The institutional research ethics committee of Kunming Medical University approved the study, and written informed consent was obtained from each subject. This study was conducted in accordance with the ethical requirements.
Intraoperative aortic samples were obtained from 12 patients with Stanford Type A acute aortic dissection as experimental group. These patients received Bentall operation in the period from April 2010 to June 2011 at the No. 1 Hospital Affiliated to Kunming Medical University. 9 were male and 3 were female (mean age, 44.7 years); 8 patients (66.67%) had a history of hypertension and 7 patients (58.33%) used to smoke. All of them had a color Doppler ultrasonic examination and computed tomography and all were diagnosed as acute aortic dissection Stanford A. Control aortic tissues were obtained from 4 cases of CAD without aorta wall damage who underwent coronary artery bypass surgery; samples were taken by punching a hole on the aortic wall tissue with a hole punch. All patients with CAD were male, aged 65.5±3.3; 2 patients had a history of hypertension and 3 patients had a history of smoking. Both groups were ruled out of the possibility of inflammation of the chest cavity, Marfan syndrome, bicuspid aortic valve abnormalities, cardiomyopathy, chronic pulmonary heart disease and infectious endocarditis possibilities.
Serum samples were obtained from 15 patients with Stanford type A acute aortic dissection as the experimental group. All patients were hospitalized in the period from April 2010 to June 2011 at No. 1 Hospital Affiliated to Kunming Medical University. Ten age-matched healthy people were included as a control group.
Recombinant human MMP-12 (GenBank: AAI43774.1) was obtained in our laboratory. Briefly, the prokaryotic plasmid pET -17×b - MMP-12 was constructed and transformed into E. coli strain BL21 (DE3). The recombinant protein expressed in E. coli consisted of 455 amino acid residues from Leu17 to Cys470 (Figure
), including the prodomain, catalytic domain, the junction between catalytic domain and hemopexin domain, and the hemopexin-like domain. Recombinant human MMP12 (54 KDa ) was expressed in the form of inclusion bodies. After refolding, it underwent self-activation to generate two active forms with molecular weights of 45 KDa and 22 KDa.
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 (more ...)
Reverse transcription-polymerase chain reaction
Aortic wall tissues (400 mg) was ground to a fine powder using a mortar and pestle in liquid nitrogen, to which a certain amount of Trizol (Gibco Brl, Rockville MD, USA) was added to extract RNA according to the manufacturer’s instructions. Approximately 1.5 μg of total RNA from each sample was used to conduct reverse transcription reaction in a 50 μl volume using the RNA PCR KitVer.3.0 (Takara Biotechnology, Dalian, China). The reaction mixture was incubated at 42°C for 2 h and the reaction was terminated by heating to 99°C for 5 min. The synthesized cDNA was used for PCR amplification or stored at −80°C for further analysis.
PCR primers (GenScript, Nanjing, China) were designed to amplify MMP-12 cDNA. The forward primer 5'-CGATGAGGACGAATTCTGGACTAC-3' is situated in the exon 4, and the reverse primer 5'-GGTTCTGAATTGTCAGGATTTGGC-3' is situated in the exon 6. The primer sequences correspond to residues Asp211 to Pro292 in the catalytic domain of human MMP-12. The PCR reaction was performed in a 50 μl volume containing 0.5 mM of each primer, 5 μl PCR buffer, 0.5U Ex-Taq DNA polymerase (Takara Biotechnology, Dalian, China). Reaction conditions included initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min, followed by a 10 min final extension at 72°C. PCR products were separated on 1.0% agarose gels and visualized by Gelview (Bioteke,Beijing,China) staining.
The quality of the total RNA was determined by RT-PCR for the house-keeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The primer sequences were as follows: GAPDH sense, 5'-CCCATCACCATCTTCCAGGAGCG-3'; anti-sense, 5'-GGCAGGGATGATGTTCTGGAGAGCC-3' (GenScript, Nanjing, China). The PCR reaction contained 10.0 μl of cDNA, 0.5 μM of each primer, 5 μl PCR buffer, 0.5 U Ex-Taq DNA polymerase in a final volume of 50 μl using the following conditions: 95°C for 5 min, 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a 10 min final extension at 72°C.
