Influence of cell density on collagen biosynthesis in fibroblast cultures

Aumailley, Monique; Krieg, Thomas; Razaka, G; Müller, Peter; Bricaud, H

doi:10.1042/bj2060505

Cited by 64 publications

(27 citation statements)

References 42 publications

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“…This phenomenon can partly be explained by dif ferences in the clinical state of the disease, the anatomical biopsy site, sex and age differences between subjects and by variations in the experimental setting [15,18,19]. It was suggested that fibroblasts from scleroderma lesions may exhibit different susceptibilities to endogenous growth fac tors and that withdrawal of such factors upon culturing would result in removal of the stimulus and so could lead to a disappearance of the activated state of the fibroblasts [20, 21 j.…”

Section: Discussionmentioning

confidence: 99%

“…Although Dobak et al [28] recently reported an increased collagen synthesis upon exposure to l,25(OH)2D3, they used subconfluent cultures and examined both proliferation and collagen synthesis simultaneously. We used confluent cultures, as it was sug gested that the relative rate of collagen synthesis was higher in quiescent than in proliferating cultures [19], Further more, in confluent cultures calcitriol did not affect cell numbers. In addition, the behaviour of normal and sclero derma fibroblasts incorporated into collagen lattices was not significantly affected in the presence of calcitriol.…”

Section: Discussionmentioning

confidence: 99%

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Effects of Calcitriol on Fibroblasts Derived from Skin of Scleroderma Patients

1995

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Background: Scleroderma is a fibrotic disorder of unknown etiology that is characterized by excessive collagen synthesis and its deposition in the skin and various internal organs. Objective: To examine whether an overproduction of extracellular matrix molecules is a result of either increased fibroblast proliferation or increased collagen synthesis. As results of clinical trials with 1,25-dihydroxyvitamin D₃ (calcitriol) have suggested beneficial effect in the treatment of scleroderma patients, the effects of calcitriol on fibroblasts derived from scleroderma and normal skin has been examined as well. Methods: Cultures of fibroblasts were established from biopsies from involved and uninvolved skin of scleroderma patients and from skin of healthy subjects, and compared with respect to proliferation, collagen synthesis and collagen lattice contraction. Results: No significant differences in cell proliferation and in the extent of fibroblast-induced collagen lattice contraction have been found between scleroderma and normal fibroblasts. Morphologically, fibroblasts derived from scleroderma patients exhibited a disorganized growth pattern in a monolayer culture in contrast to normal fibroblasts. Collagen synthesis tends to be higher in scleroderma fibroblasts as compared with controls. Calcitriol exerted an antiproliferative and antisynthetic effect on fibroblasts, which, however, did not discriminate healthy fibroblasts from fibroblasts derived from involved or uninvolved scleroderma plaques. Conclusions: Our findings suggest that collagen accumulation may not result from increased proliferation or altered dynamic properties of fibroblasts in a scleroderma lesion but from increased collagen biosynthesis. We additionally found that calcitriol does not selectively affect scleroderma fibroblasts.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Effects of Calcitriol on Fibroblasts Derived from Skin of Scleroderma Patients

1995

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“…Human embryonic skin fibroplasts [17] and the human fibrosarcoma cells HT-1080 [IS] were those used in previous studies. Smooth muscle cell cultures were established from human aorta and showed the characteristics described elsewhere for rabbit smooth muscle cells [19].…”

Section: Cell Rulturrsmentioning

confidence: 99%

Immunochemistry, genuine size and tissue localization of collagen VI

Mark

Aumailley

Wick

et al. 1984

European Journal of Biochemistry

265

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Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple-helical and globular structures. Antisera prepared against a constituent chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross-react with other collagen types I -V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide-bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers.Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross-striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.Collagens represent a family of related proteins found in the extracellular space. They include collagens I, I1 and 111, which are abundant in most connective tissues and organized in the form of distinct fibrils with diameters up to 100 nm. Collagen IV is unique to basement membranes, where it forms network-like structures. Only little is known about the macromolecular organization and ultrastructural localization of collagen V [I ,2]. A new collagenous protein, now referred to as collagen VI [2 -41, was originally discovered in pepsin extracts of aortic intima [5]. The pepsin fragment consisted of triplehelical and non-collagenous domains, which are formed from two or three constituent polypeptide chains [4 -81. Electronmicroscopical and physical studies [?,] demonstrated as the basic unit a monomer with M,= 170000 composed of a 105-nm-long triple helix and two globules located at both ends of the triple helix. These monomers are associated into overlapping dimers, tetramers and larger filaments, which are cross-linked by disulfide bridges. This peculiar macromolecular assembly suggested that collagen VI is a component of microfibrillar structures [3]. The function of collagen VI in tissues is, however, not yet understood.Well-characterized antibodies, specific for the various types of collagens, were important tools in previous studies on the structure, tissue localization and biological functions of these proteins [9 -111. We have now prepared antibodies against pepsin-solubilized collagen VI and have characterized their specificity. These antibodies we...

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“…Although Aumailley et al [16] reported that proliferating human skin fibroblasts synthesized more radiolabeled peptide-bound hydroxyproline per cell than did confluent cultures, their data were not corrected for the specific activity of intracellular proline and it has been shown, in HFL-1 cells, that proline-specific activity is higher in proliferating than in confluent cells [2]. A disparity, therefore, exists in view of the good correlation between collagen mRNA levels and collagen synthesis on the one hand and the lack of correlation between total poly(A)-rich RNA and protein synthesis on the other.…”

Section: Discussionmentioning

confidence: 99%

Regulation of type I collagen mRNA levels in fibroblasts

Voss

Börnstein

1986

Eur J Biochem

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Type 1 procollagen mRNA levels, as well as total RNA and poly(A)-rich mRNA, remain constant when rapidly growing human fetal lung fibroblasts (HFL-1 cells) are compared with quiescent cells. Polysome profiles of cells in both growth states revealed that the distribution of type I collagen mRNA in the mRNP fraction and in polysomes also remained constant even though total RNA and poly(A)-rich mRNA were shifted from polysomes to the mRNP pool in resting cells. Similar results were obtained when RNA fractions in polysomes associated with the cytoskeletal framework were examined. It is known that procollagen production is unaffected by the growth state of cells [Breul, S. D., Bradley, K. H., Hance, A. J., Schafer, M. P., Berg, R. A. and Crystal, R. G. (1980) J . Biol. Chem. 255, 5250-52601 although total protein synthesis is markedly decreased in resting cells. It would therefore appear that the translational control responsible for reduced synthesis of non-collagenous proteins in resting cells does not extend to procollagen and that transcriptional control can account for levels of type I procollagen produced by cultured human fibroblasts.

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Influence of cell density on collagen biosynthesis in fibroblast cultures

Cited by 64 publications

References 42 publications

Effects of Calcitriol on Fibroblasts Derived from Skin of Scleroderma Patients

Effects of Calcitriol on Fibroblasts Derived from Skin of Scleroderma Patients

Immunochemistry, genuine size and tissue localization of collagen VI

Regulation of type I collagen mRNA levels in fibroblasts

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