FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2077508723> ?p ?o ?g. }

• W2077508723 endingPage "1902" @default.
• W2077508723 startingPage "1895" @default.
• W2077508723 abstract "A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis. A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis. Hepatic fibrosis is a condition that results in loss of normal liver cell function due to the disorganized over-accumulation of extracellular matrix (ECM) components in the liver. It is clear that the increased production of ECM components is responsible for the altered ECM metabolism in the fibrotic liver.1Schuppan D Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins.Semin Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (324) Google Scholar However, deregulation of the enzymatic machinery involved in ECM degradation may also be an important contributing factor in the pathogenesis of hepatic fibrosis and cirrhosis. The main components of the extracellular matrix in normal liver are collagen types I, III, IV, V, and VI, although other types of collagen are present in smaller proportions. There are also many noncollagenous components, including fibronectin, laminin, tenascin, undulin, and entactin.1Schuppan D Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins.Semin Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (324) Google Scholar In a normal liver, these matrix components are constantly remodeled by matrix-degrading enzymes leading to a controlled deposition of matrix components. Of the several families of ECM-degradative enzymes, the matrix metalloproteinases (MMPs) are the most important, as collectively MMPs can degrade all of the protein components of the ECM.2Matrisian L Metalloproteinases and their inhibitors in matrix remodeling.Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1532) Google Scholar, 3Birkedal-Hansen H Moore WGI Bodden MK Windsor LJ Birkedal-Hansen B DeCarlo A Engler JA Matrix metalloproteinases: a review.Clin Rev Oral Biol Med. 1993; 4: 197-250PubMed Google Scholar, 4Woessner JF Matrix metalloproteinases and their inhibitors in connective tissue remodelling.FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3091) Google Scholar MMPs are classified into four groups: 1) collagenases, which cleave collagen at a specific site in triple helical collagen fibrils resulting in fragments that are susceptible to thermal denaturation into gelatin, which can then be acted on by other groups of MMPs, predominantly the gelatinases, 2) gelatinases, which include gelatinase A (MMP-2) and gelatinase B (MMP-9), 3) stromelysins, and 4) The RXKR-motif-containing sub-family, which includes the membrane-type MMPs (MT-MMPs).5Sato H Takino T Okada Y Cao J Shinagawa A Yamamoto E Seiki M A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature. 1994; 370: 61-65Crossref PubMed Scopus (2379) Google Scholar The activities of MMPs are regulated at three levels, namely, transcription and zymogen activation and through the action of a family of inhibitory proteins, ie, the tissue inhibitors of metalloproteinases (TIMPs). The TIMPs interact with a 1:1 stoichiometry with MMPs to inhibit their activity.2Matrisian L Metalloproteinases and their inhibitors in matrix remodeling.Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1532) Google Scholar, 3Birkedal-Hansen H Moore WGI Bodden MK Windsor LJ Birkedal-Hansen B DeCarlo A Engler JA Matrix metalloproteinases: a review.Clin Rev Oral Biol Med. 1993; 4: 197-250PubMed Google Scholar There have been four members of the TIMP family identified so far, designated TIMP-1, -2, -3, and -4.6Leco KJ Khoka R Pavloff N Hawkes SP Edwards DR Tissue inhibitor of metalloproteinases (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar, 7Apte SS Olsen BR Murphy G The gene structure of tissue inhibitor of metalloproteinases (TIMP-3) and its inhibitory activities define the distinct TIMP gene family.J Biol Chem. 1995; 270: 14313-14318Crossref PubMed Scopus (283) Google Scholar, 8Stetler-Stevenson WG Krutzsch HC Liotta LA TIMP-2: identification and characterization of a new member of the metalloproteinase inhibitor family.Matrix Suppl. 