17 - Lecture 18 November 17, 2008 Role of Fibrosis in...

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Unformatted text preview: Lecture 18 November 17, 2008 Role of Fibrosis in Diabetic Cardiomyopathy Role Dr. Christopher McCulloch Canada Research Chair in Matrix Dynamics Director, CIHR Group in MATRIX DYNAMICS The Heart Works as a Pump The Heart Failure Heart Heart Failure Heart • • • In USA: heart failure In accounts for 400,000700,000 deaths a year, 700,000 $20-40 billion/year in health care costs Is the leading hospital Is discharge diagnosis Defined as functionally Defined insufficient pumping capacity to meet metabolic needs of tissue Heart Failure: Heart A Common Complication of Diabetes Underlying mechanism Underlying Cardiac Ischemia Cardiac Hypertension Hypertension Diabetic Cardiomyopathy Diabetic cardiotoxic triad Pathogenesis of Diabetic Pathogenesis Cardiomyopathy FFA- free fatty acid CPC- cardiac progenitor cells RAAS- renin-angiotensin-aldosterone system Effects of Hyperglycemia Effects on Diabetic Myocardium on RAAS- renin-angiotensin-aldosterone system Diabetes-induced Alterations of Myocardial Diabetes Matrix and Impact on Cardiac Function 2.4 X risk of heart failure in diabetic men 5.1 X risk increase in women Increase in AGE macromolecules Interacts with AGE receptors (RAGE) Stimulate release of pro-fibrotic growth factors, promote collagen deposition, increase inflammation leading to diastolic dysfunction and atherosclerosis Increased collagen cross-linking Increased rigidity of heart decreased contractility and compliance heart failure Extracellular Matrix Components Extracellular of the Myocardium (Baudino, 2006) Not A Great Pump!!! Not Diabetic Modifications of Collagen Diabetic Glycation: Effects on Physical Glycation: Properties of Collagen Increased stiffness and breaking load of collagen Increased denaturation temperature Decreased susceptibility to degradative enzymes: blocking enzyme cleavage sites closer molecular packing in fibres due to cross-linking Biological Manifestations of Biological Collagen Glycation Perturbation in interactions of collagen and integrins Impaired collagen phagocytosis Impaired collagen turnover Increased collagen deposition in ECM Increased rigidity of myocardium MGO-induced Glycation as MGO an in vitro Model of Diabetes in Methylgloxygal (MGO) Methylgloxygal Reactive dicarbonyl compound Breakdown of Reactive Breakdown triosephosphates, acetone, ketones and glucose. Present in minute amounts in tissue Present Very reactive, therefore only require short Very incubation times Causes cross linking of arginines and lysines Causes Effect of MGO on Collagen Effect W n er GO m en co n ig M r Tx n nd olla M en BS lage Ge m P ol De C AP llag 10 ollag x C T C Co γ l ro t M β 170 kDa 130 95 72 α2 α1 Collagen monomers Loss of α monomer bands; less distinct protein bands Cardiac Fibroblasts and Myofibroblast Cardiac Differentiation Modifications to Collagen? – chemical/physical Modifications to Collagen? – chemical/physical + (Tomasek, 2002) Hypothesis: Hypothesis: Differentiation of cardiac fibroblasts into Differentiation myofibroblasts is regulated by glycation of collagen matrices Phenotype of Human Cardiac Fibroblasts Vimentin Vimentin SMA SMA Desmin Pro - Collagen Desmin Collagen PBS 1 mM MGO Effect of MGO-Collagen on α-Smooth Muscle Actin Effect Counted m arkers under 40X 35 30 25 20 15 10 5 0 M M PB S M 1m 01 1m AP G m DAPI non alphaSMA expressing cells alpha SMA expressing cells Rhodamine Phalloidin 40.57% Conditions 23.72% 42.86% 56.12% 0. 0. 