Jürgen Rauterberg

In vitro differentiation of fat-storing cells parallels marked increase of collagen synthesis and secretion

Fat-storing cells were isolated and purified from livers of normal adult rats and maintained in primary culture. By light and electron microscopy it was established that they underwent phenotypic changes into cells with the ultrastructural characteristics of myofibroblasts, between the third and sixth day in culture. These morphological changes were accompanied by a 2-fold increase of L-[3H]proline incorporation into secretory proteins and an 11-fold increase into secreted collagenase-sensitive proteins. In contrast, incorporation into cell layer-associated proteins and into cell layer-associated collagenase-sensitive proteins was not significantly elevated. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with fluorography, demonstrated that the main collagen type secreted by the myofibroblast-like cells was collagen type I. Collagen types III and IV, and fibronectin were present in lesser amounts. The similarity between the well known in vivo alterations of fat-storing cells under pathological conditions and the spontaneous in vitro differentiation described in this study, makes primary cultures of fat-storing cells a valuable tool for studying their role in chronic liver disease.

Rupture of the Atherosclerotic Plaque. Does a Good Animal Model Exist

Abstract—By its very nature, rupture of the atherosclerotic plaque is difficult to study directly in humans. A good animal model would help us not only to understand how rupture occurs but also to design and test treatments to prevent it from happening. However, several difficulties surround existing models of plaque rupture, including the need for radical interventions to produce the rupture, lack of direct evidence of rupture per se, and absence of convincing evidence of platelet- and fibrin-rich thrombus at the rupture site. At the present time, attention should therefore focus on the processes of plaque breakdown and thrombus formation in humans, whereas the use of animal models should probably be reserved for studying the function of particular genes and for investigating isolated features of plaques, such as the relationship between cap thickness and plaque stability.

Primary cultures of rat hepatocytes synthesize fibronectin.

Summary Fibronectin was detected by indirect immunofluorescence on primary cultures of rat hepatocytes maintained in the presence or absence of fetal calf serum. [ 14 C]-Fibronectin synthesized in the presence of [ 14 C]-glycine was isolated by immunoprecipitation and visualized by fluorography.

Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque

Type VIII collagen is a short‐chain collagen that is present in increased amounts in atherosclerotic lesions. Although the physiological function of this matrix protein is unclear, recent data suggest an important role in tissue remodeling. Type VIII collagen in the atherosclerotic lesion is mainly derived from smooth muscle cells. We now show that macrophages in the atherosclerotic vessel wall and monocytes in adjacent mural thrombi also express type VIII collagen. We demonstrated this using a novel combined fluorescence technique that simultaneously stains, within the same tissue section, specific RNAs by in situ hybridization and proteins by indirect immunofluorescence. In culture, human monocyte/macrophages expressed type VIII collagen at all time points from1 h to 3 wk after isolation. Western blotting and immunoprecipitation also revealed secretion of type VIII collagen into the medium of 14‐day‐old macrophages. Because this is the first report of secretion of a collagen by macrophages, we tested the effect of lipopolysaccharide (LPS) and interferon γ, substances that stimulate macrophages to secrete lytic enzymes, on macrophage expression of type VIII collagen. LPS and interferon γ decreased expression of type VIII collagen. By contrast, secretion of matrix metalloproteinase 1 (MMP 1) was increased, indicating a switch from a collagen‐producing to a degradative phenotype. Double in situ hybridization studies of expression of type VIII collagen and MMP 1 in human coronary arteries showed that in regions important for plaque stability, the ratio of MMP 1 RNA to macrophage type VIII collagen RNA varies widely, indicating that the transition from one phenotype to the other that we observed in vitro may also occur in vivo.—Weitkamp, B., Cullen, P., Plenz, G., Robenek, H., Rauterberg, J. Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque. FASEB J. 13, 1445–1457 (1999)

Localization of collagen types I, III, IV and V, fibronectin and laminin in human arteries by the indirect immunofluorescence method.

The distribution of types I, III, IV and V collagen and of the glycoproteins fibronectin and laminin in sections of human aortas, arteries and atherosclerotic plaques were studied using monospecific antibodies and indirect fluorescence microscopy. Types IV and V collagen and laminin were present in a narrow zone, representing the basement membrane, apposed to the endothelial layers of all these tissues. Types I and III collagen and fibronectin were located in the interstitial spaces of the intima and the media of blood vessels walls, whereas types IV and V collagen and laminin were found in the basement membranes underlying smooth muscle cells in these areas. Two types of atherosclerotic plaques were observed. Lipid-rich plaques contained less collagen and reduced amounts of the glycoproteins. Fibrous plaques consisted of regions deficient in types I and III collagen and collagen-rich regions with elevated levels of these two collagens as well as more fibronectin. The collagen-rich regions of fibrous plaques contained, however, little type IV and type V collagen and little of the glycoproteins laminin and fibronectin. This may be due to the reduced number of cells involved in the biosynthesis of these basement membrane proteins.

Distribution of collagen type I and type III and of two collagenous components of basement membranes in the human liver.

