ComBio: Flii and the elephant in the room

Allison Cowin will lead off the session with her group’s latest findings on the regulation of cellular adhesion and migration during wound repair. Cowin hails from England originally, where she did her PhD and postdoctoral training at the University of Manchester. In 1996, she saw the light and headed for Australia, where she now runs the wound healing group at the Women’s and Children’s Health Research Institute in Adelaide.

Cowin’s overall research aim is to understand the mechanisms involved in wound healing and scar formation. A lot of the players involved are known, but how do they all work together at the cellular and signalling level as cells migrate and fill in a wound site, and what are the best targets for therapy?

The goal for Cowin and for the field in general is to develop new therapies for wounds and burn injuries. Currently, no effective mechanism-driven products exist for countering wound healing-related health problems: that is, something that will make the cells do a better job.

Treatments still rely on bandaging and compression measures, and just trying to stop the associated tissue oedema from taking over. So, there is a huge market out there for something that would work to fix or alleviate the problem from the source.

The major applications for this work are to make chronic wounds heal and to reduce scarring in burns and other injuries. Chronic wounds tend to be something that people don’t think about but they are an increasingly large problem in our society, particularly in the elderly and in patients with diabetes.

“They get these wounds that just will not heal,” Cowin says. Chronic wounds and scarring are really two sides of the same coin, and the same molecules are probably regulating both.

Cowin has wanted to know what regulates wound healing for many years. She is interested in the mechanisms behind cells migrating to and repopulating a wound site under different physiological and pathological conditions.

“We knew already that the cytoskeleton was important in this whole process, in the ways cells travel into a wound and in bringing the wound margins together to re-epithelialise the site,” she says. In particular, Cowin was looking at the roles of actin and actin-remodelling proteins such as gelsolin and paxillin, which are important mediators of cell adhesion and migration.

As is often the case, a serendipitous conversation (and subsequent collaboration) with some scientists at the Australian National University in Canberra turned Cowin’s attention to a cytoskeletal protein she had previously not investigated. Called Flightless I (Flii), it belongs to the same family of actin-remodelling molecules as gelsolin.

“So, I looked at it in our experimental systems, and it now turns out that Flii might be more important than any of the other family members in the wound healing process.”

Using some transgenic and knockout mouse models from the ANU labs and an incisional wounding assay, Cowin identified Flii as a negative regulator of wound healing, with the findings published in the Journal of Pathology in 2007.

Over-expression of Flii impaired the healing process, while knocking it down made the incisions heal much better. “It was quite a fundamental discovery, that this important protein is actually a bad guy in wound healing. Since then we have been designing methods to reduce its activity within a wound in the hope of improving wound healing.”

---PB--- Fetal wound repair

One system that Cowin has always been interested in is fetal wound healing. Up to a certain point in utero, humans and other animals regenerate damaged tissue rather than repair it, thus no scar tissue is generated. This phenomenon of fetal wound repair and the loss of it at some stage late in gestation is both fascinating scientifically and potentially important clinically for what it might teach us about the healing and scarring process.

For instance, when keyhole surgery is performed on a fetus in utero, that wound will completely regenerate in contrast to the scarred mess that we oldies get. So, why do we lose that ability? Current thinking says this is least partly because out of the sterile environment of the womb, the biggest threat to damaged tissue is infection.

So, the body wants to fix it up as quickly as possible without caring about unattractive and often painful scar formation. “Now that we mostly live in an environment where we can control infection, we would quite like to get rid of the scarring element. That is one of our research aims – to decrease scarring and make wounds heal in a more coordinated fashion so that the collagen is laid down correctly rather than in a rapid form that turns into a scar.”

One of the aspects of this work that Cowin will be talking about at ComBio is the project of a PhD student in her lab, Zlatko Kopecki. Using in vitro assays with cells grown out from animals with modulated levels of Flii expression, he showed that loss of Flii make cells adhere better, migrate faster and proliferate more profusely.

Conversely, Flii over-expression impairs all these same functions. Electron microscopy showed that Flii over-expression renders the skin of the experimental animals thinner and weaker, with the hemidesmosome cell junctions between adjacent epithelial cells showing a markedly different structure and formation.

He also stained the skin for different integrins, which connect the epidermis to the basement membrane of the skin and which are important in the migration of cells across the wound. The integrin expression profiles differed significantly depending on the level of Flii expressed.

The features in these animals indicate major changes to skin physiology with changes in Flii expression, particularly illustrated by the disrupted hemidesmosomes, and confirm Flii as a negative regulator of wound healing.