1992; 1: 299-306PubMed Google Scholar, 9Leco KJ Apte SA Taniguchi GT Hawkes SP Khokha R Schultz G Edwards DR Murine tissue inhibitor of metalloproteinases-4 (TIMP-4): cDNA isolation and expression in adult mouse tissues.FEBS Lett. 1997; 401: 213-217Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar The action of the TIMPs is considered to be quite broad, as TIMP-1, -2, and -3 are indistinguishable in their MMP-inhibitory abilities in solution-based assays.7Apte SS Olsen BR Murphy G The gene structure of tissue inhibitor of metalloproteinases (TIMP-3) and its inhibitory activities define the distinct TIMP gene family.J Biol Chem. 1995; 270: 14313-14318Crossref PubMed Scopus (283) Google Scholar Although there appears to be a similarity in activity of the TIMPs, there are differences in localization and regulation. TIMP-1 and TIMP-3 are inducible in response to phorbol myristate acetate (PMA) and many growth factors.6Leco KJ Khoka R Pavloff N Hawkes SP Edwards DR Tissue inhibitor of metalloproteinases (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar, 10Edwards DR Waterhouse P Holman ML Denhardt DT A growth responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constitutive transcripts.Nucleic Acids Res. 1986; 14: 8863-8878Crossref PubMed Scopus (123) Google Scholar, 11Edwards DR Rocheleau H Sharma R Wills AJ Cowie A Hassel JA Heath JK Involvement of AP-1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-a) transcription.Biochem Biophys Acta. 1992; 1171: 41-55Crossref PubMed Scopus (129) Google Scholar, 12Ponton A Coulombe B Steyaert A Williams BR Skup D Basal expression of the gene (TIMP) encoding the murine tissue inhibitor of metalloproteinases is mediated through AP1- and CCAAT-binding factors.Gene. 1992; 116: 187-194Crossref PubMed Scopus (17) Google Scholar Alternatively, TIMP-2 is largely constitutively expressed in most cell types. During the course of fibrosis and cirrhosis, all of the liver matrix proteins increase in abundance, but to varying extents.13Arthur MJP Matrix degradation in the liver.Semin Liver Dis. 1990; 10: 47-55Crossref PubMed Scopus (57) Google Scholar, 14Arthur MJP Iredale JP Hepatic lipocytes, TIMP-1 and liver fibrosis.J R Coll Physicians Lond. 1994; 28: 200-208PubMed Google Scholar, 15Milani S Herbst H Schuppan D Hahn E Stein H In situ hybridization for procollagen types I, III, and IV mRNA in normal and fibrotic rat liver: evidence for predominant expression in nonparenchymal liver cells.Hepatology. 1989; 10: 84-92Crossref PubMed Scopus (261) Google Scholar Collagen I is the most up-regulated, with the percentage composition in the liver increasing 5- to 10-fold.1Schuppan D Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins.Semin Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (324) Google Scholar One recent study has focused on the expression levels of interstitial collagenase with respect to those of TIMP-1 in the bile duct ligation (BDL) and carbon tetrachloride models of experimental fibrosis.16Iredale JP Benyon C Arthur MJP Ferris WF Alcolado R Winwood PJ Clark N Murphy G Tissue inhibitor of metalloproteinases-1 messenger RNA in experimental liver injury and fibrosis.Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar This study found an increase in TIMP-1, whereas interstitial collagenase levels remained unchanged, suggesting that increased matrix deposition occurs due to the disruption of the balance between MMPs and TIMPs in favor of the inhibitors. Another group has analyzed the involvement of gelatinases in the dynamic alterations occurring during fibrosis,17Takahara T Furui K Funaki J Nakayama y, Itoh H, Miyabayashi C, Sato H, Seiki M, Ooshima A, Watanabe A: Increased expression of matrix metalloproteinases-II in experimental liver fibrosis in rats.Hepatology. 1995; 21: 787-795Crossref PubMed Google Scholar finding an increase in both active and latent MMP-2 protein by gelatin zymography. These results collectively imply that normal maintenance of the ECM is disrupted, resulting in a disorganized and overabundant extracellular scaffold with a consequent impairment of liver cell functions. To obtain a more detailed analysis of the dynamic interactions occurring between the MMPs and TIMPs during experimental hepatic fibrosis, we have systematically studied expression levels of MMP-2, -3, -9, and -13 and protein activity of MMP-2 and -9 as well as TIMP-1, -2, and -3 protein activity and expression at the mRNA level. Our data demonstrate that changes in the gelatinolytic activities, and TIMPs, occur rapidly after BDL and are sustained during the initial