Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

低氧促进大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ胶原mRNA表达

目的:研究低氧(2%氧)对成年Wistar大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ型胶原前a肽链表达的影响.方法:分离培养成年Wistar大鼠心脏成纤维细胞,采用液体闪烁计数方法检测心脏成纤维细胞的DNA合成速率,采用原位杂交技术检测Ⅰ、Ⅲ型胶原前α肽链mRNA的表达.结果:成年Wistar大鼠心脏成纤维细胞在低氧第6h、12 h时3H-TdR掺入量较常氧组显著增加,分别增加34%(P＜0.05)和36%(P＜0.01);低氧第4 h、8 h、12 h Ⅰ型胶原前α肽链mRNA表达显著高于常氧培养的细胞;低氧第2 h,Ⅲ型胶原前α肽链mRNA表达显著高于常氧培养的细胞.结论:低氧能够直接促进体外培养的成年Wistar大鼠心脏成纤维细胞DNA合成和Ⅰ、Ⅲ型胶原前α肽链表达,提示低氧对心脏成纤维细胞生长和胶原表达的直接调节可能是低氧性心肌纤维化的重要机制.     

Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts

The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e., with 10% newborn calf serum. The selective decrease was first observed after 6 h exposure to 1 μM cortisol. In depleted medium conditions, i.e., with 2% newborn calf serum, the initial response was a stimulatory one, followed after about 12 h by a decrease in the procollagen mRNA activity. The results suggest that the selective inhibitory effect of cortisol on the cellular concentration of translatable procollagen mRNA species needs an optimal serum concentration. Furthermore, the results give support to the hypothesis that the decrease in the procollagen mRNA concentration after cortisol administration is a secondary response, preceeded by the induction of some intracellular regulation system.     

Effects of HEMA on type I collagen protein in human gingival fibroblasts

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations on specific proteins such as type I collagen has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen protein. Primary lines of human gingival fibroblasts were exposed to 3 mmol/L HEMA for different periods of time (24 h, 72 h, 96 h). The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. The presence and localization of type I collagen was determined by immunofluorescence in HGFs treated with HEMA for the same period of time. The vitality of the cells decreased after 72 h of exposure. The HGFs grown in monolayer and observed by field emission in-lens scanning electron microscopy demonstrated a preserved surface morphology after 24 h of treatment, while they showed an altered morphology after 96 h of treatment. Immunofluorescence demonstrated a reduction of type I collagen due to HEMA exposure after 96 h. From these results, we conclude that low concentrations of HEMA can significantly alter the morphology of human gingival fibroblasts and interfere with the presence of type I collagen protein.     

Effect of electromagnetic stimulation with different waveforms on cultured chick tendon fibroblasts

An energy efficient electromagnetic stimulator device for fracture healing was compared to a commercially available device in stimulating cell growth in tissue cultures. The energy efficient device, which conserves energy by using a bidirectional time-dependent magnetic wave form, and the commercially available stimulator, which uses a unidirectionaltime-dependent magnetic wave form, were tested on chick tendon fibroblasts in primary culture. Comparing non-stimulated control and cells electromagnetically stimulated with unidirectional and bidirectional waveforms showed that at the growth phase between days 2 and 3, both electrical stimulation techniques increased cell division as measured by DNA synthesis. When cells were dividing rapidly, collagen synthesis was reduced. When the cells reached the confluence there was no difference among the groups (control, unidirectionally stimulated, and bidirectionally stimulated) in terms of number of cells or collagen produced. © 1994 Wiley-Liss, Inc.     

