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J Viac

Publications and source records attributed to J Viac.

At least 19 recordsLinked to original sources
PubMed2004

Comparative expression of vascular endothelial growth factor family members, VEGF-B, -C and -D, by normal human keratinocytes and fibroblasts.

The vascular endothelial growth factor (VEGF) family includes the related polypeptides VEGF-B, -C and -D, which contribute to endothelial and lymphatic vessel development. The parental VEGF molecule, VEGF-A, has been widely described in the skin, but the other members of the VEGF family have not yet been reported. The aim of our study was to determine whether the two main skin cells, keratinocytes and fibroblasts, expressed VEGF-B, -C and -D in basal condition and after stimulation by either growth factors or the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). Reverse-transcription polymerase chain reaction (RT-PCR) analysis on cultured normal human keratinocytes (NHKs) and normal human fibroblasts (NHFs) allowed the detection of different levels of VEGF-B, -C and -D mRNA, in both cell types with similar RT-PCR products in the skin cells. A semi-quantitative evaluation of the VEGF family proteins by dot blot, using the different human recombinant VEGFs, showed different levels of VEGF-B, -C and -D, in NHKs and NHFs. After cell stimulation by growth factors (epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) for NHKs and NHFs, respectively), a significant up-regulation of the VEGF family member proteins was observed in NHFs but not in NHKs. Conversely, TNF-alpha did not exert a significant effect. However, we could not detect any transcriptional modification in stimulated cells, whatever the stimulation duration. The addition of cycloheximide to the cell cultures strongly inhibited the increase of VEGF proteins in TGF-beta1-stimulated NHFs. Taken together, the results underline the major role played by NHFs in the elaboration of the VEGF family proteins known to regulate wound healing, chronic inflammation and tumour angiogenesis and lymphangiogenesis.

Cells, Cultured↗
PubMed2003

Use of human reconstructed epidermis to analyze the regulation of beta-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS.

Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.

Blotting, Western↗
PubMed2003

Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.

The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.

Calcium↗
PubMed2002

IL-4 and interferon-gamma differentially modulate vascular endothelial growth factor release from normal human keratinocytes and fibroblasts.

IL-4 and interferon-gamma (IFN-gamma) are crucial modulators of the immune system and are reported as active antitumor agents and potent inhibitors of angiogenesis. We investigated the effects of these two cytokines on the expression of vascular endothelial growth factor (VEGF), a mediator of major importance in the angiogenesis associated with inflammation, wound healing and tumor invasion and expressed by activated keratinocytes and dermal fibroblasts. Human keratinocytes (HK) and fibroblasts (HF) derived from foreskins, were further cultured during 24 h in defined medium, supplemented or not with the selected growth factors, EGF and TGF-beta1, respectively, before receiving the addition of either IL-4 or IFN-gamma during 24 and 48 h. In basal conditions, fibroblasts produced smaller amounts of VEGF than keratinocytes; the addition of growth factors to the skin cells induced a drastic increase of VEGF secretion. In HF, the basal level of VEGF secretion was reduced by IFN-gamma and slightly increased by IL-4 whereas in HK, IFN-gamma enhanced the secretion of VEGF after 48 h and IL-4 either tended to reduce VEGF secretion or did not exert any effect. Similar but more significant results were observed in skin cells activated by growth-stimulating factors. The association of IL-4 and IFN-gamma mimicked the effects of IFN-gamma alone both in HF and HK. Taken together, these results indicate opposite effects of IFN-gamma and IL-4 on VEGF expression from normal and activated HF and HK. IL-4 may be considered as a poor modulator of VEGF secretion by dermal and epidermal cells. Conversely, IFN-gamma appears as a prominent and versatile mediator in the desregulated angiogenesis associated with inflammatory skin reactions characterized by a T-helper type 1 cell-mediated response.

Cells, Cultured↗
PubMed2001

UV radiation and prostaglandin E2 up-regulate vascular endothelial growth factor (VEGF) in cultured human fibroblasts.

