Could you provide an insight into what first led you to research the process of skin healing?

I have always been fascinated by the mechanisms that control the adhesion and polarisation of epithelial cells in human tissue – and the skin is an excellent model to study these biological events. I developed an interest in the wound healing process because of the major defects in epidermal cell adhesion seen during skin healing after deep burns. Indeed, the renewal and correct reorganisation of proteins in the dermal-epidermal junction is particularly problematic when the wounds are deep or large. Therefore, after analysing adhesion mechanisms in quiescent epidermis, I decided to investigate them in the dynamic process of wound repair.

My lab belongs to the Tissue Biology and Therapeutic Engineering Unit at the Institute of Protein Biology and Chemistry in Lyon – which is one of the major centres in France specialising in the extracellular matrix and tissue repair field of research. In addition, I have long-term collaborations with Lyon Hospitals and connections have been created between my lab, the Cell and Tissue Bank and the Burn Centre. The scientists in my lab undertake research projects in conjunction with clinicians on the topic of skin repair and regeneration at both in vitro and in vivo levels.

What have been the greatest successes of your most recent project and how are you planning to maximise their impact?

Concerning wound healing studies, an important success was accurately mimicking – through a short decapeptide – the biological activity of an entire protein domain and recapitulating its pro-migratory activity. Another success came with the results of our preliminary in vivo studies, which showed accelerated wound epidermisation. Currently, together with the pharmaceutical company SYMATESE, we are working to achieve peptide stability in the wound deleterious environment and, to this end, we are designing a specific formulation to be used in superficial and partial-thickness acute wounds. We foresee further successes in this area in the future. For instance, at a more fundamental level, we’ve had some very interesting and innovative mechanistic results concerning the involvement of the receptor syndecan-1 in the maintenance of epidermal homeostasis. These findings open challenging perspectives.

Have you encountered any difficulties carrying out your investigations?

Like any cell biologists, we have encountered difficulties in performing our experiments and interpreting our results, particularly regarding the study of cell migration. The development of in vitro models that mimic or closely resemble the epithelialisation phase of wound healing is necessary to elucidate the molecular mechanisms underlying the keratinocyte migration phase of the epidermal regeneration event. Considering the high level of complexity of the mechanisms involved, the development of molecular tools is vital for visualising dynamic adhesion processes and associated intracellular events through time-lapse video microscopy in living cells.

Who have been your main collaborators? How have these multidisciplinary partnerships helped you build a thorough picture of peptides in treating acute wound healing?

Over the past 10 years, I have conducted fundamental research in cooperation

with my group, including postdoctoral fellows and PhD students. We started a multidisciplinary partnership when SYMATESE became interested in the potential impact of the peptide in wound healing. Furthermore, Professor Fabienne Braye and surgeons from the Burns Centre at Lyon Hospital are now involved in the project, where they are designing pre-clinical experiments aimed at testing the peptide efficacy in hard-to-heal wounds, which are a major therapeutic challenge.

Additionally, I am currently establishing a French National Center for Scientific Research (CNRS) research group on the theme of scarring, skin regeneration and mucous membranes. The goal will be to develop a national network that enables partnerships and collaborations between scientists from various disciplines – molecular and cellular biologists, physiologists, chemists, physicists and engineers – clinicians with different specialities, surgeons and veterinarians. The meetings of this group will also be open to industrial partners. So far, at least 50 partners have displayed interest in participating in this multidisciplinary consortium.

Which are your preferred methods of dissemination to ensure a successful impact on interested stakeholders and the broader public?

My preferred methods are scientific communication such as peer-reviewed publications and participation in conferences, symposia and international meetings such as the European Tissue Repair Society Meeting, Wound Healing Society Meeting and the International Society for Burn Injuries Meeting. We also communicate in various society meetings, including the European Society for Dermatological Research, the European Society for Matrix Biology and the French Societies for Extracellular Matrix or Cell Biology research. Furthermore, several SKINHEALING project partners participate annually in student training and give lectures in skin biology, wound repair, tissue engineering and skin ageing for numerous university programmes.

Dr Patricia Rousselle is a biologist with expertise in crosstalks between cells and the microenvironment in the context of tissue repair. Here, she discusses insights from her research into the complex and dynamic processes of skin regeneration in the wound healing process

Epidermal endeavours DR PATRICIA RO

USSELLE

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WHILE THE PRECISE global burden of chronic wounds is unknown, increasing numbers of patients across the world are being referred to physicians for wound care. Indeed, it has been estimated that 5 million people every year need dressings for wounds as a result of burns, pressure ulcers and diabetic leg ulcers, among other things. In view of factors such as the ageing population, growing incidence of diabetes and prolonged life expectancies of fragile individuals, the likelihood is that the prevalence and impact of these chronic wounds will only increase in the future.