Aorta wall tissues (4 cm)were obtained at the aortic dissection site, The blood was washed from the newly obtained sample with 0.9% sodium chloride, dipped the sample into 10% Formalin solution for 24 h for serial sections of 4 μm thick, then, stained with hematoxylin for observation. The antibody for MMP-12 (Epitomics, Inc, CA, USA) was a rabbit monoclonal antibody generated by immunizing rabbit with the synthetic peptide corresponding to residues on the C-terminus of human MMP-12; After incubating with Biotin-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 15 min at room temperature(RT), the streptavidin-alkaline phosphatase-biotin complex solution was applied to all sections for 15 min according to the manufacturer’s instructions. Positive results of immunohistochemistry indicated yellowish brown or brown particles presented in the cytoplasm. A random selection 10 fields were viewed under a 400× microscope and calculated the percentage of positive cells was calculated. Rating criteria: no positive expression scored 0 point (negative), positive expression less than 25% scored 1 point (weak positive), positive expression higher than 25% and lower than 50% scored 2 points (positive) and positive expression higher than 50% scored 3 points (strong positive).
Aorta wall tissue (400 mg) was obtained and ground into powder in liquid nitrogen, to which 4 ml Trizol was added and mixed, then it was centrifuged at 11,000 rpm for 15 min. The supernatant was then spun for an additional 10 min and collected. The protein (15 μl) from the tissue samples was immersed in boiled water for 15 min. The total protein was separated on 12% gels. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Recombinant human MMP-12 was used as positive control. Briefly, after blocking for 1 h at 37°C in 10 mM Tris–HCl pH 7.5, 100 mM NaCl, triethanolamine-buffered saline (TBS) containing 3% bovine serum albumin and 0.1% Tween 20, the membrane was incubated with an antibody (rabbit anti-human MMP-12 mAb, Epitomics) at 1:1000 dilution for 2 h at RT . After repeated washes, the membrane was incubated with Biotin-conjugated goat anti- rabbit IgG (Santa Cruz, American) at 1:1000 dilution for 1 h at RT , followed by incubation with Streptavidin-alkaline phosphatase (R&D, USA) for 1 h at RT. Finally, the membrane was incubated with BCIP/NBT (Calbiochem, Bad Soden, Germany) in the darkness.
MMP-12 FRET activity assay
The activity of MMP-12 was assessed using a peptide-based FRET assay (EnzoLyte MMP-12 Assay Kit, AnaSpec, Inc, Fremont, CA,USA) in accordance with the manufacturer’s protocols and fluorescence intensity was determined using a fluorescence analyzer (Fluoroskan Ascent 2.6, Thermo) at excitation/emission wavelengths of 340 nm/490 nm. Briefly , aorta wall tissues (200 mg ) in assay buffer(0.5 ml component D, AnaSpec) were ground in liquid nitrogen, centrifuged at 12,000 rpm for 10 min at 4°C and the supernatant collected and stored at −70°C until use. The blood samples(5 ml, no anticoagulant added) were centrifuged at 3000 rpm for 20 min at 4°C and the serum collected and stored at −70°C until use. Samples were incubated with APMA (4-aminophenyl- mercuric acetate, in component C, AnaSpec) at a final concentration of 1 mM in the assay buffer for 2 h at 37°C to activate MMP-12 prior to the experiment. After activating MMPs with APMA for 2 h , blood samples were incubated with 1 μM MMP Inhibitor v (Merck KGaA Frankfurter Str. 250 64293 Darmstadt, Germany), an orally active non-peptidyl hydroxamate compound that acts as an effective broad-spectrum inhibitor against MMP-2, -3,-8,-9, -12, -13, for 1h at 37°C. The assay was carried out in black 96-well microplates. Fluorescence intensities were recorded using a Fluoroskan Ascent fluorimeter at an interval of 5 min over a period of 1 h until peak fluorescence was achieved. The total volume of the assay was 100 μl assay buffer (SensoLyte TM 490 mmp-12 Assay Kit, ANA SPEC) containing various optimized sample volumes.
Enzyme-linked immuno sorbent assay
Protein samples were prepared by the methods mentioned above. An ELISA kit for MMP-12 detection is commercially available from EIAab (Wuhan EIAab Science Co.,Ltd,China). This assay employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for MMP-12 had been pre-coated onto a microplate. Standards, samples and the antibody that is bound by horseradish peroxidase were pipetted into the wells, which were then incubate and washed. Peroxidase can catalyze the oxidation of (3,3',5,5'-tetramethylbenzidine)TMB into two colored products. The first product is a blue which, if sufficient acid exists, the blue product will be further oxidized to a yellow diimine, that is stable in acidic conditions and has a maximal absorption wavelength of 450 nm. The depth of the color is positively correlated with the concentration of MMP-12. Samples were diluted with the appropriate amounts of sample dilution buffers provided with the kits. All tests were performed in duplicate for each sample. The optical density OD was determined using a microplate reader set to 450 nm, the average OD was calculated for each set of standards.
Analysis was performed by using SPSS version20.0. Descriptive statistics analysis was done. An independent sample t test was used to find out the difference between the means in two groups. Values were given as mean±s.e.m. If the difference is P<0.05, then there is statistical significance.