Fibronectin-α4β1 Integrin Interactions Regulate Metalloproteinase-9 Expression in Steatotic Liver Ischemia and Reperfusion Injury
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060456
ISSN1525-2191
AutoresCarolina Moore, Xiu‐Da Shen, Feng Gao, Ronald W. Busuttil, Ana J. Coito,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoIschemia/reperfusion injury is a major cause of the highly dysfunctional rate observed in marginal steatotic orthotopic liver transplantation. In this study, we document that the interactions between fibronectin, a key extracellular matrix protein, and its integrin receptor α4β1, expressed on leukocytes, specifically up-regulated the expression and activation of metalloproteinase-9 (MMP-9, gelatinase B) in a well-established steatotic rat liver model of ex vivo ice-cold ischemia followed by isotransplantation. The presence of the active form of MMP-9 was accompanied by massive intragraft leukocyte infiltration, high levels of proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, and impaired liver function. Interestingly, MMP-9 activity in steatotic liver grafts was, to a certain extent, independent of the expression of its natural inhibitor, the tissue inhibitor of metalloproteinases-1. Moreover, the blockade of fibronectin-α4β1-integrin interactions inhibited the expression/activation of MMP-9 in steatotic orthotopic liver transplantations without significantly affecting the expression of metalloproteinase-2 (MMP-2, gelatinase A). Finally, we identified T lymphocytes and monocytes/macrophages as major sources of MMP-9 in steatotic liver grafts. Hence, these findings reveal a novel aspect of the function of fibronectin-α4β1 integrin interactions that holds significance for the successful use of marginal steatotic livers in transplantation. Ischemia/reperfusion injury is a major cause of the highly dysfunctional rate observed in marginal steatotic orthotopic liver transplantation. In this study, we document that the interactions between fibronectin, a key extracellular matrix protein, and its integrin receptor α4β1, expressed on leukocytes, specifically up-regulated the expression and activation of metalloproteinase-9 (MMP-9, gelatinase B) in a well-established steatotic rat liver model of ex vivo ice-cold ischemia followed by isotransplantation. The presence of the active form of MMP-9 was accompanied by massive intragraft leukocyte infiltration, high levels of proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, and impaired liver function. Interestingly, MMP-9 activity in steatotic liver grafts was, to a certain extent, independent of the expression of its natural inhibitor, the tissue inhibitor of metalloproteinases-1. Moreover, the blockade of fibronectin-α4β1-integrin interactions inhibited the expression/activation of MMP-9 in steatotic orthotopic liver transplantations without significantly affecting the expression of metalloproteinase-2 (MMP-2, gelatinase A). Finally, we identified T lymphocytes and monocytes/macrophages as major sources of MMP-9 in steatotic liver grafts. Hence, these findings reveal a novel aspect of the function of fibronectin-α4β1 integrin interactions that holds significance for the successful use of marginal steatotic livers in transplantation. Orthotopic liver transplantation (OLT) is an effective therapeutic modality for end-stage liver disease. The critical shortage of human donor livers has provided the rationale to identify methods that would allow successful utilization of marginal steatotic donor grafts, which are characterized by higher dysfunction rate compared with nonsteatotic OLTs.1Alexander JW Vaughn WK The use of "marginal" donors for organ transplantation: the influence of donor age on outcome.Transplantation. 1991; 51: 135-141Crossref PubMed Scopus (249) Google Scholar, 2Lehmann TG Wheeler MD Schwabe RF Connor HD Schoonhoven R Bunzendahl H Brenner DA Jude SR Zhong Z Thurman RG Gene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the rat.Hepatology. 2000; 32: 1255-1264Crossref PubMed Scopus (67) Google ScholarIschemia/reperfusion (I/R) insult, an antigen-independent event associated with leukocyte adhesion/migration and release of cytokines and free radicals, plays a major role in post-I/R organ injury suffered by OLTs.3Koneru B Dikdan G Hepatic steatosis and liver transplantation current clinical and experimental perspectives.Transplantation. 