Atomic level structure of Extracellular Matrix (ECM): spectroscopic approaches to the systems biology of intact tissue

Lead Research Organisation: University of Cambridge
Department Name: Chemistry

Abstract

The extracellular matrix (ECM) is a network of solid protein and other substances which supports the cells that make up our various tissues. The maintenance of healthy tissues into older age has huge implications for the economy and our quality of life. Moreover, the design of tissue replacements is of vital importance when tissues wear out or become damaged, and the ECM is critical to the faithful integration of these replacements into the host tissue. This project will develop methods for growing ECMs from cells in the laboratory that on molecular and nano-lengthscales look as far as possible like the ECM found in bone. These ECMs may then be used to grow bone tissue in the laboratory for grafting purposes and for further basic science research into the detailed structure of bone and its reaction to drugs and other chemicals, for instance. In order to do this, we will first develop ways of examining the molecular structure of the ECM using a form of spectroscopy, solid-state nuclear magnetic resonance (SSNMR). To test these methods, we will use molecules which resemble those found in the ECM of bone, but which can be synthesised by normal chemical reactions in the laboratory. We will then use various cell culturing methods to grow ECMs from a several different cell types that are capable of producing bone-like material, supporting the cells on different materials and under a variety of different conditions in order to investigate what features are necessary to produce an ECM which most closely resembles that in bone, as judged by the SSNMR methods we have developed in the first part of the project and electron microscopy to examine the nanostructure of the material. Finally we will apply the SSNMR methods we have developed to study bone into which we have introduced NMR-active isotopes as 'reporters' of molecular structure in order to show that the material we have produced using cell culture represents a valid model of the real material.

Technical Summary

The ECM is of paramount importance as a cell scaffold, for cell signalling and for cellular nutrition. Equally important is its material role in giving strength and structural integrity to tissues. The maintenance of healthy tissues into older age has huge implications for the economy and our quality of life. Concurrently, the design of biocompatible tissue prostheses is of vital importance in the replacement or repair of degenerated or traumatized tissue, and the ECM is critical to the faithful integration of these devices into the host tissue. The purpose of this application is twofold: first to develop a solid-state NMR toolkit for determining the molecular-level structure of the ECM, focussing particularly on that in bone and the interactions between its various components and then to use this to examine how ECM structure depends on the differentiation and type of the osteogenic cells producing the matrix. Different cell scaffolds and culture conditions will be used to produce cell populations with different differentiation and specifically, different levels of expression of the various proteins important in ECM production. Correlating the resulting ECM molecular structure (via NMR) and nanostructure (via electron microscopy) with the details of cell differentiation and protein expression will yield essential information on the structural relevance of key components in the mechanism of ECM production.

Publications

10 25 50
 
Description We have now developed a "heavy mouse", that is a mouse whose proteinaceous tissues are enriched in the stable isotopes, 13C and 15N allowing state-of-the-art solid-state NMR spectroscopy experimetns to be performed on its tissues, in turn allowing detailed molecular structure determination within a tissue, with the molecule in its native surroundings. Without this development, the only approach and that generally adopted, is to extract the molecules of interest from the tissue, purify them and where possible, crystallise them. The structures determined via such routes may have limited applicability to the protein in its native environment. We have then used the detailed (multidimensional) NMR data as "fingerprints" of the underlying molecular structures of a tissue and developed an in vitro cell culture method to produce bone-like tissue which when assessed via NMR in the same way as the mouse tissues, gives near identical spectra, and thus a tissue which at a molecular level is truly native-like. Electron microscopy shows that the scaffold structures formed by the in vitro tissue on a length scale of tens to hundreds of nanometres is also like native tissue. We are now able to use the in vitro tissue to study details of tissue structure and what may happen to it through ageing or disease, confident that this tissue is representative of native tissues and to do this without further use of animals.



One of the first discoveries we made using the new cultured tissue was the extent to which excess sugar, in this case glucose, present in the tissue, disrupted the tissue structure. This has led to further funding (from the MRC) to further understand the secondary effects of diabetes on tissues.



Another discovery has been that the structure of tissue collagen protein molecules is not as previously thought. The previously accepted features of collagen protein structure have come mainly form the study of model proteins. It is now clear that these do not pack together as native collagen molecules do, resulting in significantly different aspects of their structures.



Publications on all aspects of this work are currently under review.
Exploitation Route The in vitro cell culture model could be used as a simple route to toxicity testing without the use of animals. The 13C, 15N-enriched mouse can now be used to study the details of tissue diseases; the same protocol that generated our heavy mouse can be used on mouse models of diseases, potentially allowing insight into the mechanism of disease progression and into ageing of tissues.



The in vitro cell culture tissue can be used to examine where and how long potential drug molecules bind into tissues. We are currently sing it to understand the differences in molecular structure of tumour tissue compared to normal tissues, with a view to developing new methods of targeting tumour tissue and cells.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.ch.cam.ac.uk/person/mjd13
 
Description We have used them to uncover the details of vascular calcification and undertaken proof-of-principle experiments to demonstrate that a class of drugs may be re-purposed to inhibit vascular calcification. We have now screened potential generic drugs and have been successful in identifying drugs to take forward to patient trials. A US patent has been granted for the use of any PARP inhibitor for the treatment of vascular calcification (WO/2017/216563). A proof-of-principle study for a specific PARP inhibitor (minocycline) in patients is planned for this year.
First Year Of Impact 2015
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Non-enzymatic glycation of collagen: role in metabolic disease and ageing
Amount £283,034 (GBP)
Funding ID MR/J007692/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2013 
End 09/2014
 
Title BRMB 
Description NMR data for proteins, DNA, RNA for 1H, 13C, 15N 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Access to 13C and 15N chemical shifts for proteins in intact tissues for e.g. development of in vitro tissue models 
 
Title PARP inhibitors for the treatment of vascular calcification 
Description Discovery by the NMR approach developed in this project that developing bone contains poly(ADP ribose) as a structural component of the mineral phase of bone led us to investigate the same possibility for vascular calcification. We discovered that poly(ADP ribose) performed the same role in both atherosclerotic and medial vascular calcification. Thus we formed the hypothesis that inhibitions the biosynthesis of poly(ADP ribose) would inhibit vascular calcification. We found that this was indeed the case and so then screened generic and modern PARP inhibitors as potential therapeutic options to treat vascular calcification. The patent application describes the effective drugs we discovered in that process. 
IP Reference WO2017216563 
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact Patient trials planned; due to begin this year