Pectin from transgenic flax shives regulates extracellular matrix remodelling in human skin fibroblasts

Pectin, a polysaccharide polymer from the plant cell wall, is an underestimated natural resource with many potential applications in the food and medical industries. Here we present, for the first time, the chemical composition of pectin obtained from flax shives, a by-product of flax fibre processing. The shives from transgenic flax overexpressing β-glucanase were analysed, revealing that genetic modification caused an increase in content of lignin, hemicellulose and pectin, without changes to cellulose, rearrangement of the structure of pectin and cellulose, a decrease in the content of phenolic compounds associated with the cell wall, and an increase in antioxidant capacity of the pectin CDTA fraction. The influence of pectin extract on the extracellular matrix remodelling process was verified. In fibroblast skin cells with induced oxidative stress, addition of pectin caused a reversal of the decrease of mRNA collagen genes, an increase of matrix metalloproteinase, interleukin 6 and MCP-1 gene expression, and a reduction in levels of TIMP-1 and SOCS-1 mRNA. The obtained results, in particular strong antioxidant properties of flax shives pectin from the CDTA-soluble fraction and its significant influence on genes participating in extracellular matrix remodelling, suggest the possible application of flax shives pectin in the wound healing process.     

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts

Background

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results

Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions

microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.     

Lipid A-associated proteins from periodontopathogenic bacteria induce interleukin-6 production by human gingival fibroblasts and monocytes

Abstract The aim of this study was to determine whether lipid A-associated proteins (LAP) from two periodontopathogenic species of bacteria were able to stimulate interleukin-6 (IL-6) release from human gingival fibroblasts and myelomonocytic cells. LAP and lipopolysaccharide (LPS) were extracted from Porphyromonas gingivalis and Prevotella intermedia and added to cultures of human gingival fibroblasts and mono-mac-6 monocytic cells. Release of IL-6 into the culture supermatants was determined by ELISA. LAP and LPS from Por. gingivalis , but not from Prev. intermedia , stimulated IL-6 release from both cell types in a dose-dependent manner although LPS was less potent than LAP in inducing IL-6 release from the fibroblasts. IL-6 was detectable in cultures of both cell types following stimulation with LAP from Por. gingivalis at a concentration as low as 10 ng/ml. In response to LAP from Prev. intermedia , IL-6 was produced by mono-mac-6 cells but not by fibroblasts. Our results show that bacterial cell wall components other than LPS can induce IL-6 release from cells of the periodontium in vitro. The production of such potent immunomodulatory agents in vivo may contribute to the connective tissue breakdown characteristic of chronic periodontitis.     

RNA interference in human foreskin fibroblasts within the three-dimensional collagen matrix

The technique of RNA interference (RNAi) was trialed in primary human foreskin fibroblasts, both in monolayer culture and in the fibroblast-populated collagen matrix. Knockdown of lamin A/C, p53, and FAK was possible with low-confluency (<50%) monolayer fibroblasts, a transfection vehicle concentration of 1%, and an siRNA concentration of 25–50 nM. Knockdown also was possible in the collagen matrix using similar reagent concentrations and a cellular density of one million fibroblasts per ml of matrix. Optimization of transfection conditions appeared to be important to increase knockdown efficiency. Consistent with prediction, knockdown of FAK induced apoptosis in the fibroblast-populated collagen matrix.     

Interleukin-6 negatively regulates Porphyromonas gingivalis fimbria-stimulated fibronectin expression in human gingival fibroblasts

Our previous study demonstrated that fibronectin (FN) is a negative regulator of Porphyromonas gingivalis fimbria-induced pathogenesis in the initiation and development of chronic periodontal diseases. We show herein the regulatory action of interleukin-6 (IL-6) on FN expression in fimbria-treated human gingival fibroblasts. Interestingly, the decrease in FN expression in the cells treated with fimbriae at a high dose (8 microg of protein ml(-1)) was negated by treatment with anti-IL-6 antibody. Also, the increase in FN expression in cells treated with fimbriae at a low dose (1 microg of protein ml(-1)) was inhibited by exogenous IL-6. These results suggest that P. gingivalis fimbria-stimulated FN expression in human gingival fibroblasts is negatively regulated by endogenous IL-6.     

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.     