2002; 73: 325-330Crossref PubMed Scopus (93) Google Scholar, 4Strasberg SM Howard TK Molmenti EP Hertl M Selecting the donor liver: risk factors for poor function after orthotopic liver transplantation.Hepatology. 1994; 20: 829-838Crossref PubMed Scopus (479) Google Scholar Leukocyte recruitment to sites of inflammatory stimulation in liver, which is a venous-driven vascular bed with slow flow rates, is poorly understood and may require distinct cascade of adhesive events compared with other organs with higher flow rates. Fibronectin (FN), a large glycoprotein with a central role in cellular adhesion and migration, is likely a key extracellular matrix (ECM) protein involved in these events.5Coito AJ de Sousa M Kupiec-Weglinski JW Fibronectin in immune responses in organ transplant recipients.Dev Immunol. 2000; 7: 239-248Crossref PubMed Scopus (19) Google ScholarThe role of FN in leukocyte adhesion, migration, and activation has been extensively reported.6Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin.J Immunol. 1990; 145: 59-67PubMed Google Scholar, 7Hauzenberger D Klominek J Sundqvist KG Functional specialization of fibronectin-binding β1-integrins in T lymphocyte migration.J Immunol. 1994; 153: 960-971PubMed Google Scholar, 8Coito AJ Binder J Brown LF de Sousa M Van De WL Kupiec-Weglinski JW Anti-TNF-α treatment down-regulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats.J Immunol. 1995; 154: 2949-2958PubMed Google Scholar Moreover, we have previously shown that the so-called cellular FN, which is the most potent form of FN in promoting cell spreading and migration,9Xia P Culp LA Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA): complementarity to plasma fibronectin functions.Exp Cell Res. 1995; 217: 517-527Crossref PubMed Scopus (40) Google Scholar, 10Manabe R Oh-e N Sekiguchi K Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction.J Biol Chem. 1999; 274: 5919-5924Crossref PubMed Scopus (92) Google Scholar is virtually absent in naïve steatotic livers, and it is expressed very early on the sinusoidal endothelium during cold ischemia, before steatotic OLT and leukocyte recruitment at the graft site.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google ScholarLeukocyte transmigration across endothelial and ECM barriers is dependent on both adhesive and focal matrix degradation mechanisms.12Bianchi E Bender JR Blasi F Pardi R Through and beyond the wall: late steps in leukocyte transendothelial migration.Immunol Today. 1997; 18: 586-591Abstract Full Text PDF PubMed Scopus (92) Google Scholar The matrix metalloproteinase (MMP) family, which comprises more than 24 well-characterized proteolytic enzymes, plays key roles in the responses of cells to their microenvironment.13Lee MH Murphy G Matrix metalloproteinases at a glance.J Cell Sci. 2004; 117: 4015-4016Crossref PubMed Scopus (81) Google Scholar Of the MMPs, a specific subset, the gelatinases (MMP-2 and MMP-9), which are characterized by fibronectin-like domain of three type II repeats, are responsible for the turnover and degradation of several ECM proteins, including FN and type IV collagen, the major component of basement membranes.14Yoshizaki T Sato H Furukawa M Pagano JS The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1.Proc Natl Acad Sci USA. 1998; 95: 3621-3626Crossref PubMed Scopus (193) Google Scholar, 15Nagase H Woessner Jr, JF Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21494Crossref PubMed Scopus (3853) Google Scholar Indeed, there is a growing body of evidence that gelatinases play important functions in processes requiring basement membrane disruption, such as tumor invasion and arthritis16Bergers G Brekken R McMahon G Vu TH Itoh T Tamaki K Tanzawa K Thorpe P Itohara S Werb Z Hanahan D Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.Nat Cell Biol. 2000; 2: 737-744Crossref PubMed Scopus (2251) Google Scholar, 17Yu Q Stamenkovic I Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis.Genes Dev. 2000; 14: 163-176PubMed Google Scholar, 18Giannelli G Erriquez R Iannone F Marinosci F Lapadula G Antonaci S MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in patients with rheumatoid arthritis and psoriatic arthritis.Clin Exp Rheumatol. 2004; 22: 335-338PubMed Google Scholar, 19Dong Z Bonfil RD Chinni S Deng X Trindade Filho JC Bernardo M Vaishampayan U Che M Sloane BF Sheng S Fridman R Cher ML Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue.Am J Pathol. 