Assessing the Role of Carbonyl Adducts, Particularly Malondialdehyde Adducts, in the Development of Dermis Yellowing Occurring during Skin Photoaging.

Solar elastosis is associated with a diffuse yellow hue of the skin. Photoaging is related to lipid peroxidation leading to the formation of carbonyl groups. Protein carbonylation can occur by addition of reactive aldehydes, such as malondialdehyde (MDA), 4-hydroxy-nonenal (4-HNE), and acrolein. All the proteins concerned with this modification, and the biological consequences of adduct formation, are not completely identified. The link between yellowish skin and dermal carbonylated proteins induced by aldehyde adducts was investigated. The study was carried out on ex vivo skin samples from sun-exposed or sun-protected areas and on in vitro dermal equivalent models incubated with 5 mM MDA, 4-HNE, or acrolein. The yellow color and the level of MDA, 4-HNE, and acrolein adducts were evaluated. Yellowish color differences were detected in the dermis of sun-exposed skin compared to sun-protected skin and in in vitro models following addition of MDA, 4-HNE, or acrolein. The yellowing was correlated with the carbonyl adducts increasing in the dermis and in in vitro models incubated with aldehydes. The stronger yellowing seemed to be mediated more by MDA than 4-HNE and acrolein. These observations suggest that dermal carbonylation especially induced by MDA result in the yellow hue of dermis and is involved, in part, in the yellowing observed during skin photoaging.

UVA Exposure Combined with Glycation of the Dermis Are Two Catalysts for Skin Aging and Promotes a Favorable Environment to the Appearance of Elastosis.

Skin aging is the result of superimposed intrinsic (individual) and extrinsic (e.g., UV exposure or nutrition) aging. Previous works have reported a relationship between UV irradiation and glycation in the aging process, leading, for example, to modified radical species production and the appearance of AGEs (advanced glycosylation end products) in increasing quantities, particularly glycoxidation products like pentosidine. In addition, the colocalization of AGEs and elastosis has also been observed. We first investigated the combination of the glycation reaction and UVA effects on a reconstructed skin model to explain their cumulative biological effect. We found that UVA exposure combined with glycation had the ability to intensify the response for specific markers: for example, MMP1 or MMP3 mRNA, proteases involved in extracellular matrix degradation, or proinflammatory cytokine, IL1α, protein expression. Moreover, the association of glycation and UVA irradiation is believed to promote an environment that favors the onset of an elastotic-like phenomenon: mRNA coding for elastin, elastase, and tropoelastin expression is increased. Secondly, because the damaging effects of UV radiation in vivo might be more detrimental in aged skin than in young skin due to increased accumulation of pentosidine and the exacerbation of alterations related to chronological aging, we studied the biological effect of soluble pentosidine in fibroblasts grown in monolayers. We found that pentosidine induced upregulation of CXCL2, IL8, and MMP12 mRNA expression (inflammatory and elastotic markers, respectively). Tropoelastin protein expression (elastin precursor) was also increased. In conclusion, fibroblasts in monolayers cultured with soluble pentosidine and tridimensional in vitro skin constructs exposed to the combination of AGEs and UVA promote an inflammatory state and an alteration of the dermal compartment in relation to an elastosis-like environment.

Author Correction: Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fibroblasts from the Human Skin Dermo-Hypodermal Junction are Distinct from Dermal Papillary and Reticular Fibroblasts and from Mesenchymal Stem Cells and Exhibit a Specific Molecular Profile Related to Extracellular Matrix Organization and Modeling.

