Skin-specific Hyaluronan Knockdown In Mice By An Optimized Topical 4-methylumbelliferone Formulation
Emily H. Steen1, Hui Li1, Xinyi Wang1, Natalie Templeman1, Alexander Blum1, Paul Bollyky2, Sundeep G. Keswani1, Swathi Balaji1.
1BCM, Houston, TX, USA, 2Stanford University SOM, Stanford, CA, USA.
Background: Hyaluronan (HA) is prominently abundant in the skin; while HA can be synthesized by the HAS1-3 enzyme family, HAS2 is the leading contributor. Dysregulation and accumulation of HA is implicated in the pathogenesis of several diseases such as keloid scarring and metastatic melanoma. To understand how HA expression contributes to the development of fibrotic disorders, we propose the development of a skin-specific HA knockdown model, which tests an optimal delivery system of topical 4-methylumbelliferone (4MU). Methods: Skin HA content was measured in male and female mice (n=30, M/F) at 1,4,7,14, and 24w. A design-of-experiments(DOE) approach was employed to develop an optimal 4MU skin-delivery formulation comprising a combination of propylene glycol(PG), ethanol(EtOH), and water(n=40; 7w M/F). This was topically applied twice daily for 7days to compare HA knockdown levels between topical 4MU, topical control, 4MU chow, and diet control (n=24; 6w M/F). Serum and skin samples were harvested to analyze HA content (HA-ELISA, immunohistochemistry) and HAS1-3 expression (qRT-PCR). Average+/-SD; p<0.05 were assessed by ANOVA. Results: HA content in dorsal (344.3+/-67.2ng/mg of skin) and ventral (324.7+/-46.8ng/mg) skin was comparable at all ages and between sexes. Consistent with our prediction of 70% knockdown, the optimal formulation of 0.82 mM 4MU in PG(16.32%;v/v)+EtOH(15.71%;v/v)+water(67.97%;v/v) resulted in 60% reduction of HA in dorsal skin(p<0.05), with 68% HA knockdown(p<0.01) in female mice compared to 22%(p<0.05) in males. 4MU topical application resulted in a significant decrease in dermal HAS2 expression (3-fold M+F;p<0.05). No significant effect of topical 4MU was observed in HA serum levels compared to controls. Histologic analyses showed thicker dermis in 7w male mice, whereas female mice had a predominant adipose layer; topical 4MU resulted in a significant breakdown in HA expression pattern. Conclusions: Our data suggest a 4MU formulation model that can be invaluable in elucidating the sex-specific and skin-specific effects of hyaluronan in normal and pathologic states of wound healing.
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