Isolation and initial characterization of human basement membrane collagens

Neutral solutions of pepsin extracted human collagens derived from glomeruli, kidney, aorta, lung, heart, bowel, spleen, skeletal muscle and skin were subjected to heat gelation at 37°C. Centrifugation of the gel provided two fractions: gelled pellet and non-gelled supernatant. Analysis of these two fractions by gel electrophoresis, molecular sieve and ion exchange chromatography, and amino acid and carbohydrate determinations indicated that the non-gelled supernatant contained a substantial enrichment of basement membrane like collagen. The initial characterization of lung derived basement membrane collagen indicated close similarities with those derived from glomeruli and whole kidney and differences with that obtained from the spleen.     

Collagen crosslinks: occurrence in basement membrane collagens

Basement membrane preparations from anterior lens capsule, Descemet's membrane, and renal glomerulus were reduced with NaB³H₄ in order to label carbonyl-derived crosslinks. Quantitative incorporation of tritium into the basement membranes was found, similar to the levels observed in fibrous collagens. A considerable proportion of the label was chromatographically identical with the reduced aldehydes and crosslinks of collagen, suggesting that is is the collagenous portions of basement membrane which contain these compounds. This was substantiated by showing that the isolated collagenous proteins contained substantial amounts of reduced aldehydes and crosslinks.     

The biosynthesis of proteoglycans and interstitial collagens by bovine pericardial fibroblasts

The biosynthesis of interstitial collagens (types I and III) and proteoglycans was studied in fibroblasts isolated from the parietal layer of bovine pericardium. Confluent cultures were labeled with Na2 35SO4 for proteoglycans or 14C-proline for collagens. The proteoglycans synthesized by pericardial fibroblasts were purified by DEAE-Sephacel chromatography and further fractionated into three components by gelfilitration. Two minor high molecular weight proteoglycans were shown by SDS-PAGE to be resistant to chondroitinase ABC and AC, and partially degraded by nitrous acid. The major, low molecular weight proteoglycan had a core protein of 45 kDa and is considered to be a dermatan sulfate/chondroitin sulfate proteoglycan since it was resistant to nitrous acid, but digested partially by chondroitinase AC and completely by ABC. The pericardial fibroblasts synthesized predominantly type I collagen and low amounts (about 10%) of type III collagen which was detected by delayed reduction on SDS-PAGE. The data show that pericardial fibroblasts synthesize the same macromolecules that can be extracted from the intact tissue and suggest that the proteoglycan may play a structural as well as physiological role.     

Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein

Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM- dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride- galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.     

Proteomic identification of collagens and related proteins in human fibroblasts

Summary. Fibroblasts are used for diagnosis of a series of metabolic diseases and are particularly suitable for the diagnosis of collagen disorders. We aimed to generate a skin fibroblast map that would be suitable for the concomitant determination of collagen and collagen-related proteins.A human skin fibroblast cell line was cultivated, homogenised, proteins extracted and subject to two-dimensional gel electrophoresis with subsequent in-gel-digestion of protein spots and mass spectrometrical identification (MALDI-TOF).Collagen alpha1 (I) chain precursor, collagen alpha1 (III) chain precursor, collagen alpha2 (VI) precursor and collagen modifying enzymes prolyl 4-hydroxylase alpha-2-subunit precursor, procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 and 2, protein disulfide isomerase ER-60 precursor and peptidyl-prolyl cis-trans isomerase were among the abundant proteins.The finding of collagen and collagen-related structures as well as the identification of other metabolic enzyme systems on one 2D gel may propose the use of this proteomic method for further characterization of collagen and collagen-related proteins or for preliminary screening of metabolic disorders.     

Oxidative damage in human gingival fibroblasts exposed to cigarette smoke

Cigarette smoke, a complex mixture of over 7000 chemicals, contains many components capable of eliciting oxidative stress, which may induce smoking-related disorders, including oral cavity diseases. In this study, we investigated the effects of whole (mainstream) cigarette smoke on human gingival fibroblasts (HGFs). Cells were exposed to various puffs (0.5-12) of whole cigarette smoke and oxidative stress was assessed by 2',7'-dichlorofluorescein fluorescence. The extent of protein carbonylation was determined by use of 2,4-dinitrophenylhydrazine with both immunocytochemical and Western immunoblotting assays. Cigarette smoke-induced protein carbonylation exhibited a puff-dependent increase. The main carbonylated proteins were identified by means of two-dimensional electrophoresis and MALDI-TOF mass spectrometry (redox proteomics). We demonstrated that exposure of HGFs to cigarette smoke decreased cellular protein thiols and rapidly depleted intracellular glutathione (GSH), with a minimal increase in the intracellular levels of glutathione disulfide and S-glutathionylated proteins, as well as total glutathione levels. Mass spectrometric analyses showed that total GSH consumption is due to the export by the cells of GSH-acrolein and GSH-crotonaldehyde adducts. GSH depletion could be a mechanism for cigarette smoke-induced cytotoxicity and could be correlated with the reduced reparative and regenerative activity of gingival and periodontal tissues previously reported in smokers.     

DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.     

Production of both interstitial and basement membrane procollagens by fibroblastic WI-38 cells from human embryonic lung

The synthesis and secretion of type IV procollagen, in addition to that of procollagen types I and III, was detected in cells derived from human embryonic lung (WI-38) by immunofluorescence, metabolic labeling, immunoprecipitation, collagenase digestion and the characteristic polypeptide sizes of both intact procollagen type IV chains and their initial pepsin-resistant fragments as determined by polyacrylamide gel electrophoresis. Locally obtained human embryonic lung cells secreted the same procollagens, but neither embryonic nor adult human skin fibroblasts were found to secrete type IV procollagen in amounts detectable by the same methods.     

