Expression of matrix metalloproteinase-12 in aortic dissection

Abstract

Background: Aortic dissection(AD) is an acute process of large blood vessels characterized by dangerous pathogenic conditions and high disability and high mortality. The pathogenesis of AD remains debated. Matrix metalloproteinase-12 (MMP-12) participates in many pathological processes such as abdominal aortic aneurysm, atherosclerosis, emphysema and cancer. However, this elastase has rarely been assessed in the presence of AD. The aim of the present study was to investigate the expression of MMP-12 in aortic tissue so as to offer a better understanding of the possible mechanisms of AD.

Methods: The protein expression levels of MMP-12 were analyzed and compared in aorta tissue and the blood serum samples by reverse transcription polymerase chain reaction(RT-PCR), Western blotting, immuno-histochemistry, fluorescence resonance energy transfer ( FRET ) activity assay and enzyme-linked immuno sorbent assay ( ELISA ), respectively. Ascending aorta tissue specimens were obtained from 12 patients with an acute Stanford A-dissection at the time of aortic replacement, and from 4 patients with coronary artery disease (CAD) undergoing coronary artery bypass surgery. Meanwhile, serum samples were harvested from 15 patients with an acute Stanford A-dissection and 10 healthy individuals who served as the control group.

Results: MMP-12 activity could be detected in both AD and CAD groups, but the level in the AD group was higher than those in the CAD group (P < 0.05). MMP-12 proteolysis existed in both serum samples of the AD and healthy groups, and the activity level in the AD group was clearly higher than in the healthy group (P < 0.05). For AD patients, MMP-12 activity in serum was higher than in the aorta wall (P < 0.05). MMP-12 activity in the aortic wall tissue can be inhibited by MMP inhibitor v (P < 0.05).

Conclusion: The present study directly demonstrates that MMP-12 proteolytic activity exists within the aorta specimens and blood samples from aortic dissection patients. MMP-12 might be of potential relevance as a clinically diagnostic tool and therapeutic target in vascular injury and repair.

Figure 1

The domain structures of human MMP-12. The latent form of human MMP-12(top), the active form of MMP-12 with molecular weight 45 KDa (middle), and the catalytic domain of MMP-12 with molecular weight 22 KDa (bottom) are illustrated. The precise amino acid residues are known from protein sequencing.

Figure 2

Agarose gel electrophoresis analysis of PCR fragment obtained from human aorta. MMP-12 gene fragments were amplified from human aorta wall by RT-PCR. PCR amplifications were then analyzed by electrophoresis using 1% agarose gel stained with Gelview. The gel was viewed on a Tanon 3500R Gel Documentation System and recorded. (A) cDNA corresponding to MMP-12 in human aorta was PCR-amplified using primers for MMP-12. (B) cDNA corresponding to GAPDH in human aorta was PCR-amplified using primers for GAPDH. Lanes 1–2: Aorta wall of coronary heart disease patient; Lanes 3–10: AD; Lane M: DNA ladder; Lane 11: negative control.

Figure 3

Immunohistochemical staining. Ascending aorta specimen in the entry site of the dissection from a AD patients were sectioned and labeled with MMP-12 antibodies as indicated. Sections were developed with alkaline phosphatase anti-alkaline phosphatase (APAAP) techniques and counterstained with hematoxylin. Total cells and positive cells were counted under the microscope. Arrows indicate examples of MMP-12 positive cells, A (magnification×200), B, C, D (magnification×400).

Figure 4

MMP-12 expression in the human aorta wall. Lysates from tissues samples collected from AD and non-AD patients were separated on 12% SDS-PAGE gels and were analyzed by immunoblotting using an antihuman MMP-12 monoclonal antibody. Recombinant MMP-12 expressed in E. coli was used as positive control. Lane 1 : molecular weight markers; Lane 2–7: Western blot products from the aorta walls of AD patients; Lanes 8–9 : Western blot products from the coronary disease patient’s aorta walls; lane 10: Recombinant human MMP-12.

Figure 5

Effects of different concentration of Ca²⁺ ions on MMP-12 activity. The MMP-12 assay was based on the MMP-12 hydrolysis of FRET substrate ( AnaSpec, Inc, Fremont, CA,USA) .A total of 100 μl reaction mixture consisting of 1 μl (100 ng) MMP-12, different concentrations of Ca²⁺ ions (0-40 mM) and 50 μl of buffer ( AnaSpec, Inc, Fremont, CA,USA)were incubated at 37°C for 10 min. Fluorescence was read at Ex/Em wavelengths = 340 nm/490 nm for 60 min. RFU: Relative Fluorescence Unit.

Figure 6

Continuous assay for MMP-12. Continuous fluorometric assay of MMP-12 in (A) aortic specimen from AD(n=12) and CAD(n=4), (B) blood serum sample from AD (n=15) and healthy(n=10) , (C) aortic specimens and sera from AD, (D) aortic specime n from AD incubated with MMP Inhibitor v. Assays were performed at RT in a total of 100 μl assay buffer containing various optimized sample volumes, by activating MMPs with 1mM APMA , inhibiting MMPs with 1 μM MMP Inhibitor v, or 100 ng of recombinant MMP-12 served as a control. Fluorescence was read at Ex/Em wavelengths = 340 nm/490 nm for 60 min. RFU: Relative Fluorescence Unit; AD: aortic dissection; CAD: coronary artery disease.

Figure 7

MMP-12 levels in the aortic specimens and sera. MMP-12 levels were measured using ELISA as described. (A) Level of MMP −12 in the aorta wall. Cases 1–10 are the aorta tissues of AD, and Cases 11–16 are aorta tissues of CAD. (B) level of MMP-12 in sera. Cases 1–15 are the sera of AD, and cases 16–25 are sera of healthy patients.