Nitric Oxide Induces the Progression of Abdominal Aortic Aneurysms through the Matrix Metalloproteinase Inducer EMMPRIN
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080845
ISSN1525-2191
AutoresTania R. Lizarbe, Carlos Tarín, Mónica Gómez, Begoña Lavin, Enrique Aracil, L Orte, Carlos Zaragoza,
Tópico(s)Aortic Disease and Treatment Approaches
ResumoNitric Oxide (NO) is involved in the development and progression of abdominal aortic aneurysms (AAA). We found that inhibition of inducible NO synthase (iNOS) protects mice in an elastase-induced AAA model, significantly inhibiting the production of matrix metalloproteinase-13 (MMP-13). The extracellular MMP inducer (EMMPRIN; CD147) was increased in human AAA biopsies and in wild-type murine AAA but not in AAA from iNOS null mice. In cells overexpressing ectopic EMMPRIN, MMP-13 secretion was stimulated, whereas silencing of EMMPRIN by RNA interference led to significant inhibition of MMP-13 expression. In addition, elastase infusion of MMP-13 null mouse aortas induced a significant increase of EMMPRIN but reduced aortic dilatation when compared with wild-type mice, suggesting that NO-mediated AAA may be mediated through EMMPRIN induction of MMP-13. These findings were further verified in elastase-infused iNOS null mice, in which daily administration of NO caused a significant aortic dilatation and the expression of EMMPRIN and MMP-13. By contrast, in iNOS wild-type mice, pharmacological inhibition of iNOS by administration of 1400 W induced a reduction of aortic diameter and inhibition of MMP-13 and EMMPRIN expression when compared with control mice. Our results suggest that NO may regulate the development of AAA in part by inducing the expression of EMMPRIN and modulating the activity of MMP-13 in murine and human aneurysms. Nitric Oxide (NO) is involved in the development and progression of abdominal aortic aneurysms (AAA). We found that inhibition of inducible NO synthase (iNOS) protects mice in an elastase-induced AAA model, significantly inhibiting the production of matrix metalloproteinase-13 (MMP-13). The extracellular MMP inducer (EMMPRIN; CD147) was increased in human AAA biopsies and in wild-type murine AAA but not in AAA from iNOS null mice. In cells overexpressing ectopic EMMPRIN, MMP-13 secretion was stimulated, whereas silencing of EMMPRIN by RNA interference led to significant inhibition of MMP-13 expression. In addition, elastase infusion of MMP-13 null mouse aortas induced a significant increase of EMMPRIN but reduced aortic dilatation when compared with wild-type mice, suggesting that NO-mediated AAA may be mediated through EMMPRIN induction of MMP-13. These findings were further verified in elastase-infused iNOS null mice, in which daily administration of NO caused a significant aortic dilatation and the expression of EMMPRIN and MMP-13. By contrast, in iNOS wild-type mice, pharmacological inhibition of iNOS by administration of 1400 W induced a reduction of aortic diameter and inhibition of MMP-13 and EMMPRIN expression when compared with control mice. Our results suggest that NO may regulate the development of AAA in part by inducing the expression of EMMPRIN and modulating the activity of MMP-13 in murine and human aneurysms. Abdominal aortic aneurysm (AAA) is an age-related multifactorial cardiovascular disease and is usually asymptomatic until rupture or diameter size requires medical intervention.1Baril DT Jacobs TS Marin ML Surgery insight: advances in endovascular repair of abdominal aortic aneurysms.Nat Clin Pract Cardiovasc Med. 2007; 4: 206-213Crossref PubMed Scopus (9) Google Scholar, 2Ernst CB Abdominal aortic aneurysm.N Engl J Med. 1993; 328: 1167-1172Crossref PubMed Scopus (691) Google Scholar AAA is characterized by local chronic inflammation, degradation of the medial elastin, and components of the extracellular matrix. Resistance to rupture depends on tensile strength by interstitial collagens, and therefore, collagen-degrading enzymes may play a pivotal effect.3Mao D Lee JK VanVickle SJ Thompson RW Expression of collagenase-3 (MMP-13) in human abdominal aortic aneurysms and vascular smooth muscle cells in culture.Biochem Biophys Res Commun. 