acute or subacute dissection (OR=2.9; 95% CI, ), but not chronic dissection, was more frequent in the highest compared with lowest quartile of fibrillin-1 concentration. Neither TAA nor dissection was associated with fibrillin-2 or fibulin-4. Conclusion: Circulating fibrillin-1 fragments represent a new potential biomarker for TAA and acute aortic dissection. Keywords: Aneurysm, dissection, fibrillin, microfibril, Marfan syndrome Nonstandard Abbreviations and Acronyms: AAA CV ECM ELISA LoD TAA abdominal aortic aneurysm coefficient of variation extracellular matrix enzyme-linked immunosorbent assay limit of detection thoracic aortic aneurysm DOI: /CIRCRESAHA

3 INTRODUCTION Aortic aneurysm and dissection are significant causes of cardiovascular morbidity and mortality, resulting in more than 10,000 deaths in the United States annually. 1 Degeneration of the extracellular matrix (ECM) of the aortic media and consequent weakening of the aortic wall lead to aneurysm formation and dissection. 2,3 Aneurysms are most common in the abdominal aorta, but they also occur in the thoracic aorta, where they are more prone to dissection. Thoracic aortic aneurysm (TAA) and dissection are the main causes of morbidity and mortality in patients with the Marfan syndrome. 4 Genetic studies have elucidated the biological causes of aortic aneurysmal disease, particularly TAA. Mutations in the gene for fibrillin-1, the major structural component of elastic fiber microfibrils, 5 cause Marfan syndrome. 6 Fibrillin-1 genotype has also been linked to TAA, dissection, and other aortic abnormalities in patients without Marfan syndrome Furthermore, a recent genome-wide association study identified a susceptibility locus for sporadic TAA and dissection in a region containing the fibrillin- 1 gene. 11 Mutations in fibrillin-1 have been shown to destabilize fibrillin-1 protein, making it more susceptible to proteolytic degradation. 12 In Marfan syndrome, proteolytic susceptibility might cause fragmentation of the aortic microfibrils and elastic lamellae, characteristic histological findings of aortic aneurysm. We hypothesized that fragments of fibrillin-1 and other elastic fiber proteins could be released into the circulation in Marfan syndrome and other TAA. To test this possibility, we generated sandwich enzyme-linked immunosorbent assays (ELISAs) that use various epitope-mapped monoclonal antibodies specific for fibrillin-1, fibrillin-2 (which also contributes to microfibril structure 13 ), and fibulin-4 (which leads to TAA and dissection in fibulin-4 deficient mice 14 ). Because there are no reliable circulating biomarkers for thoracic aortic disease, imaging is the only modality currently used to detect TAA, monitor aneurysm size, and determine when surgical repair should be performed to prevent aortic dissection or aneurysm rupture. The reliance on cross-sectional imaging techniques to diagnose and monitor aortic disease has several limitations. For example, aneurysm size does not appear to be a reliable indicator of the risk for dissection. 15 Therefore, biomarkers of aortic ECM degradation could be extremely valuable in the diagnosis and management of patients with thoracic aortic disease. 16 We used our sandwich ELISAs to measure concentrations of fragments of fibrillin-1, fibrillin-2, and fibulin-4 in plasma samples obtained from a large registry of patients evaluated for aortic aneurysm. We tested two hypotheses in this cross-sectional study of aneurysm samples: (1) patients with the highest concentrations of microfibril fragments would be more likely to have TAAs, particularly aneurysms involving both the thoracic and abdominal regions, than patients with the lowest concentrations; (2) among patients with TAAs, those with the highest microfibril fragment concentrations would be more likely than those with the lowest concentrations to have dissection. Results reported here indicate that circulating fibrillin-1 fragments are good candidate biomarkers for TAA and dissection. METHODS Study sample. A registry of patients who were referred to the surgical service at the Baylor College of Medicine (Houston, TX) for evaluation and management of aortic aneurysm and dissection provided the basis for this study. The Baylor College of Medicine Institutional Review Board approved the registry protocol. All patients provided written informed consent. For all subjects, whole blood was drawn into heparin DOI: /CIRCRESAHA

4 tubes, processed immediately into plasma, and stored at 80 C. Blood draws occurred in the operating room just before surgery for surgical patients and, during the hospital stay for nonsurgical patients. Data regarding patient characteristics and details about surgical repair were collected prospectively from medical records during the patient s hospital stay, confirmed by the study physicians, and recorded in the registry database as per our registry protocol. A patient s aorta was considered aneurysmal when its diameter was greater than 1.5 times normal diameter, based on the patient s gender, age, and body size. Variables selected from the registry for analysis included gender, age, ethnicity, smoking status, hypertension, diabetes, body mass index (BMI), inflammatory diseases, connective tissue disorders, bicuspid aortic valve, previous aortic operation, aneurysm location, extent and acuteness of aortic dissection, aneurysm diameter, aneurysm symptoms, aortic rupture, and urgency of operation. We used the registry database to select all available patients who had a documented thoracic or abdominal aortic aneurysm and had a blood sample drawn before surgery or during non-operative management. Patients who received packed red blood cells, whole blood, or platelets within 10 days before the blood draw and those who had aortic trauma, pseudoaneurysm, dissection without aneurysm, or active cancer were excluded from selection. We identified 1265 patients who were enrolled between March 2003 and October Using the same registry, controls without aortic aneurysm or dissection were selected from 297 potentially eligible subjects who had chest and/or abdominal imaging, underwent non-aortic cardiac surgery, or volunteered for a blood draw. Potential controls were listed in random order within strata of age, sex, race, smoking and imaging status; 125 were then selected to match the frequency distributions of these characteristics in the aneurysm sample. Of these, 84 had no TAA or dissection confirmed by chest imaging, 35 underwent non-aortic cardiac surgery, and 6 were volunteers. Aneurysm patients were grouped according to the location of their aneurysms at the time of enrollment. This categorization was applied only to existing aortic disease; previously repaired aneurysms were not considered when patients were assigned to groups. We defined the thoracic aorta as the portion of the vessel located above the diaphragm, including the ascending aorta, the aortic arch, and the descending thoracic aorta, and we defined the abdominal aorta as the portion of the vessel located below the diaphragm, including the suprarenal and infrarenal segments. By these definitions, 174 patients (13.8%) had aneurysms involving only the abdominal aorta (AAA), 614 (48.5%) had aneurysms involving only the thoracic aorta (TAA-only), and 477 (37.7%) had aneurysms in both the abdominal aorta and the thoracic aorta (A+TAA). We classified acuteness of aortic dissections according to the time between the onset of symptoms and the blood draw: patients whose blood was drawn within 14 days of symptom onset were classified as having acute dissection, those with blood drawn between 15 and 60 days after onset were classified as having subacute dissection, and those with blood drawn beyond 60 days of onset were classified as having chronic dissection. In unusual circumstances, the timing of the onset of symptoms was unclear and the acuteness of dissection was determined from other clinical features, such as the characteristics of the dissecting membrane on imaging and the appearance of the aortic wall during operation. Sandwich ELISAs. Concentrations of fibrillin-1, fibrillin-2, and fibulin-4 fragments were measured in plasma samples by using a modified sandwich ELISA. Eight antibody pairs consisting of a biotinylated (B) capture antibody and an alkaline phosphatase (AP)-conjugated detector antibody were used: 4 for fibrillin-1 (B15-AP26, B15-AP78, B15-AP201, B201-AP78), 3 for fibrillin-2 (B48-AP60, B72-AP143, B205-AP143), and 1 for fibulin-4 (B492-AP347) (Online Figure I). The epitopes for fibrillin-1 and fibrillin-2 antibodies have been described 17,18 ; epitopes recognized by the fibulin-4 antibodies are contained within the N-terminal half of the molecule. DOI: /CIRCRESAHA