OBJECTIVE AND DESIGN: Exposure to UV radiation is responsible for skin erythema and inflammation. PGE2 is an important inflammatory mediator involved in this process and vascular endothelial growth factor (VEGF) is a potent vascular permeability factor mainly produced by epidermal keratinocytes. This study was aimed at determining whether UVB/A1 radiation and prostaglandin E2 (PGE2) could modulate the production of VEGF by cultured dermal human fibroblasts (HF) in comparison to keratinocytes (HK). MATERIALS AND METHODS: The skin cells derived from foreskin, were cultured in defined medium before treatment by either UVB/A1 radiation, or stimulation by addition of PGE2 (10(-8) to 10(-5) M). The expression of VEGF in cultured fibroblasts and keratinocytes was evaluated at the mRNA (RT-PCR) and protein levels (ELISA). RESULTS: The basal level of VEGF was lower in HF than in HK. Both UVB and UVA1 radiation strongly up-regulated VEGF mRNA and protein in HF whereas UVB but not UVA1 radiation induced a VEGF increase in HK only at the protein level. UVA1, when associated with UVB radiation, showed an additive effect on VEGF secretion in HF but not in HK. PGE2 increased in a dose-dependent manner the expression of VEGF in HF but not in HK. Indomethacin as well as the antioxidant alpha-tocopherol did not reduce UV-induced enhanced secretion of VEGF by both fibroblasts and keratinocytes whereas pyrolidine dithiocarbamate exerted an inhibition of this overexpression. CONCLUSIONS: These results indicate different signaling pathways in the PGE2 and UV-induced regulation of VEGF in dermal fibroblasts and epidermal keratinocytes. They also suggest a role for VEGF from both fibroblasts and keratinocytes in the UV-induced erythema, independent of PGE2. A dermal overexpression of VEGF by fibroblasts from UV-irradiated skin may contribute to dilated microvasculature, a feature of skin photoaging and more generally, to a more permissive stroma to tumor formation than unexposed skin.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Modulation of endothelin-1 in normal human keratinocytes by UVA1/B radiations, prostaglandin E2 and peptidase inhibitors.

In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.

Cells, Cultured↗
PubMed2000

Transforming growth factor-beta1 and ultraviolet A1 radiation increase production of vascular endothelial growth factor but not endothelin-1 in human dermal fibroblasts.

BACKGROUND: Normal and dysregulated wound healing involves fibroblast activation and angiogenesis, in which polypeptide factors such as transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) play an important part. Ultraviolet (UV) A1 (365 nm) has recently received attention as a possible treatment for some dermal fibrotic disorders. OBJECTIVES: The aim of this study was to evaluate the effects of TGF-beta1 and UVA1 radiation, as well as that of cobalt chloride, reported to mimic hypoxia both in vivo and in vitro, on the expression of VEGF and ET-1 by cultured human dermal fibroblasts. METHODS: Levels of VEGF and ET-1 were measured by enzyme-linked immunosorbent assay and expression of neutral endopeptidase (NEP, CD10), known to degrade ET-1, was quantified by flow cytometric analysis after cell trypsinization. RESULTS: Our results showed that the cells released minor amounts of VEGF and ET-1. Both TGF-beta1 and UVA1 strongly increased VEGF secretion in a dose- and time-dependent manner, without significantly affecting ET-1 release. Irradiation of TGF-beta1-stimulated fibroblasts resulted in a synergistic effect on increasing levels of VEGF but not ET-1 after 48 h. Cobalt chloride stimulated the secretion of VEGF by fibroblasts; the effects of TGF-beta1 and cobalt were additive. However, no significant effect of cobalt chloride on ET-1 secretion was observed, suggesting that ET-1 production in fibroblasts is not oxygen-sensitive. The expression of NEP was not modified by TGF-beta1 or UVA1 radiation. Addition of a neutralizing anti-CD10 antibody to fibroblast cultures downregulated CD10 expression at the cell surface without changing ET-1 levels in cell supernatants after 24 or 48 h. This suggests that membrane-bound NEP has minimal or no activity against secreted ET-1. CONCLUSIONS: Taken together, these results underline the major role played by TGF-beta1 in increasing VEGF secretion by fibroblasts. This, as well as the documented effect of UVA1 on increasing VEGF production, may have implications for wound healing in vivo.