1999; 261: 904-910Crossref PubMed Scopus (104) Google Scholar Matrix metalloproteinases (MMPs) are extracellular matrix degrading enzymes involved in the development and progression of AAA in humans and mice. The effect of collagenases MMP-83Mao D Lee JK VanVickle SJ Thompson RW Expression of collagenase-3 (MMP-13) in human abdominal aortic aneurysms and vascular smooth muscle cells in culture.Biochem Biophys Res Commun. 1999; 261: 904-910Crossref PubMed Scopus (104) Google Scholar and MMP-13,4Abdul-Hussien H Soekhoe RG Weber E von der Thusen JH Kleemann R Mulder A van Bockel JH Hanemaaijer R Lindeman JH Collagen degradation in the abdominal aneurysm: a conspiracy of matrix metalloproteinase and cysteine collagenases.Am J Pathol. 2007; 170: 809-817Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar gelatinases A and B,5Longo GM Xiong W Greiner TC Zhao Y Fiotti N Baxter BT Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.J Clin Invest. 2002; 110: 625-632Crossref PubMed Google Scholar and membrane type MMPs6Eagleton MJ Ballard N Lynch E Srivastava SD Upchurch Jr, GR Stanley JC Early increased MT1-MMP expression and late MMP-2 and MMP-9 activity during Angiotensin II induced aneurysm formation.J Surg Res. 2006; 135: 345-351Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar have been extensively analyzed. In particular, collagenases are responsible of cleavage of type I and III collagens, which are involved in keeping the vessel tensile strength, and therefore, increased production of collagen-degrading enzymes may be determinant for the progression of AAA. However, the molecular mechanisms leading to MMP expression and activation are not fully understood. The expression of MMPs have been recently associated to cardiovascular disease through the activation of the extracellular MMP inducer (EMMPRIN; Basigin and CD147).7Schmidt R Bultmann A Ungerer M Joghetaei N Bulbul O Thieme S Chavakis T Toole BP Gawaz M Schomig A May AE Extracellular matrix metalloproteinase inducer regulates matrix metalloproteinase activity in cardiovascular cells: implications in acute myocardial infarction.Circulation. 2006; 113: 834-841Crossref PubMed Scopus (157) Google Scholar EMMPRIN is an immunoglobulin, which induces the expression of MMP-2, MMP-9, and MT1-MMP.8Caudroy S Polette M Nawrocki-Raby B Cao J Toole BP Zucker S Birembaut P EMMPRIN-mediated MMP regulation in tumor and endothelial cells.Clin Exp Metastasis. 2002; 19: 697-702Crossref PubMed Scopus (166) Google Scholar, 9Caudroy S Polette M Tournier JM Burlet H Toole B Zucker S Birembaut P Expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) and the matrix metalloproteinase-2 in bronchopulmonary and breast lesions.J Histochem Cytochem. 1999; 47: 1575-1580Crossref PubMed Scopus (109) Google Scholar, 10Haseneen NA Vaday GG Zucker S Foda HD Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN.Am J Physiol Lung Cell Mol Physiol. 2003; 284: L541-L547Crossref PubMed Scopus (89) Google Scholar, 11Kanekura T Chen X Kanzaki T Basigin (CD147) is expressed on melanoma cells and induces tumor cell invasion by stimulating production of matrix metalloproteinases by fibroblasts.Int J Cancer. 2002; 99: 520-528Crossref PubMed Scopus (288) Google Scholar, 12Norgauer J Hildenbrand T Idzko M Panther E Bandemir E Hartmann M Vanscheidt W Herouy Y Elevated expression of extracellular matrix metalloproteinase inducer (CD147) and membrane-type matrix metalloproteinases in venous leg ulcers.Br J Dermatol. 2002; 147: 1180-1186Crossref PubMed Scopus (95) Google Scholar, 13Sameshima T Nabeshima K Toole BP Yokogami K Okada Y Goya T Koono M Wakisaka S Glioma cell extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) stimulates production of membrane-type matrix metalloproteinases and activated gelatinase A in cocultures with brain-derived fibroblasts.Cancer Lett. 2000; 157: 177-184Crossref PubMed Scopus (172) Google Scholar Acute and chronic inflammation induces the expression of EMMPRIN by immune cells and vascular endothelial cells, and its role on MMP expression was related to atherosclerosis.14Schmidt R Redecke V Breitfeld Y Wantia N Miethke T Massberg S Fischel S Neumann FJ Schomig A May AE EMMPRIN (CD 147) is a central activator of extracellular matrix degradation by Chlamydia pneumoniae-infected monocytes: implications for plaque rupture.Thromb Haemost. 