HSA, WW, YW, QZ, MAK and DJL are full-time employees of Nordic Bioscience. MAK holds stock in Nordic Bioscience. Other authors have no competing interests. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: HSA REC MAK DJL. Performed the experiments: HSA REC. Analyzed the data: HSA REC LMR DJL MAK. Contributed reagents/materials/analysis tools: MRL AN WW YW QZ LMR MBP BV DK LS HM JL ACPD FJM CMH MH. Wrote the paper: HAS REC DJL MAK LMR.
Elastin is a signature protein of the arteries and lungs, thus it was hypothesized that elastin is subject to enzymatic degradation during cardiovascular and pulmonary diseases. The aim was to investigate if different fragments of the same protein entail different information associated to two different diseases and if these fragments have the potential of being diagnostic biomarkers.
Monoclonal antibodies were raised against an identified fragment (the ELM-2 neoepitope) generated at the amino acid position ‘552 in elastin by matrix metalloproteinase (MMP) −9/−12. A newly identified ELM neoepitope was generated by the same proteases but at amino acid position ‘441. The distribution of ELM-2 and ELM, in human arterial plaques and fibrotic lung tissues were investigated by immunohistochemistry. A competitive ELISA for ELM-2 was developed. The clinical relevance of the ELM and ELM-2 ELISAs was evaluated in patients with acute myocardial infarction (AMI), no AMI, high coronary calcium, or low coronary calcium. The serological release of ELM-2 in patients with chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) was compared to controls.
ELM and ELM-2 neoepitopes were both localized in diseased carotid arteries and fibrotic lungs. In the cardiovascular cohort, ELM-2 levels were 66% higher in serum from AMI patients compared to patients with no AMI (p<0.01). Levels of ELM were not significantly increased in these patients and no correlation was observed between ELM-2 and ELM. ELM-2 was not elevated in the COPD and IPF patients and was not correlated to ELM. ELM was shown to be correlated with smoking habits (p<0.01).
The ELM-2 neoepitope was related to AMI whereas the ELM neoepitope was related to pulmonary diseases. These results indicate that elastin neoepitopes generated by the same proteases but at different amino acid sites provide different tissue-related information depending on the disease in question.
Elastin is an integral component of the extracellular matrix and enables tissues to repeatedly distend and relax over a lifetime. Elastin is predominantly expressed in connective and vascular tissues, in the lungs and in skin. Elastin makes up the core of elastic fibres and is surrounded by a mantle of fibrillin-rich microfibrils
Extracellular matrix (ECM) components are degraded by a number of different proteases including matrix metalloproteinases (MMPs). Human neutrophil elastase (HNE), MMP-9 and MMP-12 in particular have been associated with elastin degradation and hence with cardiovascular
Macrophages are one of the main sources of MMPs in adult tissue. MMP-12 knock-out mice subjected to cigarette smoking do not have increased numbers of macrophages in their lungs and do not develop emphysema
The cross-linked nature and extreme hydrophobicity of elastin makes it one of the most stable proteins in the body with a half-life of decades and subject to very little remodelling in healthy tissue.
Atherosclerosis is the underlying basis of most cardiovascular diseases (CVD) and degradation of extracellular proteins is a critical step for the development of atherosclerosis
The hypothesis for this research was that elastin is subject to pathologic remodeling during cardiovascular and pulmonary diseases, thus the aim was to investigate if two different fragments of the same protein entail different information associated to two different diseases and if these fragments have the potential of being diagnostic biomarkers. In the current study, a human elastin neoepitope, ELM-2, located at glycine ‘552 was identified and characterized. Its clinical relevance when assessed in serum was evaluated by ELISA in a cardiovascular study and in patients with COPD or IPF. Another elastin neoepitope, ELM, located at alanine ‘441, has recently been shown by our group to be elevated in patients with COPD or IPF
Purified elastin from human aorta (Sigma Aldrich), prepared using the method described by Starcher et al
The proteases were activated according to the manufacturers' instructions. Each cleavage was performed in a solution of protein/proteinase at a ratio of 100∶1. The activated proteinase was added to a MMP-buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl2, 2 mM ZnOAc, pH 8.0)), a cathepsin buffer (50 mM NaOAc, 20 mM L-cystine, pH = 5.5) or a aggrecanase buffer (50 mM tris-HCl, 10 mM NaCl, 10 mM CaCl2, pH = 7.5). The final concentration of elastin before cleavage was 0.33 mg/mL. Each aliquot was incubated for 2, 4, 24 and 48 hours at 37°C. All MMP cleavages were terminated using GM6001 (Sigma-Aldrich) and all cathepsin and aggrecanase cleavages using E64 (Sigma-Aldrich). Finally the cleavage was visually verified by using the SilverXpress® Silver Staining Kit (Invitrogen) according to the manufacturers' instructions.
