An inflamed response

Paul Martin, Professor of Cell Biology in the University of Bristol Departments of Physiology and Pharmacology, and Biochemistry, talked to Cherry Lewis about the discovery made in his lab that speeds up wound healing and reduces the size of the wound scar. She was interested not only in the discovery itself, but also in the processes and years of research that led up to it.

CL: Given that scarring is a natural process, what made you think you could improve on it?

PM: Well, we know that the natural process isn't perfect because wounds don't heal very rapidly and they often leave a nasty scar. However, we also know that in an embryo you get tissue repair that is perfect. I'm a developmental biologist and we often do what we call cut-and-paste surgery on mice and chick embryos. What we see is that within an hour of making a cut, you can't see any evidence of having done so. The repair and regenerative capacities of an embryo are amazing, and what it tells you is that perfection is achievable. Indeed, human foetal surgeons take advantage of this. If they go in early enough, they can do major corrective surgery in the womb without leaving any sign of a scar.

CL: At what point does scarring start, then?

PM: It has been known for some time that there is a transition moment during development - embryogenesis - when you start to scar. Some 15 years ago, in my lab in Oxford, I discovered that you get scarring from the first time in development when inflammatory cells are drawn to the wound - that is, the first time an inflammatory response was activated upon wounding. So it seemed very likely that inflammation was causing scarring.

CL: But isn't it important to our survival to have an inflammatory response?

PM: Indeed, it's been evolutionarily selected for in order to kill microbes wherever there's a breach in your skin. The key thing is to not die of septicaemia because bugs get into a cut. So as soon as the embryo starts generating inflammatory cells, it's practising raising an inflammatory response.

CL: So what is it about the inflammatory response that causes scarring?

PM: Scarring is rather like laying down a rushed, slightly excessive and poorly organised collagen matrix. Collagen is a protein that is rich in all connective tissues, but in normal skin it's laid down in a special way. What happens when tissue repairs is that it's laid down badly in dense bundles, which is why scars feel hard and matted. So we set about wondering how you might stop them forming in the first place.

As I explained, there seemed to be evidence that it was inflammatory cells rushing into the wound and releasing signals that somehow told the local wound fibroblasts to make a scar. To test this theory, we scratched a mouse that had a gene 'knocked out' that it needs in order to make the white blood cell types that are the inflammatory response. If you scratch mice that are missing this PU.1 gene much later than the transition moment in embryogenesis when you should start getting scars, it doesn't scar. This demonstrates that inflammation leads to scars.

CL: And you were able to identify which gene was responsible for scarring?

PM: Well, not exactly. There are hundreds of genes that are switched on when you wound skin and many of them are needed to close the wound hole. But by subtracting the genes that are switched on in the PU.1 knockout mouse where there's no inflammation when you wound it, from the larger pool of genes that are switched on in a normal mouse we were able to identify the extra ones that were present due to inflammation. One of these was a gene called osteopontin.

CL: How did you know it was the culprit?

PM: We didn't. The next step took ages to design. We could have just made a knock-out mouse that didn't have the osteopontin gene, but while that might have told us osteopontin was the culprit, it wouldn't have said anything about how to counter its scarring effects. Instead, what we decided to do was allow the inflammatory response to happen and osteopontin to be switched on, but then to dampen down its effect and see what happened.

We already knew of a Pluronic gel that acted as a delivery system. The great thing about this gel is that it is liquid at low temperatures so you can squirt it into a wound, but as soon as it reaches body temperature it goes hard. So it moulds itself into the wound and then slowly releases whatever is in the gel.

CL: And what is in the gel?

PM: When the genetic sequence of a particular gene is known to cause a disease, or, as in this case, an inflammation, it is possible to synthesise a strand of DNA that will bind to the MRNA encoded by that gene and inactivate it, effectively turning that gene off. This synthesised nucleic acid is termed an 'anti-sense oligonucleotide'. So what we put in to the gel was an anti-sense oligonucleotide against osteopontin. What happens is that the inflammatory cells are still rushing in to the wound, they're still telling the fibroblasts to make osteopontin but all the osteopontin is being shut down by the anti-sense oligonucleotide. And sure enough, what we found is that if you block osteopontin, the wound heals faster and you block scarring. We don't fully understand yet how and why osteopontin causes scarring, but what we do know is that if you reduce osteopontin, you reduce scarring.

CL: And there are no indications of side effects?

PM: Good question and one I can't answer yet. However, my guess is that there's no benefit to having osteopontin switched on in the wound and therefore switching it off shouldn't cause side effects, but I might be wrong. And it's important to realise that it's not only external scarring of the skin that will be improved. Any kind of surgery - heart operations and caesarean sections, for example - will benefit from reduced scarring.

CL: So might we eventually see this as a treatment?

PM: Hopefully, but it is at an early stage in development and will require further research, followed by human clinical trials. This takes considerable funding and particular skills, so we have to have an industrial partner. We therefore worked closely with the University's Technology Transfer team who have been invaluable in helping us patent our findings. Furthermore, we have just granted an exclusive licence to an established pharmaceutical company who will take the development forward. So watch this space!

Physiology and Pharmacology

If you scratch mice that are missing this PU.1 gene much later than the transition moment in embryogenesis when you should start getting scars, it doesn't scar. This demonstrates that inflammation leads to scars.

Paul Martin

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