The distribution of collagen type I and III and of the basement membrane collagens type IV and alphaA (alphaB)2 in normal and fibrotic human livers has been studied by indirect immunofluorescence using type specific antibodies. Collagens type I as well as type III are present in normal livers in the blood vessel walls and the wall of biliary ducts of the portal tracts. In the parenchyma both types are visible along the sinusoids. Basement membrane collagens are only present in the connective tissue of portal canals and of central veins whereas in the parenchyma both type IV and alphaA (alphaB)2-collagen are nearly absent. In the tissue of fibrotic livers basement membrane collagens are also found in the parenchyma in similar distribution as type I and III.

Collagen synthesis in cultured aortic smooth muscle cells. Modulation by collagen lattice culture, transforming growth factor-beta 1, and epidermal growth factor.

We investigated the effects of transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the protein synthesis and production of collagen in cultured smooth muscle cells (SMCs) from the aortic media of pigs. SMCs were cultured as monolayers on plastic as well as in three-dimensional collagen lattices to gain some information about the influence of a preexisting collagenous matrix on the growth factor-induced effects. A 48-hour exposure of SMCs to TGF-beta 1 at concentrations of 5 ng/ml in the presence of 1% serum caused a marked enhancement of the production of collagen and noncollagen proteins. The rate of net collagen production by SMCs exposed to TGF-beta 1 was approximately threefold higher than that of control cells. Moreover, TGF-beta 1 specifically stimulated collagen synthesis, resulting in a greater proportion of collagen in total proteins synthesized compared with controls. The preexisting matrix of collagen lattices affects the response of SMCs to TGF-beta 1 and EGF. In monolayer cultures the collagen proportion increased twofold under the influence of TGF-beta 1, whereas in collagen lattices the specific stimulation of collagen synthesis was lower. We found that EGF enhanced TGF-beta 1-induced protein production in collagen lattices but not in monolayer cultures. In addition, the protein production by SMCs was influenced differently by EGF in these culture systems. Taken together, these data show a mutual influence of growth factors and extracellular matrix components on collagen production in SMCs, thus indicating that TGF-beta 1 may be an important pathophysiological regulator of collagen metabolism in the vessel wall.

Type-VI collagen in the human iris and ciliary body.

SummaryThe distribution of type-VI collagen in the human iris and ciliary body was investigated by means of immunohistochemical techniques and compared with that of type-IV collagen, fibronectin and laminin. As has been described for other tissues, type-VI collagen surrounds type-I and-III collagen fibers. The aggregated from of type-IV collagen (the “long-spacing” or “curly” collagen), which has already been described in the trabecular meshwork and sclera, was also observed at the ciliary muscle tips surrounding the anterior elastic tendons of this muscle. In addition, staining for type-VI collagen was seen directly adjacent to the basement membranes of the ciliary muscle cells, the iris muscles, the uveal vascular endothelia and nerves, but not adjacent to the epithelial basement membranes. The staining did not form a discrete line like the immunoreaction for type-IV collagen, but bundles of marked fibrils extended into the surrounding connective tissue. We assume that type-VI collagen similar to type-VII collagen forms part of an anchoring system for these tissues. As type-VII collagen has been described only in connection with epithelial basement membranes, both type-VI and type-VII collagens may represent anchoring fibrils, however for different tissue components.

The primary structure of a triple-helical domain of collagen type VIII from bovine Descemet's membrane

We have isolated and sequenced a fragment of 469 amino acid residues from bovine type VIII collagen. The sequence was composed of a series of Gly‐X‐Y repeats which was interrupted 8 times by short imperfections. The number and relative location of these interruptions were similar to those of chicken α1(X) and rabbit αl(VIII) chain triple‐helical domains. Comparison to published N‐terminal sequences to two triple‐helical fragments of bovine type VIII collagen and to the cDNA derived sequence of the rabbit αl(VIII) chain showed that this fragment was the triple‐helical domain of a second type VIII collagen chain which we designate α2(VIII).

Responsiveness of aortic smooth muscle cells to soluble growth mediators is influenced by cell-matrix contact.

Excessive proliferation and overexpression of collagens by smooth muscle cells (SMCs) are important features of atherogenesis. To understand the role of the extracellular matrix in the regulation of these processes, we examined proliferation and protein/collagen synthesis of SMCs in contact with a collagen matrix. Adult pig SMCs were isolated from the aortic media by collagenase digestion, subcultured as monolayers, and then embedded into a three-dimensional network of type I collagen, ie, a collagen lattice. Cells were subsequently exposed to growth-promoting media, and their behavior was observed in comparison with monolayer cultures on plastic. Treatment of monolayers with increasing concentrations of fetal calf serum resulted in activation of the cell cycle, onset of cell proliferation, and increased protein/collagen synthesis. In contrast, similar treatment of collagen lattice-cultured SMCs failed to influence cell proliferation and protein/collagen synthesis. However, stimulation of proliferation of lattice-cultured SMCs by platelet-derived growth factor-A/B was feasible; nevertheless, the rate of proliferation was modest compared with monolayers. In addition, the onset of proliferation was accompanied by a decrease in collagen synthesis of the cells. Thus, a collagenous matrix appears to suppress the responsiveness of SMCs to soluble growth mediators. It is speculated that interactions between SMCs and the extracellular matrix may modify proliferation and protein/collagen synthesis of cells not only in vitro but also in vivo during atherogenesis by making and breaking binding sites between extracellular collagen and matrix receptors.