Vital Epidermis Research Articles

Dermal peptide delivery using enhancer molecules and colloidal carrier systems. Part III: Tetrapeptide GEKG

Dermal application of peptides as drugs is an interesting and growing field in therapeutics. Besides therapy, they are increasingly used as cosmetic agents. Peptide delivery into the skin is highly challenging since they provide disadvantageous properties like a high molecular weight, hydrophilicity, polarity and susceptibility to enzymatic degradation. The aim of the presented study was to improve the bioavailability of a dermal administered tetrapeptide GEKG (amino acid sequence in one-letter notation). A nano-sized carrier system (microemulsion, ME) of w/o type was therefore developed since ME provide excellent penetration enhancing properties. Furthermore, enhancers were used to increase the penetration capacity of GEKG. The penetration of GEKG from the ME and enhancer-containing emulsions was compared to the penetration from a standard formulation. For this purpose ex vivo penetration studies with human skin were performed, which provide insights into dermal penetration and also localization and distribution of the active substances in each skin layer as well as the influence of vehicle variations.Experiments with three incubation times (30, 100, 300 min) and 5000 ppm GEKG in the formulations clearly show that the nano-sized carrier system is significantly better suited to transport GEKG rapidly into upper and deeper vital skin layers as well as through the skin compared to the standard cream. Studies with o/w emulsions containing 5% glyceryl caprylate/caprate 2.0× or 1.5× as enhancer and 50 ppm peptide revealed only a tendency to increased penetration into the SC and for the formulation with glyceryl caprylate/caprate 1.5× into the vital epidermis compared to the standard cream without additional enhancer. Significant better penetration rates were observed for the ME with 50 ppm GEKG.It can be concluded that liquid nano-sized systems are significantly more effective as carriers for extremely hydrophilic peptides used in cosmetics and also in therapeutics than classic cosmetic emulsions or enhancer-containing emulsions. Chemical compoundsTetrapeptide-21 (CAS: 960608-17-7)glyceryl caprylate/caprate (CAS: 73398-61-5)sorbitan oleate (CAS: 1338-43-8)polyglyceryl-4 laurate (CAS: 75798-42-4)butylene glycol (CAS: 107-88-0)isopropyl palmitate (CAS: 142-91-6)

Hyaluronan metabolism enhanced during epidermal differentiation is suppressed by vitamin C.

Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.

Abstract B76: Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma

Abstract 3D in vitro skin models are valuable research tools to investigate normal keratinocyte as well as skin carcinoma cell interaction with dermal fibroblasts. Currently, the models lack either a structured vital human extracellular matrix or long-term stability and longevity. Therefore, we aimed at a new 3D in vitro skin model for normal human epidermis and tumorigenic stages of skin cancer. This model is based on dermal equivalents containing a highly structured cell-derived matrix (CDM) produced by human dermal fibroblasts. First, we show that the CDM dermal equivalent supports a high structural integrity of the skin model and maintains a vital epidermis for six months. The epidermis is well-stratifying and differentiates in a regular orthokeratotic pattern. Transmission electron microscopy reveals ultrastructural entities as basement membranes, hemidesmosomes, desmosomes, keratohyalin granulae and lamellar bodies. In line, the molecular differentiation markers are properly distributed. Second, five non-tumorigenic and tumorigenic HaCaT keratinocyte variants are tested for their proliferation, differentiation and invasion capacities. After four weeks of co-cultivation the tumorigenic HaCaT variants show hyperproliferative epithelia and lose the capability of an ordered stratification and differentiation. Furthermore, they destroy the basement membrane as well as the extracellular matrix and invade the dermal equivalent. Moreover, seven squamous cell carcinoma (SCC) cell lines were co-cultivated with the CDM dermal equivalent. All SCC cells form hyperproliferative epithelia that heavily invade the extracellular matrix. Remarkably, the epithelia are individually distinct for their invasion and differentiation capacity. Thus, the CDM-based skin cancer model is suitable for demonstrating the heterogeneity of SCC tumors and their interaction with the microenvironment. In conclusion, the CDM model for normal skin and tumorigenic keratinocytes provides a novel tool to investigate keratinocyte-fibroblast interactions and microenvironment modulations during SCC development. Therefore, this system will provide further insights into mechanistic processes and the interaction of SCCs with their tumor microenvironment. Citation Format: Manuel Berning, Petra Boukamp. Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B76. doi:10.1158/1538-7445.CHTME14-B76

Demonstration of free fatty acids in the integument of semi-aquatic and aquatic mammals

The sensitive red fluorescence dye BODIPY® 665/676, and embedding in the water-soluble resin Technovit® 7100 were used to demonstrate free fatty acids in the epidermis of seven semi-aquatic and aquatic mammalian species with a sparse or dense hair coat. The staining generally marked lipid layers of varying thickness between the lamellae of the Stratum corneum, as found particularly in very densely haired species (otter), but also in rather sparsely haired animals (beaver, nutria), and especially in the seal. The very sparsely haired capybara contained no free fatty acids in the corneal layer system, but exhibited an accumulation of such substances in the vital epidermis. All haired species showed a strongly positive reaction staining of the sebaceous glands. In the hairless species, a distinct intracellular staining was restricted to cells of the thick vital epidermis in the hippopotamus, whereby in the Str. corneum positive intercellular reactions appeared. In the dolphin, on the contrary, positive intercellular reactions became visible in the vital epidermis, whereas in the Str. corneum the lipids concentrated in large longitudinal intracellular vesicles.

