Steady-state mRNA levels of interleukin-1, integrins, cJun, and cFos in hairless mouse skin during short-term chronic UV exposure and the effect of topical tretinoin

We have proposed that UV activation of cytokine and integrin signaling pathways may initiate the photoaging process and that one of the effects of tretinoin treatment may be to alter the cytokine and integrin patterns. In previous results, steady-state mRNA levels of interleukin-1alpha, tumor necrosis factor alpha, transforming growth factor beta, collagenase, stromelysin, collagen, and integrins (alpha1 and alpha2) were increased in the skin of hairless mice that were either UV treated or concurrently treated with UV followed by topical tretinoin for 5 weeks. The aim of this study was to focus on the expression of alpha1, alpha2 and alpha5 integrins, IL-1alpha, IL-1beta, cJun, and cFos at an earlier time point (3 weeks). Animals were UV irradiated thrice weekly for 3 weeks and were treated topically with either 0.05% tretinoin or the vehicle immediately after each exposure. Total RNA was prepared and used in RT-PCR with radiolabeled dCTP and specific primers. UV slightly increased steady-state mRNA levels for alpha1, alpha2 and alpha5 integrins whereas UV + tretinoin increased their expression (3-, 2- and 7-fold respectively). Steady-state mRNA levels for IL-1alpha, IL-1beta and cJun were increased with UV (3-, 12- and 6-fold respectively) and with UV + tretinoin (6-, 7- and 9-fold respectively). In contrast, cFos expression was unchanged. In situ staining for IL-1alpha mRNA was slightly more abundant in mice treated for 3 weeks with UV and UV + tretinoin than in controls whereas 5 weeks of UV + tretinoin treatment gave strongly positive staining. Results are consistent with cytokines and integrins mediating the effects of UV on the skin, with modulation of these effects by tretinoin.     

Gene expression of retinoic acid receptors and cellular retinoic acid-binding proteins in rhino and hairless mouse skin

The rhino mouse comedolytic model and the hairless mouse photoaging model are established animal models for screening the in vivo activity of retinoids. However, the expression of the retinoic acid receptors (RARs) and cellular retinoic acid-binding proteins (CRABPs), known to regulate retinoid activity, is not completely understood in these mouse mutants. For this purpose, mRNA was isolated from rhino and hairless mouse skin and the gene expression of the RARs and CRABPs was measured by Northern blot hybridization. Results showed that RAR was the predominantly expressed RAR in both mouse strains. Two isoforms of RAR, RAR1 and RAR2, were detected with RAR1 being the more strongly expressed. RAR was also detected, but to a lesser degree than RAR. RAR expression was not detectable by our methodology. Additionally, topical treatment of these mice with 0.1% all-trans-retinoic acid (tRA) cream resulted in no significant alteration in the expression of the RAR genes. By contrast, CRABP-II was induced 2–4 fold by topical tRA treatment. CRABP-I, expressed to a lesser degree than CRABP-II, was not inducible. The relative expression of the RARs, CRABPs, and inducibility of CRABP-II by tRA in both rhino and hairless mouse skin paralleled that reported for human and mouse skin. These observations suggest that the altered phenotype observed in the rhino mouse most likely does not result from an altered expression level of these genes. The results also support these two animals as models for evaluating the therapeutic potential of retinoids.     

Keratinocyte degeneration in human facial skin: Documentation of new ultrastructural markers for photodamage and their improvement during topical tretinoin therapy

