Connective Tissue Response in IBD
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1 Connective Tissue Response in IBD Dr I C Lawrance MB BS, PhD FRACP School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital
2 Intestinal response to Chronic Inflammation Control UC CD Trichrome Staining of Collagen
3 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
4 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
5 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
6 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
7 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Degradation Deposition
8 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Degradation Deposition
9 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Degradation Deposition
10 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
11 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
12 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
13 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
14 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
15 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
16 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
17 Balancing ECM deposition and degradation Collagens Elastin Fibronectin Laminin TIMPs PAI Proteoglycans Plasmin/PA MMPs Collagenases Gelatinases Stromeolysins Deposition Degradation
18 Immunohistochemisrty Collagen III and I LP Lamina Propria Control SM Submucosa ME Muscularis Externa Inflamed UC Ratio Collagen type III:I LP SM * SM ME A A Adverntia Fibrosed CD ** Collagen III Collagen I 0.00 LP SM ME A Lawrance et.al. IBD 2001
19 Isolated IBD Intestinal Fibroblasts-like like cells Phase contrast light microscopy Electron microscopy Rough endoplasmic reticulum (thick arrows) Collagen fibrils (thin arrows). Lawrance et.al. IBD 2001
20 Intestinal Fibroblasts 200 bfgf 10 ng/ml 150 Scintillation CPM x10 3 /μgdna Doubling times in days IGF-1 10 ng/ml Control Bowel Fibrosed CD Inflamed CD Inflamed UC 25 0 Unstim Stim Unstim Stim Unstim Stim Control CD UC Lawrance et.al. IBD 2001
21 Fibroblast TGF-β1 IGF-1 bfgf Myofibroblast Smooth Muscle Cell
22 Regulators of ECM - TGF-β1 Plasmin - + PAI-1 PA TGF-β COL I TIMP-1 - IGF MMP-1 MMP-3 MMP-9 PDGF Recruitment Macrophages Leukocytes Recruitment Fibroblasts Myofibroblasts
23 150 TGF-β1 1 and IGF-1 1 expression in IBD TGF- β1 In situ hybridisation TGF- β1 Immunohistochemistry No. Positive cells per HPF LP SM ME A IGF-1 Insitu hybridisation LP SM ME A IGF-1 Immunohistochemistry Control Inflamed UC Fibrosed CD LP - Lamina Propria SM - Submucosa ME - Muscularis Externa A- Adverntia 25 0 LP SM ME A LP SM ME A Lawrance et.al. IBD 2001
24 Regulators of ECM - SPARC SPARC (Secreted Protein Acidic and Rich in Cysteine) Transiently secreted in actively inflamed tissue Plasmin - PAI PDGF Retinoic acid TGF-β1 SPARC COX-2 - Indomethacin COX-2 2 inhibitors COL I TIMP-1 - PGE2? + Blocks TGF-β1 1 effects Fibroblast MMP-1 MMP-3 MMP-9 Macrophage MMP-1 MMP-3
25 Microarray analysis of ECM-related genes UC & CD compared to Control UC 24 genes 5 genes CD 15 genes 0 genes 7 genes in common Inflamed UC Fibrosed CD Mucin Trefoil factor 1 Mucin 4 MMP-12 MMP-9 MMP-1 MMP-3 Elafin Cystatin A Fibronectin 1 COL4A2 COL3A1 COL1A2 COL1A1 COL6A3 COL6A2 Proteoglycan 1 Epican Amelogenin Vimentin a2-sma g2-sma PMP22 vwf APM2 TGFbI OSF-2 SPARC Hevin Adducin 2 Zygin 2 Trichohyalin Actin-related Cytokeratin 20 Paralemmin Caldesmon 1 Tropomyosin Lawrance et.al. Human Mol Genetics 2001 Fold Change c/w Control
26 SPARC Immunohistochemistry Control Intestine Neural bundles Fibroblast-like like cells Lawrance et.al. Gastro 126 (4): Suppl
27 SPARC Immunohistochemistry Inflamed UC Inflamed Ischaemic Colitis Neo-endothlial endothlial cells Inflammatory cells
28 SPARC Immunohistochemistry Fibrosed CD Fibrosed Diverticular Disease Neo-endothlial endothlial cells Fibroblasts-like like cells Inflammatory cells
29 Methods TNBS murine model of Chronic Inflammation-induced induced intestinal fibrosis 12 week old female CD-1 1 and BALB/c mice sensitised to TNBS. Weekly enemas for 6-88 weeks Colons removed cleaned & weighed Lawrance, Wu et.al. Gastroenterology 2003
30 Histology H & E Trichrome Water control TNBS & ETOH 2/52 TNBS & ETOH 8/52 Lawrance, Wu et.al. Gastroenterology 2003
31 Methods TNBS murine model ± Indomethacin or Retinoic Acid 12 week old female CD-1 mice sensitised to TNBS. Retinoic Acid 50ug/day or Indomethacin 0.2mg/kg/day Weekly enemas for 6 weeks Colons removed cleaned & weighed Lawrance et.al. Gastroenterology 126 (4): Suppl
32 Histology at 2 weeks TNBS alone or with 0.2mg/kg/day indomethacin or 50ug RA/day TNBS TNBS + Indo TNBS + RA Nil Mild Mod Severe Inflammation Nil Mild Mod/Severe Fibrosis
33 SPARC mrna at 2 weeks Ratio to control mice SPARC/ βactin Control p=0.047 TNBS High Fibrosis p= p=0.016 TNBS Low Fibrosis p=0.005 p=0.016 p=0.047 RA/TNBS Indo/TNBS
34 SPARC Immunohistochemistry Trichrome SPARC
35 SPARC +ve+ Cells per High Power Field 150 p=0.007 p=0.005 Cell count p= Water Control n=10 RA Control n=5 Indo Control n=5 TNBS High n=6 TNBS Low n=6 RA n=6 Indo n=10
36 Regulators of ECM - SPARC Indomethacin SPARC COX-2 - PGE2 PGE2? + Fibroblast MMP-1 MMP-3 MMP-9 Macrophage MMP-1 MMP-3
37 SPARC-null and WT mice TNBS murine model Weekly enemas for 2 weeks and 6 weeks Indomethacin 0.2mg/kg/day.. SPARC-null 11 week female C57bl6 x 129SV mice SPARC WT ± Indomethacin 11 week female C57bl6 x 129SV mice Lawrance et.al. Gastroenterology 128 (4): Suppl. 2 Apr 2005
38 SPARC-null and WT mice at 2 weeks SPARC +/+ mice TNBS ± with 0.2mg/kg/day indomethacin SPARC -/- mice TNBS alone Wild type TNBS alone Wild type TNBS + Indo SPARC -/- TNBS alone Nil Mild Mod Severe Inflammation Nil Mild Mod/Severe Fibrosis
39 IGF-1 SPARC TGF-β1 Myofibroblast Epithelial cells SPARC IGF-1 Fibroblast TGF-β1 SPARC Smooth Muscle Cells Macrophage Collagen III IGF-1 Collagen I TGF-β1 Extracellular Matrix (ECM)
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