Investigation of the role of cellular senescence in skin aging using in vivo and in vitro approaches

Human populations throughout the world are ageing with the numbers of people over the age of 60 increasing three times as fast as the number of people under the age of fifteen. These demographic changes have significant impacts on the way we live and in particular the types of products that we need in order to adapt to and cope with ageing populations. Ageing is a complex phenomenon and we don't really understand how or why it occurs. However, we do know the features of ageing in certain organs in the body and in particular the features of ageing in skin are quite well characterized. Skin ageing actually starts relatively early in life with a gradual loss of elasticity at around thirty years of age. This loss of elasticity occurs in a part of the skin underneath its surface called the dermis and at the same time as elasticity is impaired there is a gradual accumulation of aged cells that enter a state of senescence. The accumulation of senescent cells has several adverse effects including a reduction in the ability of skin to deal with oxidative stress and an increase in the production of inflammatory molecules. This in turn leads to adverse effects in the upper layers of the skin, which protect us from our environment, and eventually this leads to typical characteristics of skin in the elderly, including dryness and a propensity to wounding. Many skin care products attempt to deal with these problems and some do have very positive effects. However, until we understand in detail how ageing occurs and what are the key drivers of skin ageing it will be difficult to either slow it down or to reverse it. We will undertake a number of studies in this project that aim to map in detail the changes that occur in all parts of human skin over period of sixty years and use this information in model systems of skin to investigate how skin ageing starts and whether we can identify or design products that can either slow down or even reverse the skin ageing process. To achieve these objectives we will work with multinational companies to devise new, safe and affordable skin products that will be available for everyone. Importantly, we aim to carry out this research without recourse to testing these products on animals.

Transcriptomic studies comparing chronologically and photo-aged skin have shown that convergent pathways implicating loss of ROS homeostasis, increased expression of pro-inflammatory markers, reduced expression of epidermal differentiation markers and impaired cholesterol lipid biosynthesis are key features of skin ageing. We propose to refine this data by performing a six decadal cross sectional analysis of human skin ageing. We will use laser capture micro-dissection in combination with transcriptomic and immunohistochemical analyses to map and then model the interaction of age dependant changes in all compartments of human skin. The data already available to us are consistent with our hypothesis that the gradual accumulation of senescent dermal fibroblasts contributes to skin ageing via a senescence associated secretory pathway (SASP) including leakage of ROS and secretion of pro-inflammatory molecules that causes bystander effects in both the dermis and the epidermis. To test this hypothesis we have developed a 3D organotypic skin equivalent that recapitulate several aspects of skin ageing, including epidermal thinning, delayed development of a cornifed envelope and altered cell cycle kinetics within transit amplifying cells. Importantly these epidermal changes are caused by the presence of aged or prematurely aged fibroblasts in the dermal equivalent. We will refine this model so that it can be used to both test the hypothesis that a SASP in the dermal equivalent of the model drives epidermal senescence via a bystander effect and to investigate which elements of skin ageing are caused by dermal senescence. Importantly, we will use to use inhibitors of SASP, already available in our laboratories, as a possible means of either slowing or even reversing ageing phenotypes in the model. In the long term we will use the model to develop and test new skin rejuvenation products.

Hutchison and von Zlignicki have each over twenty years research experience working in instrinsic and extrinsic mechanisms of ageing. Maatta, Karakesisoglou and Di Colandrea have all trained in the leading skin laboratories in the world and have worked on models of skin development and barrier function for more than ten years. The track record of the team suggests that multiple high impact publications will emerge as the principle pathways to impact from this study.

The team have recently focused on pathways that lead to ROS generation and have demonstrated in multiple publications that free radical scavengers and drugs that inhibit pro-inflammatory pathways have potential as anti-ageing agents. These products are available immediately for proof of principle anti-ageing studies in complex culture models.
Procter & Gamble already exploit anti-oxidants in their skin ageing and beauty consumer products for which they hold substantive market shares. They also have proprietary rights on thousands of similar products. This research program has the potential to inform the development of more effective rejuvenation products that are better able to promote healthy skin ageing through the generation of ectopically applied products. The juxtaposition of the Durham teams to the P&G Open Innovation Partnership within Durham University's Chemistry Department will positively influence this outcome.

Life Technologies Ltd has been named as one of the most innovative companies in the world and the company has a significant UK manufacturing and R&D presence. The reporter constructs, culture media and supplements that will be developed in order to support the 3D model, will have immediate value in both the academic research market and in product development within the cosmetic industry.

ReInnvervate Ltd. is now positioned for flotation and the use of its 3D cell culture products as an industry standard for product testing in 3D organotypic skin models will further enhance its reputation and value. Similarly, Ruskinn Life Sciences has gained a worldwide reputation for generating engineering solutions for long term culture models and adapting their lab in-a-box technologies for multicellular 3D cultures will further develop their capacity to serve the needs of Biotechnology industry in the 21st Century. The recent acquisition of Ruskinn by Baker, has enhanced the capacity of the company for product development and to reach a world wide market.

Over 57,000 thousand animals are were used in studies relating to skin n the UK in 2011. Many of the procedures used were severe or required the use of transgenic animals which develop severe phenotypes (Home Office Statistics). The generation of organotypic models that are completely reproducible between laboratories and could replace these animals will make significant contributions to the 3Rs agenda of RCUK. In addition, we are fully aware of the EU directives on chemical safety testing (REACH) and the demand within this directive to reduce the need for animal use in safety testing. In the long term we intend to apply for accreditation of our model in compliance with REACH.

Other pathways to impact include the training of personnel in complex and long term organotypic cultures as well as in industry standards. Ageing research and replacement of animals in research are of considerable interest to the general public and Hutchison has extensive experience of public engagement activity including radio and television work, he presented the Society of Biology Charter Lecture in 2010 and supported the BBSRC funded program 'Stemistry' via workshops. The team will participate in two public events each year via each Universities Science Festivals and one special platform event organized through the International Centre for Life. Throughout the study the team will use timely and judicious release of information through public media, under the guidance of the media teams in Durham and Newcastle.