Using the same procedure as described above, purified elastin from lung and aorta (Sigma Aldrich), prepared using the method described by Starcher et al
Human vascular tissue (atheroma-aorta, Biocat, Heidelberg, Germany) was cleaved by MMP-9 as described by Zhen et al
The peptides were purified and desalted using reversed phase (RP) micro-columns (Applied Biosystems) prior to nanoLC-MS-MS analysis as described in the literature
The first six amino acids of each free end of the sequences identified by MS were regarded as a neoepitope generated by the protease in question. All protease-generated sequences were analyzed for homology and distance to other cleavage sites and then blasted for homology using the
Six 4–6 week old Balb/C mice were immunized subcutaneously in the abdomen with 200 μL emulsified antigen (50 μg per immunization) using Freund's incomplete adjuvant (KLH-CGG- GVAPGIGPGG (Chinese Peptide Company, Beijing, China)). Immunizations were continued until stable titer levels were obtained. The mouse with the highest titer was selected for fusion and boosted intravenously with 50 μg immunogen in 100 μL 0.9% sodium chloride solution for three days before isolation of the spleen for cell fusion. The fusion procedure has been described elsewhere
The sequence GVAPGIGPGG, named ELM-2, was selected for antibody generation. Native reactivity and peptide-binding of the generated monoclonal antibodies were evaluated by displacement of human serum in a preliminary indirect ELISA using biotinylated peptides (Biotin- GVAPGIGPGG) on a streptavidin-coated microtitre plate and the supernatant from the growing monoclonal hybridoma. Tested were the specificities of clones towards the free peptide (GVAPGIGPGG), a non-sense peptide, and the elongated peptide (GVAPGIGPGGV). Isotyping of the monoclonal antibodies was performed using the Clonotyping System-HRP kit (Southern Biotech). The selected clones were purified using Protein G columns according to manufacturer's instructions (GE Healthcare Life Science).
Paraffin-embedded lung tissue from patients (n = 5) with fibrotic pulmonary diseases were provided by Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark and carotid arteries (n = 5) were provided by the University Hospital, Odense, Denmark. The carotid arteries are from internal carotid endarterectomy and are intima-media tissue. The tissues were from anonymous subjects.
After paraffin melting and rehydration, the sections were pretreated with citrate buffer (citrate acid monohydrate, pH 6.0) overnight at 60° C. The sections were blocked for 30 minutes with 0.5% TBS-casein (Casein, Thimerosol, TBS). The antibodies Anti-Elastin (monoclonal BA-4, Sigma-Aldrich, diluted 1∶200), ELM mAb (2 µg/mL), ELM-2 mAb (2ug/mL), CD68 (Sigma Aldrich) diluted 1∶100, negative control Mouse IgG1 (Dako) 15ug/mL diluted in TBS were incubated overnight at 4° C. The ELM and ELM-2 monoclonal antibodies were mixed with the related free peptide 1∶10. Immunoreactivity was detected using Envision+ System-HRP (Dako). Sections were counterstained with Mayer's Haematoxylin. Pictures were taken with an Olympus DP71 digital camera on an Olympus BX60 microscope using 4 or 10× magnification.
The selected monoclonal antibody was labelled with horseradish peroxidase (HRP) using the Lightning link HRP labelling kit according to the instructions of the manufacturer (Innovabioscience). A 96-well streptavidin plate was coated with 100 µL of 2.5 ng/mL Biotin- GVAPGIGPGG (Chinese Peptide Company, Beijing, China) dissolved in assay buffer (50 mM PBS, 1% BSA, 0.1% Tween-20, pH 7.4) and incubated for 30 minutes at 20°C. After washing, 20 µL of free peptide calibrator or sample were added in duplicate to appropriate wells, followed by 100 µL of 65 ng/mL conjugated monoclonal antibody, and incubated for 1 hour at 20°C. Finally, after washing, 100 µL tetramethylbenzinidine (TMB) (Kem-En-Tec) was added and the plate was incubated for 15 minutes at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. After each incubation step the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The TMB reaction was stopped by adding 100 µL of stopping solution (1% HCl) and measured at 450 nm with 650 nm as the reference. A master calibrator prepared from the synthetic-free peptide accurately quantified by amino acid analysis was used as a standard curve and plotted using a 4-parametric mathematical fit model.
