Christian Tran

Hyaluronate Fragments Reverse Skin Atrophy by a CD44-Dependent Mechanism

Background Skin atrophy is a common manifestation of aging and is frequently accompanied by ulceration and delayed wound healing. With an increasingly aging patient population, management of skin atrophy is becoming a major challenge in the clinic, particularly in light of the fact that there are no effective therapeutic options at present. Methods and Findings Atrophic skin displays a decreased hyaluronate (HA) content and expression of the major cell-surface hyaluronate receptor, CD44. In an effort to develop a therapeutic strategy for skin atrophy, we addressed the effect of topical administration of defined-size HA fragments (HAF) on skin trophicity. Treatment of primary keratinocyte cultures with intermediate-size HAF (HAFi; 50,000–400,000 Da) but not with small-size HAF (HAFs; <50,000 Da) or large-size HAF (HAFl; >400,000 Da) induced wild-type (wt) but not CD44-deficient (CD44−/−) keratinocyte proliferation. Topical application of HAFi caused marked epidermal hyperplasia in wt but not in CD44−/− mice, and significant skin thickening in patients with age- or corticosteroid-related skin atrophy. The effect of HAFi on keratinocyte proliferation was abrogated by antibodies against heparin-binding epidermal growth factor (HB-EGF) and its receptor, erbB1, which form a complex with a particular isoform of CD44 (CD44v3), and by tissue inhibitor of metalloproteinase-3 (TIMP-3). Conclusions Our observations provide a novel CD44-dependent mechanism for HA oligosaccharide-induced keratinocyte proliferation and suggest that topical HAFi application may provide an attractive therapeutic option in human skin atrophy.

Topical calcineurin inhibitors decrease the production of UVB-induced thymine dimers from hairless mouse epidermis.

Background: An increased incidence of ultraviolet-light-related skin tumours is a well-known problem in patients undergoing posttransplantation immunosuppression with systemic calcineurin inhibitors such as cyclosporine A or tacrolimus. UV-related carcinogenesis as a consequence of long-term treatment of sun-exposed sites with topical calcineurin inhibitors is therefore of theoretical concern. Results: In this study, we show that tacrolimus acts as a UVB filter when incorporated into liposome membranes. In hairless mice pretreated with 1% pimecrolimus cream, 0.1% tacrolimus ointment or vehicle, the amount of epidermal thymine dimers, measured 1 h after 1 J/cm2 of UVB irradiation, was decreased by 89, 84 and 47%, respectively, as compared to untreated mice. Forty-eight hours after UVB irradiation, 97, 89 and 93% of epidermal thymine dimer levels were removed in pimecrolimus-, tacrolimus- or vehicle-treated mice, respectively. In contrast, 69% of thymine dimers, originally present in much higher amounts than in treated mice, were removed from untreated controls. UVB-induced apoptosis was less pronounced in treated mice. Conclusion: Taken together, these results suggest that topical calcineurin inhibitors prevent DNA photodamage due to a filter effect of both vehicle and active components, whereas they do not affect the clearance of DNA photoproducts.

Retinol and retinyl ester epidermal pools are not identically sensitive to UVB irradiation and anti-oxidant protective effect

Background: UV irradiation can deplete epidermal vitamin A, thus the hypothesis that UV-induced depletion of vitamin A in sun-exposed skin is involved in the pathogenesis of skin cancers and skin ageing. Objectives: In this study we addressed two questions: (1) Are retinol (ROL) and retinyl esters (RE) – the two predominant forms of vitamin A – equally sensitive to the action of UVB, and (2) could the depletion be prevented by anti-oxidants? Methods: Hairless mice were irradiated with a single UVB dose, corresponding to the maximum of ROL and RE absorption. Retinoid content, enzyme activities catalysing the esterification of ROL (ARAT and LRAT) and the hydrolysis of RE (REH), as well as retinol-binding protein (CRBP-1) expression were determined in the epidermis. Results: A single UVB dose induced a rapid, dose-dependent decrease in both ROL and RE in the epidermis of hairless mice, with partial replenishment after 24 h. The dose-response curve for ROL showed a high sensitivity to UV at doses not exceeding 200 mJ/cm2, followed by a plateau, whereas RE underwent a continuous dose-dependent decrease at UVB doses up to 1 J/cm2. A topical anti-oxidant mixture containing 0.5% ascorbate, 0.25% tocopherol and 0.25% melatonin failed to protect epidermal RE from UVB-induced depletion, whereas it did prevent ROL depletion. ARAT and REH, as well as CRBP-1, were not affected by UVB in these conditions. Conclusion: Vitamin A storage in the epidermis comprises two forms, ROL and RE, that do not show similar sensitivity to acute UVB exposure. ROL stores comprise a UVB-resistant (possibly by CRBP) portion and a UVB-sensitive portion that can be protected by anti-oxidants. RE stores do not show such a pattern.

Cutaneous vitamins A and E in the context of ultraviolet- or chemically-induced oxidative stress.