71.42% α SMA Effect of MGO-Collagen on α-Smooth Muscle Actin Expression 35 C o u n te d m a rk e r s u n d e r 4 0 X Collagen MGO-Coll SMA 30 25 20 15 10 5 0 PBS 0.01mM 0.1mM Conditions alpha SMA expressing cells non alphaSMA expressing cells GAPDH 1mM Effect of MGO Collagen on SMA Promoter Activity 2 Days Treatment Normalized Alpha-SMA Luciferase Value 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Collagen MGO-Col Cellular Interactions with Collagen Cellular Collagen Receptors and Function Integrins For cell adhesion to extracellular matrix Collagen fibril binding integrins are α2β1 and α10β1 (Gullberg) Interact with sequences GFOGER on collagen MGO Adducts on Collagen MGO Receptors (Avery, 2006) MGO Treatment of Collagen Blocks b1 Integrin Activation Rhodamine Phalloidin 12G10 Antibody Staining PBS-treated collagen 0.01 mM MGO-treated collagen 0.1 mM MGO-treated collagen 1 mM MGO-treated collagen Effect of MGO on Cell Attachment Strength to Collagen 1 0 .9 0 .8 Proportion of cells remaining [MGO] 0 0 0 1 1 mM .0 1 m M .1 m M mM 0m M 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 11 110 220 Shear stress levels applied (dyn cm-2) for 4 minutes 4 8 12 Number of Washes Importance of Integrins on MGO-Induced SMA Expression Integrin Control PBS 0.1 mM MGO 1 mM MGO Cell Suspension α SMA – 42kDa GAPDH – 37kDa Integrin Activator Cell Suspension PBS 0.1 mM MGO 1 mM MGO α SMA – 42kDa GAPDH – 37kDa Integrin Inhibitor PBS 0.1 mM MGO 1 mM MGO Positive control α SMA – 42kDa GAPDH – 37kDa Impact of Integrins on MGO-Induced SMA Expression Human Cardiac Fibroblasts PBS 0.01mM MGO 0.1mM MGO 1mM MGO α SMA – 42kDa GAPDH – 37kDa GD-25 – Integrin β1 Mutant PBS 0.01mM MGO 0.1mM MGO 1mM MGO α SMA – 42kDa GAPDH – 37kDa DDR1 Mutant PBS 0.01mM MGO 0.1mM MGO 1mM MGO α SMA – 42kDa GAPDH – 37kDa Effect of MGO on Cell Migration on Collagen 80 70 # migrations cells/unit area Modified Boyden Chamber Coated in F Collagen 60 50 40 30 20 10 0 PBS 0.01 mM MGO 0.1 mM MGO 1mM MGO 75 μm Mechanosensing and Responses (Vogel, Sheetz, 2006) Effect of MGO on Collagen Stiffness Focal Adhesion Formation on MGO-Collagen PBS 0.1 mM MGO 1 mM MGO BSA Vinculin 116kDa Paxillin 66kDa Focal Adhesion Proteins 45 a ea e o n / el vr gc u t cl 40 35 30 25 20 15 10 5 0 Vinculin Paxillin PBS 0.1mM 1mM A PBS treated collagen 0.1mM MGO treated collagen 1mM MGO treated collagen Paxillin Rhodamine Phalloidin B PBS treated collagen 0.1mM MGO treated collagen 1mM MGO treated collagen Vinculin Rhodamine Phalloidin Collagen Gel Models Collagen Grinnell, 2002, Trends in Cell Biology Floating Collagen Gel Contraction Assay Floating 8 Collagen gel diameter (mm) 6 4 2 0 1 2 3 4 5 1mM MGO treated collagen gels - no cells 1mM MGO treated collagen gels - 10^6 cells n o cell control non treated collagen gel - 10^6 cells Stress-Relaxed Collagen Gel Stress Contraction Assay PBS 1 mM MGO DAPI 14 12 maximal diameter (mm) 10 8 6 4 2 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Rhodamine Phalloidin Time (hours) PBS treated collagen gels - HCF cells 1mM MGO treated collagen gel - HCF cells 1mM MGO treated collagen gel - No cells α SMA Cadherins can regulate many cellular activities Wheelock, M. J. et al. Regulation of SMA by Actin Assembly Regulation and the Transcriptional Co-Activator MAL and actin monomers bound to MAL MAL MAL MAL MAL MAL MAL MAL MAL MAL MAL SRF MAL SMA Expression actin assembly SRF MAL SRE HCF on reg collagen HCF on MGO collagen HCF on reg collagen HCF on MGO collagen MGO-Collagen Enhances MGO β-Catenin Expression GAPDH β−catenin Glycated Collagen-Induced Glycated Collagen Myofibroblast Formation is Not Dependent on Rho Kinase SMA EDA Fibronectin pan Cadherin MGO-Collagen Reg. coll. + Y27632 MGO-coll + Y27632 GAPDH MGO-Collagen + Y Reg. collagen MGO-collagen Summary Summary focal adhesion formation and cell adhesion strength •Collagen glycation enhances myofibroblast differentiation, cell migration and tractional remodelling of collagen remodelling of •MGO-collagen-induced myofibroblast differentiation is independent of Rho kinase but requires β1 integrins •MGO-collagen may induce intercellular adhesions, which enable N-cadherin and βcatenin induced myofibroblast differentiation catenin •MGO-collagen inhibits integrin activation, Role of Fibrosis in Diabetic Cardiomyopathy Amy Yuen Juliana Birek (UWO) (UWO) Carol Laschinger Pam Arora Derek Watt (IBBME) Matthew Chan Wilson Lee Craig Simmons (IBBME) CIHR Heart and Stroke Foundation of Ontario ...
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