Acid phosphatase activity of human gingival fibroblasts measured using a simultaneous coupling semi-permeable membrane technique

Summary Acid phosphatase activity has been measured in cultured human gingival fibroblasts using a validated histochemical simultaneous coupling semi-permeable membrane technique. The histochemical reaction was linear over a three hour incubation period and had a pH optimum of 5.0. The activity was not increased by prior exposure to hypotonic acetate buffer and was inhibited by fluoride and molybdate but not by formaldehyde. These results indicate that the semi-permeable membrane technique described may be used for observing and measuring acid phosphatase activity in cultured fibroblasts. From results obtained using inhibitors, it appears that in these cells most of the acid phosphatase observed is lysosomal. The absence of any activation of activity following pre-incubation with hypotonic buffer indicates that the method is not suitable for monitoring lysosomal membrane function.     

The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions

Mouse entactin derived from the extracellular matrix of M1536-B3 cells and from insect cells infected with a recombinant virus containing entactin sequences were shown to promote the attachment of mouse mammary tumor, human melanoma, and other cells. The cell attachment was inhibited by antibodies against mouse entactin but not by anti-fibronectin or anti-laminin antibodies. On a weight basis entactin was as effective as laminin in promoting the attachment of mouse mammary tumor cells. The attachment of cells to entactin was in part mediated by the integrin recognition RGD peptide sequence. This was demonstrated by the cell attachment properties of peptides derived from entactin which contained this sequence. Furthermore, the peptide RGDS could inhibit the attachment of mouse mammary tumor cells to entactin to approximately 60% of control. It is suggested that additional cell recognition sequences may be present in entactin. The direct binding of calcium ions to entactin was observed. It is probable that the binding sites reside in peptide sequences located toward the NH2 terminus region of entactin. This conclusion was supported by the demonstration that synthetic peptides, containing potential calcium binding sequences derived from entactin, bound calcium. In addition, a recombinant peptide containing the amino-terminal 330 amino acids of entactin also bound calcium ions. The significance of these properties of entactin is discussed.     

Alkyllysophospholipid influenced melanoma cell morphology is associated with decreased attachment to basement membrane.

The alkyllysophospholipid analog 1-0-octadecyl-2-0-methyl-3-phosphorylcholine (ET-18-OCH3) was examined for possible anti attachment effects on B16-F10 murine melanoma cells in vitro. At sub-lethal lipid concentrations B16-F10 cells were inhibited from attaching to reconstituted basement membrane (Matrigel) during a 45 min assay. This type of inhibition was also imparted by the isoprenoid farnesol but not by egg lysophosphatidylcholine (LPC) at concentrations up to 10 micrograms/ml. Both lipids were toxic to B16-F10 cells in the absence of bovine serum albumin (BSA), BSA (0.1%) completely protected the cells from lysis except when both lipids were combined as a mixture. Light and electron microscopy, as well as electronic sizing of cells, gave evidence of alkyllysophospholipid induced reduction in cell size which correlated well with attachment inhibition. The results suggest that alkyllysophospholipid induced reduction of cell surface area leads to inhibition of cell attachment to basement membrane which 8 with our experimental conditions, was not permanent since cells eventually attach within 24 h after treatment. The enhanced lytic effect the lysophospholipid imparts on the alkyl compound, in conjunction with the anti-attachment properties should be important areas for future research.     

Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro

Summary To study the role of phagocytosis in periodontal tissues, internalization of fibronectin-coated latex beads by Gin-1 fibroblast populations was investigated. Demonstration of phagocytosis by internalization of beads was confirmed by immunofluorescence microscopy, electron microscopy, and flow-cytometry. The percent of cells phagocytosing beads measured by flow-cytometry was negligible at 4° and 23°C, but increased to approximately 17% at 37°C. As measured by automated image analysis, the percentage of phagocytosing cells increased linearly from 8 to 22 with increasing fibronectin concentration of the incubation solution from 30 ng to 300 g/ml. Similar linear increases in the percentage of phagocytosing cells were observed when beads were incubated with cells for periods ranging from 2 h to 2 days. To examine the role of the Arg-Gly-Asp receptor in mediating phagocytosis, fibronectin-coated beads were first coated with either Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro peptides at concentrations of 0.125, 0.5, and 1 mg/ml, or with control vehicle, and then incubated with cells. Phagocytosis was completely blocked at 1 mg/ml of the Gly-Arg-Gly-Asp-Ser-Pro peptide, but the Gly-Arg-Gly-Glu-Ser-Pro peptide showed no significant inhibition compared to control values. Blocking antibodies to the cell attachment domain of the fibronectin molecule also reduced the percentage of phagocytosing cells significantly. The data show that these phagocytic assays are sensitive enough to detect the influence of incubation temperature and time, cellular heterogeneity, ligand type, and ligand concentration on the percentage of phagocytosing cells. Further, the mechanisms which determine internalization of fibronectin-coated beads rely in part on the initial binding of ligand to the Arg-Gly-Asp receptor present on fibroblasts.     

Sericin enhances attachment of cultured human skin fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.     

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).     

A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation

Recent advances in tissue engineering technology suggest its application in different medical fields, including periodontology. There are some reports of new non-enzymatic methods of isolating human gingival fibroblast for short-time cultivation in vitro to be used in autologous gingival augmentation. The aim of this study was to obtain a simple and established method of culturing human gingival fibroblasts. The authors developed a recurrent method that can be successfully used in autologous gingival augmentation.