2005; 166: 1173-1186Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar; therefore, it is reasonable to postulate that gelatinases may have an important function in leukocyte recruitment at the graft site.We have previously shown that blockade of the interactions between FN and the integrin α4β1, the integrin receptor expressed on leukocytes, profoundly improved liver function and recipient survival of steatotic OLTs.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The present study shows that MMP-9 is a novel identified player in steatotic OLT and that FN-α4β1 interactions regulate its expression by infiltrating leukocytes.Materials and MethodsAnimals and Grafting TechniquesGenetically obese (fa−/fa−) male Zucker (230–275 g), lean (fa/−) Zucker (260–300 g), and male Sprague Dawley (250–300 g) rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Syngeneic OLTs were performed using steatotic and nonsteatotic livers that were harvested from obese Zucker and from normal Sprague Dawley rats, respectively. Steatotic and lean livers were then stored at 4°C in UW solution for 4 and 24 hours, respectively, before being isotransplanted into lean Zucker or Sprague Dawley recipients. The standard techniques of liver harvesting and orthotopic transplantation without hepatic artery reconstruction were performed according to the previously described Kamada's cuff technique and an anhepatic phase of ∼16 to 20 minutes.20Kamada N Calne RY Orthotopic liver transplantation in the rat: technique using cuff for portal vein anastomosis and biliary drainage.Transplantation. 1979; 28: 47-50Crossref PubMed Scopus (678) Google Scholar, 21Kamada N Calne RY A surgical experience with five hundred thirty liver transplants in the rat.Surgery. 1983; 93: 64-69PubMed Google Scholar Animals were fed a standard rodent diet, with water ad libitum, and cared for according to guidelines approved by the American Association of Laboratory Animal Care. Blood was collected for serum glutamic oxaloacetic transaminase (SGOT) levels. Oil red O staining confirmed the high content of fat in the steatotic donor livers, and fatty Zucker rats of 230 to 275 g body weight have >30% (≅40%) liver steatosis, which sets them as marginal donors.Treatment with CS1 PeptidesCS1 peptides were synthesized on a Beckman System 990 peptide synthesizer (Beckman Instruments, Inc., Fullerton, CA) and purified by high-performance liquid chromatography, as described.22Coito AJ Onodera K Kato H Busuttil RW Kupiec-Weglinski JW Fibronectin-mononuclear cell interactions regulate type 1 helper T cell cytokine network in tolerant transplant recipients.Am J Pathol. 2000; 157: 1207-1218Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar The sequence of the peptides used in the study corresponded to the alternatively spliced CS1 variant of FN (25 mer: DELPQLVTLPHPNLHGPEILDVPST) and has been shown in vitro and in vivo to block the interaction between α4β1 integrin and its FN ligand.23Elices MJ Osborn L Takada Y Crouse C Luhowskyj S Hemler ME Lobb RR VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.Cell. 1990; 60: 577-584Abstract Full Text PDF PubMed Scopus (1509) Google Scholar, 24Elices MJ Tsai V Strahl D Goel AS Tollefson V Arrhenius T Wayner EA Gaeta FC Fikes JD Firestein GS Expression and functional significance of alternatively spliced CS1 fibronectin in rheumatoid arthritis microvasculature.J Clin Invest. 1994; 93: 405-416Crossref PubMed Scopus (130) Google Scholar, 25Makarem R Newham P Askari JA Green LJ Clements J Edwards M Humphries MJ Mould AP Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin α4β1.J Biol Chem. 1994; 269: 4005-4011Abstract Full Text PDF PubMed Google Scholar, 26Wahl SM Allen JB Hines KL Imamichi T Wahl AM Furcht LT McCarthy JB Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.J Clin Invest. 1994; 94: 655-662Crossref PubMed Scopus (111) Google Scholar CS1 peptides (500 μg/rat) were administered to livers intraportally during procurement and before OLT. In addition, OLT recipients received a 3-day course of CS1 peptides (1 mg/rat/day, i.v.) and were followed for survival and SGOT levels. Control recipients received scrambled peptides or remained untreated. The chosen therapeutic regimen was based on previous work, in which CS1 peptide therapy improved function/histological preservation of steatotic liver grafts and prolonged their 14-day survival in lean recipients from 40% in control to 100% in CS1-treated OLTs.