Circadian rhythms and the control of NRF2-based antioxidant signalling as a therapeutic target in dermal tissue repair and pathological wound healing

Pathological wound healing is associated with ageing and many chronic diseases. It is one of the major medical burdens in the developed world, contributing towards leading causes of death and disease worldwide. The stages of wound healing normally progress in a predictable, timely manner; if they do not, healing may progress inappropriately to either a chronic wound such as diabetic ulcer or fibrotic scarring such as scleroderma. The prevalence of chronic wounds and fibrotic pathologies arrives at the number of ~80 million, which increases to >100 million if we include fibrosis associated with surgical procedures. Current treatments for chronic wounds and fibrotic scarring have limited success, so better understanding of biological mechanisms that promote healing whilst preventing scarring is urgent to achieve better therapies.

Research carried out by scientists around the world as well as our own laboratory has recently discovered a crucial role for biological clocks in optimal wound healing and in curbing fibrotic scarring following injury. Biological clocks are timing mechanisms in our body that generate 24h rhythms in physiology and behavior, such as sleep/wake cycles, body temperature and hormone levels. They exist in almost all tissues and cells in our body. Their disruption due to shift work and ageing is a strong risk factor for chronic diseases. Our new pilot data using rodent models shows that there is a robust day/night variation in skin wound healing rates so that skin heals faster when injured during the night then when injured during the day. This faster healing at night is associated with increased levels of genes involved in production and organisation of matrix, a very important component for closing the wound. We have also discovered that antioxidant protection in our cells varies between day and night, and has an important role in faster night-time healing following injury. Even more, small, drug-like molecules capable of boosting antioxidant levels in our cells have a greater efficiency when used at the right time of day. These observations suggest exciting new ways to tackle pathological wound repair mechanisms.

In this new project, we will answer the main question as to whether the biological clock present in key cells involved in wound healing (called fibroblasts) is critical for daily variation in repair capacity that we observed in our pilot studies. We will make use of two genetically modified rodent models 1) one in which we can visualise (by fluorescent tags) a matrix gene (called collagen) specifically activated in fibroblast cells and 2) the other in which we have genetically deleted the clock gene specifically in fibroblast cells. Using these unique rodent models, we will be able to monitor temporal changes in clock gene activity and matrix organisation within wounds. This research will uncover critical importance of robust clocks within key wound healing cells for time-of-day variation in healing rate and organisation of optimal wound repair.

Furthermore, we will take advantage of advanced molecular biology tools to find out which genetic mechanisms biological clocks use to regulate matrix genes important in healing such as collagen. Using high-tech biochemical approaches, we will further find out whether biological clocks help our cells 'tell the time' when to produce the right amounts of antioxidants to fight off rises in dangerous free radicals following injury. Finally, we will test whether antioxidant-based chronotherapy (giving treatments according to one's body clock) has beneficial healing effects in chronic wound or scarring conditions such as diabetes and scleroderma. To test this, we will use rodent models of diabetes as well as skin tissue biopsies and cells from patients with Scleroderma. These new studies will provide crucial evidence to support future studies pinning down biological clocks as a new therapeutic target for the management of chronic wounds and scarring.

This proposal will determine whether and how genetic targeting of circadian clocks in fibroblasts modulates healing responses during cutaneous wound repair, and test novel time-scheduled pharmacological interventions for efficacy in treating pathological wound repair.

Using unique fluorescent collagen 1 reporter as well as fibroblast-specific clock-deficient mice, we will monitor temporal changes in fibroblast activation during wound repair. Wounds from mice injured at different circadian phases will be analysed for a range of histological parameters (e.g. collagen deposition) and matrix gene expression. Purified fibroblasts from wounds will be used for ex vivo culture. Parallel analyses will include clock gene expression, proliferation/migration assays as well as analysis of candidate signalling pathways by targeted PCR arrays. To identify circadian elements that drive transcriptional regulation of matrix genes such as collagen 1, we will perform advanced in vitro molecular studies to determine collagen 1 promoter activity following over-expression or knockdown of clock transcription factors and NRF2. We will confirm in vitro findings both in mouse tissues and in human Scleroderma fibroblasts.

To test whether circadian gating mechanism in fibroblasts couples redox signalling to NRF2 antioxidant program during wound repair, we will use state-of-the-art techniques for measuring reactive oxygen species (ROS), NRF2 activity and oxidative damage markers in wound tissues. To determine sub-cellular sites of ROS production, in situ imaging with specific ROS probes will be used in explanted wound fibroblasts.

Finally, we will test whether chronotherapy is more effective in improving wound repair in both animal and human translational models. Following in vitro optimisation studies, we will test whether time-scheduled NRF2 activator improves delayed healing in a preclinical model of type II diabetes and in human fibroblasts from Scleroderma patients.

Chronic tissue injuries associated with pathological wound healing, including chronic wounds, result in pain and disability for millions of people around the world. As our population continues to age and the incidence of diabetes increases, it is important that we address the pathological wound healing so that people can lead their lives in a more productive manner. This work aims to better understand circadian control of skin tissue repair and, in doing so, develop novel chrono-therapeutic antioxidant approaches to treat chronic wounds and connective tissue diseases such as scleroderma. This will benefit other scientists in the fields of dermal repair, skin biology, redox signalling, connective tissue diseases, ageing, diabetes and circadian physiology. Furthermore, skin tissue engineers will be able to use and build on the knowledge of dermal repair that the project will define. The genes and cells examined in the project perform essential roles in tissues throughout the body, which will be of interest to biological researchers in these diverse areas. These impacts will occur over a period of months or years as the work is disseminated at conferences and in scientific literature.

The project will impact upon society. Improved understanding of circadian tissue repair mechanisms will be the basis upon which treatments for individuals suffering from chronic wounds will be developed. This applies particularly to those likely to suffer from leg/foot chronic ulcers (older people and patients with diabetes or scleroderma). Such treatments will relieve pain, immobility and reduce risk of amputation, vastly improving quality of life. A further consequence of better treatments for chronic wounds include lower treatment costs and a reduced care burden within the National Health Service. In addition, the UK economy would benefit through savings in disability and mobility benefit payments as would employers in the public and private sectors as a result of reduced sickness pay and lost working hours. These are long-term societal impacts (years or decades), but stakeholders can be informed of the pipeline for the development of these treatments in the shorter term (months/years), which could shape policy. Stakeholders will include politicians, industry leaders, clinicians, healthcare managers and charities such as Diabetes UK, The Scleroderma Society, Age UK. Impact of the work on the general public can occur continuously and will raise awareness of scientific research. This can be implemented through open days where the public can meet scientists, through hosting work experience placements and by taking part in outreach activities in local schools.

The mechanisms determined in this work will be of interest to industry sectors (pharmaceutical, personal care) that are keen to develop products that can improve skin tissue repair and treat chronic wounds as well as fibrotic scarring. The project will also develop novel antioxidant-based chrono-therapeutic approaches. The findings have the potential to be commercially exploitable leading to the production of new spin out companies and to partnership with or expansion of existing enterprise. The project will employ a post-doctoral research associate and a research technician for three years, training them in numerous molecular and in vivo techniques. In addition, they will gain transferable skills such as science writing, data presentation, project management and commercialisation. This will result in individuals who possess skillsets that would benefit employers in the UK public or private sector.

Timescales for these impacts could be measured in months and years for public engagement and academic beneficiaries. Applying the findings to clinical treatments and commercialisation to a level that may benefit the general public could take 10 years or more and the further development of these treatments to a scale where they are able to affect welfare on a national scale may take decades