Effects of density and cellular aging on collagen synthesis and growth kinetics in keloid and normal skin fibroblasts

Summary Cell proliferation and collagen synthesis were analyzed in high, medium, and low density keloid and normal skin fibroblasts and also at various times during the course of in vitro aging to expand previous findings of increased collagen synthesis in keloid compared to normal skin fibroblasts. It was found that both keloid and normal fibroblasts (<20 population doublings) responded similarly to high, medium, and low initial plating densities; however, sparsely plated keloid fibroblasts exhibited a loss of replicative capacity earlier in their in vitro lifespan than did sparsely plated normal skin fibroblasts. When analyzed at population doubling levels 2 to 38, collagen synthesis was elevated in keloid compared to normal skin fibroblasts but decreased at the same rate in both cell types throughout this in vitro interval. Supported by NIH Grant GM-20298.     

Development of cell-based immunoassays to measure type I collagen in cultured fibroblasts

Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new cell-based assays to directly quantify the amount of collagen I incorporated into the extracellular matrix of primary human lung fibroblasts. Utilizing a monoclonal antibody specific to native human collagen I, we optimized conditions and parameters including incubation time, specificity and cell density to demonstrate dose-dependent induction of collagen I by transforming growth factor beta, as measured by in-cell enzyme linked immunosorbent assay. The results obtained by this assay were mimicked by an “In situ Quantitative Western Blot” on cultured cells using the same antibody. Results from these assays were comparable to those obtained with a commercial assay for collagen I N-propeptide, which is an index of collagen formation. These assays have been optimized for a 96-well format and provide a novel and useful approach for screening of anti-fibrotic agents in vitro. The assays described here also offer a significant improvement in throughput and specificity over conventional methods that primarily measure soluble collagen.     

Collagen synthesis by cultured skin fibroblasts from siblings with hydroxylysine-deficient collagen

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both α1(I) and α2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal and mutant cells is severely depressed without ascorbate but in all cultures collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by α,α′-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.     

Comparative effects of four surfactants on growth,contraction and adhesion of cultured human fibroblasts

A range of surfactants, including the anionic sodium dodecyl sulfate, the cationic cetyltrimethylammonium bromide and the nonionics octylphenoxy polyethoxyethanol (Triton X100) and polyoxyethylene 20 sorbitan monoleate (Tween 80) was studied for effects on proliferation, contractibility and attachment of cultured human fibroblasts. Only ionic surfactants exhibited a stimulatory effect on fibroblast proliferation, whereas all the surfactants tested increased the contraction of collagen gels containing fibroblasts, with the greatest effect from the non-ionic surfactants. This activity was not correlated with an increase of cell population or cell attachment within the collagenous matrix. The activity of the surfactants was seen only at levels close to their LD₅₀ values and in a narrow range of concentrations. Thus, we consider that they are the result of the so-called hormesis phenomenon.Abbreviations CTAB cetyltrimethyl-ammonium bromide - FCS fetal calf serum - LD₁₀₀ dose lethal to 100% of exposed - MCD maximal contraction dose - PDL population doubling level - SDS sodium dodecyl sulfate     

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

Synergistic enhancement of type I and III collagen production in cultured fibroblasts by transforming growth factor-β and ascorbate

Transforming growth factor-β (TGF-β) is a prototype of a family of polypeptides that regulates cellular growth and phenotypic differentiation [(1986) Science 233, 532-534; (1987) Cell 49, 437-438]. TGF-β injection induces angiogenesis and fibrosis locally [(1986) Proc. Natl. Acad. Sci. USA 83, 4167-4171; (1987) Science 237, 1333-1336] and stimulates the synthesis of extracellular matrix proteins, fibronectin, collagens, and proteoglycans in vitro in many cell types [(1986) J. Biol. Chem. 261, 4337-4345; (1987) Biochem J. 247, 597-604]. Ascorbate is also known to induce collagen synthesis and to promote wound healing [(1988) J. Invest. Dermatol. 90, 420-424; (1986) Coll. Rel. Res. 6, 455-466]. We report that in cultured human skin fibroblasts, ascorbate and TGF-β synergistically enhance the biosynthesis of type I and III collagens and their steady-state mRNAs. TGF-β alone has no enhancing effect on type III collagen synthesis. The cooperation between ascorbate and TGF-β may be of significance in wound healing and in disorders of fibrosis.