Matrix Metalloproteinase-2 Isoforms Differ within the Aortic Wall of Ascending Aortic Aneurysms Associated with Bicuspid Aortic Valve

The pathogenesis of ascending thoracic aortic aneurysm (aTAA) is thought to differ between patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV), and one of the causes is different hemodynamics. Influenced by hemodynamics, the tissue levels of proteins associated with aTAA might differ between aTAAs with BAV and TAV and between different localities within the aortic wall. We therefore analyzed aTAA tissue levels of MMP-2 (matrix metalloproteinase-2) isoforms (Pro-MMP-2, active MMP-2, and total MMP-2) and tissue levels of MMP-14, TIMP-2 (tissue inhibitor of metalloproteinase-2), MMP-9, and TIMP-1 in 19 patients with BAV and 23 patients with TAV via gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. TAV and BAV groups’ protein levels did not differ significantly. Whereas the TAV group exhibited no significant differences in protein levels between the aneurysm’s anterior and posterior parts, the BAV group revealed significantly higher levels of Pro-MMP-2, total MMP-2, and TIMP-2 in the aneurysm’s posterior parts (mean Pro-MMP-2 200.52 arbitrary units (AU) versus 161.12 AU, p=0.007; mean total MMP-2 235.22 AU versus 193.68 AU, p=0.002; mean TIMP-2 26.90 ng/ml versus 25.36 ng/ml, p=0.009), whereas the other proteins did not differ significantly within the aortic wall. Thus, MMPs are distributed more heterogeneously within the aortic wall of aTAAs associated with BAV than in those associated with TAV, which is a new aspect for understanding the underlying pathogenesis. This heterogeneous protein level distribution might be attributable to differences in the underlying pathogenesis, especially hemodynamics. This result is important for further studies as it will be essential to specify the location of samples to ensure data comparability regarding the main goals of understanding the pathogenesis of aTAA, optimizing treatments, and establishing a screening method for its potentially deadly complications

Acute, Chronic, and Treated Aortic Diseases Present Distinguishable Serum Proteome Fingerprints with Protein Profiles That Correlate with Disease Severity

Aortic diseases are a rare but potentially life-threatening condition. We present a serum proteomic study for a spectrum of aortic diseases including thoracic aortic aneurysms (n = 11), chronic dissections (n = 9), acute aortic dissections (n = 11), and surgically treated dissections (n = 19) as well as healthy controls (n = 10) and patients of coronary heart disease (n = 10) to represent non-aortic cardiovascular disease. In total, we identified and quantified 425 proteins across all 70 samples. The different aortic diseases represented distinguishable proteome profiles. We identified protein clusters that positively or negatively correlate with disease severity, including increase of cytosolic tissue leakage proteins and decrease of components of the coagulation and complement system. Further, we identified a serum proteome fingerprint of acute aortic dissections, consisting, among others, of enriched inflammatory markers such as C-reactive protein and members of the S100 protein family. The study underlines the applicability of serum proteomics for the investigation of aortic diseases and highlights the possibility to establish disease-specific prognostic markers

MMP-2 Isoforms in Aortic Tissue and Serum of Patients with Ascending Aortic Aneurysms and Aortic Root Aneurysms