5 The personnel who performed the assays were blinded to patient information. An assay consisted of eight 96-well plates with a single antibody pair per plate. Each plate contained a standard curve that included a blank (all components except sample peptide), 22 patient samples measured in triplicate, and 2 pooled control samples. Plates were coated for 18 hours at 4 C with 5 µg/ml streptavidin (Thermo Fisher Scientific, Rockford, IL) in 0.05M Na 2 CO 3, 0.05M NaHCO 3, ph 9.6. Biotinylated monoclonal antibodies (15, 201, 205, 72, 48, and 492 at 0.25 µg/ml in 50mM Tris, 150 mm NaCl, ph 7.4, 0.025% Tween20 [TBST] supplemented with 5% fetal bovine serum [FBS]) were incubated for 1 hour at 25 C. Patient samples were diluted 1/10 or 1/25 in TBST and incubated for 20 hours at 4 C. Standard curves were generated by serial dilution of recombinant peptide rf11 (fibrillin-1), rf37 (fibrillin-2), or rfib4-1 (fibulin-4) in TBST with 10% FBS for 20 hours at 4 C. Formation of the sandwich was completed by incubating AP-conjugated monoclonal antibodies (201, 78, and 143, 0.05 µg/ml; 26 and 60, 0.2 µg/ml; 347, µg/ml in TBST with 5% FBS) for 1 hour. Each step was followed by washing unbound materials away with TBST. The Invitrogen ELISA Amplification System (Invitrogen, Carlsbad, CA) substrate was used according to the manufacturer s protocol. The average absorbance of each sample triplicate was computed and converted to μg/ml with an equation generated from the standard curve. A 4-parameter log logistic curve was fit to the standards. The goodness of curve fit was determined by percent recovery, and standards were required to fall within 15% of the calculated recovery value. The lower limit of detection (LoD) was defined as the value 2 standard deviations above the mean blank absorbance. Although the actual concentrations in µg/ml or ng/ml of the circulating fragments are unknown, fragments are quantitated by using µg/ml equivalents of the protein standard used in the assay. Conversion of μg/ml to nm was performed by using the molecular weight (MW) of the recombinant peptides (rf11 MW 190,000; rf37 MW 100,000; rf4-1 MW 30,000). Pooled plasma obtained from 10 individuals with no known disease was used in each assay plate. There was little variation among the 10 individual samples that formed the pooled plasma sample, and fibrillin fragment concentrations were mostly below LoD (data not shown). Based on the pooled plasma samples, means of intra-assay coefficients of variation (CV%) ranged from 2.7 to 5.8 for the different fragments. Statistical analyses. Demographic and medical factors between aneurysm and control groups, or according to aneurysm location among aneurysm patients, were compared descriptively with chi-square or Fisher exact tests for categorical variables and 1-way analysis of variance. Chi-square or Fisher exact tests were also used to compare proportions of aneurysm and control subjects with fragments above LoD. Concentrations of the B15-AP78 and fibulin-4 fragments were compared between aneurysm patients and controls with the nonparametric Wilcoxon rank sum test because of non-normal distributions. Concentrations of the remaining 6 fragments had 0 values for medians and interquartile ranges (25 th -75 th percentile) (IQR) in one or both subject groups. Therefore, it was technically impossible to perform statistical comparisons of these fragment concentrations between aneurysm and control subjects. We conducted further exploratory analyses to determine how best to characterize the overall fibrillin-1 concentration for comparison between aneurysm types. Spearman rank sum correlations showed that the B15-AP26, B15-AP78, and B15-AP201 concentrations were moderately to highly correlated; correlation coefficients (r) ranged from r=0.6 to 0.8. However, the B201-AP78 fragment, which had the largest percentage of concentrations below the LoD, was only moderately correlated (r= ) with the other fibrillin-1 fragments. Based on this information, we computed a single summary variable from the B15- AP26, B15-AP78, and B15-AP201 fragments as follows. Each fragment concentration was first scaled by subtracting the median value of its distribution and dividing by the IQR to approximate a z- distribution. We then computed a single summary fibrillin-1 variable as the mean of the 3 scaled DOI: /CIRCRESAHA