month of fibrogenesis. We discuss the implications of these findings and the effects of imbalance of gelatinase/TIMP levels in reference to hepatic fibrosis in a BDL model. The BDL protocol was approved by the University of Calgary Faculty of Medicine Animal Care Committee, and all animals were treated humanely in accordance with guidelines established by the Canadian Council on Animal Care. Male Sprague-Dawley rats weighing 300 to 350 g were divided into three groups (three animals in each): 1) normal, 2) bile duct ligated, sacrificed at 2, 5, 10, 20, and 30 days after surgery, and 3) sham-operated rats, sacrificed at the same intervals. The animals were fed and watered ad libitum throughout the experiment. BDL is a well characterized and widely used model of experimental fibrosis and was performed in this study as previously described.18Lee SS Huang M Ma Z Rorstad O Vasoactive intestinal peptide in cirrhotic rats: hemodynamic effects and mesenteric arterial receptor characteristics.Hepatology. 1996; 23: 1174-1180Crossref PubMed Google Scholar In brief, under halothane anesthesia, through a midline laparotomy, the extrahepatic common bile duct was double ligated with 30 silk and sectioned between the ligatures. The abdominal incision was sutured with silk, and the rats were allowed to recover in individual cages. Sham operated rats were treated in the same manner except that the bile duct was merely identified and gently manipulated but not ligated or sectioned. On the day of the studies, the rats were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally). The abdomen was opened through a midline incision, and before sacrifice, 1 ml of blood was obtained from the inferior vena cava using a 20-gauge needle. The blood was collected in heparinized tubes without EDTA and centrifuged, and the plasma was aliquotted and frozen at −70°C until testing. The livers were flushed with 10 ml of cold phosphate-buffered saline through the portal vein to eliminate MMPs and TIMPs present in the blood. Liver fragments of 0.5 cm3 were dissected out fresh and snap-frozen in plastic vials in liquid nitrogen. Frozen liver tissue in protein extraction buffer (1% Triton X-100, 500 mmol/L Tris/HCl, pH 7.6, 200 mmol/L NaCl, and 10 mmol/L CaCl2) was homogenized using an IKA-Ultra-Turrax T25 homogenizer. The homogenate was centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant was transferred to a clean tube and stored at −70°C. Homogenate protein content was determined using the Bradford procedure.19Bradford MM A rapid and sensitive method for the quantification of microgram quantities of protein utilizes the principle of protein dye binding.Anal Biochem. 1980; 102: 196-202Crossref PubMed Scopus (1851) Google Scholar Proteins were analyzed for gelatinolytic activity by gelatin zymography using the previously described procedures. A total of 20 μg of protein extract or 5 μl of plasma (1/25 dilution) was separated by a 10% polyacrylamide gel, prepared as described.20Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1989Google Scholar Gelatin was included to a final concentration of 1 mg/ml. After electrophoresis, the gel was washed once for 15 minutes and then overnight in wash buffer (2.5% Triton X-100, 50 mmol/L Tris/HCl, pH 7.5, and 5 mmol/L CaCl2) to remove the sodium dodecyl sulfate (SDS). The gel was then rinsed three times in water, followed by a 24-hour incubation at 37°C in incubation buffer (50 mmol/L Tris/HCl, pH 7.5, and 5 mmol/L CaCl2). The gel was then stained for 4 hours in Coomassie Brilliant Blue. Reverse zymography was similarly performed with the exception that conditioned medium from baby hamster kidney (BHK) cells, which express gelatinase A, was included in the gel mix with the gelatin (1 ml of conditioned medium was added to 15 ml of the gel mix). After regeneration, the gelatinase A degrades the gelatin in all regions of the gel except in regions where there is TIMP activity. All washes and incubations were the same as zymography. As a positive control for zymography, conditioned medium from BHK cells expressing MMP-2 and transfected with MMP-9 was used. For reverse zymography, conditioned medium from BHK cells transfected with mouse TIMPs was used. Complete gels were photographed, and the photos then digitized on a Hewlett Packard Scanjet 4c scanner. The images were then analyzed and quantified