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google ScholarHistology and ImmunohistochemistryLiver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Sections were made at 4 μm and stained with H&E or Masson's trichrome. The previously published Suzuki's criteria27Suzuki S Toledo-Pereyra LH Rodriguez FJ Cejalvo D Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury: modulating effects of FK506 and cyclosporine.Transplantation. 1993; 55: 1265-1272Crossref PubMed Scopus (659) Google Scholar were modified to reflect the histological severity of I/R injury in our OLT model. In this classification, sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded from 0 to 4.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The absence of necrosis, congestion, or centrilobular ballooning is given a score of 0, whereas severe congestion, ballooning degeneration as well as >60% lobular necrosis is given a value of 4.OLTs were also examined serially by immunohistochemistry for mononuclear cell infiltrate, activation, and MMP detection.28Coito AJ Brown LF Peters JH Kupiec-Weglinski JW Van De WL Expression of fibronectin splicing variants in organ transplantation: a differential pattern between rat cardiac allografts and isografts.Am J Pathol. 1997; 150: 1757-1772PubMed Google Scholar In brief, liver tissue was embedded in Tissue Tec OCT compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −70°C. Cryostat sections (5 μm) were fixed in acetone, and then endogenous peroxidase activity was inhibited with 0.3% H2O2 in phosphate-buffered saline (PBS). Appropriate primary mouse antibodies against rat T cells (R73), monocyte/macrophages (ED1), IL-2R+ cells (CD25) (Harlan Bioproducts, Indianapolis, IN), cellular FN (IST-9) (Accurate Chemical, Westbury, NY), metalloproteinase-2 (MMP-2, gelatinase A), and metalloproteinase-9 (MMP-9, gelatinase B) (NeoMarkers, Fremont, CA; Oncogene, San Diego, CA; and EMD Biosciences, La Jolla, CA) were added at optimal dilutions. Bound primary antibody (Ab) was detected using biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG and streptavidin peroxidase-conjugated complexes (Dako, Carpinteria, CA). Negative controls included sections in which the primary Ab was replaced with either dilution buffer or normal mouse or rabbit serum. Control sections from inflammatory tissues known to be positive for each stain were included as positive controls. The peroxidase reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (DAB) Substrate Kit (Vector Laboratories, Burlingame, CA). Dual immunostaining was performed by confirming peroxidase-based immunostaining for cellular markers with alkaline phosphatase-based immunohistochemistry for MMP-9. The alkaline phosphatase reaction was developed using the Vector Red Substrate Kit (Vector Laboratories). The sections were evaluated blindly by counting the labeled cells in triplicates within 10 high-power fields per section. In the case of continuous labeling, some antigens were analyzed in a semiquantitative fashion where the relative abundance of each one was judged as negative [−, little (+), moderately abundant (++), or very abundant (+++, >200 cells/10 high-power fields].RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)For evaluation of cytokine and metalloproteinase gene expression, OLTs were harvested serially, and RNA was extracted with Trizol (Life Technologies Inc., Grand Island, NY) using a Polytron RT-3000 (Kinematica AG, Littau-Luzem, Switzerland) as previously described.30Basbaum CB Werb Z Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.Curr Opin Cell Biol. 1996; 8: 731-738Crossref PubMed Scopus (292) Google Scholar Reverse transcription was performed using 4 μg of total RNA in a first-strand cDNA synthesis reaction with SuperScript II RNaseH reverse transcriptase (Life Technologies Inc.) as recommended by the manufacturer. The cDNA product was amplified by PCR using primers specific for rat cytokines and β-actin as previously described29Coito AJ Shaw GD Li J Ke B Ma J Busuttil RW Kupiec-Weglinski JW Selectin-mediated interactions regulate cytokine networks and macrophage heme oxygenase-1 induction in cardiac allograft recipients.Lab Invest. 2002; 82: 61-70Crossref PubMed Scopus (26) Google Scholar or for rat MMP-9, MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2, which were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Relative quantities of mRNA were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY).Western Blot and Zymography AnalysesSnap-frozen liver tissue was immediately homogenized as previously described.