04/19/10 EXTRACELLULAR MATRIX PROTEINS AND PROTEINASES By, Raghu Ambekar Photonics Research of Bio/nano Environments Department of Electrical & Computer Engineering University of Illinois Urbana - Champaign BioE 506

04/19/10 Outline  Extracellular matrix proteins  Collagen  Classification  Fibril assembly and collagen diseases  Extracellular matrix proteinases  Role of MMP in metastasis  Modification of tumor collagen for therapeutics

04/19/10 Extracellular matrix (ECM)  Surrounds cell  Provides mechanical support  Controls the flow of nutrients and signals to the cells  Consists of  Fibrous: collagen, elastin, fibronectin, laminin  Non-fibrous: Proteoglycans and polysaccharides

04/19/10 Collagen  Collagen : most abundant protein found in the human body. About 1/3 rd of the total proteins.  Found abundantly in tendon, cartilage, bone and skin  Functions:  cell migration  cell adhesion  molecular filtration  tissue repair

04/19/10 Structure of collagen  It has a triple-helix structure containing three α-polypeptide chains arranged in right-handed supercoil  Glycine, proline, hydroxyproline  1.5 nm diameter  At least 28 different collagens found  The three α-chains could be same (collagen II) or different (collagen I) Collagen molecule

04/19/10 Classification of collagen  No interruptions in triple helix  Regular arrangement results in characteristic “D” period of 67 nm  Diameter : nm  Example : Types I, II, III, V, XI 1. Fibril-forming collagens

04/19/10 Classification of collagen  Forms network in basement (Collagen IV) and Descemet’s membrane (Collagen VIII)  Molecular filtration  Example : Types IV, VIII, X 2. Network-forming collagens

04/19/10 Classification of collagen  Short collagens with interruptions  Linked to collagen II and carries a GAG chain  Found at the surface of fibril-forming collagens  Example : Types IX, XII, XIV 3. Fibril-associated collagens with interrupted triple helices (FACITs)

04/19/10 Classification of collagen  Provides functional integrity by connecting epithelium to stroma  Example : Type VII 4. Anchoring collagens

04/19/10 Classification of collagen  Form structural links with cells  Example : Type VI  Collagen VI crosslink into tetramers that assemble into long molecular chains (microfibrils) and have beaded repeat of 105 nm 5. Beaded-filament-forming collagens

04/19/10 Type I Fibril assembly Chain recognition sequence Fibril assembly is determined by chain recognition sequence in C-propeptide Fish scale Bone osteon Tendon Skin

04/19/10 Diseases associated with collagen Diseases caused by mutations  Subtypes of osteogenesis imperfecta (collagen I)  Ehlers-Danlos syndrome (collagen I and V)  Alport syndrome (collagen IV)  Certain arterial aneurysms (collagen III)  Ullrich muscular dystrophy (collagen VI)  Certain chondrodysplasias (collagen IX and XI)  Kniest dysplasia (collagen II)

04/19/10 Role of MMP in metastasis Metastasis  Spread of cancer from a primary tumor to distant sites of the body  A defining feature of cancer

04/19/10 Role of MMP in metastasis  Understanding the molecular mechanisms of metastasis is crucial for the design of therapeutics  Extracellular matrix metalloproteinases (MMP) associated with metastasis  MMPs are capable of digesting ECM and basement membrane under physiologic conditions  Collagenases degrade fibrillar collagen  Stromelysins degrade proteoglycans and glycoproteins  Gelatinases degrade nonfibrillar and denatured collagens  At tumor sites, experiments have found  Increased number of MMPs  Increased levels of MMPs  Reduced levels of TIMPs (Tissue inhibitors of metalloproteinases)

04/19/10 Role of MMP in metastasis  Major role of MMPs was to facilitate the breakdown of physical barriers, thus promoting invasion, intravasation, extravasation and migration  MMPs targeted for antimetastasis therapies

04/19/10 Role of MMP in metastasis  Clinical trials of inhibiting MMPs to cure cancer have failed  Metastasis is a complicated process  MMPs contribute to every stage in tumor progression at both primary and metastatic sites  Specific MMPs play a role in each stage of metastasis  MMP 13, 14 – invasion  MMP 9– angiogenesis  Understand the role of the MMPs in each cancer setting

04/19/10 Modification of collagen for therapeutics  Structure and content of collagen governs the delivery of therapeutic molecules in tumors  Penetration of therapeutic molecules improved by developing agents that modify ECM and increase diffusion  Detect tumor collagen noninvasively to quantify collagen content and estimate drug delivery characteristics

04/19/10 Modification of collagen for therapeutics Uses Second-harmonic generation (SHG) for imaging only collagen fibers Conditions :  Non-centrosymmetric (collagen, microtubules)  Lasers (high intensity) Advantages :  No staining  3D imaging  No photobleaching SAMPLE Red Wavelength=800 nm SHG: Blue Wavelength=400 nm Collagen stained red and imaged by fluorescence microscopy Collagen imaged by SHG microscopy

04/19/10 Modification of collagen for therapeutics  SHG intensity collected from live imaging of collagen fibers provides an good estimate of diffusion coefficient in tumors

04/19/10 Modification of collagen for therapeutics  Chronic relaxin treatment degrades tumor matrix and improve macromolecular diffusion in tumors 0 th day3 rd day6 th day9 th day12 th day THANK YOU!