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In case of an oil spill, dispersant application represents a response option, which enhances the natural dispersion of oil and thus reduces coating of seabirds and coastal areas. However, as oil is transferred to the water phase, a trade-off of potential harmful effects shifted to other compartments must be performed. This paper summarizes the results of a workshop on the current knowledge on risks and benefits of the use of dispersants with respect to specific conditions encountered at the German sea areas. The German North Sea coast is a sensitive ecosystem characterised by tidal flats, barrier islands and salt marshes. Many prerequisites for a potential integration of dispersants as spill response option are available in Germany, including sensitivity maps and tools for drift modelling of dispersed and undispersed oil. However, open scientific questions remain concerning the persistence of dispersed oil trapped in the sediments and potential health effects., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.

The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.

During atherogenesis vascular smooth muscle cells are converted from a contractile into a synthetic phenotype characterized by enhanced matrix production. The transcription factors Gax and GATA-6 are considered negative, and Oct-1 positive regulators of the synthetic phenotype. Since the phenotype transition can be induced by culturing the cells with serum, we followed the expression of Gax, GATA-6 and Oct-1, integrins and matrix genes in quiescent porcine vascular smooth muscle cells after serum application. Comparisons were made between enzymatically released primary smooth muscle cells and cells grown out from explants of the medial layer of porcine aorta. The serum-mediated down-regulation of Gax was more intense than that of GATA-6, and stronger in explant-derived than in primary cells. Serum was without influence on the expression of Oct-1. Changes in the expression of the transcription factors preceded the induction of integrin alpha2 and the down-regulation of decorin, while mRNAs for laminin beta1 and osteopontin rose immediately after serum stimulation. Primary cells reacted more rapidly than explant cells with respect to changes in laminin isoforms. Studies with a Gax-expressing adenovirus indicated that among all the gene products tested only the expression of integrin alpha2 responded to Gax induction. Thus, our data show that i) Gax should be considered a transcription factor being directly responsible for only few aspects of the phenotypic conversion of smooth muscle cells and that ii) explant cells may represent a subpopulation of smooth muscle cells, which differ from the total population of smooth muscle cells, as obtained in primary culture, in their response to serum stimuli.

Several members of the ATP-binding cassette (ABC) transporter family are involved in cholesterol efflux from cells. A defect in one member, ABCA1, results in Tangier disease, a condition characterized by cholesterol accumulation in macrophages and virtual absence of mature circulating high-density lipoproteins. Expression of a second member, ABCG1, is increased by cholesterol-loading in human macrophages. We now show that ABCG1, which we identified by differential display RT-PCR in foamy macrophages, is overexpressed in macrophages from patients with Tangier disease compared to control macrophages. On examination by confocal laser scanning microscopy, ABCG1 was present in perinuclear structures within the cell. In addition, a combination of in situ hybridization and indirect immunofluorescence microscopy revealed that ABCG1 is expressed in foamy macrophages within the atherosclerotic plaque. These data indicate that not only ABCA1 but also ABCG1 may play a role in the cholesterol metabolism of macrophages in vitro and in the atherosclerotic plaque., (Copyright 2001 Academic Press.)

Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (P). Among the protected tripeptides FmocP(tBu)PG, FmocPP(tBu)G, and FmocGPP(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP(tBu)Gly was best suited for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(3) trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39. 4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PPG(6-8) follows a two-state, pseudomonomolecular reaction.

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 from 1 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 gamma, substances that stimulate macrophages to secrete lytic enzymes, on macrophage expression of type VIII collagen. LPS and interferon gamma 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.

The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and type VIII collagen was studied in human arteries. GM-CSF and type VIII collagen were codistributed in all layers of the walls of nondiseased arteries and during early atherogenesis with up to type V lesions. The number of cells expressing both mRNAs increased during the development of advanced atherosclerotic lesions. Whereas type VIII collagen expression increased further in complicated lesions, GM-CSF was downregulated. During early atherogenesis smooth muscle cells (SMC) and endothelial cells were the principal GM-CSF and type VIII collagen mRNA-expressing cell types. In advanced lesions monocytes/macrophages also expressed the mRNAs. In complicated lesions the number of GM-CSF mRNA-expressing SMC was markedly reduced. In in vitro experiments transforming growth factor-beta1, platelet-derived growth factor, and GM-CSF, but not basic fibroblast growth factor, stimulated the expression of type VIII collagen mRNA by SMC. GM-CSF transiently stimulated type VIII collagen transcription. Thus GM-CSF is a prominent component of the regulatory network influencing collagen metabolism during atherogenesis. By modulating the synthesis of type VIII collagen in SMC, GM-CSF may influence the course of plaque development and may govern processes such as cell movement, plaque stability, and thrombus organization.

The functional properties of the perivascular matrix facilitate the movement, protection and the nutrition of the blood vessels. At the same time the matrix deliniates the vessels to the organs. Different vascular diseases of the vascular system implicate the perivascular matrix consisting of a network of structural and cellular connective tissue components. Impairment of its structural integrity predominantly occurs either from primary or secondary affections. Monospecific antibodies directed against different structural proteins enable detailed histomorphological examinations of the inflammatory tissue reaction in the perivascular space. Since myofibroblasts, fibrocytes, endothelial cells, and macrophages participate in tissue repair mechanisms, in vitro studies on the collagen synthesis may contribute to the understanding of the perivascular tissue reaction according to injury. The present report summarizes perivascular diseases and the in vitro synthesis of procollagen, collagen, and fibronectin of endothelial cells, fibroblasts, smooth muscle cells, and macrophages.