using the image analysis program NIH Image (National Institutes of Health, Bethesda, MD). For Western blotting, 20 μg of protein from each sample were boiled for 3 minutes in the presence of SDS gel-loading buffer with 0.1 mol/L dithiothreitol and electrophoresed through a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The gel was run at 75 mA at room temperature. The proteins were transferred to nitrocellulose, blocked with 5% nonfat milk in Tris-buffered saline/Tween 20 (TBS-T), and incubated with 1:5000 diluted anti-mouse TIMP-1 antibodies (gift of Dr. G. Murphy, School of Biological Science, University of East Anglia, Norwich, UK) in TBS-T for 1 hour at room temperature. After washing, the blots were incubated with 1:5000 diluted rabbit anti-sheep antibody (catalog item 31480, Pierce, Rockford, IL) in TBS for 1 hour at room temperature. After repeated washes, the membranes were incubated in equal volumes of detection reagents 1 and 2 (ECL Western blotting detection reagents from Amersham Life Science, Arlington Heights, IL) for 1 minute at room temperature and immediately exposed to x-ray film. To confirm the specificity of binding, the anti-mouse TIMP-1 antibodies were preincubated for 2.5 hours with a Western blot containing 500 μl of 25X concentrated conditioned medium from BHK cells transfected with TIMP-1. Two Western blots were prepared under identical conditions. One was incubated with nonabsorbed antibodies and the other with the preabsorbed antibodies as described above. Total RNA was isolated by the acid-guanidinium-phenol-chloroform procedure of Chomczynski and Sacchi.21Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar Ten micrograms of total RNA was electrophoresed on a 0.8% denaturing agarose gel and then transferred to nylon membranes (Duralon-UV, Stratagene, La Jolla, CA). Equal loading was determined by methylene blue staining of the membrane for 15 minutes and analysis of 28 S and 18 S rRNA. The membranes were then prehybridized in buffer containing 5X SSC, 50% (v/v) formamide, 10X Denhardt's solution, and 0.1% SDS and then hybridized with nick-translated probes (specific activity, >108 cpm/μg) and washed in SDS/SSC solutions at 42°C (the highest stringency was 0.2X SSC/0.1% SDS). The probes used were generated from mouse TIMP-1, -2, and -3 plasmids, which have been described previously.6Leco KJ Khoka R Pavloff N Hawkes SP Edwards DR Tissue inhibitor of metalloproteinases (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar, 22Leco KJ Hayden LJ Sharma RR Rocheleau H Greenberg AH Edwards DR Differential regulation of TIMP-1 and TIMP-2 mRNA expression in normal and Ha-ras-transformed murine fibroblasts.Gene. 1992; 117: 209-217Crossref PubMed Scopus (93) Google Scholar A partial mouse 72-kd gelatinase (MMP-2) clone was obtained by reverse transcription polymerase chain reaction from RNA isolated from mouse C3H 10T1/2 fibroblasts. The following oligonucleotides were used: 5′-GGCCCTGTCACTCCTGAGAT, corresponding to nucleotides 1337 to 1356 of the human MMP-2 sequence published by Collier et al23Collier IE Wilhelm SM Eisen AZ Marmer BL Grant GA Seltzer JL Kronberger A He C Bauer EA Goldberg GI H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen.J Biol Chem. 1988; 14: 6579-6587Google Scholar and 5′-oGCATCCAGGTTATCGGGGA, which is the reverse complement of the sequence from 1791 to 1810 of the same human sequence. The 475-bp polymerase chain reaction product was cloned into the EcoRI/HindIII sites of pGEM2 with the 5′end of the cDNA at the T7 promoter end of the multipurpose cloning site. The mouse gelatinase B (MMP-9) 3160-bp cDNA clone was a gift of Dr. Kenji Sugita, Division of Oncology, Department of Microbiology, Shionogi Research Laboratories, Osaka, Japan, and was described previously.24Tanaka H Hojo K Yoshida H Yoshioka T Sugita K Molecular cloning and expression of the mouse 105-kd gelatinase cDNA+.Biol Biophys Res Commun. 