Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 37 records · Page 2Linked to original sources

PubMed1999

Bilayered biodegradable poly(ethylene glycol)/poly(butylene terephthalate) copolymer (Polyactive) as substrate for human fibroblasts and keratinocytes.

The purpose of this study was to find an optimal polymer matrix and to optimize the culture conditions for human keratinocytes and fibroblasts for the development of a human skin substitute. For this purpose porous, dense bilayers made of a block copolymer of poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT; Polyactivetrade mark) with a PEGT/PBT weight ratio of 55/45 and a PEG molecular weight (MW) of 300, 600, 1000, or 4000 Da were used. The best performance was achieved with PEGT/PBT copolymer with MW of PEG 300 D (300PEG55PBT45). When fibroblasts were seeded into the porous underlayer and cultured for 3 weeks in medium supplemented with 100 microg/mL ascorbic acid, all pores were filled with fibroblasts and with extracellular matrix, which was judged from the presence of collagen types I, III, and IV, and laminin. When seeded onto the dense top layer of the bilayered (cell free or fibroblast populated) copolymer matrix, human keratinocytes grew out into confluent sheets. After subsequent lifting to the air-liquid interface, a multilayered epithelium with a morphology corresponding to that of the native epidermis was formed. Some differences could still be observed: the expression and localization of some differentiation specific proteins was different and close to that seen in hyperproliferative epidermis; a basal lamina and anchoring zone were absent.

Biocompatible Materials↗

PubMed1999

TR146 cells grown on filters as a model of human buccal epithelium: permeability of fluorescein isothiocyanate-labelled dextrans in the presence of sodium glycocholate.

The aim of the present study was to characterize the TR146 cell culture model as an in vitro model of human buccal epithelium with respect to the permeability of test substances with different molecular weights (M(w)). For this purpose, the apparent permeability (P(app)) values for mannitol and for fluorescein isothiocyanate (FITC)-labelled dextrans (FD) with various M(w) (4000-40000) were compared to the P(app) values obtained using porcine buccal mucosa as an in vitro model of the human buccal epithelium. The effect of 10 mM sodium glycocholate (GC) on the P(app) values was examined. To identify the pathways by which FD of M(w) 4000-40000 were transported, the confocal laser scanning microscope (CLSM) was used. The P(app) values obtained with the TR146 cell culture model in the absence of a permeability enhancer linearly decreased with increasing M(w) of the test substance from 0. 65+/-0.055x10(-8) to 44+/-7.5x10(-8) cm/s, as the P(app) values obtained with porcine buccal mucosa. In the presence of the permeability enhancer, GC, the permeability of the FD across the cultured TR146 cell culture model increased in a parabolic manner, reaching maximum at M(w) 10000. In the absence of the enhancer, only paracellular localization of FD was observed while, in the presence of GC, FD also could be detected in the cytosol of some of the superficial cells. The GC-induced enhancement of FD permeation may be partially attributed to changed permeation pathways. The present results indicate that the TR146 cell culture model is a suitable in vitro model for mechanistic permeability studies of human buccal drug permeability.

Animals↗

PubMed1999

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.

Animals↗

PubMed1999

In vivo human skin permeability enhancement by oleic acid: a laser Doppler velocimetry study.

Topical application of a skin permeation enhancer such as oleic acid (OA) can result in: (i) higher skin permeability for many exogenous substances (ii) an irritation reaction. Laser Doppler velocimetry (LDV) is one of few techniques which can assess both effects using appropriate protocols. Direct LDV measurement, measuring cutaneous blood flow, has been preferred as a tool to evaluate skin irritation. By comparing the LDV value of an irritant-treated site with an untreated site, an irritation index for the irritant can be obtained. Occlusive application of 0.16 M OA in propylene glycol (PG) for either 3 or 24 h produced irritation in form of redness and slight swelling. Using LDV, we obtained an irritation index of 2 and 4, respectively. The vehicle, PG alone, produced an index of around 1, which corresponded well to the slight to almost no irritation observed visually. The duration of the high irritation index assessed by LDV after OA-PG application is comparable to the duration of the increase in transepidermal water loss following the same application. This indicates a correlation between skin irritation and barrier perturbation caused by OA. LDV can also be used to elucidate the effect of enhancers on skin using hexyl nicotinate (HN) as a model drug, since its vasodilative effect can be clearly assessed by LDV. Pre-treatment of PG for 3 h significantly reduced the t0 and tmax of HN. No further reduction could be observed when OA was added into PG, suggesting that OA-PG is not more effective than PG alone in enhancing the permeation of HN.