Basic structural and functional characteristics of the epidermal barrier in wild mammals living in different habitats and climates

Based on the combination of standard light and transmission electron microscopy, cryo-SEM, immunohistochemistry and a new sensitive glycolipid histochemical technique (5-hexadecanoylaminofluorescein staining, laser scanning microscopy), including densitometrical evaluation, our approach gives for the first time an overview of the specific biology of the epidermal permeability barrier in wild mammals (20 species from five orders), living under varying (aquatic or moist to dry) habitat conditions. The results obtained emphasised that the barrier region in most of the species studied is a continuous zone (thickness, 0.1 and 3 μm) between the upper cells of the stratum granulosum and the inner cells of the stratum corneum conjunctum, normally present as a homogeneous glycolipid layer originating from fusion of lamellar body contents after exocytotic activities of the granular cells. However, this finding did not apply to all of the species studied, i.e., the Wild boar, the Common seal and the three large species with a very thick vital epidermis, the African elephant, the hippopotamus and the common dolphin, exhibited variations from the basic scheme. Densitometric evaluation of the 5-hexadecanoylaminofluorescein staining revealed that reaction intensity was not only generally related to the habitat conditions but also to vital epidermis thickness and hair density. The immunohistochemical demonstration of Na+/H+ exchanger 1 corroborated for all wild mammals studied that this important regulator of pH conditions during barrier formation is continuously produced in the epidermis. The variations in barrier biology observed for some species obviously had to be developed in relation to animal size (or body size area) and hair coat density, but, particularly, by the specific adaptation of certain mammalian groups to the aquatic environment. In the latter case, the typical barrier zone system was lost, as in the hippopotamus or the cetaceans.

Demonstration of substances of innate immunity in the integument of the Malayan pangolin (Manis javanica)

Using immunohistochemistry, the study demonstrates substances of the innate immunity in the skin of the Malayan pangolin (Manis javanica), referring mainly to the epidermis. The results obtained showed clear reaction differences between the dorsolateral body region with its strong cover of hard horny scales and the abdominal body part with a thick soft stratum corneum and a dense cover of fine hairs. Regarding pathogen recognition receptors, positive reactions for Toll-like receptors were generally weak for TLR2, in contrast to TLR4, that exhibited strong reactions in the epidermis of both body regions, with increasing staining intensities towards the stratum corneum; ß-glucan receptors showed positive reactions only for l-ficolin and mannose-binding lectin, but not for dectin-1, and only at the abdomen. A generally positive staining for both body regions was obtained for all cationic antimicrobial peptides tested, whereby cathelicidin exhibited strong reaction intensities in all epidermal layers, ß-defensin 2 staining was often limited to the stratum basale and the stratum spinosum, and positive reactions for ß-defensin 3 appeared distinctly only in the epidermis of the abdomen; for these peptides, positive reaction staining could also be found in the outer epithelial root sheath of hair follicles, their glandular annexes, and free cells of the dermis. Lysozyme was found in the vital epidermis of both body regions studied, with strong staining limited to the dorsum; strong reactions were also visible in the hair follicles.

Possible antimicrobial defense by free sugars on the epidermal surface of aquatic vertebrates

Biochemical, lectin histochemical, and densitometrical methods demonstrated contents and spectra of free sugars in the skin of aquatic vertebrates (fishes: cod, marine trout, freshwater trout, perch; amphibians: Koa-Tao-Island caecilian, smooth newt, edible frog, South-African clawed toad; mammals: northern fur seal, common seal, otter, Capybara). Total free sugar contents, particu- larly sialic acids, were relatively higher in freshwater species (perch, Capybara) than in marine species. In amphibians, the caecilian and the newt showed higher amounts of free sugars than the anurans; strong reactions indicated a specific involvement of α-L-fucose in mucus functions. Lectin histochemistry demonstrated positive reactions in the stratum superficiale, the outer stratum spinosum, and specific cells of the fish epidermis. The integumental free sugar spectrum of the amphibians was narrow, with generally lower reaction intensities, except for mucus glands. In the mammals, the epidermal cells exhibited rather weak lectin histochemical staining, except for the northern fur seal and the Capybara, where the upper vital epidermis and corneal cells reacted strongly. The mammalian apocrine skin glands, particularly, exhibited positive reaction staining in their secretory endpiece. The free sugar spectrum was similar in all species: α-D-mannose, β-D-N-acetylglucosamine, α-D-N-acetylgalactosamine, α-D-galactose, β-D-galactose, α-L-fucose, sialic acids (mainly in fish epidermis and skin glands). High concentrations of free sugars on the skin surface possibly impede attacks of commensal skin micro-inhabitants (bacteria, fungi) against the integrity of the epidermis. This features a basic or general biological mechanism that operates before the specific immune system is activated.

