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M Ponec

Publications and source records attributed to M Ponec.

At least 55 records · Page 3Linked to original sources
PubMed1997

The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.

Our analysis of epidermal lipids revealed that (glucosyl)ceramide profiles in various human skin equivalents are different from those of native tissue. The main difference is the reduced content in skin equivalents of ceramides 4-7 and especially the very low content of the most polar ceramides 6 and 7, which contain hydroxylated sphingoid base and/or fatty acid. To facilitate hydroxylation, the culture medium was supplemented with vitamins C and E. Although in vitamin E-supplemented medium lipogenesis was not affected, in vitamin C-supplemented medium the content of glucosylceramides and of ceramides 6 and 7 was markedly increased, both in the presence and absence of serum and irrespective the substrate used (inert or natural, populated or not with fibroblasts). The improvement of the lipid profile was accompanied by a marked improvement of the barrier formation as judged from extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/stratum corneum interface, and the formation of multiple broad lipid lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle x-ray diffraction. Some differences between native and reconstructed epidermis, however, were noticed. Although the long-range lipid lamellar phase was present in both the native and the reconstructed epidermis, the short lamellar phase was present only in native tissue. It remains to be established whether these differences can be ascribed to small differences in relative amounts of individual ceramides, to differences in fatty acid profiles, or to differences in cholesterol sulfate, pH, or calcium gradients. The results indicate the key role vitamin C plays in the formation of stratum corneum barrier lipids.

Ascorbic Acid↗
PubMed1996

The role of ceramides 1 and 2 in the stratum corneum lipid organisation.

A mixture of ceramide 1 and ceramide 2 (CER(1 + 2)) was isolated from pig stratum corneum and mixed in various molar ratios with cholesterol (CHOL) or with CHOL and palmitic acid (PA). The mixtures were hydrated in a buffer solution of pH 5.0 and their phase behaviour was studied by wide- and small-angle X-ray diffraction. The small-angle diffraction curve of the CHOL/CER(1 + 2) mixture at a molar ratio of 0.4 revealed the presence of only one peak at a spacing of 6.7 nm. Increasing the amount of CHOL to a molar ratio of 0.6 was accompanied by a shift of this peak to a smaller spacing (5.7 nm) and the appearance of two weak peaks at 11.8 and 4.1 nm spacings. Increasing the CHOL content to an equimolar ratio resulted in the appearance of two lamellar phases with periodicities of 5.5 and 12 nm, respectively. In a CHOL/CER(1 + 2) mixture at a molar ratio of 2 the periodicities of the two phases were 5.6 and 12 nm, respectively. From these observations it was concluded that the CHOL/CER(1 + 2) mixtures exerted similar phase behaviour, as reported earlier for intact SC (Bouwstra et al. (1995) J. Lipid Res. 36, 496-504) and for mixtures (Bouwstra et al. (1996) J. Lipid Res., in press) prepared from CHOL and total ceramide fraction (CER) isolated from pig stratum corneum. However, in the CHOL/CER mixtures a lower relative amount of CHOL was required to acquire these lamellar phases, indicating that at low CHOL contents, CER 3, 4, 5 and 6 play a crucial role in the formation of the lamellar phases. Furthermore, the solubility of CHOL in the mixtures increased in the presence of CER 1, suggesting its important role for the barrier function of the skin. When palmitic acid (PA) was included, the phase behaviour of the CHOL/CER(1 + 2)/PA mixture was more complex. Next to two lamellar phases, an additional phase with a spacing of 3.77 nm was observed, never seen in intact stratum corneum. In the absence of CHOL, the wide-angle diffraction pattern of the CER(1 + 2) revealed one sharp reflection at 0.456 nm and two diffuse reflections at 0.430, 0.417 nm and 0.395 nm, indicating the presence of a crystalline sublattice. In an equimolar mixture of CHOL/CER(1 + 2) no sharp 0.456 nm reflection was observed indicating a more disordered packing. Furthermore, phase separation of CHOL occurred, this conclusion is based on the presence of reflections corresponding to polycrystalline cholesterol monohydrate. These findings indicate that the lateral packing of mixtures of CHOL/CER(1 + 2) is more complex than that of the CHOL/CER mixtures that reveals a hexagonal lateral packing.

Animals↗
PubMed1996

Regulation of keratinocyte proliferation and differentiation by all-trans-retinoic acid, 9-cis-retinoic acid and 1,25-dihydroxy vitamin D3.

We studied the effect of all-trans retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun reached a maximum after 2 weeks in culture (1 week postconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simultaneous treatment with both retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation and stimulated cell differentiation resulting in acceleration of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly by inhibition of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were maintained. These results show that modulation of the composition of the medium by the addition of various vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation of the culture lifespan.

Alitretinoin↗
PubMed1996

Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1.

In the epidermis, the keratinocytes are the first cells to be encountered by external stimuli and they are able to promote the inflammatory response by increased production and release of various cytokines. In their turn, these cytokines may directly affect the production of proinflammatory cytokines in human dermal fibroblasts. In addition, in both epithelial and mesenchymal cells cytokine production may be modulated by their mutual interaction, and thereby regulate the inflammatory response. The present study aimed to examine the role of fibroblasts in the regulation of proinflammatory IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. The data show that in fibroblasts exposed to conditioned media derived from cultures of normal human keratinocytes or squamous carcinoma cells (SCC-4), both the IL-8 and IL-6 mRNA expression as well as protein production were elevated. In addition, it was shown that these effects were induced by IL-1 alpha. The IL-1 alpha-induced increase in IL-8 and IL-6 production, both on the protein level as well as on the mRNA level, were concentration dependent and occurred almost simultaneously. While the induction of IL-6 and IL-8 occurred simultaneously, the IL-6 mRNA remained elevated for longer. In contrast to increased IL-6 and IL-8 production the IL-1 alpha levels markedly decreased upon culturing of fibroblasts in keratinocyte-derived conditioned medium. From internalization experiments it could be concluded that binding of IL-1 to IL-1 receptors, and its subsequent internalization and intracellular degradation is the most likely mechanism involved in the reduction of IL-1 levels by fibroblasts. Comparing the rate of IL-1 reduction in the presence of various cell types indicated that the rate of IL-1 reduction is directly related to the number of IL-1 receptors found on these cell types. In conclusion, these results indicate that the release of IL-1 alpha by activated keratinocytes may act as an inducer of IL-8 and IL-6 production in neighbouring fibroblasts. This may be an important pathway for the amplification of the inflammatory response. The amounts of both cytokines produced by fibroblasts were at least two to three orders of magnitude higher than those produced by keratinocytes, suggesting an important role of fibroblasts in the general inflammatory response. Furthermore, fibroblasts might be involved in turning off this inflammatory response by reducing IL-1 levels, most likely via IL-1 receptor-mediated uptake.

3T3 Cells↗
PubMed1996

Impaired desquamation in the in vitro reconstructed human epidermis.

A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at the air-liquid interface shares many similarities with native tissue. However, in contrast to native epidermis, its stratum corneum consists of a larger number of compactly packed corneocyte layers, indicating that the processes involved in corneocyte desquamation are disturbed. Although the tri-lamellar appearance of desmosomes in viable cell layers of both types of epidermis is similar, abnormalities in the transformation of desmosomes into the corneosomal plug in the stratum corneum of reconstructed epidermis have been observed. In the native epidermis, desmosomes are transformed into corneosomes at the stratum granulosum/stratum corneum interface. This process is retarded in vitro, since desmosomal structures with preserved lamellar appearance are present in the lower parts of the stratum corneum. Moreover, the corneosome frequency in reconstructed epidermis is significantly higher than in native human skin. A comparison of reconstructed epidermis with hyperproliferative epidermis, such as UV-irradiated epidermis, psoriatic epidermis and recently healed burn-wounds treated with cultured epidermal autografts, has revealed that only the structure of the stratum corneum derived from psoriatic skin is similar to that of reconstructed epidermis. The stratum corneum organization in the UV-treated epidermis and in recently healed burn-wound is, however, close to that seen in native skin.

Adult↗
PubMed1996

Phase behavior of stratum corneum lipids in mixed Langmuir-Blodgett monolayers.

The lipids found in the bilayers of the stratum corneum fulfill the vital barrier role of mammalian bodies. The main classes of lipids found in stratum corneum are ceramides, cholesterol, and free fatty acids. For an investigation of their phase behavior, mixed Langmuir-Blodgett monolayers of these lipids were prepared. Atomic force microscopy was used to investigate the structure of the monolayers as a function of the monolayer composition. Three different types of ceramide were used: ceramide extracted from pigskin, a commercially available ceramide with several fatty acid chain lengths, and two synthetic ceramides that have only one fatty acid chain length. In pigskin ceramide-cholesterol mixed monolayers phase separation was observed. This phase separation was also found for the commercially available type III Sigma ceramide-cholesterol mixed monolayers with molar ratios ranging from 1:0.1 to 1:1. These monolayers separated into two phases, one composed of the long fatty acid chain fraction of Sigma ceramide III and the other of the short fatty acid chain fraction of Sigma ceramide III mixed with cholesterol. Mixtures with a higher cholesterol content consisted of only one phase. These observations were confirmed by the results obtained with synthetic ceramides, which have only one fatty acid chain length. The synthetic ceramide with a palmitic acid (16:0) chain mixed with cholesterol, and the synthetic ceramide with a lignoceric acid (24:0) chain did not. Free fatty acids showed a preference to mix with one of these phases, depending on their fatty acid chain lengths. The results of this investigation suggest that the model system used in this study is in good agreement with those of other studies concerning the phase behavior of the stratum corneum lipids. By varying the composition of the monolayers one can study the role of each lipid class in detail.

Animals↗
PubMed1996

Phase behavior of isolated skin lipids.

Ceramides were isolated from the pig stratum corneum (SC) and mixed in varying molar ratios with either cholesterol or with cholesterol and free fatty acids. The phase behavior of the mixtures was studied by small-(SAXD) and wide-angle (WAXD) X-ray diffraction. Ceramides alone did not exhibit a long range ordering. Upon addition of cholesterol to ceramides, lamellar phases were formed and a hexagonal lateral packing was detected similar to that seen in intact SC. At a cholesterol/ceramide molar ratio of 0.1, only one reflection at 5.9 nm was observed. At a cholesterol/ceramide molar ratio of 0.2, three reflections corresponding to 12.3, 5.56, and 4.26 nm appeared. The reflections were based on two phases. Increasing the cholesterol/ceramide ratio to 0.4, the peak positions were slightly shifted. The diffraction pattern revealed the presence of two lamellar phases with periodicities of 12.2 and 5.2 nm, respectively. The positions of the peaks remained unchanged when the cholesterol/ceramide ratio was increased up to 1.0. At a cholesterol/ceramide molar ratio of 2.0, the intensity of various peaks based on the 12.2 nm phase decreased in intensity. The phase behavior of the cholesterol/ceramide mixtures in a ratio between 0.4 and 1.0 was very similar to that found in intact pig SC in which two lamellar phases with periodicities of 6.0 and 13.2 nm are present. Our data further indicate that the formation of the 5.2 nm lamellar phase requires a higher cholesterol content than the formation of the 12.2 nm lamellar phase. Furthermore, when the relative amount of cholesterol is very high, the 5.2 nm phase is the most pronounced one. Addition of free fatty acids increased the solubility of cholesterol, indicating the role free fatty acids may play for the skin barrier function. The phase behavior of cholesterol/ceramide/fatty acid mixtures was found to be dependent on the chain length of fatty acids used. Namely, addition of short-chain free fatty acids (C14-C18) did not change the periodicity of the 12.2 and 5.2 nm phases, but induced the formation of an additional 4.2 nm phase. In the presence of long-chain free fatty acids (C16-C26), the periodicity of the lamellar phases was slightly increased (to 13.0 and 5.3 nm, respectively) but no additional 4.2 nm phase was formed. These results indicate that the lipid phase behavior of the cholesterol/ceramide/free fatty acid mixtures closely mimics that of the intact stratum corneum only in the presence of long-chain free fatty acids.

