557 Targeted genetic alteration in hyaluronan catabolism delays wound healing in mice J Muto 1 , D Watanabe 1 and RL Gallo 2 1 Department of Dermatology, Aichi Medical University, Nagakute, Japan and 2 Department of Dermatology, University of California, San Diego, La Jolla, CA The breakdown and molecular size of Hyaluronan (HA) has been hypothesized to influence wound healing, but direct evidence of this has not been obtained. To test if the breakdown of HA acts as an endogenous signal of injury we generated transgenic mice that conditionally overexpressed Hyaluronidase 1 (HYAL1). When HYAL1 was constitutively expressed in early embryogenesis (EIIa/HYAL1), these mice showed extensive degradation of HA in the dermis yet were morphologically normal. When these mice were wounded by full-thickness 6 mm punch biopsy, wound closure was delayed by approximately 2 days in EIIa/HYAL1 compared to control mice (p<0.001). To examine the dynamic nature of epidermal response to expression of HYAL1, we next evaluated the wound healing in the tamoxifen-inducible K14-dependent Cre system and wound closure was delayed in K14CreERT/HYAL1 (p<0.001). Re-epithelialization is a key event in wound healing. To determine if delayed wound healing was due to the regulation of keratinocyte migration, we harvested the keratinocytes from the transgenicmice and performed the wound scratch assays. The keratinocytes from EIIa/HYAL1 mice showed significantly impared migration (p<0.001). Furthermore, delayed repair may result from abnormal macrophage polarization in wound tissues 48 hours after wounding as we found a significantly higher M1/M2 macrophage ratio in EIIa/HYAL1 mice (p<0.05). Histological and flow cytometirc evaluation of the skin wounds at 96 hours showed signifi- cantly more inflammatory cell infiltration in EIIa/HYAL1 mice (p<0.05). To exclude the effects that are unrelated to HA degradation, we injected the tetrasaccharide HA right after wounding and found that the injection delayed wound healing significantly. These data show that abnormal degradation of hyaluronan modulates wound healing and suggests targeting this pathway may be therapeutically useful in treatment of abnormal wound repair. 558 The effects of inaudible ultrasound on human skin regeneration H Choi, N Park, D Min, I Bae, Y Kim and J Lee Amorepacific R&D center, Yongin, Korea (the Republic of) There are some evidences that human skin cell can sense mechanical stresses or temperature changes. Furthermore, it was suggested olfactory and optical receptor present in human skin cells recently. Considering evolutionary procedure, it is conceivable that there may be un- usual sensing mechanism on human skin because sensory and neural systems originated from whole body. Likewise, it is not strange that skin has sound sensing mechanism. The hyper- sonic effect is known as a phenomenon that sounds containing ultrasonic frequencies above the human audible range affect cerebral blood flow and brain waves. It is interesting even when ultrasonic sounds were presented only to body surface, not to ears, subject’s brain took hypersonic effect. The result suggests that human skin may have mechanisms sensing ultra- sound directly. It was also reported that inaudible ultrasound at a frequency above 20 khz affected on the skin of rodents. Presenting of ultrasound on disrupted skin of rodents accel- erated significant barrier recovery which was due to increased exocytosis of lamellar body from granular layer of epidermis. In this study, we hypothesized that human skin barrier function may altered by inaudible ultrasound treatment. To elucidate that, human skin equivalent (SE) barrier was disrupted with punch biopsy followed by ultrasound exposure. As expected, ultrasound promoted epidermal regeneration of SE. We used genomic analysis to get a clue how ultrasound accelerated epidermal regeneration. Gene set enrichment analysis revealed that fatty acid biosynthesis pathway had noteworthy enrichment score. Furthermore, we confirmed that the mRNA level of elongation of very long-chain fatty acid-4 (ELOVL4), which is a gene that may contribute skin barrier function, was elevated in ultrasound treated normal human keratinocytes (NHK). These data suggest that inaudible ultrasound may have beneficial effects on human skin regeneration. 