Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/AkT signaling pathways.

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt.

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.

A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion.

Tissue microenvironment plays a critical role in tumour growth and invasion. This study established a novel 3-dimensional (3-D) cell invasion model for direct microscopic observation of oral cancer cell invasion into the underlying basement membrane and connective tissue stroma. A multilayer cell construct was developed using the OptiCell chamber, consisting of a lower layer of oral mucosa fibroblasts embedded in collagen gel and an overlaying upper layer of oral cancer cells. The two layers are separated by a basement membrane composed of reconstituted extracellular matrix. To verify the applicability of the cell invasion model, multilayer cell constructs of oral squamous cell carcinoma and oral mucosal fibroblasts were exposed to extrinsic urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor (PAI-1), which are known effectors of cell migration. In addition, the constructs were exposed to both normoxic and hypoxic culture conditions. Microscopic study showed that the presence of uPA enhanced cell invasion, while PAI-1 inhibited cell migration. Western blot and zymographic analysis demonstrated that hypoxia up-regulated uPA and matrix metalloproteinases (MMPs) expression and activity; conversely, PAI-1 level was down-regulated in response to hypoxic challenge as compared to normoxic condition. Our results indicated that the novel 3-D invasion model could serve as an excellent in vitro model to study cancer cell invasion and to test conditions or mediators of cellular migration.

Assessment of morphological and immunohistological alterations in long-term keloid skin explants.

One of the major impediments in keloid research is the lack of a keloid animal model that can mimic human keloid. This imposes investigative constraints on studying cellular interactions and biochemical processes that normally occur in vivo. Our main objective is to establish an in vitro model for maintaining long-term viable keloid dermal explants as a tool for investigating the pathogenesis of keloid scar formation. Explants of adult keloid scars were cultured in vitro by embedding them in enriched collagen gel matrix and maintaining them for up to 6 weeks, whereupon changes in tissue morphology and cellular differentiation were examined. The effects of medium enrichment, air versus liquid submersion, and different substrates on the explants were examined. Our results indicated that keloid explants embedded in a collagen gel matrix were morphologically better preserved than explants placed on a plastic substrate. Explants with epidermis at the air-liquid interface had better morphology than collagen-submerged explants, and there were no differences between serum-free and serum-supplemented explant cultures. Immunohistochemical and apoptotic analyses were performed to assess cellular viability and differentiation. In situ hybridization confirmed that keloid fibroblasts had sustained collagen type I gene expression throughout the 6 weeks in culture, thus validating the integrity of a long-term keloid culture system. In conclusion, the collagen-embedded skin explant system demonstrates that keloid tissues could be maintained for up to 6 weeks for long-term in vitro studies.

Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts.

Keloids are characterized as an "overexuberant" healing response in which disequilibrium between production and catabolism of extracellular matrix (ECM) occurs. Previous studies from our laboratory and others demonstrate an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) expression in keloid tissues and cultured fibroblasts compared with normal bordering skin. These findings support the concept that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis. Vascular endothelial growth factor (VEGF) has been implicated as a critical factor in regulating angiogenesis and inflammation under both physiological and pathological conditions. This study was designed to assess whether VEGF plays a role in keloid fibrosis. We report that VEGF was expressed at higher levels in keloid tissues and their derived fibroblasts compared with their associated normal skin. We have further demonstrated that VEGF stimulated the expression of PAI-1, but not urokinase plasminogen activator (uPA), in keloid fibroblasts at both mRNA and protein levels, in a dose- and time-dependent manner. However, treatment of normal skin fibroblasts with VEGF exerted little effects on PAI-1 gene expression. Additionally, we have characterized for the first time that the extracellular signal-regulated kinase (ERK)1/2 signaling pathway is mainly involved in VEGF-induced PAI-1 expression and have demonstrated its potential as a target molecule for modulation of scar fibrosis. These findings suggest that VEGF may play an important role in keloid formation by altering ECM homeostasis toward a state of impaired degradation and excessive accumulation.