Pep-1 as a Novel Probe for the In Situ Detection of Hyaluronan

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.

EGF Upregulates, Whereas TGF-β Downregulates, the Hyaluronan Synthases Has2 and Has3 in Organotypic Keratinocyte Cultures: Correlations with Epidermal Proliferation and Differentiation

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.

Topical retinoic acid treatment of photoaged skin: its effects on hyaluronan distribution in epidermis and on hyaluronan and retinoic acid in suction blister fluid.

Topical treatment with retinoic acid (tretinoin, vitamin A acid) has been reported to partly reverse signs of photodamage. To determine whether the histochemical distribution of hyaluronan (hyaluronic acid, HYA) in the epidermis and dermis and the amounts of HYA and retinoic acid in suction blister fluid were influenced by such topical treatment, 14 subjects healthy apart from moderate photodamage were instructed to treat the lateral forearm with 0.01-0.05% retinoic acid cream for 6 months. In a study of the short-term effects, another six subjects applied 0.05% retinoic acid cream for 2 weeks. After 6 months the thickness of the vital epidermis had increased by 23%. The HYA staining was based on a specific immunohistochemical method in which hyaluronan-binding protein is used. Before treatment HYA was seen as a meshwork around the cells in the upper half of the stratum spinosum. After 6 months of treatment this meshwork had increased in thickness by 31% compared with pretreatment specimens. The HYA staining was already intense in the papillary dermis before treatment and no difference was observed after 6 months' treatment. The mean concentration of HYA in blister fluid had increased significantly (43%) after 2 weeks of treatment whereas after 6 months there was no significant difference in this respect between the treated and untreated arm. The increase in the thickness of the epidermal HYA meshwork after 6 months and the blister fluid HYA after 2 weeks may indicate that HYA is involved in the epidermal change induced by topical retinoic acid therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topical retinoic acid treatment of photoaged skin: its effects on hyaluronan distribution in epidermis and on hyaluronan and retinoic acid in suction blister fluid.

Topical treatment with retinoic acid (tretinoin, vitamin A acid) has been reported to partly reverse signs of photodamage. To determine whether the histochemical distribution of hyaluronan (hyaluronic acid, HYA) in the epidermis and dermis and the amounts of HYA and retinoic acid in suction blister fluid were influenced by such topical treatment, 14 subjects healthy apart from moderate photodamage were instructed to treat the lateral forearm with 0.01-0.05% retinoic acid cream for 6 months. In a study of the short-term effects, another six subjects applied 0.05% retinoic acid cream for 2 weeks. After 6 months the thickness of the vital epidermis had increased by 23%. The HYA staining was based on a specific immunohistochemical method in which hyaluronan-binding protein is used. Before treatment HYA was seen as a meshwork around the cells in the upper half of the stratum spinosum. After 6 months of treatment this meshwork had increased in thickness by 31% compared with pretreatment specimens. The HYA staining was already intense in the papillary dermis before treatment and no difference was observed after 6 months' treatment. The mean concentration of HYA in blister fluid had increased significantly (43%) after 2 weeks of treatment whereas after 6 months there was no significant difference in this respect between the treated and untreated arm. The increase in the thickness of the epidermal HYA meshwork after 6 months and the blister fluid HYA after 2 weeks may indicate that HYA is involved in the epidermal change induced by topical retinoic acid therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemistry of glycoconjugates in the skin of the bovine muzzle, with special reference to glandular structures.

The distribution of glycoconjugates in the muzzle of young adult Holstein cows has been studied by means of selected light-microscopic histochemical methods, including lectin histochemistry. In the skin layers, strong reactions were confined to intercellular substances in between the cells of the vital epidermis, exhibiting neutral glycoconjugates mainly with alpha-D-galactosyl and N-acetyl-D-galactosaminyl residues. In the nasolabial glands, distinctly positive staining for neutral glycoproteins with various saccharide residues (alpha-D-galactose, alpha-N-acetylgalactosamine, D-galactose-beta(1----3)D-N-acetylgalactosamine, beta-D-galactose), and for smaller amounts of acidic glycoconjugates, was found in the secretory cells and the luminal secretion. The cells of the excretory duct system showed weak to moderate reactions (alpha-D-galactose, beta-D-galactose), only the collecting ducts reacted positively for acidic glycoproteins with sialyl residues. The results obtained are discussed in view of muzzle function, with special reference to the salivary nature of the secretion of bovine nasolabial glands.