10 research outputs found
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
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
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
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
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<sub>S</sub> = 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<sub>S</sub> = - -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<sub>S</sub> = -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<sub>S</sub> = -0.29; P = 0.17).</p
Representative zymograms showing gelatinolytic activity in protein extracts gained from aortic tissue of patients with ascending aortic/aortic root aneurysm.
<p>Protein extracts from patients with ascending aortic/aortic root aneurysms were analyzed for existence of different MMP-2 isoforms. Lane 1: Human full length MMP-2 as delivered. Lane 2: Human full length MMP-2 was activated with 2 mM APMA for 2 hours at 37°C prior to electrophoresis. Asterisk indicates 67 kDa intermediate MMP-2 isoform. <b>A.</b> 7 representative of 24 analyzed samples are shown. Signals corresponding to pro-MMP-2 were detected in every sample. Signals relatable to the active 65kDa MMP-2 were detected in all samples as well (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164308#pone.0164308.s001" target="_blank">S1 Fig</a>). <b>B.</b> pro- and active MMP-2 in patients with bicuspid and tricuspid aortic valves. 15 of the analyzed samples were taken from patients with triicuspid aortic valves and 9 of the samples were from patients with bicuspid aortic valves. In both groups, gelatinolytic activities for either pro-MMP-2 and active MMP-2 were detectable. <b>C.</b> Activation of aortic tissue MMP-2. To test if tissue MMP-2 can be activated similar to the human full length MMP-2 used as a standard, protein extracts were incubated with 2mM APMA for either 2 or 24 hours. Different final dilutions of protein extracts were analyzed as indicated. Aortic tissue MMP-2 was split into three fragments similar to the human full length MMP-2. Extending APMA incubation time did not change the band pattern obtained after activation.</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<sub>P</sub> = 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<sub>P</sub> = - 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<sub>P</sub> = - 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<sub>P</sub> = - 0.06; P = 0.8). Correlation coefficient is almost zero.</p
Representative zymograms revealing gelatinolytic activity in serum after incubation with APMA for different incubation times.
<p>Serum from patients with ascending aortic aneurysms and healthy controls was incubated with 2 mM APMA for different times to exclude that active MMP-2 can be generated from serum MMP-2 after longer incubation times. Lane 1: Human full length MMP-2 after activation with 2mM APMA for 2 hours. Lane 2: Human full length MMP-2 as delivered. <b>A.</b> Serum from patients with ascending aortic aneurysms was incubated with 2mM APMA for 15–240 minutes. All lanes show bands corresponding to pro-MMP-2 and bands corresponding to intermediate MMP-2. No signals for active 65kDa MMP-2 became visible after prolonged incubation time with APMA. <b>B.</b> Serum from healthy controls was incubated with 2mM APMA for 15–240 minutes. Lanes show bands corresponding to pro-MMP-2 and intermediate MMP-2. No signals relatable to active MMP-2 appeared after prolonged incubation times with APMA. <b>C.</b> Serum from patients with ascending aortic aneurysms was incubated with 2mM APMA for 0,25–24 hours. All lanes show bands corresponding to pro-MMP-2 and intermediate MMP-2. No signals relatable to active MMP-2 were detectable.</p