Linearity was determined by 2-fold dilutions of quality control (QC) serum and plasma samples, as a percentage of recovery of the 100% sample. The lower limit of detection was determined from 21 zero serum samples (i.e. buffer) and calculated as the mean+3× standard deviation. The inter- and intra-assay variation was determined by 12 independent runs of 8 quality control serum samples, with each run consisting of two replicas of double determinations. The stability of serum was measured using three serum samples that had undergone freeze/thaw cycle ten times.
The ELM-2 neoepitope raised antibody was characterized using the MMP-9/12 cleaved elastin described under “
The level of ELM-2 and ELM neoepitopes was assessed in serum from cardiovascular patients using the ELM-2 and ELM ELISA. The serum samples were diluted 1∶2 for the assessment. In the CVD cohort, individuals were selected and grouped on the basis of prior knowledge about the presence or absence of well-defined clinical symptoms. Importantly, pre-analytical conditions and gender- and age- distribution were similar in all groups. Hypertension was defined as patients receiving antihypertensive medical treatment and diabetes patients receiving anti-diabetic medication. Systolic and diastolic blood pressure was measured on the same day as blood sampling. Heartscore and Agatston score were calculated in the two groups of patients undergoing cardiac CT. Troponin, fibulin-1, total cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL) and triglycerides were measured prior to the blood sampling. Blood samples were drawn in tubes with EDTA and centrifuged at 200 g for 10 min. Plasma was stored at −80°C until biochemical analysis.
120 individuals with different stages of atherosclerotic heart disease were selected from two larger studies (DanRisk
The DanRisk and DEFAMI protocols were approved by the Regional Scientific Ethical Committee for Southern Denmark (S-20080140 and S-20090082) and were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each participant.
The level of the ELM-2 ELISA was assessed in serum from patients diagnosed with COPD (n = 10) or IPF (n = 29) and compared with healthy controls (n = 11). The COPD and IPF serum samples were obtained as a part of the ”Lung Tissue Research Consortium” (
Comparison of serum levels of ELM and ELM-2 in the atherosclerotic heart disease cohort and the COPD/IPF cohort was performed using the two-sided non-parametric Wilcoxon rank sum test. Correlations were made by Spearman's correlation. Demographics of each patient-group were analysed by the one-way of analysis of variance (ANOVA) non-parametric Kruskal-Wallis test.
Differences were considered statistically significant if p<0.05. Area under the Receiver Operating Characteristic Curve (AUROC) was calculated using Graphpad Prism 6 software. Prism uses the method of Henley et al
The identified protease-generated elastin peptides were investigated for homology and cross-reactivity to other proteins to select sequences that were unique for elastin and the most representative of elastin degradation. The sequence GVAPGIGPGG, generated at amino acid position ‘552, was selected since it was identified by LC-MS/MS
The requirements for selected monoclonal antibodies for ELISA development were I) IgG subtype, II) specificity towards the neoepitope and not the elongated peptide or uncleaved elastin, III) native reactivity towards diseased human body fluids and not only to the synthetic peptide and IV) acceptable dilution recoveries in human body fluids. Based on these requirements an antibody recognizing the sequence GVAPGIGPGG was selected. The subtype was determined to be an IgG2b subtype. The monoclonal antibody did not show any affinity toward either the elongated peptides or the uncleaved elastin (
A) ELISA showing percent inhibition of the signal of the free peptide, elongated peptide and three human serum samples. The human samples were run undiluted and diluted 1∶2, 1∶4 and so forth as indicated by the dotted lines. B+C) Release of ELM-2 by MMP-9 and −12 cleavages as a function of time of human elastin from B) insoluble elastin and C) soluble elastin. The cleaved material was diluted 1∶10 in the assay.
By using the developed ELM-2 ELISA it was confirmed that the fragment was generated by MMP-9 and −12 (
Results of the technical evaluation of the ELM-2 ELISA are presented in table 1and show a technically robust assay with dilution recovery within the recommended range of +/−20%. The accuracy and precision were acceptable with low inter- and intra-assay variation.