TWEAK/Fn14 signaling may function as a reactive compensatory mechanism against extracellular matrix accumulation in keloid fibroblasts

Overabundance of the extracellular matrix resulting from hyperproliferation of keloid fibroblasts (KFs) and dysregulation of apoptosis represents the main pathophysiology underlying keloids. TWEAK is a weak apoptosis inducer, and it plays a critical role in pathological tissue remodeling via its receptor, Fn14. However, the role of TWEAK/Fn14 signaling in the pathogenesis of keloids has not been investigated. In this study, we confirmed the overexpression levels of TWEAK and Fn14 in clinical keloid tissue specimens and primary KFs. The extracellular matrix (ECM)-related genes were also evaluated between primary KFs and their normal counterparts to determine the factors leading to the formation or development of keloids. Unexpectedly, exogenous TWEAK significantly reduced the levels of collagen I and collagen III, as well as alpha-smooth muscle actin (α-SMA). Additionally, TWEAK promoted MMPs expression and apoptosis activity of KFs. Furthermore, we verified that the inhibitory effect of TWEAK on KFs is through down-regulation of Polo-like kinase 5, which modulates cell differentiation and apoptosis. The TWEAK-Fn14 axis seems to be a secondary, although less effective, compensatory mechanism to increase the catabolic functions of fibroblasts in an attempt to further decrease the accumulation of collagen.Data AvailabilityAll data generated or analyzed during this study are included in this published article (and its Supporting Information files).     

Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin

The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase-digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Deposition of extracellular matrix along the pathways of migrating fibroblasts

Summary Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.     

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.     

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.     

Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts

The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.     

Clonal characterization of fibroblasts in the superficial layer of the adult human dermis

The dermis of adult human skin contains a physiologically heterogeneous population of fibroblasts that interact to produce its unique architecture and that participate in inflammatory and wound repair functions in vivo. This heterogeneity has been well documented for fibroblasts located in the superficial papillary dermis and the deep reticular dermis. However, the existence of diverse fibroblast subpopulations within a given region of the dermis has not been explored. In this study, fibroblast cultures have been established from the superficial dermis following enzymatic dissociation of the tissue. These fibroblasts have been cloned by limiting dilution and initially selected on the basis of morphology and proliferation kinetics. Fibroblasts in some of the clones selected for study express α-smooth muscle actin, a myofibroblast characteristic. Significant differences for fibroblast clones obtained from the same piece of skin have been observed with regard to their rate of collagen lattice contraction, their ability to organize a fibronectin matrix, their release of specific growth factors/cytokines into culture medium, and their response to interleukin-1α. These differences in both morphological and physiological characteristics indicate that the superficial papillary dermis contains a heterogeneous population of fibroblasts. This heterogeneity might indicate that diverse subpopulations of fibroblasts are required to interact in both homeostatic and pathological situations in skin. We thank L’Oréal Life Sciences for providing funding for these studies.     

Persistent effect of TGF-beta 1 on extracellular matrix gene expression in human dermal fibroblasts

TGF-beta, a potent inducer of the extracellular matrix, is also known to stimulate its own synthesis. In this report we have analyzed long term effects of TGF-beta 1 on its own expression and on the expression of extracellular matrix genes. We demonstrated that 24 hours of incubation of human dermal fibroblasts with TGF-beta 1 (1 ng/ml) under serum free conditions resulted in an elevated expression of TGF-beta 1, collagen alpha 2(I) and fibronectin mRNAs that persisted at least 96 hours after removal of TGF-beta 1. These data suggest the possibility of persistent in vivo activation of target cells following exposure to TGF-beta 1.     

Interleukin-6 does not support interleukin-2 induced generation of human lymphokine-activated killer cells

Summary The activity of lymphokine-activated killer (LAK) cells is supported by various cytokines. The objective of this study was to see if recombinant interleukin-6 (IL-6) either alone or in combination with interleukin-2 (IL-2) has any effect on the generation of LAK cells. Peripheral blood mononuclear cells of healthy donors were cultured for 4 or 6 days with both cytokines either alone or in combination. LAK activity against K562 and natural killer-resistant Daudi cells was assessed by a 4-h and an 18-h⁵¹Cr-release assay at various effector to target ratios. IL-6 alone in increasing concentrations did not induce LAK cell activity. Neither additive nor synergistic effects of IL-6 with IL-2 were observed. Immunofluorescence analysis with phycoerythrin-conjugated anti-CD56 antibody demonstrated that IL-6 could not maintain or increase the number of CD56-positive cells over a 6-day culture period. These results suggest that IL-6 does not support LAK cell generation by itself or increase LAK cell activity in combination with IL-2.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.