Abstract We examined epidermal impairment in photodamaged Caucasian skin by light and electron microscopy and observed two types of degeneration of the basal and suprabasal keratinocytes. The first was an electron-lucent degeneration predominantly seen in the periphery of the cells. The marked lucent degeneration occurred in 14% of the basal and suprabasal keratinocytes, predominantly in cells immediately adjacent to melanocytes. In skin specimens with a large number of such damaged keratinocytes, bleb-like keratinocytic protrusions or electron-lucent intercellular structures were also seen. Many vacuolar structures were observed just under the dermoepidermal junction, occupying 9% of the junction length. These structures were produced by hernialion of the degenerative portion of the basal and suprabasal keratinocytes, and appeared to be phagocytized by dermal macrophages. The vacuolar alterations in the basal layer and dermoepidermal junction previously reported at the light microscopic level probably represent these intercellular lucent structures, bleb-like protrusions and vacuole-like structures at the electron microscopic level. The second type, dark-staining keratinocytes, probably representing an extensive degenerative process, constituted 4% of the basal and suprabasal keratinocytes. After 12 months of topical trelinoin treatment, dramatic improvement of both degenerative processes of the keratinocytes was noted.     

MEL4B3, a novel mRNA is induced in skin tumors and regulated by TGF-beta and pro-inflammatory cytokines

Tumor-stroma interactions play a decisive role in the growth and metastasis of solid tumors, and involve signalling either by soluble mediators or direct cell-cell interaction. Here, we report the isolation and characterisation of a novel cDNA (MEL4B3), which is induced in cultured dermal fibroblasts exposed to supernatants of melanoma cell lines. MEL4B3 shares high homology with two predicted cDNA sequences for which no activity has so far been described. In situ hybridisation revealed the expression of MEL4B3 in malignant melanoma increasing with tumor depth; in basal cell carcinoma and in squamous cell carcinoma. MEL4B3 was barely detectable in normal skin or non-malignant melanocytic naevi. Furthermore, MEL4B3 was expressed at high level in the epidermis of psoriatic skin. In vitro, the expression of MEL4B3 was found to be induced by the exposure of human dermal fibroblasts to melanoma cell culture supernatants or to transforming growth factor-beta, interleukin-1 and tumor necrosis factor-alpha. The expression MEL4B3 therefore reflects closely cell activation occurring during tumor growth, metastasis and inflammation.     

Aminolaevulinic acid diffusion characteristics in 'in vitro' normal human skin and actinic keratosis: implications for topical photodynamic therapy

Background: The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT.
Methods: ALA penetration through excised normal human skin (NS) and AK lesions was evaluated using a cryostatic sectioning technique and radio-isotope counting following drug delivery using a novel, bioadhesive patch, loaded with 19, 38 or 50 mg/cm² ALA.
Results: Distinct differences in ALA concentration with respect to depth between AK and NS samples were shown, particularly within the superficial layers of the tissue structure, down to a depth of 1.0 mm. Patch application times were shown to influence ALA concentrations in tissue, but there was no clear correlation between ALA penetration in AK lesions taken from different body locations and from patients of different age. Similarly, the thickness of stratum corneum was not related to the ALA distribution profiles.
Conclusions: Sizable variation in ALA concentration was a prominent feature of profiles through AK lesions, which may explain the variation of observed protoporphyrin IX production seen in the clinical implementation of AK PDT. That said, the results of this study show sufficient ALA penetration to a depth of 1.0 mm, which should be satisfactory for successful treatment of the majority of non-hyperkeratotic, hypertrophic AK using patch-based delivery methods.     

Metabolism of topical retinaldehyde and retinol by mouse skin in vivo: predominant formation of retinyl esters and identification of 14-hydroxy-4, 14-retro-retinol

