HA-RAL Synergy in Skin Figure 2. The effect of RAL and HAFi in vivo. A. The effect of RAL and HAFi in vivo on mouse skin. Histological sections of vehicle- (a), RAL- (b), HAFi- (c) or RAL- and HAFi- (d) treated DBA/1 and vehicle (e) or RAL- (f) treated CD44-/- mouse dorsal skin. Note the significant epidermal hyperplasia in DBA/1 but not in CD44-/- mice. B. Epidermal thickness in SKH1, DBA/1 and CD44-/- mouse dorsal skin measured with an ocular micrometer. Ten measurements were performed per mouse and the average value was calculated. The results are presented as the mean epidermal thickness 6 SEM of six animals per group. ***p,0.001 versus vehicle (student’s t test). C. Ki67 staining of vehicle- (a), RAL- (b), HAFi- (c) or RAL- and HAFi- (d) treated DBA/1 and vehicle (e) or RAL- (f) treated CD44-/- mouse dorsal skin. doi:10.1371/journal.pone.0014372.g002 RAL and HAFi have a synergistic effect on the protein and mRNA expression of CD44 in mouse skin Immunostaining of vehicle-treated back skin of DBA/1 mice revealed the standard expression of CD44 in basal and suprabasal keratinocytes (Figure 3A, a). However, topical application of RAL and HAFi for 3 days significantly increased CD44 expression in follicular (data not shown) and interfollicular keratinocytes (Figure 3A, d), as shown by using an antibody recognizing all CD44 isoforms. The staining intensity for CD44 was stronger in HAFi alone- and RAL- and HAFi-treated epidermis than the one treated with RAL alone (Figure 3A, b, c, d). An antibody which specifically recognizes CD44v3 isoform revealed increased expres- sion of CD44v3 in suprabasal keratinocytes with the exception of the basal and granular layer after topical application of RAL and HAFi (Figure 3B, a, b, c, d). The staining for CD44v3 was stronger in the upper suprabasal layers in the RAL alone- and RAL- and HAFi-treated epidermis when compared to the one treated with HAFi alone (Figure 3B, b, c, d). Although RAL seemed to increase the expression of CD44 more at the RNA level (Figure 3D), the protein expression of CD44 was more augmented by HAFi (Figure 3C). CD44 expression in RAL- and HAFi-treated SKH1 hairless or DBA/1 mouse back skin was more significantly increased than in RAL alone- or HAFi alone- treated skin of these mice both at the RNA and protein levels (Figure 3C, D). RAL and HAFi have a synergistic effect on the epidermal and dermal HA production in mouse skin Quantification of epidermal and dermal HA was performed by an enzyme-linked binding protein assay on the RAL-, HAFi, and RAL and HAFi-treated epidermis and dermis of SKH1 hairless (not shown) and DBA/1 mice. RAL alone seemed to increase more epidermal HA than HAFi alone (Figure 4A). In contrast, HAFi alone resulted in more HA in dermis than RAL alone (Figure 4B). RAL and HAFi combination more significantly increased epider- mal and dermal HA than with RAL or HAFi alone (Figure 4A, B). RAL and HAFi have a synergistic effect on the protein expression of pro-HB-EGF in mouse skin To address the effect of the combination of topically-applied RAL and HAFi on the expression of CD44v3, pro- and active HB-EGF and its receptor erbB1 in vivo, we performed a western blot analysis on the protein extracts of RAL- and HAFi-treated SKH1 hairless mice. RAL alone-treated epidermis showed a slight increase of CD44v3 (data not shown), active HB-EGF and erbB1 protein expression when compared to vehicle-treated epidermis (Figure 5A, a, c). Protein content of pro-HB-EGF was not increased by RAL (Figure 5A, b). HAFi alone-treatment resulted in an increase of CD44v3 (data not shown), pro- and active HB-EGF, and erbB1 protein expression when compared to vehicle-treated epidermis (Figure 5A, a, b, c). HAFi increased active HB-EGF more than RAL (Figure 5A, a). There was no difference between the RAL and HAFi in terms of the increase of erbB1 (Figure 5A, c). There was no difference in erbB3 expression between RAL- and vehicle-treated epidermis (data not shown). No expression of erbB2 or erbB4 was detected in mouse skin (data not shown). RAL did not influence the expression of MMP-7 (data not shown). RAL and HAFi-treatment showed a synergy only on the expression of pro-HB-EGF (Figure 5A, b). RAL and HAFi show synergy in the correction of human skin atrophy Topical application of RAL and HAFi to the atrophic forearm skin of elderly dermatoporosis patients for 1 month not only corrected the skin atrophy but also caused marked skin hyperplasia. This effect was more significant than RAL alone (data not shown) or HAFi alone (Figure 5B). Clinically, the wrinkles, hematoma and pseudoscars seen in atrophic skin decreased and smoothening of the skin was observed after RAL and HAFi treatment (Figure 5C, a, b). Histologically, the epidermal hyperplasia observed after RAL and HAFi treatment was associated with a decrease of elastosis an increase of collagen in the dermis (Figure 5C, c, d). Materials and Methods Topical retinoid preparation RAL was used at the concentration of 0.05% compounded in an oil-in-water cream [11]. Preparation of HAFi HA from rooster comb (IAL) for clinical application was provided by Transbussan (Geneva, Switzerland). HAFi: Fragmentation products of HA of medium size (50’000–400’000 Da) was prepared PLoS ONE | www.plosone.org 4 December 2010 | Volume 5 | Issue 12 | e14372