Thoracic Aortic Aneurysm Frequency and Dissection Are Associated With Fibrillin-1 Fragment Concentrations in CirculationNovelty and Significance
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 from 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 (abdominal aortic aneurysm), 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 (versus abdominal aortic aneurysm) or with dissection (versus no dissection) were estimated with odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, and smoking. Compared with controls, significantly higher percentages of aneurysm patients had detectable levels of fibrillin fragments. TAA was significantly more common (than abdominal aortic aneurysm) in the highest compared with lowest quartile of fibrillin-1 concentration (OR=2.9; 95% CI, 1.6–5.0). Relative to TAA without dissection, acute or subacute dissection (OR=2.9; 95% CI, 1.6–5.3), 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.
Conclusions: Circulating fibrillin-1 fragments represent a new potential biomarker for TAA and acute aortic dissection.
Aortic aneurysm and dissection are significant causes of cardiovascular morbidity and mortality, resulting in >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.7–10 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, the 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 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 seem 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 2 hypotheses in this cross-sectional study of aneurysm samples: (1) patients with the highest concentrations of microfibril fragments would be more likely than those with the lowest concentrations to have TAAs, particularly aneurysms involving both the thoracic and abdominal regions; (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.
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 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 >1.5 times the normal diameter, based on the patient’s sex, age, and body size. Variables selected from the registry for analysis included sex, age, ethnicity, smoking status, hypertension, diabetes mellitus, body mass index, 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 TAA or abdominal aortic aneurysm (AAA) and had a blood sample drawn before surgery or during nonoperative management. Patients who received packed red blood cells, whole blood, or platelets <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 2009. Using the same registry, controls without aortic aneurysm or dissection were selected from 297 potentially eligible subjects who had chest or abdominal imaging, underwent nonaortic 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 nonaortic 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 <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 >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.
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 capture antibody and an alkaline phosphatase–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 previously17,18; epitopes recognized by fibulin-4 antibodies are contained within the N-terminal half of the molecule.
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 0.05 mol/L Na2CO3, 0.05 mol/L NaHCO3, pH 9.6. Biotinylated monoclonal antibodies (15, 201, 205, 72, 48, and 492 at 0.25 µg/mL in 50 mmol/L Tris, 150 mmol/L 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 alkaline phosphatase–conjugated monoclonal antibodies (201, 78, and 143 at 0.05 µg/mL; 26 and 60 at 0.2 µg/mL; 347 at 0.125 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 microgram per milliliter 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 SDs above the mean blank absorbance. Although the actual concentrations in micrograms per milliliter or nanograms per milliliter of the circulating fragments are unknown, fragments are quantitated by using micrograms per milliliter equivalents of the protein standard used in the assay. Conversion of micrograms per milliliter to nmol/L 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 ranged from 2.7 to 5.8 for the different fragments.
Demographic and medical factors between aneurysm and control groups, or according to aneurysm location among aneurysm patients, were compared descriptively with χ2 or Fisher exact tests for categorical variables and 1-way ANOVA. χ2 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 value for medians and interquartile ranges (25th–75th percentile; IQR) in 1 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 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=0.2–0.4) 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 fragments. As a result of these procedures, the summary fibrillin-1 variable was unitless, with a median of ≈0 and an IQR of ≈1.
Unadjusted comparisons according to aneurysm type were performed with the nonparametric Kruskal–Wallis test for the fibrillin-1 and fibulin-4 concentrations and with χ2 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 ≥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 with 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 probability values are 2-sided. Statistical analyses were performed with SAS version 9.2 (SAS Institute, Cary, NC).
Comparisons Between Aneurysm Patients and Controls
Patients with aneurysm were aged 61 years on average and primarily men, white, and current or former 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 was 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 with 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 nmol/L) than that (0.86 nmol/L) in aneurysm patients (P<0.0001). The value of 0 for concentrations of B15-AP201 and B15-AP26 medians and IQRs (Figure 2) precluded testing for differences in these fragments between aneurysm patients and 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 nmol/L) than in aneurysm patients (1.1 nmol/L; P<0.0001).