6 fragments. As a result of these procedures, the summary fibrillin-1 was unitless, with a median of approximately 0 and an IQR of approximately 1. Unadjusted comparisons according to aneurysm type were performed with the non-parametric Kruskal- Wallis test for the fibrillin-1 and fibulin-4 concentrations and with chi-square or Fisher exact tests for percentages of fibrillin-2 concentrations above LoD. Adjusted associations of aneurysm location or dissection with microfibril fragment concentrations were estimated with odds ratios (ORs) and 95% confidence intervals (CIs) with logistic regression. For modeling, we created quartiles for the B-15- AP78, B15-AP201, and summary fibrillin-1 concentrations. For the B15-AP26, B201-AP78, and fibulin- 4 fragments, we also created 4 categories: undetectable, and equal thirds (tertiles) of the detectable concentrations. The percentile distributions for these variables were based on the entire aneurysm sample. Fibrillin-2 was categorized as detectable if the patient had concentrations above the LoD in at least 2 fragments; otherwise, it was categorized as undetectable. The outcome variables, aneurysm location and dissection type, with 3 levels each, were modeled with multinomial logistic regression, which estimates ORs for 2 categories in comparison to a referent category. For example, ORs and their corresponding 95% CIs in the aneurysm sample were estimated for the TAA-only and A+TAA groups in comparison to the AAA group. Potential confounding by age, sex, body mass index, current smoking, medical history, and medication use was evaluated during model-building procedures. Confounding was defined by using the 10% change in point estimate criterion. 19 Age (4 categories), sex, and current smoking status (ever, never) met this definition and were retained in all models. All P values are 2-sided. Statistical analyses were performed with SAS version 9.2 (SAS Institute, Cary NC). RESULTS Comparisons between aneurysm patients and controls. Aneurysm patients were age 61 years on average and primarily male, Caucasian, and current or former cigarette smokers (Table 1). Because of frequency matching, the control subjects had nearly identical distributions of these characteristics. All controls reported no history of aortic aneurysms or dissection. Absence of TAA was confirmed by chest imaging in 83 control subjects (67%). The subset of 21 with absence of both TAA and AAA confirmed by complete aortic imaging were classified as imaging-negative controls. Percentages of subjects with detectable microfibril fragments varied considerably (Figure 1). Fibrillin-1 fragments were detectable in significantly higher proportions of aneurysm patients than in controls. Individual fibrillin-1 fragments were detected in most aneurysm patients, with the exception of the B201-AP78 fragment (detectable in 42%). Fibrillin-2 fragments were detectable in 25% of aneurysm patients and in 7% of controls. Fibulin-4 was detectable in all subjects, but significantly more often in controls than in aneurysm patients. Detectable percentages in imaging-negative controls were comparable to those for all controls. Detectable fragment concentrations also differed considerably between aneurysm patients and controls (Figure 2). In controls, B15-AP78 was the only fibrillin-1 fragment with median and IQR concentrations >0, and the median value was significantly lower (0.47 nm) than that (0.86 nm) in aneurysm patients (p<0.0001). Values of 0 for concentrations of the B15-AP201 and B15-AP26 medians and IQRs (Figure 2) precluded testing for differences in these fragments between aneurysm patients and DOI: /CIRCRESAHA

7 controls, as well as computation of the summary fibrillin-1 variable for controls. In contrast, median fibulin-4 values were significantly higher in controls (3.2 nm) than in aneurysm patients (1.1 nm) (p<0.0001). In further exploratory analyses among control subjects only, concentrations 1 nm were observed in 31 (25%) with B15-AP78. Those with the higher compared to lower B15-AP78 concentration were more likely to be non-caucasian (23% vs 7%, p=0.04), to have been referred for non-aortic cardiac surgery (45% vs. 28%, p=0.10), to have at least one other fibrillin-1 fragment 1 nm (23% vs 1%, p=0.0002), and to have 2 fibrillin-2 fragments above LoD (16% vs 0%, p=0.0007), but these two groups did not differ by age, sex, smoking history, or availability of chest imaging (data not shown). Comparisons between aneurysm patients. Compared to AAA patients, TAA-only patients were younger on average, more often female, more likely to have never smoked, and more likely to have Marfan syndrome or bicuspid aortic valve (Table 2). Among A+TAA patients, prevalences of these characteristics tended to be between those of TAA-only and AAA patients. Few patients had genetic conditions or inflammatory diseases other than Marfan syndrome and bicuspid aortic valve. The A+TAA patients had the largest maximum aneurysm diameter and were most likely to have aneurysms 7.0 cm or dissection, to be asymptomatic, and to have had a previous surgical repair. Patients with TAA-only most frequently had aneurysms <5.5 cm. Most patients underwent elective surgery. Detectable percentages of microfibril fragments did not differ materially according to aneurysm location (Table 3). The median fibrillin-1 B15-AP78 fragment concentration was somewhat greater in patients with A+TAA than in those with TAA-only or with AAA, whereas medians of other fibrillin-1 fragments and the summary fibrillin-1 variable did not differ between groups. Median fibulin-4 concentrations were similar between groups. Aneurysm Location According to Microfibril Fragment Concentration. Associations of TAA and of A+TAA with the individual fibrillin-1 B15-AP78, B15-AP201, and B15-AP26 fragments were of a similar magnitude (Online Table I). Multivariable ORs adjusted for age, sex, and smoking showed that frequencies of TAA-only and A+TAA were 1.5- to 2.5-fold greater than AAA in the top compared to bottom quartile. Further adjustment for fibrillin-2 and fibulin-4 did not materially alter these associations. Analyses performed with the summary fibrillin-1 variable (Online Table II) confirmed that the frequencies of TAA-only or A+TAA were significantly elevated compared to AAA in the top compared to bottom quartile. Neither TAA-only nor A+TAA were associated with fibrillin-2 or fibulin-4. Because results for the TAA-only and the A+TAA patients were comparable, we repeated the previous analyses after combining these groups into one category containing all of the patients with an aneurysm involving the thoracic aorta (TAA) (Figure 3). Patients with any TAA were nearly 3 times as frequent as those with AAA in the top compared to bottom quartile of the fibrillin-1 summary concentration. The ORs did not change materially when patients with genetic conditions associated with TAA (such as Marfan syndrome, Loeys-Dietz syndrome, and bicuspid aortic valve) were excluded or when the analysis was restricted to those without aortic dissection. The association of TAA with fibrillin- 2 and fibulin-4 remained null (data not shown). In analyses stratified by patient sex (Online Table III) and adjusted for age, results were consistent with the foregoing main analyses. Among both women and men, patients with any TAA were about 3 times as frequent as those with AAA in the top compared to bottom quartile of the fibrillin-1 DOI: /CIRCRESAHA