Animals↗
PubMed1995

Mutations of keratinocyte transglutaminase in lamellar ichthyosis.

Lamellar ichthyosis is a severe congenital skin disorder characterized by generalized large scales and variable redness. Affected individuals in three families exhibited drastically reduced keratinocyte transglutaminase (TGK) activity. In two of these families, expression of TGK transcripts was diminished or abnormal and no TGK protein was detected. Homozygous or compound heterozygous mutations of the TGK gene were identified in all families. These data suggest that defects in TGK cause lamellar ichthyosis and that intact cross-linkage of cornified cell envelopes is required for epidermal tissue homeostasis.

Base Sequence↗
PubMed1995

Visualization of diffusion pathways across the stratum corneum of native and in-vitro-reconstructed epidermis by confocal laser scanning microscopy.

Confocal laser scanning microscopy is a technique that permits the direct visualization in unfixed material of diffusion pathways and the cellular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, allows the study of changes in distribution pattern of applied compounds depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used for such studies. Using this technique the permeability properties of in-vitro-reconstructed epidermis were compared with those of the native counterpart. The epidermis was reconstructed by culturing human adult keratinocytes at the air-liquid interface either on fibroblast-populated collagen or on de-epidermized dermis. A fluorescent probe--Nile red (NR)--was applied in three different vehicles--polyethylene glycol (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and dimethyl sulphoxide (DMSO)--which perturb the SC barrier function to different extents. When NR was applied in PEG and PG on native epidermis, the amount of NR penetrating into and through the SC was very low, but was markedly increased when NR was applied in DMSO. Unlike native epidermis, the reconstructed epidermis allowed rapid NR penetration after the application in any of the solvents used. Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously between the cellular and the intercellular spaces throughout the SC, suggesting that not only intercellular lipid structures but also the properties of the cornified envelopes differed markedly from those found in native epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1995

Expression of integrin receptors and ICAM-1 on keratinocytes in vivo and in an in vitro reconstructed epidermis: effect of sodium dodecyl sulphate.

Several integrin heterodimers such as alpha 2 beta 1, alpha 6 beta 4 and alpha v beta 5 are expressed on basal keratinocytes of the epidermis. Because overexpression of these integrins as well as induction of the intercellular adhesion molecule-1 (ICAM-1) have been found in inflammatory dermatoses, we sought to determine whether these modulations could be used as markers of skin irritation. In normal epidermis, topical application of 1% sodium dodecyl sulphate (SDS) for 24 h caused an upregulation of alpha 3, beta 1, alpha 6, beta 4, alpha v, beta 5 and to a lesser extent alpha 2 integrin chains as well as an induction of ICAM-1. To investigate whether these parameters could also be used for evaluation of skin irritancy in vitro, SDS was applied for 24 h to reconstructed epidermis on de-epidermized dermis (RE-DED). In RE-DED, integrin overexpression and aberrant alpha 5 expression was seen under normal in vitro culture conditions and topical application of SDS caused only marginal additional upregulation. We could not detect any ICAM-1 reactivity on either normal or irritated RE-DED. Our results demonstrate that the modulation of integrin and ICAM-1 expression can be used as markers of irritation of the epidermis in vivo, but not in vitro.

Biomarkers↗
PubMed1995

Triglyceride metabolism in human keratinocytes cultured at the air-liquid interface.