In further exploratory analyses among control subjects only, concentrations ≥1 nmol/L were observed in 31 (25%) with B15-AP78. Those with higher, compared with lower, B15-AP78 concentration were more likely to be non-white (23% versus 7%; P=0.04), to have been referred for nonaortic cardiac surgery (45% versus 28%; P=0.10), to have ≥1 other fibrillin-1 fragment ≥1 nmol/L (23% versus 1%; P=0.0002), and to have ≥2 fibrillin-2 fragments above LoD (16% versus 0%; P=0.0007), but these 2 groups did not differ by age, sex, smoking history, or availability of chest imaging (data not shown).
Comparisons Between Aneurysm Patients
Compared with AAA patients, TAA-only patients were younger on average, predominantly women, 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 with 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 with AAA in the top compared with bottom quartile. Neither TAA-only nor A+TAA were associated with fibrillin-2 or fibulin-4.
Because results for TAA-only and A+TAA patients were comparable, we repeated the previous analyses after combining these groups into 1 category containing all of the patients with an aneurysm involving the thoracic aorta (TAA; Figure 3). Patients with any TAA were ≈3 times as frequent as those with AAA in the top compared with 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 ≈3 times as frequent as those with AAA in the top compared with bottom quartile of the fibrillin-1 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 with 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 (0.4–2.2), 1.9 (0.9–4.1), and 4.3 (2.1–8.7).
We report here new immunoassays for the quantitation of circulating fragments of fibrillin-1, fibrillin-2, and fibulin-4, the 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 in control samples, suggesting that specific increased turnover of fibulin-4, compared with 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 nmol/L, with maximum values extending >100 nmol/L. 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 in 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 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, compared with those without dissection, were more likely to have high concentrations of fibrillin-1 fragments. 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 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 comparable with 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-1 fragment concentration and thoracic aortic aneurysm was maintained 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 has been causing twice as many deaths from dissection and rupture compared with AAA,20 but further prospective studies are necessary to test this possibility.
Finally, biomarkers are needed to enable 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 al21 showed that serum concentrations of soluble elastin fragments (in nanograms per milliliter) were substantially higher in patients with acute aortic dissection and patent false lumen compared to healthy adults. Circulating concentrations of several matrix metalloproteinase types seem 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 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 think 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 concentration23 and concomitant medical conditions24 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 hence 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 because of 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 segment. These 2 limitations precluded evaluation of the use 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 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 microfibril fragments detected in this study is unknown. Because we studied aortic aneurysm patients and that fibrillin-1 is abundant in 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, and our results may not be generalizable to other aneurysm patient populations.
Our study has clinical, epidemiological, and biological implications. This study represents an initial step in evaluating the use of 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 epidemiological 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.
We 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. L.Y.S. is especially grateful to Dr Eva Engvall for suggesting the sandwich ELISA methodology.
Sources of Funding
This study was supported by the National Institutes of Health through grant RC1 HL100608 (to L.Y.S.). 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 HL083794 to Dr Dianna Milewicz). Funding (to L.Y.S.) from the Shriners Hospitals for Children and from the National Marfan Foundation supported initial studies for the development of the sandwich ELISAs.
In August 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.8 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.301498/-/DC1.
- Nonstandard Abbreviations and Acronyms
- abdominal aortic aneurysm
- extracellular matrix
- limit of detection
- thoracic aortic aneurysm
- Received April 3, 2013.
- Revision received September 10, 2013.
- Accepted September 13, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Aortic aneurysms and dissections, which often have symptoms similar to a myocardial infarction, cause >10 000 deaths annually in the United States.
Thoracic aortic aneurysms are more prone to dissection compared with 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 with percentage of 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 3 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 3 times more common in patients with acute dissections compared with those with no dissection. This study shows, for the first time to our knowledge, 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.