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.

A monoclonal antibody against keratins (KL1) from normal human stratum corneum was obtained using hybridoma techniques. Spleen cells from immunized BALB/c mice were fused with NS1, a mouse myeloma cell line, to produce hybrids. Antibody activity to epidermal keratins was tested using an indirect immunofluorescence test on cryostat sections of human epidermis and rabbit lip. A stable clone producing antikeratin antibody was isolated and an ascitic fluid was produced and used as a source of antibody (IgG1 kappa). KL1 was characterized by its immunohistochemical staining of various epithelia and by its recognition of 55-57 kilodalton (kd) keratin polypeptide from normal epidermis using the immunoblot technique. Frozen and deparaffinized sections of normal human epidermis, mucosa, and esophagus were stained by this antibody only in the upper cell layers as demonstrated by both indirect immunofluorescence and immunoperoxidase techniques. Approximatively 80% of normal keratinocytes isolated after trypsinization were labeled by KL1 whereas most negative cells showed basement membrane zone antigens. This confirmed differences in the expression of medium-sized polypeptides between basal and supra-basal cells during the course of human epidermal differentiation. All epithelial cells from other human epithelia (thymus, thyroid, bronchial mucosa, stomach, intestines) were positive with KL1 whereas nonepithelial cells and tissues did not show any staining. In view of these results KL1 promises to be a useful tool in the exploration of human epithelial differentiation.

Anti-keratin polypeptide sera (K.P.S) were obtained by immunizing guinea pigs with fibrous proteins from stratum corneum, which were acquired from normal human epidermis by m eans of S.D.S. polyacrylamide gel electrophoresis. After absorption with red blood cells and liver powder the sera were tested by indirect immunofluorescence technique on different substrates. Antibodies against polypeptides P1 and P2 of M.W. 67,000 and 62,000 dalton, respectively, were directed toward cytoplasmic Ag of keratinocytes of spinous and graunular layer of normal human and rabbit epidermis. No labeling could be detected in the basal cell layer. This finding is in favor of various differentiation stages of the keratinizing cells. P3 of M.W. 53,000 dalton induced low titre anibodies which labelled the whole epidermis, including the basal cell layer. The fourth polypeptide of M.W. 49,000 dalton seemed not to be immunogenic in such experiences. In tumors, such as basal cell carcinom,a squamous cell carcinoma, and warts, the expression of keratin antigens is markedly diminished. No analogy could be drawn between experimental keratin polypeptide antibodies and the human epidermal cytoplasmic antibodies which were detected in some patient sera.

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.

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.

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.

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.

In skin inflammation, vascular endothelial growth factor (VEGF) and IL-8 play an important role and are produced by activated keratinocytes. Recently, some polyphenols have been reported to exhibit antiinflammatory and antiangiogenic properties. We therefore evaluated the effects of green tea, its major component epigallocatechin-3-gallate (EGCG) and an isoflavone derived from soybean (genistein) on the release of VEGF and IL-8 by activated normal human keratinocytes (NHK). NHK cultured in defined medium were stimulated for 48 h with the proinflammatory cytokine TNFalpha with the addition or not of different concentrations of polyphenols. Levels of VEGF and IL-8 were measured in cell supernatants by enzyme-linked immunosorbent assays. The different constituents tested inhibited keratinocyte proliferation without inducing apoptosis. They reduced in a dose-dependent manner the basal release and the upregulation of VEGF in NHK. Green tea and EGCG were also potent inhibitors of IL-8 release by TNFalpha-stimulated NHK, whereas genistein exerted only minor effects. These results underline the divergent pathways involved in the downregulation of VEGF and IL-8 by polyphenols in activated keratinocytes. They also suggest that polyphenols may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.