Human skin dermis contains fibroblast subpopulations in which characterization is crucial due to their roles in extracellular matrix (ECM) biology. This study investigates the properties of fibroblasts localized at the frontier of deep dermis and hypodermis, i.e., dermo-hypodermal junction fibroblasts (F-DHJ), which were compared to intermediate reticular dermis (Fr) and superficial papillary dermis (Fp) fibroblasts. F-DHJ differed from Fr and Fp cells in their wider potential for differentiation into mesodermal lineages and in their absence of contractility when integrated in a three-dimensional dermal equivalent. The transcriptomic profile of F-DHJ exhibited specificities in the expression of genes involved in ECM synthesis-processing and "tissue skeleton" organization. In accordance with transcriptome data, ECM proteins, notably Tenascin C, distributions differed between the reticular dermis and the dermo-hypodermal junction areas, which was documented in normal adult skin. Finally, genome-wide transcriptome profiling was used to evaluate the molecular proximity of F-DHJ with the two dermal fibroblast populations (Fp and Fr) and with the mesenchymal stem cells (MSCs) corresponding to five tissue origins (bone marrow, fat, amnion, chorion, and cord). This comparative analysis classified the three skin fibroblast types, including F-DHJ, as a clearly distinct group from the five MSC sample origins.

Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

Clinical observations of both normal and pathological skin have shown that there is a heterogeneity based on the skin origin type. Beside external factors, intrinsic differences in skin cells could be a central element to determine skin types. This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Full-thickness skin models were constructed with site matched human keratinocytes and papillary fibroblasts to investigate potential skin type related differences. We report that reconstructed skin epidermis exhibited remarkable differences regarding stratification and differentiation according to skin types, as demonstrated by histological appearance, gene expression analysed by DNA microarray and quantitative proteomic analysis. Signalling pathways and processes related to terminal differentiation and lipid/ceramide metabolism were up-regulated in epidermis constructed with keratinocytes from Caucasian skin type when compared to that of keratinocytes from African skin type. Specifically, the expression of proteins involved in the processing of filaggrins was found different between skin models. Overall, we show unexpected differences in epidermal morphogenesis and differentiation between keratinocytes of Caucasian and African skin types in in vitro reconstructed skin containing papillary fibroblasts that could explain the differences in ethnic related skin behaviour.

Transcriptome profiling of human papillary and reticular fibroblasts from adult interfollicular dermis pinpoints the 'tissue skeleton' gene network as a component of skin chrono-ageing.

Interactions between extracellular matrix (ECM) and fibroblasts are essential for maintaining dermis integrity, and are subject to ageing. The ötissue skeleton' network connects ECM to the nucleus and DNA, impacting nuclear shape and gene expression. In a previous Mech Ageing Dev publication, we have presented a transcriptomic study of papillary (Fp) and reticular (Fr) fibroblasts, with a main focus on Fp ageing. As shown here, ageing affects ötissue skeleton' transcripts, even more clearly in Fr than in Fp. Accordingly, using circular index measurement, we show that nuclear shape is affected by ageing in both cell fractions.

Genome-wide profiling of adult human papillary and reticular fibroblasts identifies ACAN, Col XI α1, and PSG1 as general biomarkers of dermis ageing, and KANK4 as an exemplary effector of papillary fibroblast ageing, related to contractility.

Deciphering the characteristics of dermal fibroblasts is critical to further understand skin ageing. We have conducted a genome-wide transcriptomic characterization of papillary (Fp) and reticular (Fr) fibroblasts extracted from human skin samples corresponding to younger and older adult ages. From this screen, biomarkers suitable for the assessment of chronological ageing were identified, and extrapolated to the context of photo-damaged skin. In particular, KANK4, ACAN, Col XI α1, and PSG1, were expressed at an increased level in both chronologically-aged and photo-damaged skin. Notably, analysis focused on Fp identified significant transcriptional signatures associated with ageing, which included transcripts related to extracellular matrix, focal adhesion points, and cytoskeleton, thus suggesting functional consequences on tissue structure. At a cellular level, an increased contractility was identified as a property of aged Fp. Accordingly, further investigations were conducted on the KN motif and ankyrin repeat-containing protein 4 (KANK4) to explore its possible function as an original effector involved in the acquisition of aged properties in Fp, notably their increased contractility. We show that KANK4 down-modulation using siRNA led to increased Rho pathway activity, thereby reducing their contractility. As a proof-of-principle, the present study shows that targeting KANK4 was efficient to attenuate aged Fp characteristics.