Technical validation step | ELM-2 |
Target | MMP-9 and −12 degraded human elastin at amino acid number 552 |
Detection range/standard curve | 1.82–250ng/mL |
Dilution range of serum samples | 1∶2 is recommended |
Dilution range of plasma samples | 1∶2, 1∶3 and 1∶4 is recommended |
Dilution recovery of human serum |
98% |
Dilution recovery of human plasma |
97% |
Intra-assay variation** | 6.4% |
Inter-assay variation** | 12% |
Analyte stability*** | 96% |
LLOQ | 5.4 ng/mL |
Percentage dilution recovery was calculated as the mean of 4 human samples diluted 1∶2 and 1∶4. **Inter- and intra-assay validation was calculated as the mean variation between 8 individual determinations of each human serum sample. ***The stability of the analyte (human serum) was calculated as the mean of three different serum samples tested after freeze/thaw for one to 4 times.
A representative of the histological examination of adjacent sections of diseased carotid arteries is shown in
ELM and ELM-2 monoclonal antibodies were also mixed with the related free peptide as a control. A) Cross section of the plaque shoulder of a carotid artery from a 77 old man B) Left lung from a patient diagnosed with IPF. All pictures were taken with 10× magnification.
The demographics of the 120 patients in the cardiovascular cohort are shown in
Parameter | AMI | Non-AMI | CT-plusCa | CT-noCa | ANOVA |
(n = 30) | (n = 30) | (n = 30) | (n = 30) | p-value | |
Age | 64.5±8.5 | 64.2±7.9 | 60.3±0.3 | 60.3±0.4 | 0.00030*** |
Systolic blood pressure | 159±28 | 143±26 | 147±20 | 145±14 | 0.030* |
Diastolic blood pressure | 88±15 | 81±14 | 86±11 | 86±10 | 0.19 |
Agatstonscore | - | - | 1509±1070 | - | - |
Total cholesterol (mmol/L) | 4.7±1.3 | 4.9±0.90 | 5.5±1.2 | 5.2±1.2 | 0.074 |
LDL (mmol/L) | 2.9±1.2 | 2.7±0.8 | 3.3±1.0 | 3.1±1 | 0.16 |
HDL (mmol/L) | 1.2±0.3 | 1.5±0.7 | 1.4±0.5 | 1.3±0.4 | 0.073 |
Triglyceride (mmol/L) | 1.5±1.0 | 1.4±0.9 | 1.6±0.67 | 1.9±1.3 | 0.11 |
HeartscoreA | - | - | 8.3±6.2 | 6.3±4.5 | 0.061 |
Troponin I (µg/mL) | 0.62±1.20 | - | - | - | - |
Values are mean ± standard deviation. The data were analysed using the one-way of analysis of variance (ANOVA) non-parametric Kruskal-Wallis test. * p<0.05; ** p<0.01; *** p<0.001,.AHeart score is an assessment on risk of cardiovascular disease based on age, systolic blood pressure, total cholesterol in mmol/L, and smoking status. LDL = low density lipoprotein, HDL = high density lipoprotein, AMI = acute myocardial infarction, CT-plusCA = subclinical coronary calcium shown on CT scans, CT-noCa = no coronary calcium detectable on a CT scan.
The serum samples from the CVD study were measured by the newly developed ELM-2 ELISA and the formerly developed ELM ELISA (
Human serum from patients with acute myocardial infarction (AMI) (n = 30), non-AMI (n = 30), coronary calcium shown on CT scans (CT-plusCA)(n = 30) and no coronary calcium detectable on a CT scan (n = 30). Bars indicate mean level. Groups were compared by Wilcoxon rank sum test. A)ELM-2, B) ELM and C) The Spearman correlation between ELM and ELM-2. Data are shown as mean±SD with 95% confidence intervals. ** p<0.01.
While a small correlation was found between ELM and HeartScore (r = 0.19, p = 0.045), the ELM and ELM-2 showed no significant correlations with age, systolic and diastolic blood pressure, Agatston score, total cholesterol, LDL, HDL, triglyceride and fibulin-1 (data not shown). The levels of the two elastin degradation markers neither showed significant correlation with troponin I levels nor did they correlate with the risk factors of hypertension, diabetes and hypercholesteremia (
A) Spearman correlation between C-reactive protein and ELM, B) Spearman correlation between C-reactive protein and ELM-2. * p<0.05, C) Biological validations of ELM versus smoking habits, D) Biological validations of C-reactive protein versus smoking habits. * p<0.05; ** p<0.01; C-reactive protein was only measured in the acute myocardial infarction (AMI) and non-AMI groups.
