P03 Hydroxypyridone antifungals induce myofibroblast apoptosis in an in vitro model of hypertrophic scars

Abstract Despite being the most common complication following burn injury, there are no medications to prevent the formation of hypertrophic scars. Myofibroblasts are key cells involved in scar formation, responsible for producing excessive amounts of extracellular matrix and have been shown to be resistant to apoptosis. We have previously shown that hydroxypyridone antifungals can inhibit the transformation of fibroblasts to myofibroblasts, and exhibit other antifibrotic effects. This study aimed to investigate the effect of these drugs on established myofibroblasts. Primary human dermal fibroblasts, derived from scars of patients with burns, were exposed to 10 ng mL–1 transforming growth factor (TGF)-β1 to induce myofibroblast transformation. The In-Cell enzyme-linked immunosorbent assay method was used to measure α-smooth muscle actin (α-SMA), with DRAQ5 staining used to measure cell viability. Concentration–response curves for the three hydroxypyridone antifungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine) were constructed (0.03–300 µmol L–1) to see if the drugs could reverse myofibroblast transformation. TUNEL staining was used to investigate if the drugs (at 10 µmol L–1) induced apoptosis in the myofibroblasts. When applied to established myofibroblasts, the hydroxypyridone antifungals did not reverse myofibroblast transformation. Instead, the drugs exhibited a concentration-dependent decrease in myofibroblast cell viability. A 22% reduction in myofibroblast cell viability was observed at 10 µmol L–1, compared to fibroblasts treated in coincubation with TGF-β1 and the hydroxypyridones. To see if this reduction in cell viability was due to the induction of apoptosis in the myofibroblasts, TUNEL staining was used. TUNEL staining confirmed that the hydroxypyridone antifungals induced apoptosis in myofibroblasts after 72 h of treatment. This affect was not seen in the fibroblasts treated in coincubation with TGF-β1 and the hydroxypyridone antifungals. This is the first study to show that hydroxypyridone antifungals can induce apoptosis in established myofibroblasts, derived from burn scar tissue. Together with our previous results, this suggests that hydroxypyridone antifungals can prevent scar formation by tackling continuous myofibroblast transformation and myofibroblast persistence.