Contribution of In Vivo and Organotypic 3D Models to Understanding the Role of Macrophages and Neutrophils in the Pathogenesis of Psoriasis.

Psoriasis, a common chronic immune-mediated skin disease, is histologically characterized by a rapid keratinocyte turnover and differentiation defects. Key insights favor the idea that T cells are not the only key actors involved in the inflammatory process. Innate immune cells, more precisely neutrophils and macrophages, provide specific signals involved in the initiation and the maintenance of the pathogenesis. Current data from animal models and, to a lesser extent, three-dimensional in vitro models have confirmed the interest in leaning towards other immune cell types as a potential new cellular target for the treatment of the disease. Although these models do not mimic the complex phenotype nor all human features of psoriasis, their development is necessary and essential to better understand reciprocal interactions between skin cells and innate immune cells and to emphasize the crucial importance of the local lesional microenvironment. In this review, through the use of in vivo and 3D organotypic models, we aim to shed light on the crosstalk between epithelial and immune components and to discuss the role of secreted inflammatory molecules in the development of this chronic skin disease.

Biological Effects Induced by Specific Advanced Glycation End Products in the Reconstructed Skin Model of Aging.

Advanced glycation end products (AGEs) accumulate in the aging skin. To understand the biological effects of individual AGEs, skin reconstructed with collagen selectively enriched with N(ɛ)-(carboxymethyl)-lysine (CML), N(ɛ)-(carboxyethyl)-lysine (CEL), methylglyoxal hydroimidazolone (MG-H1), or pentosidine was studied. Immunohistochemistry revealed increased expression of α6 integrin at the dermal epidermal junction by CEL and CML (p<0.01). Laminin 5 was diminished by CEL and MG-H1 (p<0.05). Both CML and CEL induced a robust increase (p<0.01) in procollagen I. In the culture medium, IL-6, VEGF, and MMP1 secretion were significantly decreased (p<0.05) by MG-H1. While both CEL and CML decreased MMP3, only CEL decreased IL-6 and TIMP1, while CML stimulated TIMP1 synthesis significantly (p<0.05). mRNA expression studies using qPCR in the epidermis layer showed that CEL increased type 7 collagen (COL7A1), ß1, and α6 integrin, while CML increased only COL7A1 (p<0.05). MG-H1-modified collagen had no effect. Importantly, in the dermis layer, MMP3 mRNA expression was increased by both CML and MG-H1. CML also significantly increased the mRNAs of MMP1, TIMP1, keratinocyte growth factor (KGF), IL-6, and monocyte chemoattractant protein 1 (MCP1) (p<0.05). Mixed effects were present in CEL-rich matrix. Minimally glycoxidized pentosidine-rich collagen suppressed most mRNAs of the genes studied (p<0.05) and decreased VEGF and increased MCP1 protein expression. Taken together, this model of the aging skin suggests that a combination of AGEs tends to counterbalance and thus minimizes the detrimental biological effects of individual AGEs.

The reconstructed skin model as a new tool for investigating in vitro dermal fillers: increased fibroblast activity by hyaluronic acid.

BACKGROUND: Clinical studies on dermal fillers have essentially focused upon visible improvement of skin quality and any eventual side effects, whereas very little is known about their detailed biological effects. OBJECTIVES: New skin equivalent models were created to investigate the biological impact of hyaluronic acid (HA) fillers on the dermal compartment in vitro. MATERIALS AND METHODS: Two different reconstructed skin models were developed to incorporate HA within the collagen fibers. In the mixed model, HA was distributed throughout the whole collagen gel whereas the HA was concentrated in the center of collagen gel in the inclusion model. RESULTS: A comparison of the addition of fillers in two models of reconstructed skin has permitted a better understanding of the biological impact of HA fillers. Protein profiling of supernatants from both models suggested a regulation of MMP-1 secretion by fibroblasts as a function of HA volume, distribution in the dermis and degree of cross-linking. Immunostaining of the inclusion model revealed increased production of type I and III procollagens close to the cross-linked HA. Fibroblasts located in this area showed a fusiform morphology as well as an increase in -smooth actin expression. The observed increase in collagen production may thus result in part from tension in fibroblasts surrounding the cross-linked HA. CONCLUSION: The inclusion reconstructed skin model, as compared to the mixed model, presented here, appears to be a useful tool for investigating the properties of various fillers in vitro and closer to the in vivo situation; our results show that HA fillers promote in vitro remodeling of the dermis by fibroblasts.