Administration, Topical↗

PubMed1999

Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT).

Reconstruction of a skin equivalent using an immortalized human keratinocyte line, HaCaT, was investigated in an attempt to generate an in vitro system representative for human skin. Three different substrates were used to establish air-exposed cultures of HaCaT cells: de-epidermized dermis, collagen gels, and filter inserts. Effects of variations in culture conditions on tissue morphology, on the expression of proliferation-specific and differentiation-specific protein markers, and on lipid profiles were investigated. When grown at the air-liquid interface HaCaT cells initially developed a multilayered epithelium, but during the course of culture marked alterations in tissue architecture were observed. Ultrastructurally, a disordered tissue organization was evident as judged from the presence of rounded cells with abnormally shaped nuclei. Keratins K1 and K10 were irregularly expressed in all cell layers, including stratum basale. Staining of K6/K16 was evident in all cell layers. Locally, basal and suprabasal cells were positive for K4 and additionally expressed K13 and K19. The cornified envelope precursors were expressed only in older cultures (>2 wk after air exposure), except for transglutaminase and small proline rich protein 1, which were irregularly expressed in both early and older cultures. In addition, HaCaT cells showed an impaired capacity to synthesize lipids that are necessary for a proper barrier formation as indicated by the absence of free fatty acids and a very low content and incomplete profile of ceramides. Our data demonstrate that the ultimate steps of terminal differentiation in HaCaT cells do not occur irrespective of the type of substrate or the culture conditions.

Cell Differentiation↗

PubMed1999

Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range.

The main diffusion barrier for drugs penetrating through the skin is located in the intercellular lipid matrix in the upper layer of the skin, the stratum corneum (SC). The main lipid classes in the SC are ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). The lipids in SC are organized into two lamellar phases with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. However, one may conclude that the phase behavior of the mixtures is similar to that in SC only when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. Therefore, in the present study, the organization of the lipid mixtures has been studied in a temperature range between 20 degrees and 95 degrees C. From these experiments it appeared that at elevated temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed between 35;-55 degrees C, which is absent or only weakly formed in intact human and pig SC, respectively. As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca(2+) is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca(2+) on the lipid phase behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin surface) to 7.4 (pH at the SC;-stratum granulosum interface) and the presence of cholesterol sulfate promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm phase to higher temperatures, and makes this phase less prominent at higher temperatures. The finding that Ca(2+) counteracts the effects of cholesterol sulfate indicates the importance of a proper balance of minor SC components for appropriate SC lipid organization. In addition, when the findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid lamellar phases, which might facilitate the desquamation process.

Animals↗

PubMed1998

Hyperlipidemia and cutaneous abnormalities in transgenic mice overexpressing human apolipoprotein C1.

Transgenic mice were generated with different levels of human apolipoprotein C1 (APOC1) expression in liver and skin. At 2 mo of age, serum levels of cholesterol, triglycerides (TG), and FFA were strongly elevated in APOC1 transgenic mice compared with wild-type mice. These elevated levels of serum cholesterol and TG were due mainly to an accumulation of VLDL particles in the circulation. In addition to hyperlipidemia, APOC1 transgenic mice developed dry and scaly skin with loss of hair, dependent on the amount of APOC1 expression in the skin. Since these skin abnormalities appeared in two independent founder lines, a mutation related to the specific insertion site of the human APOC1 gene as the cause for the phenotype can be excluded. Histopathological analysis of high expressor APOC1 transgenic mice revealed a disorder of the skin consisting of epidermal hyperplasia and hyperkeratosis, and atrophic sebaceous glands lacking sebum. In line with these results, epidermal lipid analysis showed that the relative amounts of the sebum components TG and wax diesters in the epidermis of high expressor APOC1 transgenic mice were reduced by 60 and 45%, respectively. In addition to atrophic sebaceous glands, the meibomian glands were also found to be severely atrophic in APOC1 transgenic mice. High expressor APOC1 transgenic mice also exhibited diminished abdominal adipose tissue stores (a 60% decrease compared with wild-type mice) and a complete deficiency of subcutaneous fat. These results indicate that, in addition to the previously reported inhibitory role of apoC1 on hepatic remnant uptake, overexpression of apoC1 affects lipid synthesis in the sebaceous gland and/or epidermis as well as adipose tissue formation. These APOC1 transgenic mice may serve as an interesting in vivo model for the investigation of lipid homeostasis in the skin.