<div><p>Objective</p><p>The need for biological markers of aortic wall stress and risk of rupture or dissection of ascending aortic aneurysms is obvious. To date, wall stress cannot be related to a certain biological marker. We analyzed aortic tissue and serum for the presence of different MMP-2 isoforms to find a connection between serum and tissue MMP-2 and to evaluate the potential of different MMP-2 isoforms as markers of high wall stress.</p><p>Methods</p><p>Serum and aortic tissue from n = 24 patients and serum from n = 19 healthy controls was analyzed by ELISA and gelatin zymography. 24 patients had ascending aortic aneurysms, 10 of them also had aortic root aneurysms. Three patients had normally functioning valves, 12 had regurgitation alone, eight had regurgitation and stenosis and one had only stenosis. Patients had bicuspid and tricuspid aortic valves (9/15). Serum samples were taken preoperatively, and the aortic wall specimen collected during surgical aortic repair.</p><p>Results</p><p>Pro-MMP-2 was identified in all serum and tissue samples. Pro-MMP-2 was detected in all tissue and serum samples from patients with ascending aortic/aortic root aneurysms, irrespective of valve morphology or other clinical parameters and in serum from healthy controls. We also identified active MMP-2 in all tissue samples from patients with ascending aortic/aortic root aneurysms. None of the analyzed serum samples revealed signals relatable to active MMP-2. No correlation between aortic tissue total MMP-2 or tissue pro-MMP-2 or tissue active MMP-2 and serum MMP-2 was found and tissue MMP-2/pro-MMP-2/active MMP-2 did not correlate with aortic diameter. This evidence shows that pro-MMP-2 is the predominant MMP-2 species in serum of patients and healthy individuals and in aneurysmatic aortic tissue, irrespective of aortic valve configuration. Active MMP-2 species are either not released into systemic circulation or not detectable in serum. There is no reliable connection between aortic tissue—and serum MMP-2 isoforms, nor any indication that pro-MMP-2 functions as a common marker of high aortic wall stress.</p></div

Characteristics of serum control group.

<p>Characteristics of serum control group.</p

Correlation between serum MMP-2 and different tissue MMP-2 isoforms Pearson`s correlation analyses between serum MMP-2 and different tissue MMP-2 isoforms were performed to test for any a measurable linear connection between tissue and serum MMP-2.

<p>r_P = Pearson correlation coefficient; AU: arbitrary units. <b>A.</b> Pearson correlation analysis revealed no significant linear relationship between serum MMP-2 and total tissue MMP-2 (r_P = - 0.15; P = 0.5). Correlation coefficient is greater than for tissue pro-MMP-2 and tissue active MMP-2 but without a statistical significance. <b>B.</b> Pearson correlation demonstrated no significant linear relationship between serum MMP-2 and tissue pro-MMP-2 (r_P = - 0.04; P = 0.9). Correlation coefficient is close to zero. <b>C.</b> Pearson correlation analysis showed no significant linear relationship between serum MMP-2 and tissue active MMP-2 (r_P = - 0.06; P = 0.8). Correlation coefficient is almost zero.</p

Validation of gelatin zymography.

<p><b>A.</b> Dilution series of MMP-2 standard. A standard curve was generated from human full length MMP-2 from 0–2.5 ng of total MMP-2. Pixel density measured from zymograms showed a linear relationship with the MMP-2 amount. <b>B.</b> Representative zymogram of standard dilution series reveals decreasing signal strength in conjunction with declining MMP-2 amount. <b>C.</b> Dilution series of protein extracts gained from patients with ascending aortic aneurysms. Protein extracts were diluted from 2fold until 16fold. The measured pixel density in the zymograms showed a linear decrease at dilution factors between 2fold and 8fold. Measurements of 4 independent dilutions illustrated the assay`s reproducibility. Each value represents the mean of two samples at the same dilution measured in the same gel. <b>D.</b> Representative zymogram of protein extract dilution series shows decreasing signal strength in conjunction with an increasing dilution factor. <b>E.</b> Representative serum and tissue zymograms showing different gelatinolytic activities. serum zymogram shows gelatinolytic activities corresponding to pro-MMP-2. Tissue zymogram shows gelatinolytic activities corresponding to pro-MMP2 and active MMP-2. Westernblot confirmed that the detected bands were MMP-2. <b>1,2</b>: patient sample. <b>S:</b> Human full length MMP-2.</p

Serum MMP-2 in patients with ascending aortic aneurysms and healthy controls.

