MICA: The role of arginase in chronic delayed wound healing

As we age our skin takes longer to heal. In one in every twenty older people healing becomes so delayed that it fails, leading to chronic wounds (eg. diabetic foot ulcers or venous leg ulcers). These chronic wounds severely decrease quality of life and can even lead to death. Unfortunately, there are no effective treatments for chronic wounds and their management poses a major financial problem for the world's healthcare economy, particularly as the global elderly population is rapidly expanding. This lack of treatments is mainly because there is little understanding of the gene changes in different wound cells that result in poor healing. Our recent studies have indentified a new gene that may be particularly important for healing, arginase (Arg1). Arginase has critical roles in repair of other tissues and has been linked to chronic diseases, such as asthma or psoriasis. Our new data shows that a number of cell types important for skin repair contain the arginase enzyme, and that it is strongly down-regulated in delayed healing. We believe that arginase in these other cells plays crucial, as yet unidentified, roles in wound repair. In this study we will test this hypothesis using a range of techniques in both mouse wounds and wound tissue from human volunteers.

We will use specially bred mice that lack arginase in each of these important wound repair cell types (cell-specific knockout). The effect on overall healing and a range of healing parameters will reveal the role of arginase in each cell type. We will also culture the cells and directly test the effect of arginase on cell function. In parallel we will measure arginase levels and activity in human tissue. Of particular note we will be able to compare arginase level over time in human chronic wounds that either do or do not heal. The final part of the project will test the possibility of using drugs that alter arginase activation to promote healing. We will do this in mouse models of human delayed healing and directly in human skin. Upon completion of this study we will be in a strong position to move into studies on human patients with chronic wounds. In addition, given the importance of arginase in other chronic diseases, our findings will potentially have wide reaching implications for the understanding and treatment of other chronic degenerative diseases.

Chronic non-healing wounds lead to substantial morbidity and mortality imposing a major financial burden on health services. Current treatments are ineffective because the molecular and cellular correlates are poorly understood. Changes in L-arginine metabolism have been linked to tissue repair defects in multiple chronic degenerative diseases. Our data now show that a central L-arginine pathway component arginase (Arg1) plays a key role in skin wound repair, with inhibition or genetic ablation having a negative impact on healing outcome. Our data indicate widespread arginase expression in mouse and human skin cells (including keratinocytes, fibroblasts and macrophages). Surprisingly little is known about the contribution of non-macrophage arginase to pathological wound repair. We propose that arginase plays important cell-type specific and temporally-regulated roles in pathological human repair. To test this we will first characterise healing in mice conditionally lacking Arg1 in keratinocytes (K14-cre;Argfl/fl), inflammatory cells (LysM-cre;Argfl/fl) and fibroblasts (Fsp-cre;Argfl/fl). Corresponding mechanistic in vitro experiments will reveal cell intrinsic Arg1 functions. In parallel, we will confirm clinical relevance by assessing L-arginine pathway component expression/activity in a unique resource of human diabetic foot ulcer biopsy tissue with longitudinal healing outcome data; allowing us for the first time to link arginase function and healing outcome. In collaboration with our commercial co-applicant Epistem, we will test the therapeutic potential of L-arginine pathway manipulation to promote healing. We will use both validated pre-clinical mouse models of human chronic healing and ex vivo human wound assays. Our proposal will provide novel insight into the role of Arg1 in skin repair; with the potential for wider translation into other chronic degenerative diseases. By the end of this study we should be in position to move from bench to bedside.

Chronic non-healing wounds (principally diabetic foot ulcers, venous leg ulcers or ischemic pressure ulcers) represent a major area of unmet clinical need. One in twenty elderly people will develop a chronic wound at some point in their lives, with estimated treatment costs to the NHS currently exceeding £3 billion per annum. Affected individuals often experience high levels of morbidity, including pain, odour and social isolation. In many cases unhealed ulcers lead to minor or major amputations. For diabetic foot ulcers, the five year mortality risk following ulcer-associated major lower limb amputation has been reported at around 70%. An expanding elderly, and particularly diabetic, population means that chronic wounds will become an even greater future burden on society and health services unless new treatments are urgently developed.

Work outlined in our proposal is specifically designed to identify new pathways and processes involved in pathological healing. Within the proposal not only will we identify potential new drug target(s), we will validate altered expression of these target(s) in human pathological healing (tissue samples), with subsequent demonstration of therapeutic potential in validated, physiologically relevant murine pre-clinical models of delayed human healing, and in human tissue ex vivo. By the end of this project we should be in a position to translate our bench findings to the bedside. To do this successfully will require substantive links with clinicians and commercial involvement. These links are already in place with Dr Hardman currently leading a Phase 1 clinical trial in collaboration with clinical colleagues in Manchester (Prof. Boulton). Furthermore, both PIs have a longstanding collaboration (>5 years) with our industrial co-applicant Dr Booth (Epistem). The active involvement of Epistem will facilitate the translation of our research to the target population in a medium term time frame.

For Epistem specifically, this collaboration represents an exciting opportunity to further understand the clinical potential of L-arginine pathway manipulation. This will be directly relevant to ongoing CRO model development in a range of tissues/models including psoriasis, arthritis, IBD and gut repair. Improved and refined models will be financially beneficial to the company. A key priority of the wider Epistem portfolio is the development of novel therapeutics via partnering opportunities, and the L-arginine pathway has been identified as holding clear commercial potential.

In addition to potential benefits via the development of new treatments, our proposal will aim to achieve greater immediate impact with both the target clinical population and the general public. The target population will be accessed by Dr Hardman who, as an AgeUK funded Senior Research Fellow, regularly meets and presents to elderly audiences with his work featured in informational literature for older people. In addition by leading ongoing clinical trials we regularly speak to chronic wound patients who are the intended beneficiaries of this research. We will also use our research as the basis for a range of public engagement events. These events will benefit the public by increasing awareness of the concept of "healthy ageing" and the crucial role the immune system plays in protecting our bodies from harm.

The project will also benefit the research teams of Hardman and Cruickshank and the dedicated post-doctoral research associate (PDRA) and research technician. Given the multi-disciplinary nature of the work, encompassing aspects of tissue regeneration, biogerontology, immunology and cell biology, the PDRA and technician will receive extensive training in wide-ranging areas of scientific research.