Biomarkers↗
PubMed1998

Circulating vascular endothelial growth factor (VEGF) is not a prognostic indicator in malignant melanoma.

Among angiogenic peptides, vascular endothelial growth factor (VEGF) is associated with growth and metastasis of solid tumours. In order to determine whether VEGF could be involved in the clinical course of malignant melanoma, we studied 96 patients with primary or metastatic melanoma and we reported the follow-up of nine cases who initially presented with a primary melanoma and further developed metastasis over a period of 12-25 months. Circulating VEGF levels quantified by enzyme-linked immunosorbent assay were found to be elevated in patients with primary or metastatic melanoma compared to a control group (P < 0.001), but no significant difference occurred between primary and metastatic melanoma. The follow-up of patients who developed metastasis showed high initial VEGF levels (in five out nine cases) which remained increased with the course of the disease. It is conceivable that increased VEGF levels reflect an intense activation of the host immune system but the variations in the concentration of circulating VEGF were not considered as an indicator of disease evolution in malignant melanoma.

Adult↗
PubMed1998

Tumour necrosis factor-alpha is involved in the contrasting effects of ultraviolet B and ultraviolet A1 radiation on the release by normal human keratinocytes of vascular permeability factor.

Erythema and the initiation of an inflammatory response are typical features of human skin after ultraviolet (UV) radiation (UVR) exposure. Among the soluble factors that account for the induction of an erythema, the most recently discovered is vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent inducer of microvascular permeability which is expressed by keratinocytes. As epidermal cells are the first target cells of UVR, we studied the effects of UVBR (312 nm) and UVA1R (365 nm) on the secretion of VEGF by normal human keratinocytes and evaluated the role of interleukin 1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) in this process. UVBR (100 and 200 mJ/cm2) induced a dose-dependent increase in the release by normal human keratinocytes of VEGF, which is widely mediated through the release of TNF-alpha but not IL-1 alpha. Conversely, UVA1R (5 and 7 J/cm2) did not modify the basal level of VEGF and did not induce the secretion of TNF-alpha by keratinocytes. Moreover UVA1R, when associated with UVBR, inhibited the increase in VEGF induced by UVBR alone. Taken together, these findings indicate that UVBR and UVA1R have a contrasting effect on the release of VEGF, which is widely mediated by TNF-alpha. They may partly explain the minor erythematous effect of UVA1R and its beneficial role in cutaneous phototherapy.

Antibodies↗
PubMed1997

Expression of vascular endothelial growth factor in normal epidermis, epithelial tumors and cultured keratinocytes.

Vascular endothelial growth factor (VEGF), an endothelium-specific growth factor and microvessel hypermeability factor, is expressed and secreted by several kinds of cells and is implicated in angiogenesis of tumors. The present study was performed to determine the relationship between the expression of VEGF in normal skin, benign and malignant epithelial lesions and cultured keratinocytes and the proliferative activity and degree of differentiation of keratinocytes. Skin lesions were studied immunohistochemically by staining with two anti-VEGF antibodies and secretion and production of VEGF by keratinocyte cultures were evaluated using an enzyme-linked immunosorbent assay. Low to moderate VEGF expression was observed in normal epidermis. In epithelial tumors, different reactivity patterns were observed and different areas of the same tumor expressed different amounts of VEGF. A more prominent labelling occurred in proliferative layers and/or more differentiated cells of virus-induced lesions, squamous cell carcinomas and Bowen's disease, whereas basal cell carcinomas always stained weakly for VEGF. In cultured keratinocytes, the amount of cell-associated and secreted VEGF increased with time, and the constitutively produced VEGF was mostly released extracellularly. High calcium concentrations upregulated the intracellular content of VEGF but downregulated its release. Taken together, these results showed a modulated expression and release of VEGF in relation to the stage of cell differentiation and in rapidly growing or activated keratinocytes.

Cell Differentiation↗
PubMed1997

Interferon gamma-induced PNA-binding glycoproteins as markers of human keratinocyte differentiation: biological evidence using protein kinase C agonists, antagonists and retinoic acid.