8 summary concentration. No associations between TAA frequency and fibrillin-2 or fibulin-4 were observed in either sex. Dissection status and dissection acuteness according to microfibril fragment concentration. Dissection status in relation to microfibril fragment concentration was assessed in patients with any TAA (Table 4). Before adjustment, the percentage distributions of patients without and with dissection were similar among the quartiles of the fibrillin-1 summary concentration. However, in multivariable analyses, the ORs for dissection were elevated in quartiles 3 and 4 of the fibrillin-1 concentration, and the test of linear trend was statistically significant. When we further examined dissection acuity, we observed that 44% of patients with acute or subacute aortic dissection, but only 21% of those with chronic dissection, were in the top quartile of the summary fibrillin-1 concentration. In multivariable analyses, the association between high fibrillin-1 concentration and acute or subacute dissection compared to no dissection remained, but the association was null among those with chronic dissection (Table 4 and Figure 4). Dissection status was not associated with fibrillin-2 or fibulin-4 concentration. When we repeated the analyses after excluding 28 patients with subacute dissection, the results were comparable to those shown in Table 4. The ORs (95% CI) for acute dissection in quartiles 1 through 4, respectively, were 1.0 (referent), 0.9 ( ), 1.9 ( ), and 4.3 ( ). DISCUSSION We report here new immunoassays for the quantitation of circulating fragments of fibrillin-1, fibrillin-2 and fibulin-4, major components of elastic fiber microfibrils. Percentages of detectable fibrillin-1 and fibrillin-2 fragments were significantly higher in aneurysm than control plasma samples. Surprisingly, the percentage of detectable fibulin-4 fragments was lower in aneurysm than control samples, suggesting that specific increased turnover of fibulin-4, compared to fibrillin-1 and fibrillin-2, occurs during normal tissue homeostasis. For fibrillin-1 fragments, median values among aortic aneurysm samples were between 0.2 and 0.9 nm, with maximum values extending beyond 100 nm. In contrast, median fibrillin-1 fragment concentrations in control samples were mostly undetectable, with the exception of the B15-AP78 fragment whose median was substantially lower than in patients with aneurysms. The distribution of extreme values of fibrillin-1 and fibrillin-2 fragments was higher in aneurysm than control samples. Among aneurysm patients, we observed a strong association of TAA with higher fibrillin-1 concentrations. This association was independent of age, sex, and cigarette smoking and was not explained by the inclusion of patients with aortic dissection or genetically-triggered conditions such as Marfan syndrome and bicuspid aortic valve. Furthermore, TAA patients with dissection were more likely to have high concentrations of fibrillin-1 fragments than patients without dissection. Fragments of fibrillin-2 and fibulin-4 were not found to be associated with aneurysm type or dissection status. Nevertheless, because fragments of fibrillin-2 may be released into the circulation only after severe elastic fiber degradation, 13 future studies may reveal a role for these fragments as biomarkers. Overall, this study confirms our hypothesis that elastic microfibril fragments, specifically fibrillin fragments, are released into circulation in aneurysmal disease. Biomarkers for detecting or monitoring TAA and dissections are critically needed. 20 Currently, the diagnosis and monitoring of thoracic aortic disease relies entirely on imaging studies. Consequently, many patients die because of undetected aortic disease, particularly those with acute aortic dissection. Identifying a reliable circulating biomarker for TAA and dissection could enable physicians to improve the care of these patients in many respects. First, the biomarker could be a diagnostic tool to facilitate the DOI: /CIRCRESAHA

9 detection of TAA. This would be particularly useful for patients at high risk, such as those with Marfan syndrome or other genetic conditions associated with aortic disease. Second, ideally, increasing levels of a biomarker for TAA would indicate increasing disease severity. This would enable physicians to use the biomarker to monitor disease progression in patients with aneurysms that are not large enough to warrant surgical treatment. We were surprised that the most extensive form of aortic disease (aneurysm involving both the thoracic and abdominal aortic segments) was not associated with higher microfibril fragment concentrations than aneurysm confined to the thoracic region. Although our study sample included several different types of aneurysms (with and without dissection, genetically triggered, sporadic, acute, chronic) involving many different aortic segments, the association between fibrillin-l fragment concentration and thoracic aneurysm held when we restricted our analyses to patients without genetic conditions or without dissection. Thus, our results suggest the possibility that fibrillin-1 may be useful in monitoring TAA, which results in twice as many deaths from dissection and rupture as AAA, 20 but further prospective studies are necessary to test this possibility. Finally, biomarkers are needed that enable the rapid diagnosis of acute aortic dissection. Among patients with TAA, the highest fibrillin-1 levels were significantly more common in those with acute or subacute dissection than those without dissection, suggesting that these fragments may be useful in detecting acute dissection. A few other investigators have studied ECM biomarkers in patients with acute aortic dissection. Shinohara et al 21 showed that serum concentrations of soluble elastin fragments (ng/ml) were substantially higher in patients with acute aortic dissection and patent false lumen than in healthy adults. Circulating concentrations of several matrix metalloproteinase types appear to be elevated in patients with acute dissection. 22 However, previous reports are challenging to interpret because they generally had small sample sizes, sources of the comparison/control patients were often not described, and analyses were conducted with descriptive statistical methods without adjustment for other factors. Nonetheless, our results, together with those of Shinohara et al, 21 support the concept that elevated concentrations of circulating macromolecules found in aortic ECM could serve as biomarkers of thoracic aortic dissection. Further studies are necessary to evaluate the sensitivity and specificity of fibrillin-1 fragment measurement in detecting acute dissection in patients presenting with chest pain. Our study has several novel aspects. First, we believe that it provides the first evidence that a biomarker of ECM degradation is associated with aortic aneurysm location. We could find no previous work on ECM biomarkers showing differences between patients with TAA and AAA, although others have compared lipoprotein (a) concentration 23 and concomitant medical conditions 24 between these patient groups. Second, the study sample was derived from an aortic aneurysm registry with standardized procedures for blood and data collection. Importantly, blood samples were obtained before surgery and so represent active aneurysmal disease. In addition, our clinical assessments were comprehensive in that we were able to study aneurysm location, dissection status, and dissection acuity. Third, the large sample size enabled us to perform multivariable analyses to adjust for confounding by age, sex, and smoking status. In addition, we were able to perform restricted or stratified analyses, which confirmed that the strength of association between fibrillin-1 concentration and aneurysm location was not due to a preponderance of patients with genetically-triggered aneurysms or aortic dissection. Finally, although we cannot determine whether fibrillin-1 degradation is a risk factor for aneurysm development, the results support further investigation into the potential importance of fibrillin-1 degradation in the pathophysiology of sporadic thoracic aortic disease. Our study had limitations as well. First, it was cross-sectional and included primarily patients with advanced aortic disease. Thus, we cannot determine whether fibrillin-1 fragments are detectable in circulation during earlier stages of aneurysm formation. Second, the relation between aneurysm diameter and fibrillin-1 levels could not be assessed because we lacked diameter measurements for each aortic DOI: /CIRCRESAHA