1993; 190: 732-740Crossref PubMed Scopus (88) Google Scholar The rat stromelysin-1 (MMP-3) cDNA clone used was a 600-bp fragment generated by EcoRI andHindIII digestion of a full-length clone provided by Dr. Lynn Matrisian, Vanderbilt University, Nashville, TN. This fragment was cloned into pGEM-2 with the 5′ and 3′ ends of the insert in theEcoRI and HindIII sites of the vector, respectively. The mouse collagenase-3 (MMP-13) cDNA clone was a full-length construct designated pCLM11, which was a gift of Dr. Yves Eeckhout, University Catholique de Louvain, Brussels, Belgium. Fragments of livers were formalin fixed, processed for routine histology, and stained with hematoxylin and eosin, Sirius red, and Gomori's trichrome. Differences between MMP-2 and -9 protein expressions in different groups were calculated by two-tailed between-groups t-tests using SPSS 4.0 for Macintosh. We have studied the levels of expression of MMP-2, -3, -9, and -13 and TIMP-1, -2, and -3 mRNAs in liver tissue from animals that have undergone BDL (three animals per group, at 2, 5, 10, 20, and 30 days after surgery) compared with sham-operated controls (same postoperative days as BDL). The study time course (2 to 30 days) was chosen to include both early and late fibrotic events. A very weak signal was seen for MMP-2 and -9 that was below publishable level. It showed no detectable differences between any of the groups. MMP-3 and -13 mRNA transcripts were not detected in any of the samples by Northern blot analysis (data not shown). This likely reflects low mRNA expression levels. Gelatin zymography analysis showed active MMP-2 and MMP-9 proteins (representative data shown in Figure 1A). Activities of both gelatinases increased in BDL livers at day 2 after BDL, and a steady increase was noted until day 10, remaining stable afterwards (Figure 2, A and B). Activities of MMP-2 and -9 were higher in BDL livers than in livers from control animals (not treated surgically) and from sham animals. For MMP-2, statistically significant differences were observed between control animalsversus 10-day, 20-day, and 30-day BDL animals; differences were also seen between 2-day, 10-day, 20-day, and 30-day shamversus BDL animals. Additional differences were found and between 10-day, 20-day, and 30-day BDL versus 2-day and 5-day BDL animals (P ≤ 0.01). No significant differences were observed between days 10, 20, and 30 in BDL animals. For MMP-9, statistically significant differences were noted between control animals versus BDL 2-day, 5-day, 10-day, 20-day, and 30-day animals, between sham versus BDL 5-day, 10-day, 20-day, and 30-day animals, and between BDL 10-day, 20-day, and 30-day versus BDL 2-day and 5-day animals (P ≤ 0.01). A less significant difference (P = 0.012) was observed between 2-day BDLversus 2-day sham animals. No significant differences were noted between 10-day, 20-day, and 30-day BDL animals. Activated forms (lower molecular bands) of MMP-2 and -9 were observed in BDL animals (Figure 1A). The appearance of increased levels of the active forms likely reflects a general increase in expression of gelatinase proforms and is therefore not the result of MMP activation in the post-BDL liver.Figure 2MMP-2 (A) and MMP-9 (B) gelatinolytic activities in liver tissue protein extracts. Gelatinolytic activities were measured by gelatin zymography (shown in Figure 1). The photographs of the gels were digitized on a Hewlett Packard Scanjet 4c scanner. The images were analyzed and quantified using the image analysis program NIH Image. Control indicates unmanipulated animals.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Northern blot analysis of TIMP mRNA transcripts showed an increase in TIMP-1 after BDL surgery (Figure 3, A and B). TIMP-1 transcripts appeared on day 2 after BDL, increased until day 10 after BDL, and remained relatively stable afterwards (Figure 3, Figure 4). The comparison of the densitometrically measured signals on the Northern blots showed statistically significant overexpression of TIMP-1 between BDL day 2 and BDL day 5 (P ≤ 0.0025) and between BDL day 5 and BDL day 10 (P ≤ 0.043). There were no significant differences between BDL day 10 and BDL day 20 levels or between BDL day 20 and BDL day 30 levels. A statistically significant difference was seen, however, when BDL day 10 levels were compared with BDL day 30 levels (p ≤ 0.018). No TIMP-1 transcripts were detected in control and sham-operated animals. Expression of TIMP-2 increased at 10 days after BDL and was not detected at either of the two earlier points, 2 and 5 days (data not shown). Of the two transcripts (3.5 and 1.0 kb), mainly the 1.0-kb transcript was increased in BDL livers (Figure 3B). This lower molecular weight transcript was not detectable in the control animals, but it was present 10 