Although epidermis reconstructed in vitro histologically demonstrates the presence of fully differentiated tissue with cornified strata, it does not synthesize or release epidermal barrier lipids in the same proportions as does native skin, causing the barrier function to be impaired. Lipids, the content of which deviates the most, include triglycerides that are present in high amounts and stored as lipid droplets. Our recent studies have revealed that a high triglyceride content may be a reflection of a high synthetic rate and a low turnover. Therefore, the present study was undertaken to examine whether the triglyceride accumulation in the air-exposed cultures may be a result of insufficient supplementation of cells with oxygen, an excessive supplementation of cells with glucose, dysregulation of lipogenesis, or an impaired catabolism of triglycerides caused either by insufficient activity of triglyceride lipase and/or accumulation of free fatty acids due to insufficient activity of beta-oxidase. When keratinocytes were cultured at the air-liquid interface in medium containing a standard glucose concentration, both the lactate and triglyceride production was high. Lowering glucose content in the medium resulted in a decrease in both lactate production and triglyceride synthesis. However, even when grown at a low glucose concentration the triglyceride content remained higher than found in vivo and synthesized triglycerides were stored in the cells as a stable pool, suggesting that the catabolism of triglycerides was impaired. Since both lipase and beta-oxidase were found to be active in cultured keratinocytes, another factor or other factors are probably implicated in the regulation of triglyceride metabolism.

Carnitine↗
PubMed1995

Reduced skin barrier function parallels abnormal stratum corneum lipid organization in patients with lamellar ichthyosis.

Most patients with autosomal recessive lamellar ichthyosis are known to have markedly impaired skin barrier function. We hypothesize that this may be due to imperfections in the composition and fine structure of the intercellular stratum corneum lipids. The aim of the present study was to test this hypothesis. To characterize the barrier properties in three female patients with lamellar ichthyosis, the following parameters were used and compared with those of healthy volunteers: transepidermal water loss, stratum corneum lipid profiles after topical acetone/ether extraction on the flexure side of the forearm, and small-angle x-ray diffraction. The extracted lipids were separated using high performance thin-layer chromatography and quantified, and the ceramide profile was determined. Small-angle x-ray diffraction was used to obtain information on the molecular structure and organization of the intercellular lipid domains of stratum corneum using stratum corneum scales collected by scraping. Transepidermal water loss was significantly increased in all three patients. Lipid analysis showed significant differences in the relative amounts of ceramide fractions 2-3a-3b-4-5, free fatty acid-ceramide ratio, and free fatty acid-cholesterol ratio. Small-angle x-ray diffraction showed smaller repeated distances of lipid bilayers in stratum corneum samples of the patients compared with the healthy volunteers. An additional diffraction peak was found in the patients compared with the healthy volunteers, which can be ascribed to crystalline cholesterol. These data suggest that there might be a relation between the impaired barrier function and stratum corneum lipid structural and composition changes.

Adult↗
PubMed1995

Differential regulation of plasminogen activation in normal keratinocytes and SCC-4 cells by fibroblasts.

