Pilotstudie 07.02

Objectives
Determination whether different agents used to prevent fibrosis directly or indirectly in other pathological conditions than skin wound healing could be utilised to improve the quality of wound healing and prevent scar formation in the skin.

Study method
A survey of the literature revealed various agents that have been described to inhibit the fibrosis or reduce the inflammatory response in a range of different pathological conditions. Because the mechanisms of fibrosis are more or less the same in all fibrotic conditions (excessive collagen deposition, decreased collagen degradation), it is feasible that at least a subset of these agents might inhibit the fibrosis seen in scar formation.

In vitro experiments with human cell culture and an ex vivo skin burn wound model supplemented with inflammatory cells will be used to test the effect of different antifibrotic/anti-inflammatory agents with respect to extracellular matrix deposition and remodelling.

The long term outcome of healing and the effects on angiogenesis will be further evaluated in a rat study. Agents of interest are: different inhibitors of the Renin Angiotensin System (RAS) and statines. As a measure of fibrosis, α smooth muscle actin, collagen type I and the fibrosis linked crosslinking enzyme LH2 expression (RNA and on protein level) will be determined. In rat the presence of myofibroblasts, contraction and amount of blood vessels will be the endpoint parameters.

Results
The aim of our pilot study was to test potent anti-inflammatory/antifibrotic drugs with respect to their beneficial effects on wound healing. Based on the literature different drugs which were shown to have beneficial effects on the fibrotic process in different pathologies were selected.  

The candidates were inhibitors of the Renin angiotensin System (Captopril and Losartan) and a cholesterol lowering drug; Mevinolin. Contact burn wounds were inflicted on the back of rats and the drugs were administred in the drinking water duting the first week after wounding. Two experiments were performed, in the first experiment full thickness wounds (3^rd degree)  were induced and in the second experiment 2^nd degree wounds were induced.

In the 3^rd degree wounds: After 6 weeks a reduced number of myofibroblasts were detected in al treatment groups.
Systemic administration of captopril resulted in a reduction of myofibroblasts from 16% to 7% of the surface area. Moreover we saw a reduction in the fibrotic crosslinking enzyme (LH2b) in relation to collagen type I. This indicates that the scar related crosslinking is reduced and a skin-like crosslinked collagen is formed.

AngII exerts its effects by binding to one of the two main receptors (AT1 and AT2). Binding to either one of these receptors often have opposing effects. Thus the definitive outcome of Ang II action depends on the presence of one, or the balance between the two receptors. It is thought that the AT1 receptor stimulates collagen production and that binding to the AT2 receptor has antifibrotic effects (Nabeshima, Tazuma et al. 2006). Inhibition of the AT1 by the AT1 antagonist Losartan indeed reduced the number of myofibroblasts in the scar and even more than the ACE inhibitor (from 16% to 3%).

Mevinolin was shown to have anti-inflammatory effects and was able to reduce α- smooth muscle actin expression and contraction of a fibroblast populated collagen matrix (Watts, Sampson et al. 2005). In addition this drug  was shown to influence the TGBβ1 response and attenuates collagen expression (Derk and Jimenez 2006). Furthermore it is known to down regulate the AT1 receptor and stimulate angiogenesis (Kurian, Rai et al. 2006). We showed a reduction in myofibroblasts in response to mevinolin treatment as well (16 to 5 %).

Also with mevinolin we saw a reduction in the fibrotic crosslinking enzyme (LH2b) in relation to collagen type I. Indicative for a reduced scar related collagen crosslinking. The 2^nd degree wounds showed a very low percentage of myofibroblasts < 1% in all groups including the control group. Indicating that the fibrotic process is very limited in this rat model.