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

Lead Research Organisation: University of Liverpool
Department Name: Institute of Ageing and Chronic Disease

Abstract

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.

Technical Summary

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.

Planned Impact

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
 
Description training of Mres Clinical Sciences researchers
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description training of Mres Clinical Sciences researchers
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description training of Mres Clinical Sciences researchers including majority of final year Medical Degree students
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description BBSRC-Japan Partnering Award-The paraventricular nucleus of the hypothalamus: networks and mathematical models
Amount £50,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2019 
End 10/2023
 
Description Biochemical Society Travel Award
Amount £750 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 12/2017
 
Description Biotechnology and Biological Sciences Research Council (BBSRC): - The role of circadian rhythms and redox signalling in musculoskeletal stem cell ageing
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2021 
End 09/2025
 
Description CIMA MRC-Versus Arthritis equipment funding
Amount £15,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2020 
End 12/2021
 
Description EPSRC-Coupling circadian rhythms with drug metabolism: a new tool for studying chronopharmacokinetics
Amount £50,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2021 
End 04/2022
 
Description International PhD studentship
Amount £150,000 (GBP)
Organisation Government of Saudi Arabia 
Sector Public
Country Saudi Arabia
Start 10/2017 
End 04/2022
 
Description Liverpool Centre for Mathematics in Healthcare (Pump Priming) funding: Modelling of the role of Lamin A in circadian networks
Amount £15,000 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 04/2020 
End 04/2021
 
Description Liverpool Centre for Mathematics in Healthcare (Pump Priming) funding: The role of non-canonical NRF2 signalling in circadian clock regulation
Amount £20,000 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 12/2019 
End 12/2020
 
Description MRC-ARUK CIMA full PhD studentship
Amount £100,000 (GBP)
Organisation Centre for Integrated research into Musculoskeletal Ageing 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 10/2021
 
Description Rosetrees Trust partial PhD studentship
Amount £21,000 (GBP)
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 10/2021
 
Description The Biochemical Society Travel Award
Amount £750 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2019 
End 06/2019
 
Description University of Liverpool-IACD partial PhD studentship
Amount £44,000 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 10/2021
 
Description Using single cell ATAC-seq to elucidate chromatin dynamics in time and space
Amount £20,000 (GBP)
Organisation 10X Genomics, Inc 
Sector Private
Country United States
Start 10/2019 
End 10/2020
 
Description equipment funding
Amount £10,000 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 07/2019 
End 07/2019
 
Title Measuring ex vivo circadian cycles from muscle biopsies 
Description We have developed a method for measuring biological cycles in real time from muscle biopsies. This will help progress research in examining disruption of biological cycles as a result of disease, ageing or injury and can be used as a outcome measure. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2020 
Provided To Others? Yes  
Impact We are in the process of publishing this methodology but we have made it available to other researchers within our MRC-Versus Arthritis Centre for Research on Musculoskeletal Ageing. As a result of this, we have developed further collaborations and have been applying for collaborative grant funding. 
 
Title Plasmid tools for circadian gene control 
Description we have created a number of plasmid constructs with mutations of clock-regulated elements that are conserved between human and other mammalian species. These can be used to study the mechanisms of gene control in disease. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2020 
Provided To Others? No  
Impact a manuscript is in preparation with this novel set of tools. 
 
Title a transgenic mouse model 
Description We have generated a transgenic mouse model of antioxidant deficiency in which a clock reporter activity can be monitored in real time. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact This has allowed finer temporal resolution of clock activity within the background of antioxidant deficiency. We will be submitting a manuscript towards publication using data obtained from this model. 
 
Title circadian time-course samples 
Description We have harvested and preserved precious tissue samples for both biochemistry and histology from circadian time-courses from different environmental conditions, ages, genotypes and gender. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact This will help us understand the role of environment, gender and age in how tissues heal and respond to damage over time. We have started sharing these resources with other researchers which will increase our outputs and collaborations. 
 