10 segment. These two limitations precluded evaluation of the utility of fibrillin-1 fragments in monitoring disease progression. Third, some control samples displayed elevated fibrillin-1 fragment levels. The significance of this finding is not yet known. Fourth, more sensitive fibrillin assays are not currently available. Development of more highly sensitive assays allowing quantitation of a full range of concentrations, especially at the low end where we expect normal levels to occur, will be required to better discriminate aortic dissection from other causes of chest pain. Fifth, the source of the microfibril fragments detected in this study is unknown. Because we studied aortic aneurysm patients, and fibrillin-1 is abundant in the aortic ECM, there is a high likelihood that at least some of the fragments originated from the aorta. Sixth, this study did not include other ECM or inflammation biomarkers. Additional study with a larger biomarker panel is required to determine whether the associations observed for fibrillin-1 are independent of these other factors. Finally, the patients studied were seen at a single institution, so our results may not be generalizable to other aneurysm patient populations. In conclusion, our study has clinical, epidemiologic, and biologic implications. This study represents an initial step in evaluating the utility of the microfibril fragment assays for detecting and monitoring TAA in affected patients. Prospective studies will be necessary to evaluate the potential role of fibrillin-1 and fibrillin-2 fragments as quantitative measures of the extent and severity of disease, of disease progression, and of the effects of treatment. Nevertheless, few epidemiologic risk factors for TAA have been identified, particularly in patients without genetically-triggered disease. Thus, our results support further research to determine whether fibrillin-1 fragmentation is a risk factor for TAA. Finally, studies are warranted to define mechanisms that contribute to the degradation of fibrillin-1 microfibrils in aortic tissue. ACKNOWLEDGMENTS The authors thank Ludivine Russell for managing subject enrollment and data collection and Stephen N. Palmer for providing editorial support. We are also grateful to the many volunteers from the National Marfan Foundation and from the Portland Shriners Hospital for Children who allowed us to test their blood samples in initial studies. Lynn Sakai is especially grateful to Dr. Eva Engvall for suggesting the sandwich ELISA methodology. SOURCES OF FUNDING This study was supported by the NIH through grant RC1 HL (to Dr. Lynn Sakai). The Thoracic Aortic Disease Tissue Bank at Baylor College of Medicine was supported in part through the Tissue Banking Core of the Specialized Center of Clinically Oriented Research in Thoracic Aortic Aneurysms and Dissections (P50 HL to Dr. Dianna Milewicz). Funding (to Dr. Lynn Sakai) from the Shriners Hospitals for Children and from the National Marfan Foundation supported initial studies necessary for the development of the sandwich ELISAs. DISCLOSURES None. DOI: /CIRCRESAHA

11 REFERENCES 1. Xu JQ, Kochanek KD, Murphy SL, Tejada-Vera B. Deaths: final data for Natl Vital Stat Rep. 2010;58: Wassef M, Baxter BT, Chisholm RL, et al. Pathogenesis of abdominal aortic aneurysms: a multidisciplinary research program supported by the National Heart, Lung, and Blood Institute. J Vasc Surg. 2001;34: LeMaire SA, Russell L. Epidemiology of thoracic aortic dissection. Nat Rev Cardiol. 2011;8: Murdoch JL, Walker BA, Halpern BL, Kuzma JW, McKusick VA. Life expectancy and causes of death in the Marfan syndrome. N Engl J Med. 1972;286: Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol. 1986;103: Dietz HC, Cutting GR, Pyeritz RE, Maslen CL, Sakai LY, Corson GM, Puffenberger EG, Hamosh A, Nanthakumar EJ, Curristin SM, Stetten G, Meyers DA, Francomano CA. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature. 1991;352: Milewicz DM, Michael K, Fisher N, Coselli JS, Markello T, Biddinger A. Fibrillin-1 (FBN1) mutations in patients with thoracic aortic aneurysms. Circulation. 1996;94: Medley TL, Cole TJ, Gatzka CD, Wang WY, Dart AM, Kingwell BA. Fibrillin-1 genotype is associated with aortic stiffness and disease severity in patients with coronary artery disease. Circulation. 2002;105: Powell JT, Turner RJ, Henney AM, Miller GJ, Humphries SE. An association between arterial pulse pressure and variation in the fibrillin-1 gene. Heart. 1997;78: Powell JT, Turner RJ, Sian M, Debasso R, Lanne T. Influence of fibrillin-1 genotype on the aortic stiffness in men. J Appl Physiol. 2005;99: LeMaire SA, McDonald ML, Guo DC, et al. Genome-wide association study identifies a susceptibility locus for thoracic aortic aneurysms and aortic dissections spanning FBN1 at 15q21.1. Nat Genet. 2011;43: Reinhardt DP, Ono RN, Notbohm H, Müller PK, Bӓchinger HP, Sakai LY. Mutations in calciumbinding epidermal growth factor modules render fibrillin-1 susceptible to proteolysis. A potential disease-causing mechanism in Marfan syndrome. J Biol Chem. 2000;275: Charbonneau NL, Jordan CD, Keene DR, Lee-Arteaga S, Dietz HC, Rifkin DB, Ramirez F, Sakai LY. Microfibril structure masks fibrillin-2 in postnatal tissues. J Biol Chem. 2010;285: Huang J, Davis EC, Chapman SL, Budatha M, Marmorstein LY, Word RA, Yanagisawa H. Fibulin-4 deficiency results in ascending aortic aneurysms: a potential link between abnormal smooth muscle cell phenotype and aneurysm progression. Circ Res. 2010;106: Pape LA, Tsai TT, Isselbacher EM, et al. Aortic diameter 5.5 cm is not a good predictor of type A aortic dissection: observations from the International Registry of Acute Aortic Dissection (IRAD). Circulation. 2007;116: Ranasinghe AM, Bonser RS. Biomarkers in acute aortic dissection and other aortic syndromes. J Am Coll Cardiol. 2010;56: Charbonneau NL, Dzamba BJ, Ono RN, Keene DR, Corson GM, Reinhardt DP, Sakai LY. Fibrillins can co-assemble in fibrils, but fibrillin fibril composition displays cell-specific differences. J Biol Chem. 2003;278: Kuo CL, Isogai Z, Keene DR, Hazeki N, Ono RN, Sengle G, Bachinger HP, Sakai LY. Effects of fibrillin-1 degradation on microfibril ultrastructure. J Biol Chem. 2007;282: Greenland S. Modeling and variable selection in epidemiologic analysis. Am J Public Health. 1989;79: Hiratzka LF, Bakris GL, Beckman JA, et al ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the diagnosis and DOI: /CIRCRESAHA