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Protein content was determined by a colorimetric assay (Bio-Rad, Hercules, CA).For the Western blots, 50 μg of protein in sodium dodecyl sulfate (SDS)-loading buffer were electrophoresed through 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Bio-Rad). The gels were then stained with Coomassie blue to document equal protein loading. The membranes were blocked with 3% dry milk and 0.1% Tween 20 (USB, Cleveland, OH) in PBS and incubated with specific primary antibodies against MMP-2, MMP-9, TIMP-1, and TIMP-2, which were purchased from two different companies (BIOMOL and NeoMarkers). The filters were washed and then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Amersham, Arlington Heights, IL). After development, membranes were stripped and reblotted with an antibody against actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software).Gelatinolytic activity was detected in liver extracts at a final protein content of 50 μg by 10% SDS-PAGE contained 1 mg/ml gelatin (Bio-Rad) under nonreducing conditions. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 minutes each time, rinsed in water, and incubated overnight in a development buffer at 37°C (50 mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02% NaN3, pH 7.5). The gels were then stained with Coomassie brilliant blue R-250 (Bio-Rad) and destained with methanol/acetic acid/water (20:10:70). A clear zone indicates the presence of enzymatic activity. Positive controls for MMP-2 and MMP-9 (BIOMOL) and prestained molecular weight markers (Kaleidoscope Prestained Standards; Bio-Rad) served as standards. Relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software).Cell CultureMurine macrophages (RAW 267) were obtained from the American Type Culture Collection (ATCC/TIB-71; Manassas, VA) and grown in Dulbecco's modified Eagle's medium supplemented with l-glutamine (ATCC/30-2002), 10% heat-inactivated serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C, 5% CO2. RAW 264.7 cells were subcultured every 2 to 3 days when they achieved 70 to 80% confluence, at which point they were passed 1:3 to 1:6. Cells suspended in Dulbecco's modified Eagle's medium at passage 4 and at a concentration of 1 × 106 cells/ml were cultured on FN (Biocoat; BD Biosciences, San Jose, CA) or polylysine-coated plates. RAW cells were placed on 24-well coated plates at 5 × 106 cells/well and incubated at 37°C, 5% CO2 for either 6 or 18 hours. For blocking experiments, cells were preincubated in suspension with two independent anti-α4 antibodies, clones PS/2 (IgG2b; ATCC), and (R1-2; eBiosciences, San Diego, CA) for 30 minutes at 37°C before being cultured on FN-coated plates. Control rat IgG was purchased from Sigma (St. Louis, MO). In some experiments, FN-coated wells were pretreated with anti-FN antibody (Chemicon International Inc., Temecula, CA) for 30 minutes at 37°C, and unbound antibodies were removed by washes in PBS before the addition of untreated RAW 264.7 cells. Cell viability was always confirmed by trypan blue exclusion. Cells and supernatants were collected for RT-PCR and zymography analyses, respectively.Statistical AnalysesData are shown as means ± SD. Statistical comparisons between groups were performed by Prism 3.0 (GraphPad Software, San Diego, CA) and Student's t-test when data had a normal distribution. P values of less than 0.05 were considered statistically significant.ResultsCS1-Facilitated Blockade of FN-α4β1 Interactions Improved Liver Histological Preservation and Function of Steatotic OLTsOur earlier studies have shown that cellular FN expression is virtually absent in naïve steatotic livers, and it is up-regulated in the liver vasculature after 4 hours of cold storage in UW solution before liver transplantation.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Moreover, CS1 peptide therapy improved both function/histological preservation of steatotic liver grafts and recipient survival from 40% in controls to 100% in CS1-treated OLTs.