<p><b>A.</b> Serum MMP-2 levels in patients and healthy controls measured by ELISA. Two-tailed t-test revealed a significant difference in serum MMP-2 levels in both groups (P = 0,00138). MMP-2 serum levels were lower in patients than in healthy controls (190.3 +/- 48.3 vs 240.3 +/- 46.4). <b>B.</b> Representative zymogram showing gelatinolytic activity in serum from 6 patients with ascending aortic aneurysms. Lane 1: Human full length MMP-2 activated with 2 mM APMA for 2 hours. Lane 2: Human full length MMP-2 as delivered. Lane 3: one representative serum sample activated with 2 mM APMA. Lane 4–9: serum from patients with ascending aortic aneurysms. Asterisk indicates 67 kDa intermediate MMP-2 isoform present in MMP-2 standard with and without APMA incubation and in serum incubated with APMA. Lanes with serum samples show gelatinolytic activity corresponding to pro-MMP-2 only. <b>C.</b> Zymogram showing gelatinolytic activity from 6 serum samples from healthy controls. Lane 1: Human full length MMP-2 activated with 2 mM APMA. Lane 2 Human full length MMP-2 as delivered. Lane3: one representative serum sample activated with 2 mM APMA Asterisk indicates 67 kDa intermediate MMP-2 isoform present in human full length MMP-2 with and without APMA incubation and in serum incubated with APMA. Lanes with serum samples show gelatinolytic activity corresponding to pro-MMP-2 only.</p

Patient characteristics.

<p>Patient characteristics.</p

Correlation between tissue total-MMP-2, tissue pro-MMP-2 and tissue active- MMP-2 and ascending aortic diameter.

<p>Different MMP-2 isoforms were correlated to ascending aortic diameter to test, whether a certain MMP-2 isoform can represent changing mechanical properties in the expanding aortic wall. r_S = Spearman correlation coefficient; AU: Arbitrary units. <b>A.</b> Spearman correlation analysis of ascending aortic diameter and tissue total MMP-2 showed that there`s no significant relationship between both these parameters. Correlation coefficient reaches levels for a very weak negative correlation. (r_S = - -0.22; P = 0.29). <b>B.</b> Spearman correlation analysis of ascending aortic diameter and tissue pro-MMP-2 revealed no significant relationship between those two parameters. Correlation coefficient reaches levels for a very weak correlation. (r_S = -0.27; P = 0.2). <b>C.</b> Spearman correlation analysis of ascending aortic diameter and 65kDa active MMP-2 revealed no significant relationship between these two parameters. Correlation coefficient also reaches levels for a very weak correlation and is slightly higher as for tissue total MMP-2 and tissue pro-MMP-2 but without statistical significance. (r_S = -0.29; P = 0.17).</p

Representative zymograms showing gelatinolytic activity in serum from patients with ascending aortic/aortic root aneurysms and healthy controls.

<p>Increasing amounts of total protein were analyzed to detect possible signals close to detection limit. Lane 1: Human full length MMP-2 as delivered. Lane 2: Human full length MMP-2 activated with 2 mM APMA for 2 hours. Asterisk indicates 67 kDa intermediate MMP-2 isoform present in MMP-2 standard with and without APMA incubation and in serum incubated with APMA. <b>A.</b> Serum from patients with ascending aortic aneurysms was analyzed in total protein amounts from 15 μg to 80 μg. Whereas signals corresponding to pro-MMP-2 increased, no signals relatable to active MMP-2 became visible. <b>B</b>. Serum from patients with ascending aortic aneurysms was incubated with 2mM APMA for 2 hours at 37°C and analyzed in increasing amounts of total protein from 15 μg to 40 μg. Signals related to pro-MMP-2 and intermediate MMP-2 became stronger with increasing amount of total protein. But no signal relatable to the 65kDa active MMP-2 appeared. <b>C.</b> Serum from healthy controls was analyzed in total protein amounts of 15 μg until 80 μg. signals increased in the same way as described for the serum samples from patients with ascending aortic aneurysms. No signals corresponding to active MMP-2 appeared. <b>D.</b> Serum from healthy controls was analyzed in total protein amounts of 15 μg until 40 μg after incubation with APMA for 2 hours at 37°C. Signals for pro-MMP-2 and intermediate MMP-2 increased with protein amount but no signals relatable to active MMP-2 appeared.</p