Vitamins A and E are present in mammalian skin. Although the main circulating form of vitamin A in the blood is retinol, the epidermis stores it as retinyl esters. The epidermis can be easily loaded with high amounts of vitamin A by topical application of either retinol or retinaldehyde, two well-tolerated precursors of the biologically active retinoic acid, while topical α-tocopherol loads the epidermis with vitamin E. The probable physiological function of epidermal vitamin E is to contribute to the antioxidant defense of the skin, whereas that of epidermal vitamin A (retinol and retinyl esters) is not yet well understood. Besides being a precursor for retinoic acid, vitamin A also has a free radical scavenging potential. Due to their physical properties, vitamins A and E absorb ultraviolet (UV) light in the region of solar spectrum that is responsible for most of the deleterious biological effects of the sun. In the mouse, topical vitamin A has been shown to prevent the UV-induced epidermal hypovitaminosis A, while topical vitamin E prevents oxidative stress and cutaneous and systemic immunosuppression elicited by UV. Thus constitutive epidermal vitamins A and E appear complementary in preventing UV-induced deleterious cutaneous and systemic effects, and these properties can be reinforced by topical application of retinol or retinaldehyde and topical α-tocopherol.

In vivo Bio-Integration of Three Hyaluronic Acid Fillers in Human Skin: A Histological Study

Background: Hyaluronic acid (HA) formulations are used for aesthetic applications. Different cross-linking technologies result in HA dermal fillers with specific characteristic visco-elastic properties. Objective: Bio-integration of three CE-marked HA dermal fillers, a cohesive (monophasic) polydensified, a cohesive (monophasic) monodensified and a non-cohesive (biphasic) filler, was analysed with a follow-up of 114 days after injection. Our aim was to study the tolerability and inflammatory response of these fillers, their patterns of distribution in the dermis, and influence on tissue integrity. Methods: Three HA formulations were injected intradermally into the iliac crest region in 15 subjects. Tissue samples were analysed after 8 and 114 days by histology and immunohistochemistry, and visualized using optical and transmission electron microscopy. Results: Histological results demonstrated that the tested HA fillers showed specific characteristic bio-integration patterns in the reticular dermis. Observations under the optical and electron microscopes revealed morphological conservation of cutaneous structures. Immunohistochemical results confirmed absence of inflammation, immune response and granuloma. Conclusion: The three tested dermal fillers show an excellent tolerability and preservation of the dermal cells and matrix components. Their tissue integration was dependent on their visco-elastic properties. The cohesive polydensified filler showed the most homogeneous integration with an optimal spreading within the reticular dermis, which is achieved by filling even the smallest spaces between collagen bundles and elastin fibrils, while preserving the structural integrity of the latter. Absence of adverse reactions confirms safety of the tested HA dermal fillers.

UVA and UVB Decrease the Expression of CD44 and Hyaluronate in Mouse Epidermis, which is Counteracted by Topical Retinoids

Abstract The transmembrane glycoprotein CD44 is currently thought to be the main cell surface receptor for the glycosaminoglycan hyaluronate. We previously showed that (1) CD44 regulate keratinocyte proliferation; (2) topical retinoids dramatically increase the expression of CD44, hyaluronate and hyaluronate synthase (HAS)s in mouse epidermis; (3) topical retinaldehyde restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus lesions; and (4) retinaldehyde-induced proliferative response of keratinocytes is a CD44-dependent phenomenon and requires the presence of HB-EGF, erbB1 and matrix metalloproteinases. In this study, we analyzed the effect of UV irradiation on the levels of epidermal hyaluronate and CD44 in mice, as well as its potential prevention by topical retinoids. UVA (10 J/cm2) or UVB (1 J/cm2) irradiation significantly decreased the expression of CD44 and hyaluronate in the epidermis of hairless mice after 2 h. Expression of both epidermal CD44 and hyaluronate was reconstituted within 24 h. Topical application of retinaldehyde for 3 days prior to UVA or UVB irradiation prevented the decrease of CD44 and hyaluronate expression. Topical retinol and retinoic acid also increased the basal levels of epidermal CD44 and hyaluronate, although their preventive effect on UV-induced decrease of these molecules was less pronounced as compared to topical retinaldehyde. These data confirm the relationships between retinoid and CD44 pathways, although the primary target(s) of UV leading to CD44 and hyaluronate degradation remain to be elucidated.

Synergistic Effect of Hyaluronate Fragments in Retinaldehyde-Induced Skin Hyperplasia Which Is a Cd44-Dependent Phenomenon