12 management of patients with thoracic aortic disease: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery, American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society for Vascular Medicine. Circulation. 2010;121:e Shinohara T, Suzuki K, Okada M, Shiigai M, Shimizu M, Maehara T, Ohsuzu F. Soluble elastin fragments in serum are elevated in acute aortic dissection. Arterioscler Thromb Vasc Biol. 2003;23: Karapanagiotidis GT, Antonitsis P, Charokopos N, Foroulis CN, Anastasiadis K, Rouska E, Argiriadou H, Rammos K, Papakonstantinou C. Serum levels of matrix metalloproteinases -1,-2,- 3 and -9 in thoracic aortic diseases and acute myocardial ischemia. J Cardiothorac Surg. 2009;4: Schillinger M, Domanovits H, Ignatescu M, Exner M, Bayegan K, Sedivy R, Polterauer P, Laggner AN, Minar E, Kostner K. Lipoprotein (a) in patients with aortic aneurysmal disease. J Vasc Surg. 2002;36: Ito S, Akutsu K, Tamori Y, Sakamoto S, Yoshimuta T, Hashimoto H, Takeshita S. Differences in atherosclerotic profiles between patients with thoracic and abdominal aortic aneurysms. Am J Cardiol. 2008;101: DOI: /CIRCRESAHA

13 Table 1. Demographic Factors in Controls and Main Study Sample. Characteristic Main Study All Controls Imaging-negative Sample Controls* Years of age, mean (SD) 61.5 (14.3) 60.8 (14.3) 57.1 (17.1) Female, n (%) 417 (33) 40 (32) 6 (29) Race Caucasian, n (%) 1130 (89) 111 (89) 20 (95) African American, n (%) 106 (8) 11 (9) 0 (0) Asian, n (%) 23 (2) 3 (2) 1 (5) Other 6 (1) 0 0 Smoking Status Never, n (%) 419 (33) 42 (34) 7 (33) Past, n (%) 578 (46) 59 (48) 10 (48) Current, n (%) 265 (21) 22 (18) 4 (19) Unknown Control Recruitment Group Not applicable Chest and Abdominal Imaging 21 (17) 21 (100) Only Chest Imaging 62 (50) Only Abdominal Imaging 1 (<1) Non-Aortic Cardiac Surgery 35 (28) Volunteer 6 (5) *Imaging-negative controls had the absence of thoracic and abdominal aortic aneurysm and dissection confirmed with complete imaging of the chest and abdomen. 1 imaging-negative control also had nonaortic cardiac surgery. 3 controls with only chest imaging and 1 with only abdominal imaging also had non-aortic cardiac surgery. DOI: /CIRCRESAHA

14 Table 2. Characteristics of Patients Treated for Aortic Aneurysm From March 2003 to October 2009 According to Aneurysm Location AAA Only (n=174) TAA Only (n=614) A+TAA (n=477) P* Demographic and medical factors Age group, y < (6) 233 (38) 101 (21) (20) 139 (23) 116 (24) (37) 64 (37) 156 (25) 86 (14) 170 (36) 90 19) Female 38 (22) 199 (32) 180 (38) Race 0.16 Caucasian 161 (93) 555 (90) 414 (87) African American 8 (5) 48 (8) 50 (10) Asian 4 (2) 8 (1) 11 (2) Other 1 (1) 3 (1) 2 (0.4) Hispanic ethnicity 15 (9) 47 (8) 34 (7) 0.81 Smoking status < Never 31 (18) 285 (46) 106 (22) Past 104 (60) 230 (37) 244 (51) Current 39 (22) 99 (16) 127 (27) Body mass index, kg/m Normal: <25 56 (42) 182 (33) 154 (39) Overweight: (27) 203 (36) 154 (39) Obese: (31) 174 (31) 85 (22) Diabetes 18 (10) 38 (6) 37 (8) 0.16 DOI: /CIRCRESAHA

15 Hypertension 118 (68) 389 (63) 331 (69) 0.10 Marfan syndrome 5 (3) 60 (10) 53 (11) Bicuspid aortic valve (19) 3 (1) < Other concomitant genetic disease 0 7 (1) 3 (1) 0.38 Concomitant inflammatory disease 2 (1) 14 (2) 21 (4) 0.05 Aneurysm symptoms < None 77 (45) 162 (26) 134 (28) Chronic 81 (47) 350 (57) 284 (60) Acute 14 (8) 100 (16) 58 (12) Aneurysm characteristics Maximum diameter, cm# 5.9 ± ± ± 1.3 <0.001 Maximum diameter, cm# < < (17) 142 (23) 29 (6) < (40) 240 (39) 119 (25) < (22) 141 (23) 167 (35) (21) 71 (12) 156 (33) Any dissection 12 (7) 204 (33) 205 (57) < Dissection acuity < Acute 2 (17) 69 (34) 12 (6) Subacute 0 15 (7) 13 (6) Chronic 10 (83) 120 (59) 180 (88) Treatment and urgency < Non-operative management 3 (2) 19 (3) 5 (1) Elective surgery 156 (90) 500 (81) 401 (84) Urgent surgery 12 (7) 41 (7) 56 (12) DOI: /CIRCRESAHA