Unfortunately, rather than being a simple and linear process, skin wound healing is extremely complex. Research has highlighted that it is the result of several dynamic and interactive mechanisms involving the blood, extracellular matrix components, and epithelial and parenchymal cells. Indeed, the skin is made up of a multi-layered epithelium that covers a highly interactive network composed of extracellular matrix proteins. Named the dermal-epidermal junction, this network separates the dermal and epidermal compartments. In the case of skin healing following a wound, the proteins in the dermal-epidermal junction need to be renewed and reorganised in a way that facilitates the correct re-attachment of the epidermis and fuels the repair process.

SKIN REPAIR

Under normal conditions, the skin repair process can be divided into a series of overlapping and non-exclusive stages, including haemostasis, infl ammation, cell proliferation and remodelling. Importantly, many of the cellular components involved in this process undergo notable changes in gene expression and phenotype, in turn fuelling cell differentiation and migration. While acute wounds (primarily the result of mechanical injuries and burns) tend to heal in between eight and 12 weeks with minimal scarring, chronic wounds (usually the result of pressure, venous, arterial and diabetic ulcers) generally do not heal properly due to disruptions in the healing sequence that cause a permanent infl ammatory state with high proteolytic activity. Unfortunately, although there are many advanced skin-healing dressings available, dressings that promote the effective healing of chronic wounds do not currently exist.

One researcher who is addressing this gap in the market is Dr Patricia Rousselle. Based in a laboratory at the Institute of Protein Biology and Chemistry in France, Rousselle’s broad expertise lies in the complex and dynamic processes that underpin wound repair. With an impressive track record of published research, she has led many cutting-edge collaborative research projects on the theme of skin repair and regeneration.

A PROMISING PEPTIDE

Following the completion of extensive in vitro studies, Rousselle and her team have recently produced an innovative peptide with a sequence derived from a specifi c domain in laminin-332, a dermal-epidermal junction protein that plays a key role in the adhesion and migration of epidermal keratinocytes. Excitingly, their studies have demonstrated that both the laminin domain and its biomimetic peptide are able to induce human primary keratinocyte adhesion and migration when added to the culture medium. “This suggests that keratinocytes may have been switched from a static phenotype to an activated phenotype that is accompanied by cell movement,” explains Rousselle. “Our results imply that in addition to growth factors and cytokines, extracellular matrix proteins are crucial in all phases of wound repair.”

As the researchers prepare to move their experiments to the in vivo stage, they are concentrating on conducting preliminary studies using a partial-thickness cutaneous wound porcine model. Promisingly, in comparison to current widely used treatments for cutaneous wounds, the application of the peptide has been shown to accelerate the re-epithelialisation process by promoting wound healing in its early stages. “The clinical and histological observations of the wounds on day four revealed that the length of the epithelial outgrowths were longer in the treated wounds,” Rousselle outlines. “Seven days post-injury, the epidermal layer was already entirely renewed in the peptide-treated wounds while it only covered 65 per cent of the lesion in the vehicle-treated wounds. In addition, the use of the peptide seemed to regulate the infl ammatory response, leading to an improved scar quality when compared to the vehicle-treated wounds.”

Importantly, Rousselle’s research efforts in this area suggest that the capacity of the innovative peptide to improve skin healing could be connected to the transmembrane receptors of the syndecan family. The precise action mechanisms, however, are currently unclear, presenting the researchers with a challenge for the future: “Our ongoing research objectives aim to determine the nature of intracellular signals triggered by the laminin peptide and, in turn, to identify the role of the signalling molecules mobilised to orchestrate this remarkable adaptation and cell plasticity,” Rousselle elucidates.

FUTURE STEPS

Funded by the French National Research Agency (ANR), Rousselle and her team are currently participating in an innovative project entitled SKINHEALING, in which they are aiming to optimise their novel peptide for use in the clinic. In conjunction with partners from the pharmaceutical company SYMATESE and from the Veterinary School of Lyon, the researchers will assess the effectiveness of the peptide through animal models of acute and chronic wounds. They are also aiming to optimise the in vivo stability of the peptide and its resistance to proteolysis, as well as forging a deeper understanding of its action mechanisms. Looking ahead, the project has the potential to accelerate the completion of proof-of-concept studies, successfully bringing the peptide to trial in a human clinical setting where it will improve the lives of many patients with large and/or chronic wounds.