2006; 95: 151-158Crossref PubMed Google Scholar, 15Yoon YW Kwon HM Hwang KC Choi EY Hong BK Kim D Kim HS Cho SH Song KS Sangiorgi G Upstream regulation of matrix metalloproteinase by EMMPRIN: extracellular matrix metalloproteinase inducer in advanced atherosclerotic plaque.Atherosclerosis. 2005; 180: 37-44Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Here we show the association of EMMPRIN and MMP-13 in human AAA and during the NO-mediated AAA in mice, providing evidence about the effect of EMMPRIN on MMP-13 expression in vascular cells. General cell culture supplies were from BD Biosciences (Spain), calf serum was from BioWhittaker (Verviers, Belgium), and cell culture gelatin, antibiotics, and trichrome staining (Masson) was from Sigma-Aldrich (St. Louis, MO). Autoradiography film was from Kodak; polyvinylidene difluoride protein transfer membranes were from Millipore (Iberica, Spain); and horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence immunoblot detection system were from GE HealthCare (Spain). EDTA-free protease inhibitor mixture tablets were from Roche (Spain). Optimem and Lipofectamine were from Invitrogen. Fluorsave coverslip mounting solution was from Calbiochem (CN Biosciences, UK). EMMPRIN small interfering RNAs were from Qiagen (Qiagen Iberia, Spain). Porcine elastase (specific activity 5 U/mg; E1250) was from Sigma-Aldrich. Primary antibodies were obtained as follows: rabbit anti-MMP-13 from Calbiochem; goat-anti-human-MMP-13 and rabbit anti-inducible NO synthase (iNOS) from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-MMP-2, mouse anti-MMP-9, rabbit anti-MMP-12, and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Chemicon (Millipore, Iberica, Spain); anti-FLAG, and goat-anti-EMMPRIN from Sigma-Aldrich; anti-ICAM-2, anti-von Willebrand factor, and anti-Mac-3 from Transduction Laboratories (BD Biosciences). Rat-anti-mouse polymorphonuclear from Serotec (Oxford, UK). Mouse-anti-human EMMPRIN was donated by Prof. Francisco Sánchez (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain). The institutional ethics committee previously approved, and written consent was obtained for the animal procedures performed in this work. Wild-type C57BL/6 mice, C57BL/6 iNOS null mice, and C57BL/6 apoE null mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 MMP-13 null mice were donated by Dr. Stephen Krane (Department of Medicine, Harvard Medical School, Boston, MA). All animals were housed in our animal facilities in isolated rooms. AAA experiments were performed in 45- to 50-week-old mice, with the exception of experiments conducted to determine changes in aortic diameter (AD), in which 12-week-old mice were used for this purpose. Atherosclerosis was induced in mice fed with Western type diet for 16 weeks (42% fat, Harland Teklad, TD88137). Institutional ethics committee approval for the study was obtained, and written consent was granted from all patients. Tissue from 18 patients under surgery was obtained intraoperatively from the anterior aneurysm wall. Biopsies were embedded in paraffin for immunohistochemistry purposes. Morphometric analysis of AAA was evaluated by preoperative computed tomography. Additional information of patients was recorded and summarized in Supplemental Table 1 (see http://ajp.amjpathol.org). In addition, ascending aortic tissue was collected from non-AAA-diagnosed patients subjected to coronary intervention for control purposes.Table 1Changes in AD Over TimeStrainAD priorAD afterAD 14 daysElastase-infused mice (12-week-old mice) C57BL/60.5 ± 0.020.77 ± 0.031.3 ± 0.2 apoE null0.5 ± 0.20.78 ± 0.031.4 ± 0.5 iNOS null0.5 ± 0.010.75 ± 0.011.2 ± 0.2 apoE/iNOS null0.5 ± 0.20.78 ± 0.0.41.3 ± 0.02Elastase-infused mice (40- to 50-week-old mice) C57BL/60.5 ± 0.010.75 ± 0.021.8 ± 0.04 apoE null0.5 ± 0.120.79 ± 0.031.9 ± 0.04 iNOS null0.5 ± 0.010.77 ± 0.011.02 ± 0.03 apoE/iNOS null0.5 ± 0.20.82 ± 0.0250.92 ± 0.02Sham-operated mice (40- to 50-week-old mice) C57BL/60.5 ± 0.020.76 ± 0.020.8 ± 0.04 apoE null0.5 ± 0.020.77 ± 0.020.8 ± 0.04 