Characterisation of LaNt regulation of Basement Membrane organisation in wound repair and angiogenesis.

In this work I propose to study the LaNt family of proteins which were recently identified and which I believe are important for processes such as wound repair, blood vessel growth and the spread of tumours. Through these studies, a deeper understanding of these processes will be obtained and this, in turn, may lead to identification of new treatment approaches for conditions such as chronic or slow-healing skin ulcers and cancer.

The different tissues of the body are composed of defined combinations of specialised cell types and a mixture of proteins and sugars outside the cells, termed the extracellular matrix (ECM). Some of the cell types reside in and contribute to the production of this ECM, whereas others cover the outer (epithelia) and inner (endothelia) surfaces of regions of ECM as sheets of cells. Directly beneath these cell sheets, as well as surrounding nerves and muscles, there is an organised region of ECM termed basement membrane (BM). BMs provide the anchorage point for cells and are therefore important for stress resistance and structural integrity. In addition, BMs support the different behavioural requirements of a wide range of cell types at different times, including acting as the road upon which the skin cells migrate to close wounds.

A major component of all BMs is the laminin family of proteins. Laminins assemble into cross shaped molecules that associate with one another to form a network. Formation of this network has been shown to involve a small region at the very end of the short arms of the laminin cross, which is termed a LN domain. The importance of this interaction is exemplified by a number of genetic diseases where specific defects in LN domains impact the laminin network and BM organisation resulting in skin blistering, eye defects, kidney failure or muscular dystrophy. However, despite this knowledge, the ways in which laminin networks form, how network organisation changes during different cellular processes and what drives those changes is yet to be fully understood.

This project will focus on the LaNts which have been demonstrated to play a role in cell attachment and migration and which my preliminary data indicate is likely to be through regulating BM formation. Like the laminins, the LaNt (Laminin N-terminus) also contain a LN domain, this suggests that they can interact directly with laminins and modify the ways in which laminin networks are organised. Importantly, there are also tissue specific differences within the laminin family and these differences are likely to mean that the impact of the LaNts is cell type specific. This may also mean that LaNts play different roles during blood vessel growth or wound repair than during normal tissue function.

In order to characterise the roles of LaNts in BM formation and the impact they have on cell behaviour and tissue function, this project will pursue 3 aims.

In Aim 1, I will use proteins in solution to directly assess the ability of LaNts to interact with laminins and other BM proteins and to determine their impact on network formation.

In Aim 2, I will use skin and corneal epithelial cells and blood vessel endothelial cells to study the impact of changing LaNt protein levels on cell behaviour. Specifically, we will determine if the BM deposited by cells changes in response to increasing or decreasing LaNt levels and we will assess cell movement speeds, how strongly they attach and how rapidly they divide on the different substrates.

In Aim 3, I will use three dimensional models of skin, eye and of blood vessel growth to study LaNt roles in these more complex tissue models.

Together the data obtained from these studies will dramatically expand what is known about LaNts, about laminin network formation, about BM organisation and ultimately about wound repair, blood vessel growth and tumour progression. In the longer term, this may lead to identification of new treatment strategies or new drug targets.

The LaNts are a recently identified family of ECM proteins which preliminary studies have demonstrated to be important for cell-matrix attachment and cell migration. Based on their conserved domain architecture, the LaNts are hypothesised to elicit these effects through binding to laminins in the ECM and modulating their assembly into higher-order networks. In turn, these LaNt mediated changes in matrix organisation are predicted to directly influence cell behaviour supported by those substrates.

To test these hypotheses, and to tease out the contribution of different aspects of LaNt biology to these processes, this proposal will use a combination of purified protein studies, cultured cells and finally three dimensional model systems to investigate LaNt roles in BM formation and organisation. Initially biochemical analyses will be used to identify and characterise LaNt - laminin interactions as well as LaNt interactions with other ECM proteins, and to determine the impact of LaNts upon laminin polymer formation. In the second part, epithelial and endothelial cells in culture will be used to analyse LaNt modulated matrix changes and to determine their impact on cellular functions such as proliferation, attachment and migration. Further mechanistic insight will be garnered through analysis of adhesion complex assembly, dynamics and ability to generate forces. Finally, tissue equivalent model systems of skin, cornea, limbal outgrowth and angiogenesis will be used to assess LaNt roles in more complex but also more physiologically relevant settings.

The results garnered from these studies are likely to have widespread implications in expanding the understanding of processes associated with human biology and disease progression, for example the regulation of wound repair, tumour invasion and metastasis, in tissue morphogenesis and for processes involved in angiogenesis.

This research will have impact upon a wide range of academics working across multiple disciplines. These impacts will become available over a period of months or years as the work is disseminated at conferences and in scientific publications. Also the PDRA performing these studies will develop transferrable skills that will benefit employers in the UK public or private sector. Additionally, and particularly as a new investigator, the applicant will himself experience significant impact in supervisory and project management experiences, in developing national and international contacts and collaborations, and in aiding reintegration back into the UK science community after an extended period in the US.

Pharmaceutical industry
Exogenous addition of LaNt proteins, peptides derived from LaNts, small molecule inhibitors, antibodies or shRNAs targeting LaNts may have therapeutic potential. Depending on the data obtained in this proposal they could act directly, for example, to promote reepithelialisation through enhancing BM deposition or, by blocking LaNt function, or to inhibit angiogenesis or tumour invasion. Conversely they could act indirectly, through modulating responses to netrins in tumour progression. Importantly, as LaNts are a relatively minor (in terms of mass) component of undamaged BMs, targeting their function in pathogenesis may have minimal off-target effects compared with targeting more abundant components. These approaches may be appropriate for commercialisation.

Tissue engineering, bioscaffolds
One specific and expanding area with direct potential application for these studies is tissue engineering and generation of bioscaffolds. The fundamental increase in understanding of normal BM formation and function will be of widespread direct benefit to these areas and could be commercially exploited. Providing the appropriate microenvironment for the culture and expansion of desired cell types of specific cellular characteristics could be achieved through coating of the bioengineered scaffold with appropriate laminin/LaNt ratios, or bioactive peptides designed from these proteins, designed to enhance de novo BM deposition. These could be used for in vitro studies, for example to specifically expand stem cells, or for ex vivo expansion of cells in culture for transplantation. The data obtained in this proposal will provide appropriate starting combinations / ratios from which to begin optimising such an approach and therefore this impact will be available in months to years from publication or meeting presentation.

NHS clinicians and the general public
The impact from novel therapeutics or bioengineered tissue improvement, ultimately leading to clinical use, will be felt in a 15-20 year time frame. This will contribute to economic productivity and prosperity by reducing the economic burden of ill health as well as lead to positive societal impact through improved quality of life.

Patient support groups
Although not directly focused on genetic disease, this research will impact specific patient groups with laminin-related disorders for example, junctional epidermolysis bullosa, Pierson syndrome, congenital muscular dystrophy type 1A, cardiomyopathy and glomerular sclerosis, as well as other conditions where changes in laminin expression are associated with disease progression such as chemotherapy induced alopecia or chronic diabetic ulcers. In the case of these patients groups, the knowledge that research is progressing in the specific disease area will have a positive psychological impact ahead of any further downstream clinical applications.