Rousselle and her team have

produced an innovative peptide

with a sequence derived from

laminin-332, a dermal-epidermal

junction protein that plays a key

role in the adhesion and migration

of epidermal keratinocytes

The healing processManaged by the French National Centre for Scientifi c Research, a laboratory in the Tissue Biology and Therapeutic Engineering Department of the Institute of Protein Biology and Chemistry is carving new insights into epidermal regeneration and repair in both skin healing and ageing

DR PATRICIA ROUSSELLE

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SKINHEALING

OBJECTIVES

• To develop an extracellular matrix peptide derived from laminin-332 for use in clinical medicine to encourage or trigger skin healing in various types of wounds

• To favour epidermal regeneration in skin ageing by targeting basement membrane proteoglycans

KEY COLLABORATORS

Lab members: Dr Guila Dayan; Dr Caroline Cluzel-Grangeasse; Anna Michopoulou; Fanny Waltz; Alysée Moulard; Marine Montmasson • Dr Odile Damour, Cell and Tissue Bank, Hospices Civils de Lyon, France • Professor Fabienne Braye, Burn Centre, Hospices Civils de Lyon, France

PARTNERS

SYMATESE, Chaponost, France • Veterinary School of Lyon, Marcy l’Etoile, France • BASF Beauty Care Solutions, Lyon, France

FUNDING

French National Research Agency (ANR), grant number n° ANR-13-RPIB-0003-01

Rhône-Alpes région (ARC 2), French National Centre for Scientifi c Research (CNRS)

CONTACT

Dr Patricia RousselleDirector of Research

Tissue Biology and TherapeuticEngineering UnitInstitute of Protein Biology and ChemistryUMR 5305 – CNRS / University of LyonSFR BioSciences Gerland-Lyon Sud7 passage du Vercors, 69367 Lyon Cedex 07, France

T +33 4 72 72 26 39E patricia.rousselle@ibcp.fr

http://bit.ly/1ItCx0fhttp://bit.ly/1uf5W3L

PATRICIA ROUSSELLE obtained a doctorate in Pharmacy and pursued a four-year residency in clinical biology at Lyon Hospitals, where she also gained an MSc in Molecular and Cellular Biology in 1990. After doctoral training at the Shriners Hospital For Crippled Children in Portland, USA, she obtained her PhD in 1994. She worked at the Cutaneous Biology Research Center in Boston, USA, before joining CNRS to set up her own group.

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Rousselle and her team are also focusing on epidermal regeneration in the context of skin ageing. Both the appearance and the mechanical functions of the epidermis change dramatically over time as a result of ageing and increased exposure to damaging stimuli such as ultraviolet radiation. Not only does the ageing process lead to thinning of the dermis and epidermis, but it also changes the dermal-epidermal junction, with decreased contact with the basal keratinocytes disrupting epidermal renewal. “In genetically programmed ageing, the progressive decrease in epidermal cell renewal results from decreased proliferation and a declined regenerative potential that is believed to be infl uenced by microenvironmental cues such as modifi cations in the extracellular matrix composition,” Rousselle explains. In order to investigate these mechanisms in more detail, her lab has established a collaborative partnership with BASF Beauty Care Solutions. Together, the researchers involved in this collaboration are aiming to explore whether the heparan sulfate proteoglycans expressed in the dermal-epidermal junction are implicated in ageing mechanisms and, as a result, develop innovative strategies that repair ageing defects.

Image 1: Partially hydrated hyaluronic acid fi bres produced by SYMATESE.

Image 2: Collagen dressings produced by SYMATESE

Image 3: Image of a regenerating epidermis. Immunostaining with an anti-keratin 14 antibody. Direction of migration is shown by the white arrow. The 5-day-old-wounding skin biopsy was provided by Fabienne Braye at the Centre des Grands Brûlés, Pavillon I-5, Hôpital Edouard Hérriot, Lyon, France. Ba = 50 µm.

Image 4: Image of a migrating keratinocyte in vitro. The actin cytokeleton of a migrating keratinocyte was revealed with phalloidin coupled to FITC. To achieve migration, keratinocytes develop actin rich membrane protrusions such as fi lopodes and lamellipodes at their leading front. The initiation and formation of these adhesion structures depends on the precisely regulated polymerisation, convergence and crosslinking of actin fi laments.

Image 5: Images of a skin equivalent model. In vitro skin equivalents are composed of a dermal compartment made of fi broblasts seeded in a porous substrate (made of collagen, glycosaminoglycans and chitosan), on top of which keratinocytes are seeded. The histological staining of paraffi n-embedded skin equivalent (a) and the immunofl uorescence detection of the differentiation marker involucrin reveals that culturing keratinocytes in presence of the peptide improved the thickness and quality of the epidermis suggesting that the epidermal regeneration process was favoured. The experiment was performed in the Skin Substitutes Laboratory headed by Odile Damour, Hôpital Edouard Hérriot, Lyon, France. Scale bars = 50 µm.

Addressing the ageing effect

cell direction

epidermis

epidermis

dermis

Ki67

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INTELLIGENCE

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