Animals↗

PubMed1998

Temperature-sensitive regulation of epidermal morphogenesis and the expression of cornified envelope precursors by EGF and TGF alpha.

Epidermis reconstructed on de-epidermized dermis was used to investigate the effects of growth factors and culture temperature on epidermal morphogenesis and the expression of cornified envelope precursors. Cultures grown at 33 degreesC or 37 degreesC in the absence or presence of transforming growth factor alpha (TGFalpha), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), or insulin-like growth factor (IGF) show a similar morphology to that of native epidermis. Loricrin and SPRR2 are expressed in the stratum granulosum and SPRR3 is absent. Cultures grown in epidermal growth factor (EGF)-supplemented medium at 37 degrees C have a normal morphology, whereas cultures grown at 33 degrees C have a disorganized basal layer, no stratum granulosum, and nuclei are present in the stratum corneum. Loricrin is absent, and SPRR2 and SPRR3 expression extend into the spinous layers. Irrespective of the culture condition used, involucrin is aberrantly expressed in all suprabasal layers. EGF stimulated keratinocyte proliferation and migration to a greater degree than TGFalpha. Epidermis reconstructed on fibroblast-populated collagen gels at 33 degrees C led to the same disturbances in keratinocyte differentiation as seen in cultures grown on de-epidermized dermis at 33 degrees C in the presence of EGF, whereas parallel cultures grown at 37 degrees C have a similar morphology to that of native epidermis.

Cell Division↗

PubMed1998

pH, cholesterol sulfate, and fatty acids affect the stratum corneum lipid organization.

Lipid mixtures prepared from cholesterol (CHOL), isolated ceramides (CER), and free fatty acids can serve as attractive tools to study the role various stratum corneum (SC) lipids or microenvironmental conditions play in the SC lipid organization, as the phase behavior in these mixtures and in SC are similar: two lamellar phases with periodicities of approximately 6 and 13 nm are present. Because pH and cholesterol sulfate (CSO4) gradients exist in SC and may affect the local SC lipid organization, the effects of pH and CSO4 on lipid phase behavior was examined. X-ray diffraction studies with CHOL:CER mixtures revealed that the lamellar ordering at pH 5 and 7.4 were similar: both the short and the long periodicity phases were present. Upon addition of free fatty acids the phase behavior became pH dependent; the long periodicity phase being more dominant at pH 7.4 than at pH 5. Similar observations have been made upon addition of CSO4. Furthermore, only in the presence of CSO4 did phase-separated CHOL disappear, indicating that CHOL completely dissolves in the lamellar phases. A major phase change from an hexagonal to an orthorhombic lateral packing has been observed in the presence of free fatty acids. Furthermore, in the presence of CSO4 next to orthorhombic also liquid lateral packing could be detected. In contrast to lamellar ordering, changes in pH did not affect the lateral packing in any of the lipid mixtures studied.

Animals↗

PubMed1998

Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice.

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4-7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin.

Animals↗

PubMed1998

Mechanistic and quantitative prediction of aminopeptidase activity in stripped human skin based on the HaCaT cell sheet model.

HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed. HaCaT sheets were cultured on porous membranes. Diffusion and concurrent metabolism were studied under reflection and permeation conditions. Numerical simulations of simultaneous diffusion and saturable Michaelis-Menten metabolism were based on a physical model and a fixed set of independently obtained parameters (diffusion coefficient D, distance x, partition coefficient P, Michaelis constant Km, maximum metabolic rate Vmax). Under reflection conditions, cleavage of Ala-MNA in HaCaT sheets was very close to stripped skin. In contrast, in permeation studies substrate only permeated through HaCaT whereas passage through stripped skin led to full cleavage of Ala-MNA to MNA. All experimental data were in reasonable to excellent agreement with numerically generated data. Differences between HaCaT and stripped skin could be quantitatively and mechanistically explained by the thickness of the metabolically active layer, i.e., approximately 10 microm in HaCaT and approximately 40 microm in stripped skin. Full cleavage of permeating Ala-MNA in stripped skin was predicted to occur within the upper approximately 20 microm of viable epidermis. Thus epidermal aminopeptidase activity may act as an efficient metabolic barrier to fully block the permeation of aminopeptidase labile xenobiotics. Within the settings of this study the kinetics of metabolism in the viable epidermis of skin is predictable from HaCaT sheets.