Abstract We have previously shown that retinaldehyde (RAL), a natural metabolite of (β-carotene and retinol (ROL), can be used topically in human skin and exerts biological activity: it may be a convenient way to deliver multipotential vitamin A activity in epidermis. RAL can be converted enzymatically into 2 pathways: one leads to ROL (and then retinyl esters), the other to retin-oic acid (RA). The aim of the present study was 2-fold: (i) to see if RAL is metabolised in vivo when topically applied on mouse skin, and (ii) if so. to an-alyse the occurrence and relative importance of the 2 metabolic pathways as compared to ROL. We studied by HPLC the metabolites detectable in mouse tail skin upon topical application of RAL and ROL. As compared to vehicle-treated controls, RAL-treated mouse skin contained low amounts of all-trans RA and 13-cis-RA, whereas ROL content increased 10-fold and retinyl esters 30-fold after RAL application. As compared to RAL. ROL-treated mouse skin showed no detectable RA, slightly less retinyl esters but a significant amount of 14-hydroxy-4, 14-retro-ROL (14-HRR). a metabolite not previously reported in the skin. 14-HRR was the predominant polar metabolite of ROL. These data indicate that keratinocytes metabolise topical RAL. thus confirming the concept of using RAL as a precursor. Both pathways are used but in significantly different proportions. Thus, only a low proportion of RAL is metabolised into all-trans-RA, which may explain the low irritancy profile of topical RAL and supports the concept of a controlled delivery of ligands. That keratinocytes predominantly channel RAL into storage forms indicates that RAL, should also be considered as a convenient way to load the epidermis with vitamin A. The detection of 14-HRR. a metabolite not previously reported in skin, that promotes growth of B lymphocytes and activation of T lymphocytes, suggests distinct potentials of topical ROL and RAL.     

Applicability of an exaggerated forearm wash test for efficacy testing of two corticosteroids,tacrolimus and glycerol,in topical formulations against skin irritation induced by two different irritants

Background/Purpose: Alternatives to corticosteroids in the treatment of irritant contact dermatitis (ICD) are needed and may include glycerol and topical immunomodulators like tacrolimus. Because the efficacy of different treatments in experimentally induced ICD may vary depending on the irritant applied, we tested the efficacy of four anti‐irritant compounds using the two different irritants sodium lauryl sulfate (SLS) and nonanoic acid (NON). Methods: In a randomized, double‐blind, controlled trial, healthy volunteers were exposed to 5% SLS and 50% NON (the right and the left forearm, respectively) in a cumulative wash test. Induction of ICD was obtained by three daily washings for 7 days, followed by a maintenance phase with two daily washings for 12 days. Treatment (triamcinolone acetonide, clobetasol propionate, tacrolimus and glycerol ointment) was started at day 7 and applied immediately after washing. Vehicle and no treatment served as the control. Reactions were evaluated clinically and instrumentally. Results: No treatments were significantly better than the other treatments and controls. There was a tendency toward a dose‐dependent response to corticoid treatment, and a trend toward worsened irritancy by tacrolimus on SLS‐irritated skin. Explained variance in the experiment by anova revealed a very small effect of treatments compared with an immense and significant subject effect. Conclusion: No claims of effective anti‐irritant properties for any of the ointments can be maintained. Application of the present wash test as a tool for anti‐irritant efficacy testing may be complicated by the small observed variance explained by treatment.     

Novel nanocapsule of α‐lipoic acid reveals pigmentation improvement: α‐Lipoic acid stimulates the proliferation and differentiation of keratinocyte in murine skin by topical application

α‐Lipoic acid is amphipathic with low molecular sulphur‐containing fatty acid and has strong antioxidant effects. It has been used at the purposes of anti‐ageing, treatment of diabetic neuropathy, and supplement as antioxidant. Though α‐lipoic acid is normally administered in oral or injection, it has not been used in a topical use via skin because of its bad penetration. We developed the novel nanocapsule of α‐lipoic acid, named α‐lipoactive (nLA), to improve skin permeability. The nLA is constructed as micelles of α‐lipoic acid mixed with the non‐ionic surfactant, and its surface of the micelles was coated with inorganic metal salts. It is water soluble and has a diameter of approximately 8‐15 nm. After nLA was applied to the murine skin, epidermal thickening was observed. It was confirmed that this effect is caused by α‐lipoic acid molecule, but not by the raw material used for encapsulation. In in vivo experiments, it was found that nLA is very effective for improving UV‐induced pigmentation and epidermal thickening. Our findings suggest that nanoencapsulation of α‐lipoic acid is considerably effective for topical application.