The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo-epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo-epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-beta2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-beta2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.     

Effect of cytokines and growth factors on the expression of elastase activity by human synoviocytes, dermal fibroblasts and rabbit articular chondrocytes

Human synoviocytes, rabbit articular chondrocytes and human skin fibroblasts in culture were examined for their ability to express elastase activity. Latent enzyme activity degrading insoluble elastin was detected in the culture media of the three cell types and was completely abolished by metal chelating agents. Triton X-100 cell extracts were found to degrade a synthetic elastase substrate, N Succinyl-(Ala)3p-nitroanilide (SANA). The SANA-degrading activity of cell extracts could be attributed to a metalloprotease for fibroblasts and synoviocytes (100%) and to a metalloprotease associated with a cysteine protease for chondrocytes (70 and 30% respectively). This SANA-degrading activity was partly due to the combined action of an endo and an exopeptidase. Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) significantly enhanced the elastin degrading activity present in the culture media of both synoviocytes and chondrocytes. Interleukin-1 beta significantly increased the secretion of elastase by chondrocytes. By contrast, Transforming Growth Factor-beta (TGF-beta) reduced by 80 per cent the secretion of elastinolytic activity by chondrocytes but had not effect on other cell types.     

Lycopene inhibits PDGF-BB-induced signaling and migration in human dermal fibroblasts through interaction with PDGF-BB

In melanoma development and progression, platelet-derived growth factor (PDGF) has been suggested to modulate the microenvironment, especially stromal fibroblasts, to the benefit of melanoma growth, invasion, and metastasis. Lycopene, a natural carotenoid that is abundant in tomato, has been shown to inhibit proliferation of several types of cancer cells. However, little attention has been paid to skin fibroblasts and melanoma cells. In the present study, we determined the effects of lycopene on stromal fibroblasts and their interactions with melanoma cells. We found that lycopene inhibited PDGF-BB-induced human Hs68 skin fibroblast migration on gelatin and collagen. Further analysis showed that lycopene inhibited PDGF-BB-induced signaling in human Hs68 and primary cultured skin fibroblasts. PDGF-BB-induced phosphorylation of PDGF receptor beta (PDGFR-beta), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) was attenuated by lycopene in a concentration-dependent manner, whereas the total expression of each protein was not affected. Interestingly, dot binding assay revealed that lycopene could directly bind to human PDGF-BB in PBS and human plasma, indicating that lycopene can bind to PDGF-BB in both in vitro and in vivo conditions. In functional studies, lycopene inhibited melanoma-induced fibroblast migration in a noncontact coculture system and attenuated signaling in fibroblasts simulated by melanoma-derived conditioned medium. Our results provide the first evidence showing that lycopene is an effective inhibitor of migration of stromal fibroblasts and this effect may contribute to its antitumor activity.     

Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

Epithelial–mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.     

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.     

Surface modification of polyhydroxyalkanoate films and their interaction with human fibroblasts

A poly(3-hydroxybutylate-co-hydroxyvalerate) (PHA) film containing 34 mol.% 3-hydroxyvalerate (Biopol D600P) was prepared by the solvent cast method using a 10 wt.% chloroform solution of PHA. The PHA film was exposed to an oxygen plasma glow discharge to produce peroxides on its surfaces. These peroxides were then used as catalysts for the polymerization of acrylic acid (AA) in order to prepare carboxyl group-introduced PHA (PHA-C). Insulin-immobilized PHA was prepared using the coupling reaction of PU-C with insulin. The surface-modified PHAs were then characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis, and a contact angle goniometer. The amounts of insulin directly coupled to the carboxyl groups on PHA-C and coupled to the terminus amino groups of the grafted polyethylene oxide were 2.9 and 0.8 microg cm(-2), respectively. The PHA water contact angle (75 degrees ) decreased with AA grafting (33 degrees ) and insulin immobilization (31 degrees ), thereby exhibiting the increased hydrophilicity of the modified PHAs. When compared with PHA and PHA-C, the proliferation of human fibroblasts in the presence of serum was significantly accelerated on the insulin-immobilized PHAs.     

Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: aging in vivo or in vitro

Attachment, spreading, and microfilament reorganization have been evaluated in human dermal reticular fibroblasts isolated from the inner, upper aspect of the arm of a newborn male (RET5 cells) and a 78-year-old male (RET8 cells). Substrata were tested using a set of complementary fragments from individual polypeptide chains of human plasma fibronectin (pFN) or cellular FNs (cFN). With both cell classes, fragments containing the C-terminal heparin-binding (HepII) domain only elicited linear bundles of microfilaments in spreading cells but no stress fibers; fragments containing the RGDS-dependent cell-binding (CellI) domain elicited only partial spreading with condensations of F-actin at ruffling membranes and at other regions along the plasma membrane. The minimum sequence required to obtain responses identical to those on intact pFN (broad spreading with extensive stress fiber formation) was found in fragment 155 (F155) from the beta chain of pFN; F155 contains both HepII and CellI domains. In contrast, the analogous fragment from the alpha chain of pFN (F145) was notably less effective for generating stress fibers. This evidence along with the better attachment, spreading, and microfilament bundle formation on the HepII fragment from the beta chain than the analogous fragment from the alpha chain indicates that the extra type III homology unit permits more effective interaction of beta chain fragments with cell-surface heparan sulfate proteoglycan and possibly integrin (binding efficiency to the substratum was similar for fragments from both chains). Therefore, alternatively spliced sequences that neighbor binding domains can play significant roles in the interaction of the domain with cell-surface receptors of dermal fibroblasts. Comparison of RET5 responses with those of RET8 cells has identified changes in adhesive mechanisms as cells undergo "aging" processes. Attachment and microfilament bundle formation were far more effective for RET5 cells than for RET8 cells on any of the HepII fragments. Conversely, RET8 cells were far more sensitive to an RGDS-containing peptide in their medium on CellI fragments than RET5 cells. These results together indicate that in vivo aging leads to greater dependence upon cell-surface integrin binding and less dependence upon heparan sulfate proteoglycan binding for responses on FN matrices. When RET5 cells entered senescence (in vitro aging), they also became much more sensitive to peptide A. On several fragments and on intact pFN, RET8 cells generated very thick stress fibers that were observed only on one fragment with RET5 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor necrosis factors alpha and beta differ in their capacities to generate interleukin 1 release from human endothelial cells