2-Naphthylamine↗

PubMed1998

Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

Adult↗

PubMed1998

Expression of skin-derived antileukoproteinase (SKALP) in reconstructed human epidermis and its value as a marker for skin irritation.

For the investigation of the skin irritancy potential of chemicals in an in vitro model, it is necessary to have sensitive end-points that predict the effects on native human skin. Our aim was to investigate whether the induction of the proteinase inhibitor SKALP in reconstructed epidermis can be used as a marker. The influence of culture conditions and the effect of topical application of sodium lauryl sulfate and oleic acid on SKALP expression were evaluated using immunohistochemistry and Northern blotting. SKALP expression was induced by serum, epidermal growth factor and fibroblasts. In the presence of retinoic acid and 1,25-dihydroxyvitamin D3 SKALP expression was inhibited, whereas supplementation with ascorbic acid and a-tocopherol had no effect. Tape-stripping of excised skin and topical treatment with sodium lauryl sulfate induced SKALP protein expression. Application of sodium lauryl sulfate and oleic acid on reconstructed epidermis also induced SKALP at the protein level but no significant effects could be demonstrated at mRNA levels. In conclusion, SKALP expression, which was increased upon application of sodium lauryl sulfate and oleic acid, can be used as an in vitro end-point for skin irritancy, irrespective of the modifying effects of culture conditions.

Biomarkers↗

PubMed1998

Role of ceramide 1 in the molecular organization of the stratum corneum lipids.

The main barrier of the skin is formed by the lipids in the apical skin layer, the stratum corneum (SC). In SC mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL) are present. The CER are composed of at least six different fractions. CER 1 has an exceptional molecular structure as it contains a linoleic acid linked to a long-chain omega-hydroxy acid (C > 30). The SC lipids are organized in two lamellar phases with periodicities of approximately 6 and 13 nm, respectively. Recent studies revealed that ceramides isolated from pig SC mixed with cholesterol in confined ratios mimic stratum corneum lipid phase behavior closely (Bouwstra, J.A., et al. 1996. J. Lipid Res. 37: 999-1011). In this paper the role of CER 1 for the SC lipid lamellar organization was studied. For this purpose lipid phase behavior of mixtures of CHOL and total ceramide fraction was compared with that of mixtures of CHOL and a ceramide mixture lacking CER 1. These studies showed that in the absence of CER 1 almost no long periodicity phase was formed over a wide CHOL/CER molar ratio. A model is proposed for the molecular arrangement of the two lamellar phases. This model is based on the dominant role CER 1 plays in the formation of the long periodicity phase, electron density distribution calculations, and observations, such as i) the bimodal distribution of the fatty acid chain lengths of the ceramides, ii) the phase separation between long-chain ceramides and short-chain ceramides in a monolayer approach, and iii) the absence of swelling of the lamellae upon increasing the water content organization in SC. In this molecular model the short periodicity phase is composed of only two high electron density regions indicating the presence of only one bilayer, similar to that often found in phospholipid membranes. The molecular arrangement in the long periodicity phase is very exceptional. This phase most probably consists of two broad and one narrow low electron density regions. The two broad regions are formed by partly interdigitating ceramides with long-chain fatty acids of approximately 24-26 C atoms, while the narrow low-electron density region is formed by fully interdigitating ceramides with a short free fatty acid chain of approximately 16 to 18 C atoms.

Animals↗

PubMed1997

Expression of the SPRR cornification genes is differentially affected by carcinogenic transformation.

The small proline rich protein (SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. Here we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels of SPRR expression than normal human keratinocytes. However, the residual SPRR expression in SCC lines appears to be both gene and cell line specific. Expression of SPRR2 appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics of SPRR2 expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression of SPRR2 contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical for SPRR2 and not observed for the other SPRR genes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.

3T3 Cells↗

PubMed1997

Epidermal growth factor and temperature regulate keratinocyte differentiation.

