Thomas Krieg

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.

Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis.

The role of transforming growth factor beta 2 (TGF-beta 2) in the pathogenesis of systemic sclerosis (SSc) was investigated by in situ hybridization of skin biopsies from six patients with SSc. Two patients with acute systemic lupus erythematosis (SLE), one with acute dermatomyositis (DM), and three healthy individuals were used as controls. TGF-beta 2 mRNA was found to be co-localized with pro alpha 1(I) collagen expression around dermal blood vessels in all patients with the inflammatory stage of SSc, whereas there was no expression of either gene in the dermis of patients in the fibrotic stage, the SLE patients or the normal controls. These findings provide evidence that TGF-beta 2 released by inflammatory cells around blood vessels may play a role in mediating the collagen gene disregulation in fibrosis.

Systemic scleroderma: Clinical and pathophysiologic aspects

Systemic scleroderma is a generalized disease of connective tissue involving mainly the skin, the gastrointestinal tract, the lungs, the heart, and the kidneys. It can be present in different forms, of which acroscleroderma, with limited cutaneous and extracutaneous involvement, and diffuse scleroderma within a more rapid progression are most characteristic. Circulating antibodies against antinucleolar antigens are present in most patients with systemic scleroderma. They are helpful for establishing a classification and for determining the prognosis of the disease; their involvement in the pathogenesis, however, is still unclear. Alterations of the blood vessels and induction of fibroblasts by potent mediators are thought to play an important role in the early phase of scleroderma. Therefore early diagnosis is required, which then can initiate vasoactive therapy. In patients with systemic scleroderma, who also suffer from additional myositis, interstitial lung diseases, or arthritis, anti-inflammatory treatment with prednisolone and azathioprine is suggested. Development and progression of fibrosis cannot yet be influenced sufficiently. Only D-penicillamine affecting cross-linking of collagen has been widely used in scleroderma and has some beneficial effect.

Collagenase gene expression in fibroblasts is regulated by a three-dimensional contact with collagen

Collagenase activity in fibroblasts is regulated by cytokines and the interaction with the extracellular matrix. In this study we demonstrate that fibroblasts cultured within a three‐dimensional collagen gel show a strong induction of collagenase gene expression. In addition to increased de novo synthesis most of the secreted enzyme was found to be activated leading to a high collagenolytic activity and complete degradation of collagen matrices after removal of fetal calf serum. Collagen I gene expression was found to be reduced under these conditions. These data suggest a specific modulation of cellular metabolism in response to contact with a three‐dimensional collagenous matrix resulting in the divergent regulation of collagen and collagenase.

Localization of collagen α1(I) gene expression during wound healing by in situ hybridization

The cellular localization of collagen alpha 1(I) chain gene expression during wound healing in rats was investigated using in situ hybridization. Activation of collagen gene expression was first found within fibroblastic cells around the deep layers of the granulation tissue as early as 16 h post wounding. Heavily labeled cells were also detected near the intact wound edge and around hair follicles. At day 6 intense alpha 1(I) collagen gene expression was found within most cells of the granulation tissue and after day 8 most of the activity was localized to cells directly underneath the epidermis. 26 d after the induction of the wounds hardly any alpha 1(I) collagen gene expression could be demonstrated, which indicates a close, time-dependent control of collagen synthesis during repair processes.

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin)

Amino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM‐40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM‐40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM‐40.

Serum and Urine Analysis of the Aminoterminal Procollagen Peptide Type III by Radioimmunoassay with Antibody Fab Fragments

A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.

Synergistic effect of tumor necrosis factor-α and interferon-γ on collagen synthesis of human skin fibroblasts in vitro

Abstract The effect of tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNFα reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFNγ affected only collagen mRNA levels. The combination of TNFα (10 ng/ml) and IFNγ (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNFα and IFNγ on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.

Disorder of collagen metabolism in a patient with osteogenesis imperfecta (lethal type): increased degree of hydroxylation of lysine in collagen types I and III

Abstract. Types I, II and III collagen were isolated from calvarium, skin and cartilage from a patient with recessive lethal osteogenesis imperfecta. The distribution of the various collagen types was normal in all three tissues. The α‐chains were purified by molecular sieve and ion‐exchange chromatography and were found to differ from the corresponding α‐chains of age‐matched controls only in that the α1(I), α2 and α1 (III) chains contained higher amounts of hydroxylysine with proportionally less lysine. α1(II) was normal. The excess hydroxylysine residues were all glycosylated in the case of α1(I) chains, but only partly so for the α2 chains. Similar observations were made with collagen from fetuses at various stages of development. In these fetuses, however, the increase in the degree of hydroxylation of lysine in αl(I), α2 and αl(III) varied with age, being highest in the youngest fetus. Seen in the context of embryonic development, the collagen of the patient would correspond to that of a fetus younger than 18 weeks, and one could speculate that the defect seen in this patient is the result of a disturbed process of maturation of connective tissue.

Immunofluorescent localization of type IV collagen and laminin in human skin and its application in junctional zone pathology

Purified antibodies against type IV collagen and laminin were used to localize basement membranes by indirect immunofluorescence in various anatomical regions of normal and diseased human skin. The two proteins showed extensive codistribution. A continuous linear staining was found along the epidermal‐dermal junction and around hair follicles, sebaceous gland acini and small capillaries. The same proteins also surrounded individual cells such as those found in vessels, hair erector muscles and subcutaneous tissue.