The capacity of the tumor necrosis factors, TNF-alpha and TNF-beta, products of activated macrophages and lymphocytes, respectively, to stimulate interleukin 1 (IL-1) release from endothelial cells derived from human umbilical veins was examined in vitro. Recombinant TNF-alpha caused IL-1 release by 4 hr with maximal levels of 17 U/ml by 24 hr; half-maximal stimulation occurred at approximately 80 pM. In contrast, recombinant TNF-beta was a relatively poor stimulus for IL-1 release. Even at concentrations as high as 600 pM, only 3 U of IL-1/ml were recovered; maximal IL-1 release (10 to 12 U/ml) required up to 5 nM TNF-beta. Natural, glycosated human TNF-beta was comparable in activity to recombinant TNF-beta. TNF-beta did not directly inhibit the IL-1 comitogenesis assay, nor was there evidence that TNF-beta induced the release of an IL-1 inhibitor, in that supernatants generated in the presence of TNF-beta did not inhibit thymocyte proliferation to a recombinant IL-1 standard. Binding of the recombinant TNF to endothelial monolayers was assessed by using [125I]TNF-alpha in competition studies with cold TNF-alpha and TNF-beta. Binding of TNF-alpha was half-maximal at 80 pM with an average of 664 receptors/cell and Kd = 0.043 nM. Although TNF-beta was capable of fully competing for [125I]TNF-alpha binding, half-maximal binding occurred at 800 pM TNF-beta. These data suggest that the TNF receptors on human endothelial cells may reflect the structural differences between these two homologous cytokines.     

Hemadsorption focus assay for growth of influenza and parainfluenza viruses in human dermal fibroblasts

An assay for virus-induced hemadsorption foci in cultures of human dermal fibroblasts is described. The assay allows determination of activity of species-specific as well as nonspecific antiviral agents or biological factors, such as interferons. The assay may be used for abortive (e.g., influenza virus) and productive (e.g., parainfluenza virus) infections of human fibroblasts.     

Effect of growth factors on morphogenesis of human keratinocytes in vitro

The effect of some growth factors on morphogenetic processes in the primary culture of human epidermal keratinocytes was studied in the model of formation of tubule-like structures in collagen gel. Culturing of keratinocytes in the presence of IGF and bFGF did not induce growth of tubule-like structures, whereas EGF, KGF, and HGF promoted the growth of three-dimensional epidermal outgrowths whose shape and size varied depending on the growth factor used.     

Normal embryo fibroblasts release transforming growth factors in a latent form

Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Regulation of fibrogenic/fibrolytic genes by platelet-derived growth factor C, a novel growth factor, in human dermal fibroblasts

Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic cytokine, and PDGF-C is a novel growth factor belonging to the PDGF family. In this study, to determine whether this growth factor can contribute to fibrosis and tissue remodeling, we examined the effect of PDGF-CC on the expression of fibrogenic/fibrolytic genes such as type I collagen, fibronectin (FN), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in normal human dermal fibroblasts in vitro. PDGF elevated the levels of MMP-1 or TIMP-1 protein as well as mRNA, whereas this cytokine had no influence on the expression of type I collagen, FN, or TIMP-2. PDGF-CC also increased the levels of MMP-1 catalytic activity in the cultured media and mRNA expression, which was paralleled that on the levels of promoter activation. Additionally, PDGF-CC induced the mitogenic and migratory activity of human dermal fibroblasts in a dose-dependent manner. On the other hand, we also determined the specificity of the inhibitory effect of monoclonal antibodies against PDGF-CC generated by immunizing balb/c mice with recombinant human PDGF-CC. This antibody could inhibit the regulatory effects of MMP-1 or TIMP-1 synthesis as well as the mitogenic effects on human dermal fibroblasts induced by PDGF-CC, whereas this antibody did not affect those induced by other PDGF forms such as PDGF-AA, -AB, or -BB. These results suggest that this cytokine plays a role in the tissue remodeling.     

Contractility of single human dermal myofibroblasts and fibroblasts

Human dermal myofibroblasts, characterised by the expression of alpha-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273-283], without changing their expression of alpha-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 microN per cell) was not significantly different from that generated by fibroblasts (2.0 microN per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 microN/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures.