Published online before print August 27, 2007, doi: 10.1161/CIRCULATIONAHA.106.683722
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(Circulation. 2007;116:1396-1403.)
© 2007 American Heart Association, Inc.
Vascular Medicine |
ß2-Microglobulin as a Biomarker in Peripheral Arterial Disease
Proteomic Profiling and Clinical Studies
From the Division of Cardiovascular Medicine (A.M.W., E.K., R.K.H., N.N., B.N., K.R.B., J.P.C.), Stanford University, Stanford, Calif; Ciphergen Biosystems Inc (X.-Y.M., F.Z., E.T.F.), Fremont, Calif; and Zena and Michael A Wiener Cardiovascular Institute (J.W.O.), Mount Sinai Medical Center, New York, NY.
Correspondence to John P. Cooke, MD, PhD, Stanford University School of Medicine Division of Cardiovascular Medicine, 300 Pasteur Dr, Falk Cardiovascular Research Center, Stanford, CA 94305-5406. E-mail john.cooke{at}stanford.edu
Received December 20, 2006; accepted June 29, 2007.
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Abstract |
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Background— Peripheral arterial disease (PAD) is common but commonly unrecognized. Improved recognition of PAD is needed. We used high-throughput proteomic profiling to find PAD-associated biomarkers.
Methods and Results— Plasma was collected from PAD patients (ankle brachial index of <0.90; n=45) and subjects with risk factors but without PAD (n=43). Plasma was analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to quantify 1619 protein peaks. The peak intensity of a 12-kDa protein was higher in PAD patients. Western blot analyses and immunoaffinity studies confirmed that this protein was ß2-microglobulin (B2M). In a validation study, B2M was measured by ELISA in plasma in age- and gender-matched PAD (n=20) and non-PAD (n=20) subjects. Finally, we studied a larger cohort of subjects (n=237) referred for coronary angiography but without known PAD. Plasma B2M levels were higher in PAD patients than in non-PAD patients with coronary artery disease. Plasma B2M correlated with ankle brachial index and functional capacity. Independent predictors of PAD were diabetes mellitus, age, and the combination of B2M and C-reactive protein level.
Conclusions— In PAD patients, circulating B2M is elevated and correlates with the severity of disease independent of other risk factors. These findings might provide a needed biomarker for PAD and new insight into its pathophysiology. Further studies in other populations are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.
Key Words: beta 2-microglobulin • inflammation • peripheral vascular disease • proteomics
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Introduction |
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A therosclerotic arterial occlusive disease that affects the lower extremities is also known as peripheral arterial disease (PAD). This disorder affects 8 to 12 million individuals in the United States and is also prevalent in Europe and Asia.1–4 PAD causes limb pain with exertion, reduces functional capacity and quality of life,5 and is frequently associated with coronary, cerebral, and renal artery disease. Patients with PAD are at increased risk of myocardial infarction, cerebrovascular attack, aortic aneurysm rupture, and vascular death, as well as ischemic ulceration and amputation.6,7
Editorial p 1346
Clinical Perspective p 1403
The high risk of vascular events in PAD is reduced by aggressive risk factor modification. In these individuals, the use of statins, ACE inhibitors, and antiplatelet therapy reduces morbidity and mortality.8 Unfortunately, PAD is underdiagnosed and undertreated. Many of those affected do not manifest the classic symptomatology. Intermittent claudication (exertional leg discomfort relieved by rest) is only noted by 10% to 30% of patients.5,9 Coexisting musculoskeletal disease or neuropathy may confound the clinical picture, and almost half of patients are so sedentary as to be asymptomatic.5 Unfortunately, even asymptomatic individuals have a reduced lifespan without aggressive treatment.10
We hypothesize that bouts of ischemia and reperfusion in the legs of PAD patients release into the circulation endothelial proteins that are specific for the skeletal muscle microvasculature. This hypothesis is based on previous observations that ischemia followed by reperfusion can cause shedding of adhesion molecules and the generation of inflammatory cytokines.11 Accordingly, we used proteomic profiling in an attempt to find a useful biomarker for PAD.
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Methods |
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Subject Sample
A total of 371 subjects were investigated in 3 serial studies: a discovery study (n=88) followed by confirmation (n=40) and validation (n=237) studies. The subjects for the discovery and confirmation studies were recruited as part of the NO-PAIN (Nitric Oxide in Peripheral Arterial Insufficiency) Study.12 In the NO-PAIN Study, subjects were recruited by advertisements targeting individuals with leg pain that limited the ability to walk.12 Subjects underwent a physical examination, measurement of ankle and brachial systolic pressures, and treadmill testing with the Skinner-Gardner protocol.13 Subjects for the validation study were derived from the ongoing GenePAD (Genetic Determinants of PAD) Study. In the GenePAD Study, subjects were recruited from individuals undergoing coronary angiography at Stanford University or Mount Sinai Medical Centers. The PAD status of these individuals was not known to the investigators at the time of recruitment. Ankle-brachial index (ABI) was determined before a comprehensive clinical characterization that included coronary angiography. Patients with PAD had an ABI at rest of <0.90, or in those with noncompressible ankle arteries, a toe-brachial index of <0.60. Glomerular filtration rate was estimated by the Modification of Diet in Renal Disease Study method.14 The NO-PAIN and GenePAD studies were funded by the National Heart, Lung, and Blood Institute and approved by the Stanford University Committee for the Protection of Human Subjects.
Proteomic Profiling of Plasma Samples
Venipuncture was performed on fasting subjects, and serum and plasma samples were stored at –75°C. Plasma samples (20 µL) were denatured at pH 9 in a buffer containing 9 mol/L urea, 2% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate), and 50 mmol/L Tris-HCl, pH 9.0. The denatured sample was applied to a BioSepra Hyper Q DF anion-exchange column (Pall Corp, East Hills, NY). Proteins were eluted with buffers at pH 9, 7, 5, 4, and 3 and organic solvent. All 6 fractions of each sample were applied to 2 types of ProteinChip arrays: an immobilized metal affinity (IMAC30) chip and a cationic exchange (CM10) chip. Arrays were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF; laser intensity 1600 to 7000 nJ and a sampling rate of 800 MHz).15 Protein peaks were identified from the raw spectrum data by application of a threshold to peak intensity analysis normalized against total ion current (with the use of CiphergenExpress data manager software version 3.0 [Ciphergen Biosystems Inc, Fremont, Calif]).
Data Analyses and Statistics
Differences in peak intensities between groups were determined by application of significance analysis of microarrays.16 This analysis was confirmed by a second bioinformatics approach, prediction analysis for microarrays.17 Differences between groups in the ELISA measurements were calculated by independent t test, 1-way ANOVA corrected with Bonferroni method, or Mann-Whitney nonparametric test where appropriate. Correlations were analyzed with Spearman nonparametric correlation analysis, and multivariate regression was used for assessments of independence of correlations. A probability value of <0.05 was taken to indicate statistical significance. Data were analyzed with SPSS software.
Confirmatory Biochemical Studies
A ProteinChip array–based immunoassay with an anti-B2M antibody (Abcam, Cambridge, United Kingdom) was used to specifically capture ß2-microglobulin (B2M) from plasma samples. Subsequently, peak intensity was determined by SELDI-TOF. In addition, fractionated or nonfractionated plasma samples were subjected to a Western blot analysis for B2M. Both fractionated plasma proteins (5 µg) and 0.4 µL of unfractionated plasma were electrophoresed on 8% to 16% gradient polyacrylamide Tris-SDS gels (Bio-Rad, Hercules, Calif) and transferred to a positively charged polyvinylidene difluoride membrane. The presence of B2M was detected with rabbit anti-human B2M antibody conjugated with horseradish peroxidase and a chemiluminescence protein detection kit (Amersham, Buckinghamshire, United Kingdom). Finally, B2M was measured by ELISA in plasma and serum samples with kits obtained from Immunodiagnostik (Bensheim, Germany) and R&D Systems (Minneapolis, Minn), respectively.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Patient Characteristics
In the initial discovery study, patients with PAD (n=45) had a greater prevalence of vascular risk factors than non-PAD subjects (n=43; Table 1), as expected. However, no differences existed between the groups in terms of gender, prevalence of diabetes mellitus, smoking history, kidney disease, body mass index, or history of coronary artery disease. In the confirmatory study, a second set of non-PAD (n=20) and PAD subjects (n=20) were well matched for age and gender, although the risk factor burden remained higher in the PAD group (Table 1). In the third screening study, 237 subjects presenting for elective coronary angiography were included. In that study, patients with coronary artery disease with and without PAD were well matched for age, gender, and risk factors (Table 2).
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High-Throughput Proteomics Analysis
In each subject in the discovery study, 1619 different protein peaks, arising from SELDI-TOF with 6 different plasma fractions and 2 types of chips, were analyzed. Significance analysis of microarrays revealed that of the 1619 peaks, 11 had higher peak intensities in the PAD group (at a q value <10%; Figure 1; Table 3). Six of the 11 peaks around 12 kDa, obtained with different pH fractions, chip types, or energy states, were suspected to represent the same protein (Table 3). These peaks were putatively identified as representing B2M.18,19 Other peaks found to be higher in the PAD group were 13- and 15-kDa proteins putatively identified as cystatin C and lysozyme C, respectively. Furthermore, unidentified proteins at 22 and 36 kDa were increased in the PAD group.
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Identification of 12-kDa Protein as B2M
The peak intensities of the 12-kDa protein (putatively identified as B2M) were highly correlated with ABI (r=–0.49, P<0.001). Also, tertiles of B2M peak intensity were related to absolute claudication time (groups being normal subjects without claudication, PAD with absolute claudication time >12 minutes, or PAD with absolute claudication time <12 minutes; Figure 2B).
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0.9, n=44; 0.9>ABI To confirm that the 12-kDa protein was B2M, immunoaffinity studies were performed with anti-B2M antibody. The 12-kDa peak was most likely B2M, given the mass of the peak immunoprecipitated by the anti-B2M antibody. In addition, the 12-kDa peak could be immunodepleted by an anti-B2M antibody. Western blot analyses for B2M in fractionated or unfractionated plasma from PAD patients and control subjects revealed expression levels that were generally consistent with the 12-kDa peak intensities and that were higher in subjects with PAD (Figure 3A and 3B).
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Confirmation Study of B2M as a Biomarker for PAD
Subsequently, we performed a confirmation study in a separate set of age- and gender-matched subjects with (n=20) or without (n=20) PAD. Plasma and serum B2M were measured by ELISA. B2M levels were significantly higher in PAD patients than in non-PAD subjects both in plasma (PAD=2.17±0.63 µg/mL, non-PAD=1.72±0.42 µg/mL, P=0.014) and serum (PAD=2.91±0.88 µg/mL, non-PAD=2.36±0.67 µg/mL, P=0.026). Plasma B2M levels were inversely correlated with ABI (r=0.727, P<0.001; Figure 4). Log-transformed plasma B2M (P=0.030) and smoking (P<0.001) were independent predictors of index ABI in this validation study. Plasma B2M levels correlated with high-sensitivity C-reactive protein by univariate correlation (r=0.201, P<0.001), and this was independent of other risk factors by multivariate regression (Table 4). Other independent correlates for plasma B2M in the present study population included estimated glomerular filtration rate, diabetes mellitus, hyperlipidemia, and body mass index (Table 4).
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Validation Study in a Population at Risk for PAD
In patients undergoing coronary angiography (n=237), serum B2M was higher in patients with PAD. Also, the combination of B2M and high-sensitivity C-reactive protein levels correlated with PAD diagnosis independent of other vascular risk factors and with glomerular filtration rate by stepwise regression analysis (Table 5), consistent with the earlier observation in the smaller confirmation study. Increasing age and diagnosis of diabetes mellitus were the other independent correlates of PAD diagnosis. The odds ratio for the diagnosis of PAD for elevated B2M was 7.2 (95% confidence interval 1.6 to 31.3, P=0.009), and for high-sensitivity C-reactive protein, the OR was 1.3 (95% confidence interval 1.0 to 1.7, P=0.026).
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Discussion |
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The salient observations of the present study are that plasma levels of B2M are elevated in patients with PAD and that B2M levels independently correlate with the severity of disease as assessed by the ABI or treadmill testing. This is the first study to employ high-throughput proteomic profiling to discover a biomarker for PAD. In our initial discovery study, we used SELDI-TOF mass spectroscopy with 6 different fractionation conditions and 2 different protein chip arrays per sample to characterize 1619 protein peaks in 88 subjects, which generated almost 150 000 data points. Subsequently, we applied 2 different bioinformatics approaches (significance analysis of microarrays and prediction analysis for microarrays) that have been developed to analyze large volumes of data in relatively small cohorts of patients. The significance analysis of microarrays revealed 11 peaks of interest that represented 5 different proteins. This statistical analysis was confirmed with prediction analysis for microarrays methodology. We focused on B2M because its peak intensities differed the most in the PAD group. Thereafter, we used supplementary biochemical approaches to confirm its identity. Subsequently, we performed a confirmation study to confirm the relationship of B2M to ABI and to functional capacity using a second group of subjects. Finally, we performed a validation study in a cohort of patients undergoing elective coronary angiography. In patients with coronary artery disease, B2M levels were higher in those who also had PAD, and B2M levels were independently correlated with the ABI. This is a unique strength of the present study. The biomarker remained correlated with ABI and continued to distinguish PAD, even between 2 groups of patients with atherosclerotic disease.