days after BDL surgery, and the expression level remained constant until 30 days after BDL. Sham-operated animals had very low levels of both 3.5- and 1.0-kb transcripts. A slight up-regulation of TIMP-3 mRNA was also seen (Figure 3B), although this was not as prominent as the changes seen for TIMP-1 and TIMP-2. Quantification of free TIMP activities in liver tissue by gelatin reverse zymography (Figure 5A) revealed that the changes in TIMP mRNA were not mirrored at the level of protein activity. The most prominent TIMP activity present in livers of control, sham-operated, and BDL animals co-migrated with unglycosylated TIMP-3 standard. Samples were also run on conventional SDS-PAGE gels (without reduction) that were Coomassie Blue stained to confirm that the bands were bona fide TIMP activities rather than abundant proteins (data not shown). The activity of TIMP-3 was unaffected by BDL surgery, as was also the case for TIMP-2 (Figure 5A). No TIMP-1 activity was detected by reverse zymography (Figure 5A), and Western blot analysis demonstrated that this TIMP was mainly present in the form of complexes (Figure 6), which likely interferes with its detection by activity assays. The Western blots indicated the presence of TIMP-1 complexes, both in post-BDL and sham livers (Figure 6). Free TIMP-1 (lower band) was seen only in normal controls and in sham-operated animals. Lack of a detectable free band in the BDL groups suggests that most of the produced TIMP-1 enters the complexes. Thus, little if any free TIMP-1 is available to exert its activities. These data agrees with the reverse zymography results, which failed to demonstrate any TIMP-1-related activity. The analysis of the composition of the TIMP-1 complexes seen on the Western blot is beyond the scope of this paper.Figure 4TIMP-1 expression in BDL livers. TIMP-1 mRNA levels were measured by Northern blot (shown in Figure 3). The gels were photographed, and the photos were then digitized on a Hewlett Packard Scanjet 4c scanner. The images were then analyzed and quantified using the image analysis program NIH Image. TIMP-1 values were normalized to 28 S rRNA values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Reverse gelatin zymography analysis for inhibition of gelatinolytic activities in liver tissue protein extracts (A) and plasma (B). Proteins from liver tissue extracts and plasma were loaded onto SDS-PAGE gels and processed for reverse gelatin zymography as described in Materials and Methods. Tissue from unmanipulated rats (normal) was included as a control. Conditioned media from TIMP-1/TIMP-2-expressing BHK cells (CM) or TIMP-1/TIMP-3-overexpressing BHK cells (MT) were included as size markers for identification of TIMP proteins. The divergent migration patterns of TIMP-1 and -2 result from their different glycosylation (TIMP-1 is double glycosylated; TIMP-3 exists in glycosylated and nonglycosylated forms, hence two separate bands).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Western blot analysis of TIMP-1 expression in BDL and sham livers.A: Western blot was performed as described in Materials and Methods. Polyclonal anti-mouse TIMP-1 antibodies (gift from Dr. G. Murphy, School of Biological Science, University of East Anglia, Norwich, UK) were used. T" @default.
• W2077508723 created "2016-06-24" @default.
• W2077508723 creator A5000890608 @default.
• W2077508723 creator A5010515520 @default.
• W2077508723 creator A5015199487 @default.
• W2077508723 creator A5017568469 @default.
• W2077508723 creator A5030718819 @default.
• W2077508723 creator A5042678528 @default.
• W2077508723 creator A5050814031 @default.
• W2077508723 creator A5077334030 @default.
• W2077508723 creator A5088766199 @default.
• W2077508723 date "1998-12-01" @default.
• W2077508723 modified "2023-10-01" @default.
• W2077508723 title "Altered Balance Between Matrix Metalloproteinases and Their Inhibitors in Experimental Biliary Fibrosis" @default.
• W2077508723 cites W1583304743 @default.
• W2077508723 cites W1602981281 @default.
• W2077508723 cites W1613102183 @default.
• W2077508723 cites W1821760033 @default.
• W2077508723 cites W1894541755 @default.
• W2077508723 cites W1979390430 @default.
• W2077508723 cites W1980480613 @default.
• W2077508723 cites W1989391836 @default.
• W2077508723 cites W1996677487 @default.
• W2077508723 cites W2000175347 @default.