559 Evaluation of Snow algae extract, tocopheryl acetate, ascorbyl glucoside and niacinamide on skin aging P Bogdanowicz, C Gumez, L Duprat, S Julie ´, M Haure, H Duplan and S Bessou-Touya Department of Pharmacology, Pierre Fabre Dermo-Cosme´tique, Toulouse, France Similar to the entire organism, skin is subject to an unpreventable intrinsic ageing process due to telomere shortening (replicative senescence). The skin is also increasingly exposed to ambient ultraviolet irradiation leading to photoaging (extrinsic aging) characterized by wrinkles, loss of skin tone and solar lentigines. Solar lentigines are macular hyperpigmented skin lesions. The aim of this study was to evaluate the properties of a combination of molecules on skin aging: Snow algae extract, tocopheryl acetate (AcVitE), ascorbyl glucoside (AA2G) and niacinamide (Vitamin B3). These molecules were studied in vitro on two different models of fibroblasts senescence: Hayflick model mimicking replicative senescence, stress- induced premature senescence mimicking extrinsic aging. A formulation containing these 4 compounds was also evaluated on a photoaging ex vivo human skin model (chronic UVA radiation of skin explant). We have showed on senescent fibroblasts (Hayflick model) that Snow algae extract increased Klotho (gene involved in longevity) mRNA level up to + 365%. Furthermore, using Comet-assay, we have also showed that tocopheryl acetate protected DNA of human keratinocytes against solar exposition (UVA and UVB). We have also showed the ascorbyl glucoside enhanced type I collagen synthesis in human fibroblastes (+66%) and decreased melanin synthesis (-56%) in human melanocytes. On the other hand, confocal microscopy revealed that chronic UVA exposure (3 x 12 J/cm2 at 365 nm) strongly induced MMP1 staining in irradiated skin explants. Topical application of the anti-aging formulation containing Snow algae extract, AA2G, AcVitE and VitB3 at 5 mg/cm2 efficiently decreased MMP1 staining and thus protected skin from UVA damage. In conclusion, using different in vitro models of senescence (intrinsic and extrinsic senescence), we demonstrated the anti- aging properties of Snow algae extract, AA2G, AcVitE and VitB3. This results have been confirmed with an ex vivo human skin model of photoaging. 560 Exploring a role for regulatory miRNAs in wound healing during ageing E Aunin, D Broadley, D Ilas and NV Botchkareva University of Bradford, Bradford, United Kingdom Chronic wounds are a common problem among the elderly population. The aim of the current study was to identify miRNAs that are differentially expressed in aged mouse skin wounds and to characterise their potential functions in wound healing. Global miRNA profiling was examined during wound healing in mouse back skin of young (8 weeks old) and aged (2 year-old) animals. Several miRNAs exhibited a contrasting expression pattern in young versus aged wounds. For example, expression of miR-31, an established regulator of skin wound repair, was upregulated in young mice and downregulated in aged mice during wound closure. The expression of the miR-200 family members was markedly decreased during wound healing, with the exception of acute upregulation of miR-200c in the early phase of wound healing in aged skin. Also, miR-200c levels is elevated in unwounded aged skin in both human and mice. To study the potential involvement of miR-200c in skin wound healing, we examined the effect of miR-200c on keratinocyte migration and proliferation. miR-200c expression was induced in both primary human and mouse keratinocytes during wound healing assay in vitro. Inhibition of miR-200c resulted in significant acceleration of keratinocyte migration, associated with the increased expression of genes controlling cell motility. However, keratinocyte proliferation was not affected by altered levels of miR-200c. miR-200c mimic reduced wound closure in human skin ex vivo. Interestingly, miR-200c was reported to be involved in the regulation of BMP signalling (Cao et al., 2013). Indeed, BMP 4/7 treatment increased miR-200c expression in keratinocytes, whereas miR-200c overexpression resulted in downregulation of BMP antagonist Noggin. Furthermore, recom- binant Noggin abolished the inhibitory effect of miR-200c on keratinocyte migration. In conclusion, the differences in expression of miRNAs in young and aged mouse skin wounds suggest an involvement of various miRNAs in age-associated changes in wound healing. Specifically, miR-200c may contribute to the age-associated alterations in wound repair by controlling keratinocyte migration. 