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The present study confirms and extends our previous observations on the beneficial role of CS1 peptide-mediated blockade of FN-α4β1 interactions. Indeed, CS1 peptide therapy improved liver function compared with respective controls; SGOT levels (IU/l) were 1667 ± 186 versus 2543 ± 352, P < 0.04 at day 1, and 155 ± 57 versus 1300 ± 180, P < 0.04 at day 7 (n = 6/group). Moreover, as we have previously shown,11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar CS1 peptide-treated livers at day 1 showed good histological preservation with only mild signs of periportal ballooning or vascular congestion, contrasting with severally damaged controls, which were characterized by disruption of lobular architecture, significant periportal edema, marked leukocyte infiltration, vascular congestion, and necrosis.CS1-Mediated Blockade of FN-α4β1 Interactions Selectively Down-Regulates MMP-9 Expression in Steatotic OLTsLeukocyte migration across endothelial or ECM barriers is dependent on a coordinated series of adhesion release steps and focal matrix degradation.30Basbaum CB Werb Z Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.Curr Opin Cell Biol. 1996; 8: 731-738Crossref PubMed Scopus (292) Google Scholar Gelatinases are metalloproteinases known to promote the breakdown of several matrix proteins, including FN31Owen CA Campbell EJ The cell biology of leukocyte-mediated proteolysis.J Leukoc Biol. 1999; 65: 137-150Crossref PubMed Scopus (350) Google Scholar; thus, to determine whether CS1-facilitated blockade of α4β1-FN interactions affects gelatinase expression, steatotic liver grafts were sequentially harvested from CS1 peptide-treated and control OLTs and analyzed at mRNA and protein levels for MMP-2 and MMP-9 expression.MMP-2 was highly expressed at mRNA level in naïve steatotic livers, and its expression was virtually unchanged after transplantation in both CS1 and control OLTs (Figure 1). On the other hand, MMP-9 mRNA expression was found virtually absent in naïve steatotic livers, scarcely detectable in CS1-treated OLTs, and highly present in control OLTs (Figure 1). Indeed, CS1-targeted therapy decreased liver MMP-9 mRNA expression by 4- to 10-fold compared with respective controls. These observations were correlated at protein level by Western blots; in fact, the lack of and modest levels of MMP-9 protein deposition characterized naïve steatotic livers and CS1 peptide-treated livers, respectively (Figure 2). In contrast, MMP-9 was readily detected at protein levels in control OLTs, and its expression appeared as early as 6 hours. Indeed, with the exception of day 3, in which we were unable to detect the 92-kd (proenzyme) band corresponding to MMP-9 in both CS1 peptide and control OLTs, the densitometric ratios of MMP-9 (92 kd)/β-actin were significantly depressed in CS1-treated livers compared with respective controls at 6 hours (0.09 ± 0.08 versus 0.47 ± 0.10, P < 0.001), 24 hours (0.20 ± 0.14 versus 0.41 ± 0.16, P < 0.01), and day 7 (0.15 ± 0.11 versus 0.65 ± 0.22, P < 0.01) after transplantation (Figure 2).Figure 2MMP-9 expression in steatotic OLTs. A: Western blot analysis of MMP-9 in steatotic OLTs. MMP-9 protein levels were readily detectable in control livers as early as 6 hours post-OLT (lane 2), and this expression was sustained in day 7 control OLTs (lane 4). In contrast, CS1 peptide-treated OLTs were characterized by a significant reduction in MMP-9 protein levels at both 6 hours (lane 3) and day 7 (lane 5) post-transplantation. Naïve steatotic livers (lane 1) showed undetectable levels of MMP-9 protein. B: Densitometric analyses showed that MMP-9 expression at protein level in CS1-treated OLTs was significantly reduced compared with respective controls. Results are presented as mean ± SD (n = 5/group, *P < 0.01, **P < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)CS1-Mediated Blockade of FN-α4β1 Interactions Down-Regulates MMP-9 Expression in Cold Lean Liver I/R InjuryTo evaluate the efficacy of CS1 peptide therapy on down-regulation of MMP-9 expression in an alternate model of lean liver I/R injury, we performed OLTs using livers that were harvested from non-steatotic Sprague Dawley donors, stored at 4°C for 24 hours, and then transplanted into syngeneic recipients. In this well-established model of lean liver I/R injury, CS1 peptide-treated Sprague Dawley recipients had a 14-day survival rate of 100%, contrasting with a 50% survival rate observed in the respective controls.11Amersi F Shen XD Moore C Melinek J Busuttil RW Kupiec Weglinski JW Coito AJ Fibronectin-α4β1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury.Am J Pathol. 2003; 162: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar CS1 peptide-treated OLTs, at 24 hours after transplantation,
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