Background CD44 is a polymorphic proteoglycan and functions as the principal cell-surface receptor for hyaluronate (HA). Heparin-binding epidermal growth factor (HB-EGF) activation of keratinocyte erbB receptors has been proposed to mediate retinoid-induced epidermal hyperplasia. We have recently shown that intermediate size HA fragments (HAFi) reverse skin atrophy by a CD44-dependent mechanism. Methodology and Principal Findings Treatment of primary mouse keratinocyte cultures with retinaldehyde (RAL) resulted in the most significant increase in keratinocyte proliferation when compared with other retinoids, retinoic acid, retinol or retinoyl palmitate. RAL and HAFi showed a more significant increase in keratinocyte proliferation than RAL or HAFi alone. No proliferation with RAL was observed in CD44−/− keratinocytes. HA synthesis inhibitor, 4-methylumbelliferone inhibited the proliferative effect of RAL. HB-EGF, erbB1, and tissue inhibitor of MMP-3 blocking antibodies abrogated the RAL- or RAL- and HAFi-induced keratinocyte proliferation. Topical application of RAL or RAL and HAFi for 3 days caused a significant epidermal hyperplasia in the back skin of wild-type mice but not in CD44−/− mice. Topical RAL and HAFi increased epidermal CD44 expression, and the epidermal and dermal HA. RAL induced the expression of active HB-EGF and erbB1. However, treatment with RAL and HAFi showed a more significant increase in pro-HB-EGF when compared to RAL or HAFi treatments alone. We then topically applied RAL and HAFi twice a day to the forearm skin of elderly dermatoporosis patients. After 1 month of treatment, we observed a significant clinical improvement. Conclusions and Significance Our results indicate that (i) RAL-induced in vitro and in vivo keratinocyte proliferation is a CD44-dependent phenomenon and requires the presence of HA, HB-EGF, erbB1 and MMPs, (ii) RAL and HAFi show a synergy in vitro and in vivo in mouse skin, and (iii) the combination of RAL and HAFi seems to have an important therapeutic effect in dermatoporosis.

Penetration and Metabolism of Topical Retinoids in ex vivo Organ-Cultured Full-Thickness Human Skin Explants

The human epidermis contains endogenous retinoids [retinol (vitamin A) and retinyl esters] and carotenoids (mostly β-carotene). Previous studies in the mouse have shown that the enzymes involved in retinoid metabolism are present in the epidermis. In this study, we wanted to assess the skin penetration and metabolism of topical retinoids in the human. To do this, fresh surgically excised human abdominal skin was mounted on Franz perfusion cells. Topical retinoic acid, retinal, retinol and retinyl palmitate were applied at 2.5 mg/cm2 in oil-in-water creams containing 0.05% retinoids on the donor compartment, while the receptor compartment was filled with culture medium. The skin was incubated for 24 h at 37°C, then epidermal retinoid concentrations were determined by HPLC. The same experiment was performed with mouse back skin mounted on Franz cells. Finally, topical retinoids were applied on the back of hairless mice for 24 h; then the mice were sacrificed and retinoid concentrations were assayed in the epidermis. In all three models, retinol and its esters were found to be endogenous, as was the case in previous studies in the mouse in vivo. The four applied retinoids penetrated well into the epidermis. Topical retinoic acid did not increase endogenous retinoids, whereas the latter were greatly increased following topical retinal in the mouse. Retinal was also metabolized into retinoic acid, unlike topical retinol and retinyl palmitate, which only increased endogenous retinoids. Topical retinal and retinol did undergo a higher metabolism in both mouse models than in human skin. In summary, the penetration and metabolism patterns of topical retinoids were quite similar in the two mouse models used, indicating that the Franz cells appear to be a good model to predict in vivo metabolism of topical retinoids. When applying this concept to our results obtained in Franz cells with human skin, we conclude that topical retinol and retinal load human skin with both storage and functional vitamin A.

Production and clearance of cyclobutane dipyrimidine dimers in UV-irradiated skin pretreated with 1% pimecrolimus or 0.1% triamcinolone acetonide creams in normal and atopic patients.

Background:  Ultraviolet (UV)‐induced pyrimidine dimers are an early step in skin carcinogenesis, which is accelerated in the setting of long‐term immunosuppression with systemic calcineurin inhibitors. It is not known whether topical application of calcineurin inhibitors exposes to a similar risk.

Topical β-carotene is converted to retinyl esters in human skin ex vivo and mouse skin in vivo

Abstract:  Human epidermis contains endogenous retinoids (retinol and retinyl esters) and carotenoids (mostly β‐carotene). Previous studies have shown that the enzymes involved in retinoid metabolism are present in human epidermis. There is still a controversy about the presence in the skin of the enzymes able to convert β‐carotene into vitamin A (retinol), although a recent study demonstrated the conversion of β‐carotene into retinol in human cultured epidermal cells. In this study, we addressed the question of the possible bioconversion of topical β‐carotene into vitamin A or derivatives by human and mouse skin. Surgically excised human abdominal skin was mounted on Franz perfusion chambers to assess the cutaneous penetration of topical β‐carotene as well as its metabolism, after a 24‐h incubation period, whereas hairless mice received topical β‐carotene 24 h before assaying epidermal β‐carotene and retinoid concentrations. Epidermal retinoid and β‐carotene concentrations were determined by high‐pressure liquid chromatography. Topical β‐carotene penetrated well into human and mouse epidermis and induced a 10‐fold (human) and a threefold (mouse) increase of epidermal retinyl esters, which demonstrates that topical β‐carotene is converted into retinyl esters by human and mouse epidermis and thus appears as a precursor of epidermal vitamin A.