The plasminogen activator (PA)/plasmin system is thought to be involved in processes such as tumor invasion and wound healing, during which epithelial and mesenchymal cells come close together. However, information on regulation of the PA/plasmin system during epithelial-mesenchymal interactions is scarce. Therefore, we examined the in vitro modulation of the production and activity of the components of the PA/plasmin system in squamous carcinoma cells (SCC-4) and normal human keratinocytes in relation to cell density and the presence or absence of fibroblasts (3T3 cells). There was an inverse relation between cell density and mRNA expression for urokinase-type plasminogen activator (u-PA) and u-PA receptor in both SCC-4 cells and keratinocytes. In addition, such a relation was found for plasminogen activator inhibitor types 1 (PAI-1) and 2 (PAI-2) in SCC-4 monocultures, but not in keratinocyte monocultures. In contrast to monocultures, variation of cell density did not affect the mRNA expression of the components of the PA/plasmin system in cocultures of SCC-4 cells or keratinocytes with 3T3 cells. However, the relative expression of mRNAs in co-cultures was clearly different from that in monocultures, especially at low cell density. For most of the components of the PA/plasmin system, a decrease in mRNA expression and u-PA receptor protein was observed at most cell densities, whereas for PAI-1 only in keratinocytes a marked increase was documented. Zymography of supernatants revealed that the levels of both free u-PA and PA-PAI were increased in SCC-4/3T3 co-cultures, whereas in keratinocytes/3T3 co-cultures, only levels of the PA-PAI complex were increased, while the amount of free u-PA activity decreased. This occurred despite the increase u-PA immunoreactivity and was probably caused by the markedly elevated levels of immunoreactive PAI-1. The results of the present study reveal that the production and synthesis of various components of the PA/plasmin system in keratinocytes and SCC-4 cells depend on the density of epithelial cells and are modulated by fibroblasts, probably through a direct cell-cell or cell-matrix contact. Fibroblast-induced modulations are similar in keratinocytes and SCC-4 cells except for the regulation of PAI-1, which is markedly enhanced only in keratinocytes. This suggests that the modulation of PA activity in the direct microenvironment may be different under physiologic and pathologic conditions.

3T3 Cells↗
PubMed1995

Use of human skin recombinants as an in vitro model for testing the irritation potential of cutaneous irritants.

Two human skin recombinants, the epidermis reconstructed on the deepidermized dermis (RE-DED) or on fibroblast-populated collagen matrix (Living Skin Equivalent, LSE), were used to study the irritating effect of sodium lauryl sulfate (SLS). The extent of cytotoxicity induced after a 24-hour exposure period to increasing concentrations of SLS (0-5%) was evaluated on the basis of (1) morphological perturbations, (2) changes in the expression of differentiation-specific protein markers (keratin 1, 10, 6, 16, involucrin and transglutaminase), (3) cell membrane integrity (LDH leakage) and (4) release of proinflammatory mediators (PGE2, IL-1, IL-6 and IL-8). SLS induced significant changes in epidermal morphology and changes in the expression and localization of differentiation-specific protein markers when applied topically in concentrations higher than 1% on RE-DED and higher than 0.1% on LSE. The exposure of both human skin recombinants to SLS resulted in a dose-dependent release of LDH, PGE2 and IL-1 alpha and in an increase in keratinocyte intracellular IL-1 levels. Upon application of 5% SLS on RE-DED the total (intra- and extracellular) IL-1 levels remained high but due to cell damage the intracellular IL-1 level was markedly decreased and the extracellular IL-1 level increased. Similar observations have been made with LSE after application of 0.5% SLS. However, with LSE the extracellular IL-1 alpha levels were found to be about 100 times lower than those measured with RE-DED. Exposure of LSE to SLS induced a marked increase of IL-6 production in fibroblasts incorporated in the collagen matrix. Contrary to LSE, both intra- and extracellular levels of IL-6 were low in unexposed controls and were only marginally modulated by the exposure of the RE-DED to SLS. In addition, a dose-dependent increase in IL-8 release was observed upon application of SLS on RE-DED. The results of the present study indicate that concentrations of SLS required to induce epidermal irritancy in vitro approximate those inducing irritation in human skin in vivo. All parameters used in the present study for evaluation of toxicity can serve as useful endpoints for screening of contact skin irritancy in vitro. Compared to RE-DED, the LSE seems to be more susceptible to SLS. The differences in sensitivity between LSE and RE-DEd can be ascribed to reported differences in their stratum corneum barrier function.

Adult↗
PubMed1995

Effects of calcitriol on fibroblasts derived from skin of scleroderma patients.