Membrane glycoproteins (gps) play an important role in cell-cell interactions during epidermal maturation, and we have previously shown an up-regulation of PNA-binding gps in cultured human keratinocytes treated with interferon gamma (IFN-gamma). The protein kinase C (PKC) pathway is known to play a key role in the regulation of proliferation and differentiation of keratinocytes and is also reported to be involved in some IFN-gamma-mediated effects. In order to evaluate the cellular mechanisms and whether PNA-binding gp expression is related to the differentiative activity of the lymphokine, we studied the effects of PKC agonists and antagonists and the role of retinoic acid (RA), in the induction of these gps in cultured human keratinocytes stimulated with IFN-gamma and processed for protein analysis. The expression of PNA-binding gps was revealed by incubation of SDS-polyacrylamide gels with 125I-PNA. The PKC antagonists (H7, sphingosine) as well as RA downregulated the IFN-gamma-induced PNA-reactive gps, whereas staurosporine and TPA upregulated their expression. These results provide evidence that PNA-reactive gps are late highly IFN-gamma-sensitive markers of keratinocyte differentiation, drastically modulated through selective isoforms of PKC.

Agglutinins↗
PubMed1997

Contact allergens and sodium lauryl sulphate upregulate vascular endothelial growth factor in normal keratinocytes.

In allergic and irritant contact dermatitis, keratinocytes are major target cells that can be activated to take part in local reactions by secreting soluble mediators. Among the growth factors produced by keratinocytes, vascular endothelial growth factor (VEGF) is a powerful inducer of permeability of endothelial cells, and is involved in inflammation. We determined whether different contact allergens, dinitrosulphobenzene (DNSB), para-phenylenediamine (pPD) and the metals nickel and chromium, as distinct from cobalt, which has been shown to mimic the effects of hypoxia, can modify the basal level of VEGF in normal human keratinocytes when tested at various, non-toxic concentrations. The effects of an irritant, sodium lauryl sulphate (SLS), and of hydrocortisone were also tested. Our results showed an intense dose-dependent upregulation of VEGF release by keratinocytes after treatments by metals, pPD and SLS. DNSB induced only a moderate increase of VEGF. Hydrocortisone reduced the basal level as well as the nickel-induced upregulation of VEGF. These findings suggest that contact allergens and irritants probably upregulate VEGF in keratinocytes by different mechanisms and may contribute directly to the microvascular hyperpermeability which characterizes both contact and irritant dermatitis.

Allergens↗
PubMed1997

Differences in responses of interleukin-1 and tumor necrosis factor alpha production and secretion to cyclosporin-A and ultraviolet B-irradiation by normal and transformed keratinocyte cultures.

Among epidermal cytokines, IL-1 and TNF alpha are involved in inflammatory skin reactions and suspected of modulation by immunosuppressive treatment (e.g., cyclosporin A, CsA) or UVB-irradiation, 2 mediators probably being involved in epithelial carcinogenesis. We evaluated the effects of 8 micrograms/ml CsA and 100 J/m2 UVB-irradiation on the production and secretion of IL-1 and TNF alpha on normal human epidermal keratinocytes (NHK) and epidermal keratinocyte cell lines either spontaneously transformed (HaCaT) or transformed by human papillomavirus (HPV) type 16 or 18 (EK 16 and EK18), by using ELISA test. Normal and immortalized keratinocytes constitutively produced and released IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1RA) but IL-1 synthesis by NHK was significantly higher than by cell lines. All the cells spontaneously excreted low amounts of TNF alpha. Different responses to treatments were evidenced between NHK and cell lines. CsA modified significantly the production and secretion of IL1 in most cells whereas slight changes were observed with TNF alpha secretion. UVB irradiation had no effect on the intracellular IL1 pool of any cells but increased the release of IL1 and TNF alpha. The association CsA-UVB did not result in additive effects on synthesis and secretion of IL1; the release of TNF alpha by the cells remained poor except for EK18 cells. Taken together, these results show that, in immortalized keratinocytes, the IL-1 and TNF alpha expression was differently affected by treatments with CsA and/or UVB-irradiation as compared to NHK. In addition, spontaneously transformed keratinocytes, HaCaT, reacted differently from HPV-transformed keratinocytes, EK16 and EK18.

Cell Line, Transformed↗