The limited life-span and irregularities in epidermal differentiation and barrier function that have restricted the utility of presently available skin culture models for pharmacological and toxicological studies indicate that further modifications of culture conditions are required for optimization of these models. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of temperature and epidermal growth factor (EGF) on epidermal differentiation and lipogenesis. When cultured at 37 degrees C, keratinocytes formed a well-differentiated epidermis whether EGF was present or not. However, the thickness of the epidermis, particularly of the stratum corneum, was higher in the presence of EGF. Both the differentiation-specific protein markers (keratins 1 and 10, involucrin and transglutaminase) and lipid markers (ceramides) were synthesized. EGF-induced increases in triglyceride content caused accumulation of lipid droplets within the stratum corneum which is indicative of a hyperproliferative effect of EGF. In the absence of EGF, a well-differentiated epidermis was generated at 33 degrees C with a morphology showing a higher resemblance to native epidermis than cultures grown at 37 degrees C. The stratum corneum was less compact and with practically no lipid droplets, irregularly shaped keratohyalin granules were abundant in the stratum granulosum, lamellar body extrusion was improved and the number of stratum corneum layers was reduced to normal levels. However, EGF supplementation had a deleterious effect on epidermal morphogenesis and differentiation of cultures grown at 33 degrees C. The epidermis lacked a stratum granulosum and the stratum corneum contained a high number of nuclear remnants. The synthesis of the early specific protein differentiation markers (keratins 1 and 10) was suppressed on both the protein and mRNA levels without significant interference with the synthesis of late differentiation lipid markers, such as ceramides. From this observation it can be concluded that the synthesis of keratins associated with terminal differentiation is profoundly affected by the presence of EGF and is sensitive to temperature and that of ceramides is not. The finding that TGF alpha did not modulate the morphogenesis and synthesis of keratins 1 and 10 in cultures grown at 33 degrees C indicates possible differences between the postreceptor binding processes of these EGF receptor ligands.

Biomarkers↗

PubMed1997

Culture of reconstructed epidermis in a defined medium at 33 degrees C shows a delayed epidermal maturation, prolonged lifespan and improved stratum corneum.

In this study we compared human keratinocyte cultures grown at the air-liquid interface on de-epidermized dermis at 33 degrees C or at 37 degrees C in two different culture media: medium I--a fully defined serum- and EGF-free medium; and medium II-a serum- and EGF-containing medium. Cultures grown in medium II were initially hyperproliferative followed rapidly by senescence, and had a high triglyceride content. The hyperproliferation was ascribed to the presence of EGF in the medium. In contrast, cultures grown in medium I at 33 degrees C showed a greatly improved balance between cell proliferation and differentiation. They had a prolonged lifespan of at least 32 days without a significant decrease in the number of living cell layers, a rate of proliferation similar to that of native epidermis and a low triglyceride content. Culturing at 37 degrees C increased the rate of differentiation without affecting the rate of proliferation. Furthermore, both at 33 degrees C and at 37 degrees C, keratin 6 was expressed only in the first suprabasal layer but was expressed in all suprabasal layers in cultures grown in medium II. High keratin 6 expression was not directly linked to hyperproliferation but to deregulated terminal differentiation. Involucrin, transglutaminase and SPRR1 were abnormally expressed irrespective of the culture conditions used, whereas SKALP expression was decreased in cultures grown in medium I. The epidermal lipid profile was better in cultures grown in medium I; the relative amounts of ceramides, free fatty acids and cholesterol being comparable to native epidermis. Small-angle X-ray diffraction showed a slightly improved structural organization of stratum corneum lipids as demonstrated by the appearance of second- and third-order peaks of the 12-nm long phase and a marked reduction in the polycrystalline cholesterol peak.

Cell Culture Techniques↗

PubMed1997

Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy.

The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue. To separate the kinetics of diffusion and degradation of Leu2-R110, a lateral application mode was introduced: the probe was applied at the cutting plane of a mechanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various strata and domains of the skin was achieved. The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distributed throughout the viable part of the epidermis, with enhanced fluorescence ("hot spots") in the upper layers of the stratum granulosum, while dermis and stratum corneum showed considerably less aminopeptidase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity, mostly at the root sheath. Because of the advantage of direct visualization and localization of enzymatic activity in intact tissue, the lateral application mode of substrate administration in combination with CLSM may be beneficial to further elucidate the location and intensity of metabolic activity in other living tissues as well.

Aminopeptidases↗