iNOS null0.5 ± 0.010.77 ± 0.010.9 ± 0.03 apoE/iNOS null0.5 ± 0.20.79 ± 0.020.8 ± 0.02AD, aortic diameter; prior, prior infusion; after, after infusion; 14 days, 14 days after infusion. N = 10 animals/strain/, repeated three times. Open table in a new tab AD, aortic diameter; prior, prior infusion; after, after infusion; 14 days, 14 days after infusion. N = 10 animals/strain/, repeated three times. Murine aortic endothelial cells were grown on gelatin as described previously.16Lopez-Rivera E Lizarbe TR Martinez-Moreno M Lopez-Novoa JM Rodriguez-Barbero A Rodrigo J Fernandez AP Alvarez-Barrientos A Lamas S Zaragoza C Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration.Proc Natl Acad Sci USA. 2005; 102: 3685-3690Crossref PubMed Scopus (76) Google Scholar Mice (12 and 45 to 50 weeks) were anesthesized by i.p. injection of ketamine (100 mg/kg)/xylazine (5 mg/kg), and a horizontal laparatomy was practiced. With a surgical stereomicroscope (Nikon), the abdominal aorta was isolated. Below the renal arteries and above iliac arteries, the abdominal aorta was temporary ligated, and an aortotomy was created with a 30-gauge needle. A PE-26 polyetilene tube was introduced through the aortotomy, and the aorta was infused at 100 mmHg, either with saline buffer or with type I porcine pancreatic elastase (0.4 U/ml), for 5 minutes. The aortotomy was then repaired, ligation was eliminated, and blood flow restoration was visualized. Elastase-infused mice and sham operated (saline-infused) controls were closed, housed, and subjected to further experimentation. Photographs of abdominal aortas were recorded before infusion, right after infusion, 15 days postinfusion, and 21 days postinfusion, and ADs were measured. Epitope-tagged constructs encoding full-length EMMPRIN were expressed in endothelial cells. The cDNA encoding recombinant protein was cloned in p3XFLAG-myc-CMV-24 (Sigma-Aldrich), which tags expressed proteins with FLAG and myc epitopes at the N-terminus and the C-terminus, respectively. For exogenous EMMPRIN expression and gene and silencing, cells were transiently transfected using Lipofectamine 2000 Reagent as described previously.16Lopez-Rivera E Lizarbe TR Martinez-Moreno M Lopez-Novoa JM Rodriguez-Barbero A Rodrigo J Fernandez AP Alvarez-Barrientos A Lamas S Zaragoza C Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration.Proc Natl Acad Sci USA. 2005; 102: 3685-3690Crossref PubMed Scopus (76) Google Scholar Mice were anesthesized with 4% isoflurane/O2 gas and maintained with 1.5% isoflurane/O2 gas. A respiration triggering probe was connected to a triggering unit, and images were acquired on a Bruker Biospec BMT 47/40 spectrometer (Bruker, Ettlingen, Germany) 4.7 T, equipped with 11.2 cm actively shielded gradient system, capable of 200 mT/m gradient strength and 80-seconds rise time. Global shimming was first performed, and three scout images in axial, sagittal, and coronal direction were acquired using a T1 weighted spin echo sequence (repetition time/echo time = 200/10 ms). Then, respiratory-gated T1-weighted spin echo images in sagittal orientation were acquired. Repetition time varied from 500 to 700 ms, depending on the respiratory rate of each animal, and echo time was 15 ms. Fifteen sagittal slices of 1.0 mm were acquired with an in-plane field of view (FOV) of 4 × 2 cm2 and matrix size of 256 × 128. Respiratory-gated T1-weighted spin-echo axial images were acquired using the same parameters as above, with a FOV of 3 × 3 cm2 and a matrix of 256 × 192. Twenty-four slices were acquired, and data were zero filled to obtain images of 256 × 256 pixels. For the magnetic resonance angiography, a gradient echo with flow compensation imaging sequence was used with the following parameters: repetition time = 45 ms; echo time = 10 ms; flip angle = 80°; acquisition matrix size = 256 × 128; FOV = 3.5 × 1.75 cm2; and number of slices: 64, 0.5 mm/slice. Slices were overlapped 0.1 mm, with a FOV in the slice direction of 2.57 cm. Maximal AD was determined between the renal and the iliac arteries with the ParaVision software package (Bruker). Data set was first interpolated to obtain isotropic voxels of 137 × 137 × 137_m3. The acquired