16 Emergent surgery 3 (2) 54 (9) 15 (3) Previous surgical repair 42 (24) 143 (23) 212 (44) < Location of previous repair < Abdominal aorta 25 (60) 26 (18) 60 (28) Thoracic aorta 6 (14) 93 (65) 127 (60) Both 11 (26) 24 (17) 25 (12) Data are mean±sd or number (%). AAA indicates abdominal aortic aneurysm; TAA, thoracic aortic aneurysm; A+TAA, abdominal and thoracic aortic aneurysm. *Estimated from 1-way analysis of variance for continuous variables and from chi-square or Fisher exact tests for categorical variables. Missing for 181 patients: 42 (24%) for abdominal aneurysm only, 55 (8%) for thoracic aneurysm only, and 84 (18%) for thoracic and abdominal aneurysm. Confirmed or suspected. Turner syndrome, Ehlers-Danlos syndrome, Loeys-Dietz syndrome, or unspecified connective tissue disorder. Rheumatoid arthritis, polymyalgia arteritis, scleroderma, arteritis (any type), Takayasu aortoarteritis, giant cell arteritis, Behcet disease, lupus erythematosus, periarteritis nodosa, Reiter syndrome, Kawasaki disease, and ankylosing spondylitis. #Missing for 27 patients: 1 (0.6%) for abdominal aneurysm only, 20 (3%) for thoracic aneurysm only, and 6 (1%) for thoracic and abdominal aneurysm. DOI: /CIRCRESAHA

17 Table 3. Microfibril Concentrations among Patients Treated for Aortic Aneurysm From March 2003 to October 2009 According to Aneurysm Location AAA Only (n=174) TAA Only (n=614) A+TAA (n=477) P* Fibrillin-1 Fragments B15A78 detectable, n (%) 159 (91%) 563 (92%) 445 (93%) Median (IQR), nm 0.82 (0.61, 1.11) 0.84 (0.58, 1.19) 0.92 (0.59, 1.29) 0.09 Maximum value B15A201 detectable, n (%) 129 (74%) 469 (76%) 374 (78%) Median (IQR), nm 0.17 (0, 0.25) 0.16 (0.05, 0.28) 0.18 (0.08, 0.29) 0.19 Maximum value B15A26 detectable, n (%) 99 (57%) 366 (60%) 300 (63%) Median (IQR), nm 0.22 (0, 0.42) 0.27 (0, 0.47) 0.29 (0, 0.49) 0.15 Maximum value B201A78 detectable, n (%) 70 (40%) 256 (42%) 206 (43%) Median (IQR), nm 0 (0, 0.55) 0 (0, 0.74) 0 (0, 0.70) 0.56 Maximum value Summary Fibrillin-1 Median (IQR), unitless (-0.46,0.25) (-0.46, 0.47) 0.01 (-0.39, 0.52) 0.13 Maximum value DOI: /CIRCRESAHA

18 Fibrillin-2 Fragments B205A143 detectable, n (%) 39 (22%) 158 (26%) 128 (26%) 0.52 B72A143 detectable, n (%) 19 (11%) 86 (14%) 56 (12%) 0.40 B48A60 detectable, n (%) 10 (6%) 49 (8%) 52 (11%) Detectable Fragments 24 (14%) 109 (18%) 84 (18%) 0.45 Fibulin-4 Detectable, n (%) 105 (60%) 352 (57%) 288 (60%) Median (IQR), nm 1.13 (0, 2.29) 0.91 (0, 1.99) 1.12 (0, 2.20) 0.29 Maximum value * Estimated for fibrillin-1 and fibulin-4 fragments from Kruskal-Wallis tests for continuous variables and for fibrillin-2 detectable percentages from chi-square tests for categorical variables. DOI: /CIRCRESAHA

19 Downloaded from by guest on April 27, 2018 Table 4. Dissection Status and Dissection Acuteness in Relation to Plasma Concentrations of Fibrillin-1, Fibrillin-2, and Fibulin-4 Microfibril Fragments Among Patients With Aortic Aneurysms With Thoracic Involvement (n=1091) Status Acuteness* No Dissection (n=682) Dissection (n=409) Acute/Subacute (n=109) Chronic (n=300) Microfibril n (%) n (%) OR (95% CI) n (%) OR (95% CI) n (%) OR (95% CI) Fibrillin-1 summary (unitless) Quartile 1:-0.90-< (26) 113 (28) Referent 22 (20) Referent 75 (25) Referent Quartile 2: < (25) 102 (25) 1.1 ( ) 15 (14) 0.8 ( ) 82 (27) 1.2 ( ) Quartile 3: < (24) 97 (24) 1.4 ( ) 24 (22) 1.5 ( ) 78 (26) 1.4 ( ) Quartile 4: (26) 97 (24) 1.6 ( ) 48 (44) 2.9 ( ) 65 (21) 1.2 ( ) Fibrillin-2 summary Undetectable 560 (82) 338 (83) Referent 87 (80) Referent 251 (84) Referent Detectable 122 (18) 71 (17) 0.9 ( ) 22 (20) 0.9 ( ) 49 (16) 0.9 ( ) Fibulin-4 (nm) Undetectable 269 (39) 182 (45) Referent 43 (39) Referent 139 (46) Referent Tertile 1: 0-< (20) 82 (20) 0.9 ( ) 20 (18) 0.9 ( ) 62 (21) 0.9 ( ) Tertile 2: 1.44-< (21) 71 (17) 0.7 ( ) 21 (19) 0.8 ( ) 50 (17) 0.7 ( ) Tertile 3: (20) 74 (18) 0.8 ( ) 25 (23) 1.0 ( ) 49 (16) 0.8 ( ) OR indicates odds ratio; CI, confidence interval. *The 409 patients with dissection were further separated into acute/subacute or chronic dissection. Adjusted for age, sex, current smoking status, and the 2 other microfibril fragments shown in the table. No dissection is the comparison category for patients with acute/subacute dissection or with chronic dissection. DOI: /CIRCRESAHA