561 A potential role of cryptochromes and opsins as photoreceptors in light-based wound healing therapies NE Uzunbajakava 1 , I Castellano 1 , C Mignon 1 , M van Vlimmeren 1 , NV Botchkareva 2 , D Tobin 2 and J Thornton 2 1 Philips Research, Eindhoven, Netherlands and 2 Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom Photobiomodulation has high therapeutic potential for acute and chronic wounds. Cyto- chrome c oxidase, cryptochromes (CRYs) and opsins (OPNs) may interact with visible and near-infrared light to trigger distinct signalling pathways and physiological responses. The aim was to study the expression of photoreceptors in human skin cells and to investigate cell response to visible radiation in relation to wound healing. Human papillary and reticular dermal fibroblasts (DFs) and epidermal keratinocytes (EKs) were isolated from post-surgery skin. Expression of OPNs and CRYs was studied by qRT-PCR and immunocytochemistry. An LED prototype (450e850 nm; 2e100 J/cm 2 ) was used in in vitro cell and ex vivo wounds assays. Read-outs were: metabolic activity, cell count, proliferation, collagen production, ROS, Ca2+, migration and keratins expression. CRY1, OPN1sw and OPN3 were expressed in EKs and DFs; OPN2 was present in DFs and OPN5 - in EKs. Expression of OPN1sw and OPN5 was stratified in suprabasal and basal epidermis, respectively; a cell compartment- specific distribution was also observed. High-dose blue light suppressed EKs and papillary DFs metabolic activity and retarded scratch-wound closure, but stimulated reticular DFs. Low-dose blue light stimulated both DFs subtypes. Low- and high-dose cyan light exerted a less pronounced impact, yet even green light induced dose- and anti-oxidant -dependent ROS formation. DFs response to near-infrared light was also bi-phasic, yet at doses scaled up vs. blue, following absorption coefficient of CRYs and cyt c oxidase. In summary, we demon- strated expression of CRYs and OPNs in skin cells. Visible light exerted dose-, wavelength-, and cell-lineage specific effects, modulating metabolism, migration and collagen production, where ROS could have play a role. These findings open up a perspective for more efficient light-based therapies for wound healing. 562 MicroRNA-132, a promising target for wound therapy X Li 1 , D Li 1 , A Wang 2 , W Lohcharoenkal 1 , J Gru ¨ nler 3 , S Narayanan 3 , E Sonkoly 1 , A Pivarcsi 1 , C Sergiu-Bogdan 3 , M Sta ˚hle 1 and N Xu Lande ´n 1 1 Molecular Dermatology Research Group, Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Department of Dermatology, The Second Affiliated Hospital of Dalian Medical University, Dalian, China and 3 Department of Endocrinology, Metabolism and Diabetes, Karolinska University Hospital, Stockholm, Sweden Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs (miRNAs) during this process remains largely unexplored. We have previously shown that microRNA-132 (miR-132) inhibits keratinocyte inflammatory response but en- hances its proliferation. Inhibition of miR-132 delays wound repair. We have collected 31 chronic wound samples from Diabetes mellitus patients and miR-132 was found down-regu- lated compared with acute wound from healthy donors. In this study, we reveal the expression and function of miR-132 in human dermal fibroblasts (HDF). We found that transforming growth factor beta 1 (TGF-b1), which is a cytokine highly expressed in normal acute wounds but down- regulated in diabetic chronic wound, induced miR-132 expression in HDF. Moreover, we showed that the level of miR-132 in HDF was increased by hypoxia, but decreased by the treatment of high dose (30mM) of glucose. In line with this, we found that the expression of miR- 132 in the wounds of a mouse model of diabetes (db/db mice) is significantly lower compared to the wounds in wild-type mice. Furthermore, we showed that overexpression of miR-132 pro- moted HDF migration, whereas inhibition of miR-132 suppressed cell migration through MAPK signaling pathways. We demonstrated that RAS p21 protein activator 1 (RASA1) is a direct target down-regulated bymiR-132in HDF. Silencing of RASA1 promotes migration of HDF, mimicking the effects of miR-132 overexpression. To explore the therapeutic potential of miR-132 for hu- man skin wounds, we topically applied miR-132 mimics on human ex vivo skin wounds and we found that overexpression of miR-132 enhanced re-epithelialization. Collectively, we identify miR-132 as a key regulator promoting skin wound healing by enhancing fibroblasts migration and re-epithelialization. We propose miR-132 as a promising target for wound therapy. ABSTRACTS | Wound Healing and Tissue Remodelling S256 Journal of Investigative Dermatology (2016), Volume 136