matrix was of 256 × 128 × 64, covering a FOV of 3.5 × 1.75 × 2.57 cm3. The resulting matrix was interpolated to a 256 × 128 × 128 matrix. Maximum Intensity Projection algorithm was applied to the interpolated data to obtain a three-dimensional view of each angiography. Cell lysis and immunoblotting were performed as described previously.16Lopez-Rivera E Lizarbe TR Martinez-Moreno M Lopez-Novoa JM Rodriguez-Barbero A Rodrigo J Fernandez AP Alvarez-Barrientos A Lamas S Zaragoza C Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration.Proc Natl Acad Sci USA. 2005; 102: 3685-3690Crossref PubMed Scopus (76) Google Scholar Proteins were detected by confocal microscopy in cultured cells and paraffin-embedded tissue sections. Cell cultures were grown on coverslips and, after treatment, were washed twice with PBS. Cells were fixed for 10 minutes with cold methanol and incubated for 1 hour with 3% bovine serum albumin in PBS. Coverslips were then washed with PBS and incubated with the appropriate primary antibodies for 1 hour. Nonbound antibody was removed by washing four times with PBS, after which coverslips were incubated with an appropriate fluorescence-conjugated secondary antibody for 1 hour. Staining was visualized by confocal microscopy (Radiance 2100; Bio-Rad, Hercules, CA). Proteins were detected with secondary antibodies tagged with the flurochromes Cy3 or fluorescein isothiocyanate. Cell nuclei were stained with Hoechst. Paraffin-embedded tissue from mice and human biopsies were sliced 4 μm thick. Elastin integrity of mouse aortas was monitored by Trichrome staining and visualized by light microscopy. In human biopsies, iNOS, EMMPRIN, and MMP-13 were visualized as follows: sections were incubated with normal serum in PBS, washed 5 minutes with PBS, and then incubated for 1 hour with the corresponding primary antibody in humidified chambers. Controls were also incubated with nonimmune serum. Slices were washed three times with PBS and then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive complexes were detected with alkaline phosphatase substrate. Before visualization, sections were also stained with hematoxylin. Unless otherwise specified, data are expressed as means ± SD, and experiments were performed at least three times in duplicate. Whenever comparisons were made with a common control, comparisons were made with analysis of variance followed by Dunnett’s modification of the t-test. The level of statistical significance was defined as P < 0.05. Error bars represent ±SD. We induced AAA in mice by the infusion of porcine elastase in the infra-renal abdominal aorta and compared the variation of AD over time respect to saline-infused sham operated animals, as described previously.17Lee JK Borhani M Ennis TL Upchurch Jr, GR Thompson RW Experimental abdominal aortic aneurysms in mice lacking expression of inducible nitric oxide synthase.Arterioscler Thromb Vasc Biol. 2001; 21: 1393-1401Crossref PubMed Scopus (65) Google Scholar Changes in aortic diameter were evaluated in 12-week-old and 40- to 50-week-old wild-type mice (C57BL/6), iNOS null mice, apoE null mice, and iNOS/apoE null mice, either infused with elastase or saline as described. As shown in Table 1, we found no differences in AD in 12-week-old mice over time. However, in older animals, we found extensive aortic dilatation 14 days after elastase infusion in wild-type mice respect to mice lacking iNOS (iNOS null mice and iNOS/apoE null mice), in association with destruction of aortic lamellae, as detected by Masson Trichromic (Figure 1A) and by Verhoeff-van Gieson staining of aortic sections (Supplemental Figure S1, see http://ajp.amjpathol.org), and significant increase of iNOS expression (Figure 1B). No significant differences were detected in 40- to 50-week-old sham operated mice in AD (Table 1) and immune cell infiltration in response to NO (Supplemental Figure S1B, see http://ajp.amjpathol.org). The progression of AAA was also followed by magnetic resonance imaging over time with similar results (Figure 1C; Supplemental Figures S1C and S1D, see http://ajp.amjpathol.org). To evaluate the effect of NO in the progression of AAA in atherosclerotic mice, we