20 FIGURE LEGENDS Figure 1. Percentage of aneurysm and control subjects with detectable microfibril fragments. Imaging-negative controls are the subset of controls with no thoracic or abdominal aneurysm or dissection confirmed with both chest and abdominal imaging. Detectable percentages were significantly higher among aneurysm patients than controls for all fibrillin-1 (all p<0.0001) and all fibrillin-2 fragments (all p<0.004). The detectable percentage of fibulin-4 was significantly lower among aneurysm patients compared to controls (p<0.0001). Figure 2. Distributions of individual fibrillin-1, fibrillin-2, and fibulin-4 fragment concentrations (nm) in circulation. Values for individual aneurysm patients (A) and controls (C) are shown in gray ( ). The upper y-axis range is nM. Crossbars represent the 25th, 50th (median), and 75 th percentiles. Fibrillin-1 fragments are B15AP78, B15AP201, B15AP26, and B201AP78; fibrillin-2 fragments are B205AP143, B72AP143, and B48AP60; Fibulin-4 is Fbln4. Figure 3. Frequency of any thoracic aortic aneurysm compared with frequency of aneurysm involving only the abdominal aorta within quartiles of plasma concentrations of fibrillin-1 microfibril fragments. Odds ratios are adjusted for age, sex, smoking status, and fibrillin-2 and fibulin-4 levels. Error bars are 95% confidence intervals. *Patients without Marfan syndrome, Ehler-Danlos syndrome, Loeys-Dietz syndrome, Turner syndrome, other unspecified connective tissue disorder, or bicuspid aortic valve. Figure 4. Dissection status in relation to plasma concentrations of fibrillin-1 microfibril fragments among patients with thoracic aortic aneurysms. Odds ratios are adjusted for age and for fibrilin-2 and fibulin-4 levels. Error bars are 95% confidence intervals. Patients with dissection and aneurysms involving only the abdominal aorta were too few for separate analysis. DOI: /CIRCRESAHA

21 Novelty and Significance What Is Known? Aortic aneurysms and dissections, which often have symptoms similar to a myocardial infarction, cause more than 10,000 deaths annually in the U.S. Thoracic aortic aneurysms are more prone to dissection than abdominal aortic aneurysms. There are currently no blood biomarkers that might facilitate early, accurate diagnosis of patients with aortic aneurysms who are at risk for potentially deadly dissections. What New Information Does This Article Contribute? We show that fibrillin-1, a protein constituent of the connective tissue in the aorta and other blood vessels, can be measured in blood from patients with aortic aneurysms and dissections. A higher percentage of patients with aortic aneurysms had detectable levels of fibrillin-1 in the blood compared to percentage of the individuals without aneurysms. High levels of fibrillin-1 were more common in patients with thoracic aortic aneurysm (and with dissection) than in patients with other types of aortic aneurysms. Thoracic aortic aneurysms and dissection are life-threatening conditions caused by the breakdown of aortic tissue. New ways to detect aortic aneurysms and dissections before they become lethal would have considerable clinical benefits. Genetic studies suggest that breakdown of fibrillin-1, a protein that makes up the extracellular matrix, is important in the development of aortic aneurysms and dissection. This research had three important findings. First, fragments of fibrillin-1 could be detected in large percentages of patients with aortic aneurysms, but were mainly undetectable in patients with no known aortic aneurysms. Second, the highest blood levels of fibrillin-1 were about twice as common in patients with thoracic aortic aneurysm than in patients with only abdominal aortic aneurysms. Third, in patients with thoracic aortic aneurysms, the highest levels of fibrillin-1 were three times more common in patients with acute dissections compared to those with no dissection. This study shows for the first time that fibrillin-1 fragments can be measured in blood from patients with aortic aneurysms and dissections. Our results confirm the hypothesis that elastic microfibril fragments, specifically fibrillin-1 fragments, are released into circulation in aneurysmal disease. Development of a sensitive and specific clinical blood test for fibrillin-1 might improve the detection of aortic aneurysm and dissection. DOI: /CIRCRESAHA

22 100 Aneurysm Patients t (n=1265) All Controls (n=125) 80 Imaging-Negative g g Controls (n=21) % 0% 0% Fibrillin-1 Fragments Fibrillin-2 Fragments Figure 1 Perce nt (%)

23 Figure 2

24 Downloaded from by guest on April 27, 2018 Figure Entire Sample (n=1265) Without Genetic Conditions (n=1017)* Without Dissection (n=844) Odds Ratio (OR R) Quartile 1 (Referent) Quartile 2 Quartile 3 Quartile 4 Fibrillin-1 Quartile

25 Acute or Subacute Dissection Chronic Dissection Quartile 1 (Referent) Quartile 2 Quartile 3 Quartile 4 Fibrillin-1 Quartile Figure 4 Odds Ratio (OR)

26 Thoracic Aortic Aneurysm Frequency and Dissection are Associated with Fibrillin-1 Fragment Concentrations in Circulation Lynn M Marshall, Eric Carlson, Jean P O'Malley, Caryn K Snyder, Noe Charbonneau, Susan Hayflick, Joseph S Coselli, Scott A LeMaire and Lynn Y Sakai Circ Res. published online September 13, 2013; Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2013 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at:

27 Online Figure I. Cartoons depicting the domain structures off fibrillin-1, fibrillin-2, and fibulin-4 and the domains containing the epitopes recognized by the numbered monoclonal antibodies (indicated by black triangles under the numbers). Monoclonal antibodies used in the sandwich ELISAs as conjugated antibody pairs (B, biotinylated-ap, alkaline phosphatase) are listed. The mapped epitopes define the minimumm size of fragments captured by specific antibody pairs. For example, the minimum size of the fragment captured by B201-AP78 extends from the second calcium-binding EGF-like domain to the second 8-cysteine domain; the minimum fragment captured byy B15-AP26 consists of only two domains, the first 8-cysteine domain and the proline-rich region. The domain structures of the recombinant polypeptides rf11, rf37, and rf4-1 are shown below the full-length molecules. These recombinant polypeptides were used in the standard curves to quantitate captured fragments.