Skin aging by glycation: lessons from the reconstructed skin model.

BACKGROUND: Aging is the result of several mechanisms which operate simultaneously. Among them, glycation is of particular interest because it is a reaction which affects slowly renewing tissues and macromolecules with elevated half-life, like the dermis, a skin compartment highly affected by aging. Glycation produces crosslinks between macromolecules thereby providing an explanation for the increased age-related stiffness of the skin. Glycation products, also called AGEs (advanced glycation end products), accumulate primarily in extracellular matrix molecules like collagen or elastin. METHODS: In order to reproduce this phenomenon in vitro we have created a model of reconstructed skin modified by glycation of the collagen used to fabricate the dermal compartment. RESULTS: This system allowed us to uncover biological modifications of dermal markers, and more surprisingly epidermal markers, as well as an increase of metalloproteinases responsible for degradation of the dermal matrix. Consequently, the imbalance between synthesis and degradation that results from glycation, may contribute to skin aging, as shown in this model. Moreover these modifications were shown to be prevented by the addition of aminoguanidine, a well-known inhibitor of glycation. CONCLUSIONS: Using this experimental approach our results taken together stress the importance and possibly central role of glycation in skin aging and the usefulness of the reconstructed skin as a model of physiological aging.

In vivo and in vitro approaches in understanding the differences between Caucasian and African skin types: specific involvement of the papillary dermis.

BACKGROUND: Most of the identified differences between Caucasian and African skin types have been related to the superficial part of the skin, the epidermis. We investigated possible implications of the dermal compartment in cutaneous differences observed between Caucasians and Africans. METHODS: In vivo and in vitro comparative studies were carried out using normal human skin biopsies and the corresponding in vitro reconstructed skin. Skin equivalents were developed with papillary fibroblasts isolated from the superficial dermis of both Caucasian and African skin types. Expression of major components of the dermal-epidermal junction (DEJ) was examined as a function of ethnicity. RESULTS: Control histological examinations of skin biopsies showed that the African skin type had greater convoluted appearance of the DEJ than the Caucasian skin type. Immunostainings of type IV and VII collagens, laminin 5, and nidogen proteins at the DEJ were lower in African skin compared with Caucasian skin biopsies. CONCLUSIONS: This study brings together new elements on involvement of the papillary dermis in differences between Caucasian and African skin types. As fibroblasts from the superficial dermis cooperate with epidermal keratinocytes in producing protein of the membrane basal zone, present in vivo results suggest that papillary fibroblasts may play a part in the distinct features observed at the DEJ. In preliminary in vitro experiments, differences in several protein expressions contributing to the DEJ framework were found in reconstructed skin models made with papillary fibroblasts from both Caucasian and African skin types. Therefore, in vitro skin equivalents may be useful for better understanding of ethnic skin differences in the future.

Distinct and complementary roles of papillary and reticular fibroblasts in skin morphogenesis and homeostasis.

To study the biological properties of dermal fibroblast sub-populations, we used a reconstructed skin model with a dermal compartment populated with either papillary or reticular fibroblasts. The histological and immunohistological characterization of these reconstructed skins revealed distinct biological and structural differences, depending on the site-matched fibroblast population incorporated. Epidermal differentiation and maturation was favored and found optimum in the presence of papillary fibroblasts with little effect on ECM, as opposed to reticular fibroblasts, which had a significant positive effect on the production of the ECM molecules of the dermal epidermal junction and the dermis. In addition, the synthesis and release of MMPs and soluble factors like VEGF and KGF into the culture medium were influenced by the fibroblast population. MMP1 and VEGF were increased in the presence of papillary fibroblasts, whereas MMP3 and KGF levels were higher in the presence of reticular fibroblasts. Our results suggest that papillary and reticular fibroblasts exert distinct functions and activities in skin as revealed by the reconstructed skin model. These functional differences may have implications in wound healing and skin aging processes, considering the slow loss of papillary fibroblasts in human skin that occurs with age.