generated iNOS/apoE double-knockout animals and fed them with high cholesterol diet (see Materials and Methods for details). We found that after elastase infusion, no significant variations on aortic dilatation were detected in double iNOS/apoE null mice when compared with single apoE knockouts (Figure 1 and Table 1; Supplemental Figures S1C and S1D, see http://ajp.amjpathol.org). The expression of MMPs is associated to AAA in mice and in humans. In this regard, we evaluated the aortic expression of MMP-2, MMP-9, MMP-12, and MMP-13 during the course of AAA in iNOS wild-type and iNOS null mice, finding increased expression of MMP-2, MMP-9, and MMP-12 over time in both groups (Supplemental Figures S2 and S3, see http://ajp.amjpathol.org), with the exception of MMP-13, whose expression was significantly reduced in iNOS null mice 15 days after elastase infusion, when compared with wild-type mice, as detected by immunofluorescence (Figure 2A) and immunoblot (Figure 2B), suggesting a role for MMP-13 in the NO-mediated effect on AAA. In view of this result, and taking into account that MMP-13 is associated to human AAA,3Mao D Lee JK VanVickle SJ Thompson RW Expression of collagenase-3 (MMP-13) in human abdominal aortic aneurysms and vascular smooth muscle cells in culture.Biochem Biophys Res Commun. 1999; 261: 904-910Crossref PubMed Scopus (104) Google Scholar that NO up-regulates the expression of MMP-13 in vivo16Lopez-Rivera E Lizarbe TR Martinez-Moreno M Lopez-Novoa JM Rodriguez-Barbero A Rodrigo J Fernandez AP Alvarez-Barrientos A Lamas S Zaragoza C Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration.Proc Natl Acad Sci USA. 2005; 102: 3685-3690Crossref PubMed Scopus (76) Google Scholar, 18Zaragoza C Balbin M Lopez-Otin C Lamas S Nitric oxide regulates matrix metalloprotease-13 expression and activity in endothelium.Kidney Int. 2002; 61: 804-808Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 19Lizarbe TR Garcia-Rama C Tarin C Saura M Calvo E Lopez JA Lopez-Otin C Folgueras AR Lamas S Zaragoza C Nitric oxide elicits functional MMP-13 protein tyrosine nitration during wound repair.FASEB J. 2008; 22: 3207-3215Crossref PubMed Scopus (37) Google Scholar and that NO and MMP-13 are associated in atherosclerosis,20Tarin C Gomez M Calvo E Lopez JM Zaragoza C Endothelial nitric oxide deficiency reduces MMP-13-mediated cleavage of ICAM-1 in vascular endothelium: a role in atherosclerosis.Arterioscler Thromb Vasc Biol. 2009; 29: 27-32Crossref PubMed Scopus (38) Google Scholar we decided to evaluate the association between NO an MMP-13 at the molecular level during the course of AAA. Recently, the expression of EMMPRIN was found associated to different cardiovascular diseases, including atherosclerosis and myocardial infarction.7Schmidt R Bultmann A Ungerer M Joghetaei N Bulbul O Thieme S Chavakis T Toole BP Gawaz M Schomig A May AE Extracellular matrix metalloproteinase inducer regulates matrix metalloproteinase activity in cardiovascular cells: implications in acute myocardial infarction.Circulation. 2006; 113: 834-841Crossref PubMed Scopus (157) Google Scholar, 15Yoon YW Kwon HM Hwang KC Choi EY Hong BK Kim D Kim HS Cho SH Song KS Sangiorgi G Upstream regulation of matrix metalloproteinase by EMMPRIN: extracellular matrix metalloproteinase inducer in advanced atherosclerotic plaque.Atherosclerosis. 2005; 180: 37-44Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 21Choi EY Kim D Hong BK Kwon HM Song YG Byun KH Park HY Whang KC Kim HS Up-regulation of extracellular matrix metalloproteinase inducer (EMMPRIN) and gelatinases in human atherosclerosis infected with Chlamydia pneumoniae: the potential role of Chlamydia pneumoniae infection in the progression of atherosclerosis.Exp Mol Med. 2002; 34: 391-400Crossref PubMed Scopus (35) Google Scholar, 22Sidhu SS Mengistab AT Tauscher AN LaVail J Basbaum C The microvesicle as a vehicle for EMMPRIN in tumor-stromal interactions.Oncogene. 2004; 23: 956-963Crossref PubMed Scopus (238) Google Scholar, 23Ergul A Portik-Dobos V Hutchinson J Franco J Anstadt MP Down-regulation of vascular matrix metalloproteinase inducer and activator proteins in hypertensive patients.Am J Hypertens. 