Collagen types synthesized in dermal fibroblast cultures from patients with early progressive systemic sclerosis

Buckingham, Robert B.; Prince, Robert K.; Rodnan, Gerald P.; Miller, Edward J.

doi:10.1002/art.1780230209

Cited by 61 publications

(24 citation statements)

References 16 publications

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“…Fibroblasts, either derived from cords of Dupuytren's disease (DF) or control tissues (CF), were isolated by using the explant culture technique as described [14] and grown in DMEM (Glucose 1.0 g/l, NaHCO 3 3.7 g/l, NAcetyl-L-alanyl-L-glutamine 1.0289 g/l, BIOCHROM) supplemented with 10% FCS, 40 µg/ml Gentamycin (GIBCO/BRL) without additional growth factors in 75 cm 2 culture flasks. All cultures were maintained at 37°C, 5% CO 2 in a humidified atmosphere.…”

Section: Cell Culturementioning

confidence: 99%

N‐Acetyl‐L‐Cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF‐β signalling

Kopp¹,

Seyhan²,

Müller³

et al. 2006

J Cellular Molecular Medi

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Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-β 1 (TGF-β 1 ) and its downstream Smad signalling system is well established as a key player during fibrogenesis. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies. One such promising treatment involves the substance N-acetyl-L-cysteine (NAC), shown to have antifibrotic properties in hepatic stellate cells and rat fibroblasts. In order to investigate antifibrotic effects of N-acetyl-L-cysteine (NAC), fibroblasts were isolated from surgically resected fibrotic palmar tissues (Dupuytren fibroblasts, DF) and exposed to different concentrations of NAC and recombinant TGF-β 1 . Fibroblasts isolated from tendon pulleys served as controls (control fibroblasts, CF). Smad signalling was investigated by a Smad binding element driven reporter gene analysis. Both cell types express TGF-β 1 , indicating autocrine signalling in DF and CF. This was confirmed by comparing reporter gene activity from LacZ and Smad7 adenovirus infected cells. NAC treatment resulted in abrogation of Smad mediated signalling comparable to ectopically overexpressed Smad7, even when the cells were stimulated with recombinant TGF-β 1 or ectopically expressed a constitutively active TGF-β receptor type I. Additionally, NAC dose-dependently decreased expression of three major indicators of impaired fibrotic matrix turnover, namely alpha-smooth muscle actin (α-SMA), α 1 type I procollagen (CollA1), and plasminogen activator inhibitor-type I (PAI-1). Our results suggest that TGF-β signalling and subsequent expression of fibrogenesis related proteins in Dupuytren's disease is abrogated by NAC thus providing a basis for a therapeutic strategy in Dupuytren's disease and other fibroproliferative disorders.

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Section: Cell Culturementioning

confidence: 99%

N‐Acetyl‐L‐Cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF‐β signalling

Kopp¹,

Seyhan²,

Müller³

et al. 2006

J Cellular Molecular Medi

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“…More than a decade ago, it was observed that fibroblasts from involved skin of patients with scleroderma synthesized increased levels of collagen in vitro (1,2); today the mechanism remains unknown (3)(4)(5)(6). During this period, observations ofthe pathogenesis of scleroderma have focused on vascular and microvascular lesions; on evidence of endothelial damage; on platelet adhesion, aggregation, and release; and on the subsequent activation both of smooth muscle cells to produce the intimal proliferation and of interstitial fibroblasts to produce fibrosis (7)(8)(9)(10).…”

mentioning

confidence: 99%

Replication and phenotypic expression of control and scleroderma human fibroblasts: responses to growth factors.

LeRoy¹,

Mercurio²,

Sherer³

1982

Proc. Natl. Acad. Sci. U.S.A.