Title reporter plasmid and cell line 
Description Our team has created a reporter plasmid which can be used as an in vitro readout of matrix gene activation during wound healing and fibrosis. We have hence generated a stable cell line having this reporter which can be used for real-time bioluminescence imaging of pharmacological drugs and stimuli involved in activation of this reporter. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? No  
Impact This will enable real-time recording of reporter activity for screening drugs and compounds that can affect wound healing and fibrosis resolution. 
 
Title Modelling bi-directional interactions between the circadian clock and cell signalling 
Description The model in question is addressing a mathematical challenge that will provide a first ever model of these interactions. The identification of a suitable detailed clock model will be made was made following reviews of most recent models. A detailed model of different molecular pathways was combined to the clock model to generate a more complete model which we can then use to generate further experimental predictions. Understanding of these network interactions between transcription factors and the circadian timing system will aid future development of novel chrono-based drug therapies for a number of age-related diseases. 
Type Of Material Computer model/algorithm 
Year Produced 2020 
Provided To Others? No  
Impact This model has helped reduce the number of animals in our experimental studies and can be applied to other research to do the same. 
 
Description Chronobiology and NRF2-based antioxidant protection 
Organisation University of Liverpool
Department Department of Molecular and Clinical Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team has provided expert knowledge in chronobiology which the collaborators have been utilizing in their projects.
Collaborator Contribution We are aiming to characterize 'time-of-day' responses of pharmacological drugs targeting NRF2 pathway, and how this can be applied to optimal wound healing and fibrosis resolution. The collaborators involved have helped in both providing appropriate mouse models, drugs as well as expert knowledge and feedback on the ongoing work.
Impact We currently have two manuscripts in preparation as a result of this collaboration.
Start Year 2017
 
Description NRF2 role in ageing, obesity and cancer 
Organisation University of Belgrade
Country Serbia 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided intellectual input and technical expertise.
Impact This collaboration has resulted in a recent joint publication as well as a joint grant application to the Ministry of Education in collaborator's country (pending outcome).
Start Year 2020
 
Description NRF2 role in behavioral rhythms 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided intellectual input and technical expertise.
Impact We have a joint manuscript in preparation.
Start Year 2019
 
Description Real-time bioluminescence imaging of hypothalamus and individual muscle fibers 
Organisation Liverpool John Moores University
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided intellectual input and technical expertise.
Impact We have two manuscripts in preparation.
Start Year 2020
 
Description cellular models of clock disruption 
Organisation Erasmus MC
Country Netherlands 
Sector Hospitals 
PI Contribution Our team has utilised the provided cellular models for testing pharmacological drugs and other relevant cellular assays to generate data and perform data analyses.
Collaborator Contribution This collaboration has enabled the testing of various cell models of genetic clock disruption as a means of reducing animal usage in line with 3Rs and streamlining in vivo experiments.
Impact A manuscript will be in preparation as an outcome of this collaboration
Start Year 2017
 
Description circadian rhythms and lysosomal diseases 
Organisation Universidade de São Paulo
Country Brazil 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided human samples, intellectual input and technical expertise.
Impact We have a joint manuscript in preparation.
Start Year 2019
 
Description human cellular disease models 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team is providing data in relation to circadian aspects of individual disease patient cells which will help with determining the optimal time of day for drug treatments
Collaborator Contribution The collaborators at UCL London have provided human tissue and cell material from different patients with Systemic Sclerosis
Impact We have a manuscript in preparation as a result of this collaboration and hope to extend this to another centres of excellence
Start Year 2019
 
Description human laminopathy disease mechanisms 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Our team has provided intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided transgenic animal models, human biopsies and technical expertise.
Impact We have a joint manuscript in preparation.
Start Year 2019
 
Description real-time cell analyses 
Organisation University of Glasgow
Department Institute of Health and Wellbeing
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided cellular genetic models of clock dysfunction and expert knowledge in the field to carry out the planned experiments.
Collaborator Contribution The collaboration will enable real-time cell analyses of 'time-of-day' responses to wound healing following cellular injury. This custom-made methodology provides reliable and quantitative measure of cellular responses in vitro which has not been previously utilised in circadian studies.
Impact This collaboration will enable generation of real-time data recording of cellular responses around the clock following injury. This will contribute towards a manuscript currently in preparation.
Start Year 2017
 