2004; 17: 775-782Crossref PubMed Scopus (42) Google Scholar, 24Sluijter JP Pulskens WP Schoneveld AH Velema E Strijder CF Moll F de Vries JP Verheijen J Hanemaaijer R de Kleijn DP Pasterkamp G Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms.Stroke. 2006; 37: 235-239Crossref PubMed Scopus (169) Google Scholar Here we found EMMPRIN accumulation at the endothelial layer of aortas in which AAAs were induced (Figure 3A). Immunoblot analysis of EMMPRIN expression revealed the presence of three major immunoreactive fragments of 25, 37, and 50 kDa in wild-type resting mice, corresponding to different levels of EMMPRIN glycosylation.24Sluijter JP Pulskens WP Schoneveld AH Velema E Strijder CF Moll F de Vries JP Verheijen J Hanemaaijer R de Kleijn DP Pasterkamp G Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms.Stroke. 2006; 37: 235-239Crossref PubMed Scopus (169) Google Scholar The association between NO and EMMPRIN during AAA was evaluated in wild-type and iNOS null mouse aortas. As result of elastase infusion, a significant increase of EMMPRIN was detected in wild-type mice, whereas in iNOS null mice, we could barely detect the two major high molecular fragments of EMMPRIN, both in resting and in elastase-infused aortas (Figure 3B). By contrast, no differences were detected when AAA were induced in wild-type 12-week-old mice (data not shown), in which iNOS levels were significantly lower respect to aged animals in response to elastase infusion (Figure 1C). However, exogenous administration of the NO donor Spermidine-nonoate in young mice after infusion of elastase by i.p. injection reported a substantial increase of EMMPRIN levels in the abdominal aortas (Supplemental Figure S4A, see http://ajp.amjpathol.org). To further explore the effect of NO on EMMPRIN expression, we evaluated the progression of AAA in aged iNOS null mice, in which NO was daily administered by i.p. injection of the NO donor Spermidine-nonoate over 15 days. We found that in iNOS null mice, the administration of NO induced a significant increase of aortic dilatation (Figure 3C), collagen, and elastin fibers degradation, as detected by Masson and Verhoeff-van Gieson staining, increased protein nitration, as detected by immunofluorescence with anti-nitro-tyrosine antibody, and increased EMMPRIN and MMP-13 levels, as detected with specific antibodies (Figure 3D) over mice treated with the drug control of the NO donor (Spermidine), resembling the inflammatory response to the vascular injury elicited in wild-type mice. Additional markers of inflammation were also evaluated in these animals (Supplemental Figure S5, see http://ajp.amjpathol.org). By contrast, inhibition of iNOS by daily i.p. injection with the pharmacological inhibitor 1400 W preserved collagen and elastin fibers, and protein nitration was reduced (Figure 3E), as well as expression of EMMPRIN and MMP-13 (Figure 3F). EMMPRIN was found to induce activation of different MMPs, yet no data regarding MMP-13 was reported so far. The results presented above lead us to investigate the possible association between EMMPRIN and MMP-13, and taking into account that EMMPRIN was located in endothelial cells (Figure 3A), we overexpressed FLAG-tagged EMMPRIN in vascular aortic endothelial cells isolated from mouse aortas (Supplemental Figure S4B, see http://ajp.amjpathol.org). In cells expressing ectopic-EMMPRIN, MMP-13 expression was down-regulated as detected by immunoblot from cell lysates (Figure 4A, MMP-13 lysate panel). However, immunoblot analysis with anti-FLAG and anti-MMP-13 antibodies from cultured media revealed that FLAG-EMMPRIN induced MMP-13 secretion (Figure 4A, MMP-13 culture media), suggesting for the first time that EMMPRIN may regulate MMP-13 activity in vascular endothelium. To further investigate the association of EMMPRIN and MMP-13, we silenced EMMPRIN in aortic endothelial cells by RNA interference, finding that EMMPRIN does not only induce MMP-13 activity but in addition EMMPRIN regulates MMP-13 expression, since we found that MMP-13 was also down-regulated in these cells (Figure 4B, left panel). In addition, the stimulatory effect of NO on MMP-13 expression was inhibited in small interfer
Referência(s)