BACKGROUND: Scleroderma is a fibrotic disorder of unknown etiology that is characterized by excessive collagen synthesis and its deposition in the skin and various internal organs. OBJECTIVE: To examine whether an overproduction of extracellular matrix molecules is a result of either increased fibroblast proliferation or increased collagen synthesis. As results of clinical trials with 1,25-dihydroxyvitamin D3 (calcitriol) have suggested beneficial effect in the treatment of scleroderma patients, the effects of calcitriol on fibroblasts derived from scleroderma and normal skin has been examined as well. METHODS: Cultures of fibroblasts were established from biopsies from involved and uninvolved skin of scleroderma patients and from skin of healthy subjects, and compared with respect to proliferation, collagen synthesis and collagen lattice contraction. RESULTS: No significant differences in cell proliferation and in the extent of fibroblast-induced collagen lattice contraction have been found between scleroderma patients exhibited a disorganized growth pattern in a monolayer culture in contrast to normal fibroblasts. Collagen synthesis tends to be higher in scleroderma fibroblasts as compared with controls. Calcitriol exerted an antiproliferative and antisynthetic effect on fibroblasts, which, however, did not discriminate healthy fibroblasts from fibroblasts derived from involved or uninvolved scleroderma plaques. CONCLUSIONS: Our findings suggest that collagen accumulation may not result from increased proliferation or altered dynamic properties of fibroblasts in a scleroderma lesion but from increased collagen biosynthesis. We additionally found that calcitriol does not selectively affect scleroderma fibroblasts.

Adult↗
PubMed1995

Characterization of stratum corneum structure in reconstructed epidermis by X-ray diffraction.

The intercellular lipid regions in the stratum corneum (SC), the outermost layer of the skin, form the major barrier for diffusion of substances through the skin. The barrier function of in vitro reconstructed epidermis is still impaired. With respect to further optimization of the model, it is necessary to characterize its stratum corneum lipid structure. In this study, small and wide angle X-ray diffraction were used to characterize the lipid organization in stratum corneum isolated from 14-day-old reconstructed epidermis. The measurements were carried out at room temperature, and subsequently as a function of temperature between 25 degrees C and 109 degrees C, followed by measurements after cooling to room temperature. The results of the X-ray diffraction measurements revealed the following in reconstructed epidermis. 1) The lamellar ordering of stratum corneum lipids was much lower than that observed in native stratum corneum. 2) Crystalline anhydrous cholesterol was present. 3) Orthorhombic packing was present, but the corresponding reflections were very weak. The orthorhombic packing disappeared between 30 degrees C and 45 degrees C. 4) A hexagonal packing was present and disappeared between 60 degrees C and 75 degrees C. 5) Soft keratin is present. 6) A higher extent of lamellar ordering could be achieved by heating to 109 degrees C and cooling down to room temperature. Analysis of SC lipids revealed the presence of high amounts of triglycerides, the level of which could be decreased by lowering the glucose content. However, modulation of culture medium composition did not significantly affect lipid lamellae structures or hydrocarbon chain packing.

Cells, Cultured↗
PubMed1994

Role of extracellular calcium in the regulation of 1,25-dihydroxyvitamin D3 formation in cultured human keratinocytes.

Cultured normal human keratinocytes (NHK) provide a useful experimental model for studies of processes occurring during terminal differentiation, since the extent of keratinocyte maturation can be manipulated experimentally by modulation of extracellular calcium concentration. When NHK are maintained in low calcium (0.06 mM) medium they proliferate but do not stratify. Raising the level of calcium to 1-2 mM results within a few hours in induction of keratinocyte differentiation. Results of the present study show that formation of 1,25-(OH)2D3 is higher in NHK grown at 0.06 mM than in NHK grown at 1.6 mM calcium concentration. After 2 h exposure of low calcium cultures to 1.6 mM calcium the 1,25-(OH)2D3 production starts to decrease. On the other hand, exposure of cells cultured in 1.6 mM calcium medium to 0.06 mM calcium concentration induced already within 4 h an increase in 1,25-(OH)2D3 formation which was not accompanied by a decrease in cornified envelope formation. Thereby, the present study demonstrated that calcium can regulate 1,25-(OH)2D3 formation independently of changes in keratinocyte differentiation.

Calcitriol↗