Cowin thinks that such a structural profile may impact on the skin’s ability to re-epithelialise and to adhere. Similar skin defects are also associated with various pathologies such as painful and chronic skin-blistering conditions, in which the skin becomes fragile and vulnerable to wounding.

---PB--- Antibody application

Such pathologies are also the subject of Cowin’s research, in collaboration with a clinical group in Sydney who study patients with the skin-blistering disorder, epidermolysis bullosa. “Wound biopsies from these patients showed high levels of Flii and we are investigating whether healing can be improved in those situations by down-regulating the expression of Flii,” she says.

To this end, Cowin is drawing on previous findings that Flii is secreted in response to wounding, and that topical application of antibodies against a particular region of the Flii protein made the wounds heal much better. Cowin’s group is now developing an antibody-based strategy for reducing Flii expression in wounds as a potential new wound healing therapy.

“We have generated and tested a panel of affinity purified antibodies raised against different parts of the Flii and have shown that they can modulate Flii activity in vitro and in vivo.” This approach will hopefully have clinical benefit in a range of physiological and pathological situations involving wound repair.

The issue of chronic wounds in particular needs to be highlighted, according to Cowin. She is part of the Australian Wound Management Association, which is currently trying to raise awareness throughout Australia of just how widespread the problem is.

“They are calling it the ‘elephant in the room’,” Cowin says. “More and more patients have such wounds, but it is the story that nobody wants to talk about.”

Graphic images of unhealed wounds or disfiguring burns scars do not make for popular viewing, so the general public does not realise the extent of the problem or that it is getting worse as more people get diabetes, and suffer lifestyle-related health problems such as obesity.

Chronic wounds in particular are also an increasing problem in any ageing population, often leading to amputations and other complications, resulting in increased hospital stays and further burdens on the health system.

---PB--- Marvellous serendipity

Cowin is also very interested in the role of TGFbeta in this whole scenario. It is another key protein in scarring and wound repair, and, based on Cowin’s results, Flii seems to work upstream of TGFbeta. Cowin would love to know how this relationship fits into the wound-healing context.

The body responds to a wound by upregulating Flii and in turn TGFbeta is also turned up. “So, they are both part of this control mechanism to get wounds to heal as quickly as possible,” she says.

“What we want to do is modulate the system so wounds heal ‘the right way’ under all conditions – turn up Flii in chronic wounds to stimulate healing, but knock it down in burn wounds to regulate the process more precisely and prevent scarring.”

A paper this year by Cowin’s group also suggested that the activity of both Flii and TGFbeta in wound healing is affected by age and possibly even by gender, and specific strategies might be helpful to improve wound repair in elderly skin.

The ultimate goal of Cowin’s group is to make products for healing wounds. They are hoping that the antibody approach will be one way to get there, and are currently going through the patenting and commercialisation process with the antibodies they have developed so far.

Plans for large animal trials are underway, and this would be followed by human trials. “We would see this as a direct therapy, a cutaneous approach to healing wounds, and in five to seven years we will hopefully have a product. Serendipity is a marvellous thing at times.”

As an aside, the research Cowin will be presenting on Flii at ComBio was chosen to be included in the 2008 NHMRC "10 of the best research projects" list. Cowin was delighted: “It’s great that our research is being recognised by the NHMRC, and it helps raise the profile of wound research in Australia.”

---PB--- Cell matrices and cell migration

Other talks in the same session nicely highlight the breadth of research in the field of wound healing. Professor Zee Upton from the Queensland University of Technology will talk about new approaches to recapitulate the multidimensional nature of interactions of cells with the extracellular matrix, in order to facilitate development of effective dermal wound therapies.

“Zee is doing a lot of really good work in her field,” Cowin says. “Her talk is more about the applications for their work. Her group is making new scaffolds or dermal substitutes for potential therapeutic use.

“For instance, in a wound where there is not enough dermis left for healing to occur, these 3D human skin equivalents will help the endogenous cells migrate into the site and start to make new extracellular matrix in a regular way. Their work has direct applications to humans, particularly in the treatment of burns and chronic wounds.”

Dr Rachael Murray of the Children's Hospital Burns Research Institute in Sydney will talk about the role of SNARE proteins in regulating cell migration.

“Rachael is a really good scientist – of course, she is also English so she must be good. She heads a burns group at the Children’s Hospital and is looking at the role of macrophages when they enter a wound from a basic cell biology slant. In particular, she will talk about her studies on the intracellular trafficking pathways in this context and the role of SNARE proteins in regulating cellular migration.”