Parameter | ELM | ELM-2 | ||
All patients | All patients | |||
Sex | 0.65 | 0.31 | ||
Hypertension | 0.47 | 0.94 | ||
Diabetes | 0.22 | 0.94 | ||
HypercholesteremiaA | 0.070 | 0.88 | ||
Smoking# | - Current/Never | 0.013 |
0.34 | |
- Former/Never | 0.048 |
0.15 | ||
- Current/Former | 0.025 |
0.0064** | ||
Statins | 0.29 | 0.66 | ||
Angiotensin-converting-enzyme inhibitor | 0.63 | 0.42 | ||
Angiotensin II receptor antagonist | 0.43 | 0.28 | ||
Beta-blocker | 0.17 | 0.57 | ||
Calcium channel blockerB | 0.057 | 0.71 | ||
ThiazideC | 0.070 | 0.13 | ||
Loop-diuretics | 0.52 | 0.13 |
p<0.05; ** p<0.01; AMean of ELM release is 17,2 ng/mL for patients having hypercholesteremia and 21,3 ng/mL for patients not having it. BMean of ELM release is 25.8 ng/mL for patients taking Calcium channel blocker and 19,4 ng/mL for patients not taking the drug. CMean of ELM release is 19,6 ng/mL for patients taking thiazides and 20,6 ng/mL for patients not taking the drug. #Levels of mean of ELM versus smoking habit is shown in
No correlation was observed between the type of cardiovascular medications and ELM or ELM-2 (
To investigate the diagnostic value of ELM, ELM-2 and previously demonstrated cardiac markers, ROC values were calculated (
Parameter | AMI vs non-AMI | ||
AUROC | Std.error | p | |
Troponin | 0.92 | 0.039 | <0.0001*** |
ELM-2 | 0.70 | 0.069 | 0.0078** |
HDL | 0.69 | 0.074 | 0.020 |
Systolic blood pressure | 0.67 | 0.071 | 0.022 |
Diastolic blood pressure | 0.63 | 0.073 | 0.088 |
ELM | 0.61 | 0.074 | 0.15 |
Osteoprotegerin | 0.61 | 0.075 | 0.16 |
C-reactive protein | 0.59 | 0.10 | 0.36 |
Heartscore | 0.58 | 0.080 | 0.29 |
Triglyceride | 0.57 | 0.081 | 0.40 |
LDL | 0.56 | 0.080 | 0.45 |
Ostepontin | 0.55 | 0.076 | 0.50 |
Total cholesterol | 0.52 | 0.079 | 0.79 |
AUROC = Area Under the Receiver Operating Characteristic Curve. Data are show as the AUROC, a probability of correct diagnosis by each marker. The P value indicates significance of the AUROC diagnosis compared to the null hypothesis which is an area of 0.5.
p<0.05, ** p<0.01, *** p<0.01.
A representative of the histological examination of adjacent sections of fibrotic lung tissue is seen in
Mean levels of ELM-2 were not elevated in patients with COPD (6.9 ng/mL) or IPF (5.3 ng/mL) compared to controls (4.9 ng/mL) (
Human serum from patients with chronic obstructive pulmonary disease (COPD)(n = 10) and idiopathic pulmonary fibrosis (IPF)(n = 29) compared with controls (n = 11). Bars indicate mean level. A) ELM-2, B) ELM (Data have been published with permission from Skjøt-Arkil et al
To investigate the diagnostic potential of ELM and ELM-2 in the lung disease cohort, ROC values were calculated (
Para meter | Control vs COPD | Control vs IPF | ||||
AUROC | Std.error | p | AUC | Std. error | p | |
ELMA | 0.97 | 0.032 | 0.00025 |
0.90 | 0.048 | 0.000 11 |
ELM-2 | 0.69 | 0.13 | 0.15 | 0.59 | 0.12 | 0.40 |
p<0.01, AData modified from Skjøt-Arkil et al
This study shows that two different MMP generated fragments of elastin were differently related for two different diseases. Our data indicated that in cardiovascular tissue, elastin degradation by MMP-9 and-12 leads to the release of the ELM-2 neoepitope while the degradation in pulmonary diseases leads to the release of the ELM neoepitopes.