Aortic smooth muscle cells were cultivated as monolayers on plastic or within collagen lattices with low- and high-serum supplementation, and the expression of mRNAs specific for pro alpha 1 (I) and pro alpha 1 (III) collagen were studied by slot blot hybridization. The steady-state levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNA of cells within collagen lattices were found to be higher than those grown on plastic, although the production of collagen was lower. The degradation of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs as revealed in the presence of actinomycin D was not affected by culturing the cells within a collagen lattice. In vitro translation assays of mRNAs of monolayer- and lattice-cultured cells showed no differences in translatability. These data suggest the involvement of posttranslational control of collagen production in collagen lattice-cultured smooth muscle cells.

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.

Types III, IV, VI collagen and laminin distribution in synovial tissue of seven autopsy knee joints from old human donors (69-94 years of age) were investigated with immunocytochemical and immunohistochemical methods. The synovial intima is separated from the subintimal tissue by an intermediate fibrillar zone rich in staining for type III collagen. In the intima basement membrane-like material associated with synovial lining cells stains for type IV collagen and laminin. Fine fibrils surrounding the lining cells stain for type VI collagen. In two of the cases type VI collagen occurs mainly as long-spacing collagen, the distinct aggregated form of type VI collagen. This staining pattern was qualitatively the same in all different regions and cases investigated. However, considerable quantitative differences were seen.

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.

Adult pig smooth muscle cells (SMC) were isolated from the aortic media by collagenase digestion, subcultured as monolayer, and then re-integrated into a three-dimensional network of type I collagen. The contractile state characteristic for resident arterial wall SMC changed to the synthetic, fibroblast-like state. The cells reorganized the randomly orientated collagen fibrils causing the lattice to shrink. The influence of the extracellular matrix on the ultrastructure, the proliferation, and the collagen synthesis of these SMC embedded in the collagen lattice was investigated and compared to cells cultured in monolayer. The amount of total protein and collagens synthesized by SMC embedded in lattices was lowered as compared to monolayer cultures. Whereas total protein synthesis decreased continuously during the culture period, the proportion of collagen synthesis remained at a constant level. Although cells proliferated in lattices, proliferation was clearly slowed down as compared to monolayer cultures. The ultrastructure of entrapped synthetic state SMC was comparable to that of monolayer-cultured cells. Their cytoplasm was largely filled by elements of the endoplasmic reticulum, Golgi complexes and abundant mitochondria. With prolonged culture time, electron-dense granules as well as bodies containing whorled membranes could be found in the cytoplasm. These results indicate that synthetic state SMC can exhibit differential biosynthetic activity dependent on the actual matrix environment; cells seem to be able to sense the macromolecular composition of the extracellular matrix and to modify their production of matrix components accordingly.

Cells with enhanced levels of collagen type I and III mRNA were identified and localized in frozen tissue sections from samples of human atherosclerotic renal and common iliac arteries by in situ hybridization using complementary 35S-labeled RNA probes. Serial sections were immunohistochemically stained for smooth muscle cells, monocytes, and differentiated macrophages. In the fibromuscular intima and in the fibrous plaques, cells with enhanced transcriptional activity were located mainly in the vicinity of differentiated macrophages. In three patients, lack of enhanced transcriptional activity in a proliferated intima was connected with complete absence of macrophages, thus indicating a quiescent stage of atherosclerosis. Immunohistochemical staining of serial sections for smooth muscle cells (SMC) revealed the presence of this cell type throughout the proliferated intima in atherosclerotic arteries including those areas in which enhanced collagen mRNAs were detected. The present results support the idea that macrophages play an important role in the activation of collagen synthesis in SMC of atherosclerotic vessel walls.

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 alpha 1(X) and rabbit alpha 1(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 alpha 1(VIII) chain showed that this fragment was the triple-helical domain of a second type VIII collagen chain which we designate alpha 2(VIII).

Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen.

Fetal calf skin was solubilized by limited pepsin digestion and type III collagen separated from type I collagen by fractional salt precipitations. Cleavage of the type III collagen with CNBr gave rise to ten peptides, which were isolated by molecular sieve and ion exchange chromatography. The peptides were characterized by determination of their molecular weights and amino acid compositions. Together they account for all the amino acids and total molecular weight of the alpha1 (III) chain. Six of the peptides contain more hydroxyproline than proline residues. The two cysteinyl residues of the alpha1 (III) chain which provide sites for interchain disulfide bonding were localized in the C-terminal CNBr peptide. In addition to the ten CNBr peptides, three double peptides were isolated which still contained one methionine residue. About 0.1 residue Gal-Hyl monosaccharide and 0.8 Glc-Gal-Hyl residue disaccharide were found per alpha1 (III) chain. Almost all hydroxylysine-bound carbohydrate was located on peptide 7.

The microdistribution of type V collagen, fibronectin, and laminin on the luminal surface of perfusion-fixed normal rat aortic endothelium has been studied by an immunoelectron microscopic method using monospecific antibodies and a protein A-gold complex. Gold particles indicating the presence of these biologically active connective tissue proteins were localized in groups on and in the vicinity of the interendothelial border. They were also found on the small flaps of cell junctions as well as on certain cell projections and scattered on the cell surface. Correlative transmission electron-microscopic examinations proved the specificity of these localizations. The endothelial cells of the aorta differed markedly in the amount of scattered connective tissue proteins on their surface, suggesting that there are several types of aortic endothelial cells with distinct functional differences. The findings provide evidence that connective tissue proteins may contribute to the surface pattern of the normal endothelium, especially on cell borders. It is likely that these proteins influence functions such as the permeability and chemotactic activity of the endothelium pertinent to the development of vascular disease.

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.

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.