• W2077508723 cites W2010846405 @default.
• W2077508723 cites W2011879593 @default.
• W2077508723 cites W2021484612 @default.
• W2077508723 cites W2022704877 @default.
• W2077508723 cites W2028462451 @default.
• W2077508723 cites W2036358552 @default.
• W2077508723 cites W2053170542 @default.
• W2077508723 cites W2071226610 @default.
• W2077508723 cites W2071583399 @default.
• W2077508723 cites W2092754204 @default.
• W2077508723 cites W2094246726 @default.
• W2077508723 cites W2102164012 @default.
• W2077508723 cites W2109658199 @default.
• W2077508723 cites W2111840256 @default.
• W2077508723 cites W2123734505 @default.
• W2077508723 cites W2128635872 @default.
• W2077508723 cites W2134812217 @default.
• W2077508723 cites W2167923772 @default.
• W2077508723 cites W2309242976 @default.
• W2077508723 cites W2330621664 @default.
• W2077508723 cites W2408892807 @default.
• W2077508723 cites W279241986 @default.
• W2077508723 cites W4294216491 @default.
• W2077508723 doi "https://doi.org/10.1016/s0002-9440(10)65703-3" @default.
• W2077508723 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1866318" @default.
• W2077508723 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9846979" @default.
• W2077508723 hasPublicationYear "1998" @default.
• W2077508723 type Work @default.
• W2077508723 sameAs 2077508723 @default.
• W2077508723 citedByCount "177" @default.
• W2077508723 countsByYear W20775087232012 @default.
• W2077508723 countsByYear W20775087232013 @default.
• W2077508723 countsByYear W20775087232014 @default.
• W2077508723 countsByYear W20775087232015 @default.
• W2077508723 countsByYear W20775087232016 @default.
• W2077508723 countsByYear W20775087232017 @default.
• W2077508723 countsByYear W20775087232018 @default.
• W2077508723 countsByYear W20775087232019 @default.
• W2077508723 countsByYear W20775087232020 @default.
• W2077508723 countsByYear W20775087232021 @default.
• W2077508723 countsByYear W20775087232022 @default.
• W2077508723 countsByYear W20775087232023 @default.
• W2077508723 crossrefType "journal-article" @default.
• W2077508723 hasAuthorship W2077508723A5000890608 @default.
• W2077508723 hasAuthorship W2077508723A5010515520 @default.
• W2077508723 hasAuthorship W2077508723A5015199487 @default.
• W2077508723 hasAuthorship W2077508723A5017568469 @default.
• W2077508723 hasAuthorship W2077508723A5030718819 @default.
• W2077508723 hasAuthorship W2077508723A5042678528 @default.
• W2077508723 hasAuthorship W2077508723A5050814031 @default.
• W2077508723 hasAuthorship W2077508723A5077334030 @default.
• W2077508723 hasAuthorship W2077508723A5088766199 @default.
• W2077508723 hasBestOaLocation W20775087231 @default.
• W2077508723 hasConcept C106487976 @default.
• W2077508723 hasConcept C109523444 @default.
• W2077508723 hasConcept C126322002 @default.
• W2077508723 hasConcept C142724271 @default.
• W2077508723 hasConcept C168031717 @default.
• W2077508723 hasConcept C185592680 @default.
• W2077508723 hasConcept C2780559512 @default.
• W2077508723 hasConcept C43617362 @default.
• W2077508723 hasConcept C71924100 @default.
• W2077508723 hasConcept C86803240 @default.
• W2077508723 hasConcept C99508421 @default.
• W2077508723 hasConceptScore W2077508723C106487976 @default.
• W2077508723 hasConceptScore W2077508723C109523444 @default.
• W2077508723 hasConceptScore W2077508723C126322002 @default.
• W2077508723 hasConceptScore W2077508723C142724271 @default.
• W2077508723 hasConceptScore W2077508723C168031717 @default.
• W2077508723 hasConceptScore W2077508723C185592680 @default.
• W2077508723 hasConceptScore W2077508723C2780559512 @default.
• W2077508723 hasConceptScore W2077508723C43617362 @default.
• W2077508723 hasConceptScore W2077508723C71924100 @default.
• W2077508723 hasConceptScore W2077508723C86803240 @default.

• next