Description the circadian clock and covid-19 drugs 
Organisation University of Liverpool
Department Department of Molecular and Clinical Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided transgenic animals, intellectual input and technical expertise.
Impact We have a joint paper in submission and have submitted a grant application. Three disciplines are involved: pharmacology, biology and mathematics.
Start Year 2020
 
Description the circadian in silico modelling 
Organisation University of Liverpool
Department School of Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided transgenic animal models, intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided intellectual input and technical expertise.
Impact We have three papers in submission. We have applied for joint grant applications and some have been successfully awarded. This is a multidisciplinary collaboration between biology, maths and chemistry.
Start Year 2020
 
Description the role of clock in material-induced stem cell fate 
Organisation Nottingham Trent University
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has provided intellectual input and technical expertise.
Collaborator Contribution The collaborating team has provided intellectual input and technical expertise.
Impact We have published three joint articles and we have another publication in submission. This is a multi-disciplinary collaboration between biological sciences and material sciences.
Start Year 2019
 
Description transgenic animal approaches 
Organisation University of Liverpool
Department Department of Musculoskeletal Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team has utilised genetic mouse models provided by collaborators and generated data pertinent to the aims in the project grant. We have also provided expert knowledge in chronobiology which the collaborators are using in their current projects.
Collaborator Contribution This collaboration has enabled the use of genetic models of skin fibrosis and has provided unique reporter mouse strains to test hypotheses of the role of circadian clock within particular cell types as a contributor in wound healing an fibrosis.
Impact We will be preparing two manuscripts for publication as a result of this collaboration; one is concerned with circadian regulation using the genetic model of skin fibrosis and the other is concerned with mechanistic determination of how various genetic components of the circadian clock regulate fibrogenesis.
Start Year 2017
 
Description IWoman Academy working group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Third sector organisations
Results and Impact Iwoman Academy team has met up with several departmental leads across the University involved in Outreach and Widening Participation to start a dialogue as to further engagement activities to be organised between the two parties over the next academic year.
Year(s) Of Engagement Activity 2017
 
Description KIND Event- Radio interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Clare Roskell discussed the importance of our work with KIND during a radio interview.
Year(s) Of Engagement Activity 2017
 
Description Meet the Scientist event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We have participated in the 'Meet the scientist' event at the Liverpool World Museum. >200 people have engaged with our exhibit in which we have included a number of activities to introduce the public to the ideas of body clock and lifestyle changes to encourage better body clock function with age and in disease.
Year(s) Of Engagement Activity 2019
 
Description Museum exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We have participated in the 'Meet the scientist' event at the Liverpool World Museum with a theme 'Inside the Body Tardis'. >200 people have engaged with our exhibit in which we have included a number of activities to introduce the public to the ideas of body clock and lifestyle changes to encourage better body clock function with age.
Year(s) Of Engagement Activity 2017
 
Description Primary School visits 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My team and I held classes for primary school children to teach them about different body systems and engage in interactive play with body part models.
Year(s) Of Engagement Activity 2019
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Held classes for primary school children to teach them about DNA and base pairing.
Year(s) Of Engagement Activity 2017
 
Description Women in Science event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Women in Science event-my team and me participated in a careers' corner for schools' visit, which included >30 pupils of ages 14-18 from several local schools.
Year(s) Of Engagement Activity 2018
 
Description open day event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact As a departmental lead of the Outreach and Widening participation Group, I participated in an open day event at our research institution where general public and school children attended and was shown a number of fun activities to engage them in science.
Year(s) Of Engagement Activity 2017
 
Description posdcast interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Dr Pekovic-Vaughan has given an interview for an IWoman Academy podcast on the effects of ageing on our body clocks with a particular emphasis on the female body. iWoman Academy and Media, funded by The Wellcome Trust, is an organisation set up by BBC Radio Mersey presenter Ngunan Adamu, aimed at empowering the female global community using media and radio. The interview will be used as a podcast in IWoman Academy website due to be launched in 2018 and has also been used during the IWoman Graduation Ceremony held at the FACT, Liverpool.
Year(s) Of Engagement Activity 2017