Pericellular mechanisms of fibrillin microfibril assembly.

Fibrillin microfibrils are filamentous structures laid down by cells as part of the 'extracellular matrix (ECM)'. They form bundles of microfibrils that interact with cells and also form the framework for the formation of 'elastic fibres', which endow the essential property of elastic recoil on dynamic tissues such as blood vessels, lungs, skin and ligaments. The importance of fibrillin is clearly highlighted by the many serious diseases that are caused by genetic defects in fibrillin (e.g. Marfan syndrome, which is associated with life-threatening cardiovascular defects), and by the degeneration of microfibrils in ageing and diseases of blood vessels, lungs (emphysema), loss of skin elasticity, and certain eye defects. Despite their essential nature, it is still not understood how microfibrils form, which has severely limited treatment advances. This proposal is to build on exciting new discoveries from my group which give important new insights into their assembly and function. The study will provide new insights into how mutations in fibrillin-1 cause severe heritable diseases, and form the basis for improved strategies to restore microfibrils to tissues with compromised elasticity. The project is of great importance to regenerative medicine, which is an MRC priority because there is a major unmet clinical need to develop new treatments to restore function to compromised elastic tissues and organs. My group is ideally placed to conduct this specialised research because we are internationally-renowned for our expertise and discoveries in fibrillin/elastic fibre biology, and we will build on our have recent discovery of cell type-specific mechanisms that throw new light on this complex process. Thus it is very timely and directly addresses the MRC Priority 'Resilience, Repair and Replacement'.

We will determine how the cell-matrix interface of epithelial and mesenchymal cells directs assembly of fibrillin microfibrils, and thus elastic fibres. The objective is to deliver a step-change in ability to restore elastic tissue function following damage or disease, or degenerative ageing changes.
How HS/syndecan-4, FN and alpha5beta1 integrin enable microfibril assembly;
- Compare in vitro and cell-driven assembly of fibrillin-1 with/without HS (electron microscopy; biochemically; determine role of syndecan-4 (siRNA, mutagenesis, domain swap with syndecans-1/-2; real-time microscopy to track microfibrils); contribution of fibronectin (mutagenesis; fibronectin fragments);
- How similar mechanisms regulate LTBP-1 deposition, and thus TGFbeta bioavailability;
- How epithelial cell-cell interactions regulate ECM assembly.
How ADAMTS10 supports microfibril assembly;
- How ADAMTS10 influences fibrillin-1 assembly and higher-order microfibril organisation (electron microscopy; biochemical/biophysical analyses);
- How WMS mutations alter microfibril organisation and function (full-length mutant fibrillin-1; assembly assays; microfibril deposition by cells expressing mutant WMS fibrillin-1 (Talen; fluorescence/ real-time microscopy; light sheet microscopy for quantitative live cell imaging).
- How ADAMTS10 stabilises cell-cell junctions (effects on microfibril organisation).
Design therapeutic strategies to repair elastic tissues.
- Test receptor-activating antibodies and fibronectin/HS/ADAMTS10 products (co-culture tissue equivalents);
- Regulate receptor activity (antibodies, siRNAs, ECM products in ECM-containing peptide hydrogels, nano-constructs; with Biomaterials, Manchester). Viral transduction, non-viral cationic liposomes or direct exposure to plasmid DNA;
- Elasticity of tissue equivalents (microfibril content; nanoindentation, acoustic microscopy).

This study is to elucidate how epithelial and mesenchymal cells differentially assemble fibrillin microfibrils, and thus the elastic fibres which provide essential elastic recoil to tissues such as blood vessels, lungs, skin and ligaments. It directly addresses the MRC Priority 'Resilience, Repair and Replacement' because it will determine the mechanistic basis of their deposition, and therefore how to repair and regenerate elastic tissues. It will benefit:
(1) Biomedical scientists and the UK regenerative medicine community
The study will deliver essential new insights into how the cellular microenvironment controls the assembly of extracellular matrix, and thus how cells make and remodel tissues. The particular focus is fibrillin microfibrils which form the template for elastic fibres that provide indispensible elastic recoil to dynamic tissues. This knowledge is urgently needed, given that degeneration of elastic tissues is a prominent feature of many of the major diseases of man.
Realising the benefits: Our research will lead to translational applications that will be developed through the Manchester Academic Health Science Centre, and through the UK Regenerative Medicine Platform hub (UKRMP) 'Exploiting and engineering the stem cell niche' (Kielty is a partner). Further added value will be achieved through research collaborations in Manchester, and nationally and internationally, and data and reagent sharing, especially with UKRMP colleagues. We will also extend links with MIMIT (Manchester: Integrating Medicine and Innovative Technology; linked to CIMIT, Boston USA), which develops clinical solutions for tissue repair and related unmet clinical needs.
(2) Biopharma
Research leading to elastic fibre repair is a high priority in regenerative medicine/tissue engineering and Biopharma companies, because effective new therapeutic products are urgently needed.
Realising the benefits: University of Manchester Intellectual Property (UMIP) will support the commercialisation of research emerging from this study, e.g. selling/licensing of reagents, provision of research expertise, in-house assays/techniques, co-development of technologies or licensing of IP.
(3) Graduate training
The application of cell-matrix biology in regenerative medicine is a priority of the University of Manchester (UoM). We have proposed an integrated graduate training by research in regenerative medicine that integrates biomedical sciences with clinical application (EPSRC-MRC Centre for Doctoral Training in Regenerative Medicine; invited submission, in review). Through my internationally-leading MRC-funded microfibril/elastic fibre research and graduate opportunities, a new generation of researchers will be trained to the very highest standards in the cell-matrix biology of regenerative medicine, and its clinical applications.
Realising the benefits: Major impact will be achieved by training graduates in the scientific methods and translational applications required to understand how the microenvironment (niche) directs cells to remodel and repair elastic tissues.
(4) General Public
This research programme is a powerful platform to inform the general public about our regenerative medicine activities, in public meetings and internet fora.
Realising the benefits: I will write review articles for popular press and student science magazines; train my PhD students in communications and public engagement; participate in Manchester Science Week; develop outreach materials to inform local, national and international audiences, and patient groups.