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To explore the mechanism of increased collagen synthesis by scleroderma skin fibroblasts in vitro, control and scleroderma fibroblasts were compared in confluent monolayer cultures growth-arrested by serum deprivation; responses to optimal mitogenic doses of platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and nerve growth factor were compared. Plateletderived growth factor had a selective mitogenic effect on control skdn fibroblasts not observed with scleroderma skin fibroblasts. None of the factors studied had a selective effect on collagen synthesis independent of cell replication; scleroderma and control fibroblasts responded similarly. Therefore, the growth factors studied may not be involved in generating the activated scleroderma fibroblast directly; platelet-derived growth factor may play an indirect role in fibroblast replication in human fibrotic disorders.More than a decade ago, it was observed that fibroblasts from involved skin of patients with scleroderma synthesized increased levels of collagen in vitro (1, 2); today the mechanism remains unknown (3-6). During this period, observations ofthe pathogenesis of scleroderma have focused on vascular and microvascular lesions; on evidence of endothelial damage; on platelet adhesion, aggregation, and release; and on the subsequent activation both of smooth muscle cells to produce the intimal proliferation and of interstitial fibroblasts to produce fibrosis (7-10). A unifying hypothesis would propose the presence offactors released from plasma or platelets which act as stimuli to fibrosis; among such factors, the potent mitogen, plateletderived growth factor (PDGF), is released during endothelial injury and platelet adhesion to the subendothelium (11). Therefore, the influence of PDGF on skin fibroblast collagen synthesis was studied.Endothelial damage and platelet interaction with the exposed subendothelium, resulting in release of platelet-derived mitogenic factors (including PDGF), have been proposed, at least indirectly, to play a role in the evolution of the scleroderma vascular lesion. Events preceding endothelial injury are poorly understood; altered immunity leading to humoral or lymphocyte/monocyte mediators ofinjury has been proposed with supporting evidence (12). If PDGF is the direct mediator of fibroblast activation in scleroderma, the enhanced collagen synthesis of scleroderma fibroblasts should be accompanied by increased cell replication at some stage in the process. Thus, a PDGF mechanism in scleroderma would imply replication offibroblast populations instead of, or in addition to, the selective stimulation ofcollagen synthesis per cell; in earlier studies, increased collagen synthesis per cell was observed in vitro without evidence of increased cell replication (1, 2). If PDGF is the mediator of fibroblast activation in scleroderma, its action should account for the initial observations of increased collagen synthesis per cell.A second characteristic of scleroderma fibroblasts in culture has been ...

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“…Additional evidence supporting the concept that the collagen found in CHL cell layers is composed of B chains and information about the localization of this material in culture was obtained by staining CHL cell layers with affinity-purified antibody specific for the B chain and molecules containing B chains (20,21). The majority of the immunoreactive material synthesized by CHL cells was localized in an extracellular matrix surrounding the cells; only a small amount of intracellular staining was observed (Fig.…”

Section: Resultsmentioning

confidence: 64%

“…Immunohistochemical staining of air-dried CHL cell layers was performed by using a specific antibody prepared against the human B chain as described (20,21).…”

mentioning

confidence: 99%

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

Haralson¹,

Mitchell²,

Rhodes³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (>90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human a2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell ayer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collaren to localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form o collagen has an unusual affinity for the cell layer in this system.The class of proteins collectively referred to as collagen represents various distinct gene products. The synthesis of these proteins is somewhat tissue specific and is altered in pathological conditions (1, 2). At present, at least nine genetically distinct chains have been described as the primary constituents of various collagen molecules. Four collagen chains-the al(I), a2(I), al(II), and al(III) chains-comprise the interstitial collagens (1). Recent evidence indicates that five additional unique collagen chains are present in collagen molecules in basement membrane-like structures and in basement membranes. These are the aA and aB chains (3-6) and aC chain (7) isolated from highly vascularized tissue, and two additional chains obtained from lens capsule (8-10) and human placenta (11-13). However, little is currently known about the mechanisms that control the differential expression of the collagen genes. This paucity of information reflects, at least in part, the limited number of systems available for studying the regulation and dissecting the genetics of mammalian procollagen biosynthesis.This (5, 16). The origin of the CHL cell line as well as the clone (HT1) used in these studies has been described (14,17).Growth of Cells in Culture and Metabolic Labeling. CHL cells were maintained and grown in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum as described (14,15). For immunological studies, cells were grown in 15 X 60 mm dishes at 370C under a 10% C02/90% air atmosphere to t90% confluency. The medium was removed, the cell layer was washed with three 5-ml portions of cold phosphate-buffered saline, and the cells...

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Collagen types synthesized in dermal fibroblast cultures from patients with early progressive systemic sclerosis

Abstract: Progressive systemic sclerosis (PSS) is a generalized disease characterized by inflammatory, fibrotic, and degenerative changes of connective tissue (1). The

Cited by 61 publications

References 16 publications

N‐Acetyl‐L‐Cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF‐β signalling

N‐Acetyl‐L‐Cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF‐β signalling

Replication and phenotypic expression of control and scleroderma human fibroblasts: responses to growth factors.

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

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