The Caucasian and African skin types differ morphologically and functionally in their dermal component.

In the literature, most reported differences between African and Caucasian skin properties concern pigmentation and barrier function of the stratum corneum and related photoprotective properties. However, little is known about differences in morphology and possibly related biological functions. In this study, we investigated: (i) architectural differences of Caucasian and African mammary skin biopsies using microscopy, (ii) comparative constitutive expression of cytokines, matrix metalloproteinase 1 (MMP-1) and its inhibitors in papillary dermal fibroblast (pF) and reticular dermal fibroblast (rF) cultures in order to reveal biological features. (i) Neither epidermis thickness nor superficial dermis thickness was significantly different in African versus Caucasian subjects. However, the dermal-epidermal junction (DEJ) length in African skin was about threefold that in Caucasian skin. No differences were noticed as regards elastic and collagen fibre organization. (ii) In papillary fibroblast cultures, a significantly higher level of monocyte chemotactic peptide-1 (MCP-1) protein was found in cell cultures from African donors when compared with that from Caucasians. With regard to keratinocyte growth factor (KGF), the ratio of papillary to reticular fibroblast expression was found to be twofold greater in cell cultures from African donors compared with that from Caucasian donors. The same trend was found regarding MMP-1 and tissue inhibitor metalloproteinase protein 1 (TIMP-1) protein expression. African skin displays a greater convolution of the DEJ and a higher papillary fibroblast activity. These findings reveal that differences between African and Caucasian skin do not only affect upper epidermis but also dermal functions and dermal-epidermal cellular interactions.

Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging.

Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr) were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin.

Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules.

Glycation is a slow chemical reaction which takes place between amino residues in protein and a reducing sugar. In skin this reaction creates new residues or induces the formation of cross-links (advanced glycation end products or AGEs) in the extracellular matrix of the dermis. Formation of such cross-links between macromolecules may be responsible for loss of elasticity or modification of other properties of the dermis observed during aging. We had previously developed a reconstructed skin model which enabled us to study the consequences of matrix alteration by preglycation of the collagen and have reported several modifications of interest induced by glycation in the dermal and epidermal compartments of reconstructed skin as well as at the level of the dermal-epidermal junction. For example we showed that collagen IV and laminin were increased in the basement membrane zone and that alpha6 and beta1 integrins in epidermis were expanded to suprabasal layers. The aim of this new study was to look at the biological effects of glycation inhibitors like aminoguanidine in the skin model. Aminoguanidine was mixed with collagen in the presence of ribose as reducing sugar, and immunostaining was used to visualize its effects on AGE Products and biological markers. After aminoguanidine treatment, we found a low amount of AGE products and a possible return to the normal pattern of distribution of markers in skin constructs as compared to those treated with ribose only. Interestingly similar results were also obtained, although to a lesser extent, with a blueberry extract. In conclusion the glycation inhibitory effect has been functionally demonstrated in the reconstructed skin model and it is shown that this model can be used to assess anti-glycation agents.

[Fibroblast subpopulations: a developmental approach of skin physiology and ageing]. / Etude des sous-populations de fibroblastes: une approche "développementaliste" de la physiologie et du vieillissement de la peau.