Elastin degradation has been investigated by several groups
The histological analysis of the neoepitopes distribution revealed that ELM and ELM-2 are located in the same areas as the total elastin and that macrophages are mainly present in the plaque shoulder in arteries close to the ELM and ELM-2-stained fragmented elastic lamellae. The same co-localization was observed in the diseased lung tissue, where macrophages were located in the alveolus air spaces close to the fibres. Since macrophages are the main source of MMP-12
The cardiovascular samples relied on a well-characterized cohort in which all samples were collected under the same standard procedure and covered patients with various degrees of atherosclerosis, from seemingly none to chronically ill. The non-AMI group however, comprised of heterogenous patients whom had been suspected to have had an AMI, when first arrived at the hospital. It is not known if they instead had atherosclerosis, renal failure, myosis or sepsis. Some of the non-AMI patients had different degrees of CVD and a fraction had heart failure, all of which might have influenced their biomarker levels. The patients without any coronary calcium are the least diseased group and functioned as controls in our analysis. A borderline significant difference between the AMI group and the no calcium group suggests that a study in a larger patient group and thereby with more statistical power could confirm this difference. The level of ELM-2 was not elevated in the CT-plusCA group, which would have been expected for an atherosclerotic marker. This questions if ELM-2 might be a marker of acute heart damage. The specific activity of MMP-9 and −12 and their effect on cardiac remodeling during either physiology or pathology have to date not been described in the literature. MMP-12 expression in cardiac valves taken from patients suffering from infective endocarditis has been shown to be elevated
One thing that might differ in the four CVD groups is drug intake, which could influence the levels of a biochemical marker. The patients included in the cohort received various cardiovascular medications such as statins, beta blockers, diuretics and antihypertensives. However, none of these treatments correlated significantly with ELM or ELM-2. Calcium channels blockers, though, did show a borderline significant difference with ELM levels, but not with ELM-2, which could be explained by a different effect of the blockers on pulmonary arteries.
ELM and ELM-2 were not found to correlate with the cardiovascular risk factors which imply that ELM and ELM-2 are independent of these factors. The lack of correlation may also be attributed to the fact that the ELM and ELM-2 assays measure MMP-degraded fragments of elastin and not the total protein as do many of the other CVD biomarkers. This protein-protease combination may make the neoepitopes more disease-specific and reveals different profiles of the remodelling in cardiovascular and pulmonary diseases. This has been shown in other diseases such as in patients with ankylosing spondylitis, where the degradation markers of CRP, the CRP-MMP and CRP-Cat, had a higher diagnostic value than the total CRP
The diagnostic power of ELM and ELM-2 in the COPD/IPF cohort reinforces the different profiles of the two elastin fingerprints. In patients with pulmonary diseases, ELM levels were up to 575% higher than the levels of ELM-2. One major limitation of the current COPD/IPF study was the relatively small sample size and the limited clinical information obtained. Thus, these preliminary findings need to be validated in larger clinical settings for the diagnostic utility.
One complicating factor in the use of ELM and ELM-2 is that elastin is expressed in the arteries, lung tissues, skin and tendons
The tissue-specific alternative splicing of tropoelastin leads to variations in incorporation of exons or coding regions into mRNA and therefore in the production of elastin isoforms. This might be one of the reasons for cleavage at amino acid position ‘441 in pulmonary diseases generating ELM and cleavage at amino acid position 552 in cardiovascular diseases. The neoepitope technology, measuring specific protein degradation fragments, allows for assessment of specific proteolytic activity in given tissues, provided that the sequence is unique for one or a few proteases. Ultimately, a panel of biomarkers may be needed to characterize different aspects of cardiovascular and pulmonary diseases in patients, including prognosis, diagnosis and assessment of the efficacy of intervention.
In conclusion, the newly identified ELM-2 was highly related to cardiovascular diseases and might be a potential non-invasive biomarker of AMI. ELM is, in contrast, related to lung tissue degradation, indicating that elastin degradation by MMPs at different amino acid sites may provide different biological information and degradation profiles most likely depending of the disease in question. Further testing in a larger cohort is needed to confirm these preliminary findings.
The authors would like to acknowledge The Lung Tissue Research Consortium (LTRC) and The National Heart, Lung and Blood Institute (NHLBI) for kindly providing the COPD and IPF samples.