The present study was driven by the recognition that PAD is underdiagnosed and undertreated, as demonstrated by the PARTNERS study (PAD Awareness, Risk, and Treatment: New Resources for Survival).9 Many patients do not have classic symptoms of intermittent claudication. Furthermore, the primary practitioner lacks the specialized equipment and trained personnel to perform ABI measurements in the office setting. Accordingly, a biomarker detectable by a blood test would be a useful diagnostic adjunct. Recognition of PAD would trigger intensive risk factor modification with therapies that confer longevity in these patients, including converting enzyme inhibitors, statins, and antiplatelet agents.20
The protein B2M is an 11.7-kDa nonglycosylated polypeptide composed of 99 amino acids. It is 1 of the major histocompatibility complex class I molecules on the cell surface of all nucleated cells. It interacts with and stabilizes the tertiary structure of the major histocompatibility complex class I 
-chain.21 Because it is noncovalently associated with the -chain and has no direct attachment to the cell membrane, free B2M circulates in the blood as a result of shedding from cell surfaces or intracellular release. It is not known whether increased shedding and/or production of B2M is present with ischemia-reperfusion. Once released, B2M is almost exclusively eliminated by glomerular filtration and has been used for estimation of the glomerular filtration rate.22 In normal individuals, the serum concentration of B2M is usually <2 µg/mL, and the urinary excretion is <400 µg/24 hours.22 In patients on dialysis, B2M levels are greatly elevated and contribute to amyloid deposition, with associated cardiovascular dysfunction.23 Increased plasma levels of B2M also occur in a variety of autoimmune, neoplastic, and infectious diseases, including multiple myeloma, lymphoma, and Sjogren disease.24–26
Inflammation is a prominent component of atherosclerotic syndromes.27 Because of its probable role in immunity and inflammation, the association of B2M with PAD or with alterations in vascular structure28 could be related to vascular inflammation.29 The inflammatory modulator C-reactive peptide is increased in patients with atherosclerosis and is predictive of the development of PAD.30 Other biomarkers of vascular disease include proinflammatory cytokines such as interleukin-6 and soluble adhesion molecules such as intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin.31,32 Alternatively, it is possible that B2M may damage vessels by participating in amyloid formation in the vessel wall.
Although the proteomic profiling in the present study was comprehensive, it was not exhaustive. It is estimated that 
10 000 plasma proteins and even more protein fragments exist. The present study has examined a subset that represents <10% of all the unique protein and peptide species present in plasma; however, the systematic high-throughput approach that we used can be extended in the future to provide greater coverage of all plasma proteins.
Another limitation of the present study is that the elevation in B2M may reflect the increased burden of cardiovascular risk factors in PAD or the severity of systemic atherosclerosis, because it is relatively nonspecific. However, as with many other putative markers of atherosclerotic disease, including inflammatory markers and markers of endothelial dysfunction such as soluble cell adhesion molecules, this lack of specificity should not necessarily preclude its utility as a disease marker. In this context, subsequent studies will be needed to confirm whether B2M is related to the severity and/or the distribution of atherosclerotic disease. These studies will need to address whether measurement of biomarkers for PAD, including B2M, is additive to the measurement of ABI, especially when applied to a large number of subjects as part of community-based population screening.
To conclude, using high-throughput proteomic profiling, we found that plasma B2M was elevated in patients with PAD and was correlated with lower ABI and functional capacity. Use of the assay in diagnosing PAD in patients at high risk of atherosclerosis provided similar predictive power as high-sensitivity C-reactive protein and appeared complementary when data from both assays were used. This finding provides support to the pursuit of studies to determine the utility of B2M as a biomarker for PAD. Further studies in other populations, such as population-based studies of those at lower risk of cardiovascular disease, are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.
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Acknowledgments |
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We thank Dr C. Holweg for advice on clinical proteomic analyses; Dr B. Varasteh for support at the General Clinical Research Center; and H. Tavana and L.D. Waring for patient care and sample collection.
Sources of Funding
This study was supported in part by grants from the National Heart, Lung, and Blood Institute (R01 HL-63685, RO1 HL-75774, and T32 HL07708; Dr Cooke) and National Institutes of Health grant M01 RR 00070 (General Clinical Research Center, Stanford University School of Medicine).
Disclosures
Drs Cooke, Kimura, Wilson, and Fung are inventors of patents based on findings from this study. The patents are jointly owned by Stanford University and Ciphergen Inc. Ciphergen Inc and Ajinomoto Inc provided supplies and equipment for this study. The other authors report no conflicts.
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Footnotes |
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Clinical trial registration information—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00284076.
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