Skin is an organ whose function is far beyond a physical barrier between the inside and the outside of the body. Skin as the whole organism is subjected to ageing which concerns skin mostly in its dermal and deepest component which is also its matricial component. The dermis is a tissue rich in matricial elements and poor in cellular content and it is generally admitted that modifications occurring in the matrix are those which mostly contribute to skin ageing, by altering its biomechanical properties. Therefore it is common to address questions related to skin ageing by considering alterations in matrix molecules like collagen. Actually the dermis is a complex tissue both matricial and cellular and is divided between a superficial dermis close to epidermis and a deep dermis much thicker and histologically different. Several years ago we have undertaken investigations related to fibroblasts which are the cells responsible for the formation and maintenance of the dermis, aiming at isolation, culture and characterization of the fibroblasts from the superficial dermis also called papillary dermis and fibroblasts from the deep dermis also called reticular dermis. We were able to show that these fibroblasts in classical culture on plastic exhibit very different morphologies associated with different secretion properties and we have confirmed and expanded such observations revealing different phenotypes by incorporating these cells in reconstructed skin which allows the reproduction of a three-dimensional architecture recalling skin in vivo especially after grafting onto the nude mouse. We also raise the question of how these two dermal regions appear during the formation of the dermis and the question of their fate during ageing. Progress in solving these questions would certainly appear to be very useful for a better understanding of skin physiology and ageing and would hopefully provide new strategies in anti-ageing research.

An organotypic model of skin to study photodamage and photoprotection in vitro.

Acute or repetitive sun exposures are known to elicit cutaneous damages such as sunburn but also long-term effects such as photoaging or cancers. Determination of early biological events occurring after ultraviolet (UV) exposure is essential for photoprotection. Using skin reconstructed in vitro containing both a dermal equivalent and a fully differentiated epidermis, the effects of UV light (UVB and UVA) were investigated. UVB-induced damage was essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA radiation-induced damage was mostly located within the dermal compartment. The model and end points used for UVB- and UVA-induced damages appeared to be very useful for the in vitro evaluation of sunscreens after topical application, in particular to investigate its protective effects against the effects of UVR, and allowed us to distinguish the efficiency of absorbers depending on their absorption spectrum.

Collagen glycation triggers the formation of aged skin in vitro.

Glycation products accumulate during the aging of many slowly renewing tissues, including skin. We have developed an in vitro model of chronologic aging of skin based on reconstructed skin modified by artificially glycating the collagen used to prepare the dermal compartment. The morphology of the modified skin is close to the morphology usually observed except that the dermis is altered in its fibrillar structure. Moreover, the analysis of skin markers revealed several unexpected biological and morphological modifications, which reflect in vivo aging and could be related to glycation per se. These include the activation of fibroblasts, increase of matrix molecules (collagen type III and collagen type IV) and metalloproteinase production (MMP1, MMP2 and MMP9), thickening of the basement membrane zone, and more strikingly, the modification of alpha6 and beta1 integrin patterns especially in epidermis, in a way closely resembling aged skin in vivo. We also found that these effects could be related to the production of putative diffusible factors by the dermal fibroblasts activated by glycation. Finally, we show that all these effects are likely to be glycation specific since they could be inhibited by aminoguanidine, a well-known glycation inhibitor. We conclude that the reconstructed skin model modified by glycation of the collagen closely mimics chronologic aging of skin in vivo. Taken together, these results strengthen the importance of glycation reactions in skin aging.

Morphogenesis of dermal-epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C.

In skin, cohesion between the dermis and the epidermis is ensured by the dermal-epidermal junction which is also required for control of epidermal growth and differentiation. Here we showed that addition of vitamin C optimized the formation of the dermal-epidermal junction in an in vitro human reconstructed skin model leading to a structure closer to that of normal human skin. Compared with controls, vitamin C treatment led to a better organization of basal keratinocytes, an increase in fibroblast number and a faster formation of the dermal-epidermal junction. Vitamin C also accelerated deposition of several basement membrane proteins, like type IV and VII collagens, nidogen, laminin 10/11, procollagens I and III, tenascin C and fibrillin-1 at the dermal-epidermal junction. The mechanism of action of vitamin C was investigated by quantitative polymerase chain reaction in fibroblasts and keratinocytes respectively. Vitamin C effects passed in part through an increase in col I alpha1, col III alpha1 and fibrillin-1 mRNA levels. Effects on the other markers appeared to happen at the translational and/or post-translational level, as illustrated for tenascin C, col IV alpha2 and col VII alpha1 mRNA levels which were reduced by vitamin C in both cell types.