Biomedical subjects

H Robenek

Publications and source records attributed to H Robenek.

At least 55 records · Page 3Linked to original sources

PubMed1993

In situ immunolocalization of lipoproteins in human arteriosclerotic tissue.

The concentration of serum lipoproteins, especially those of low density (LDL) and high density (HDL) lipoprotein, are related to the pathogenesis of arteriosclerosis. However, there is a lack of data concerning lipoprotein distribution in the human arteriosclerotic plaque. To detect these lipoproteins, we performed immunogold labeling on ultrathin sections of fixed and embedded human arteriosclerotic tissue. We used a panel of specific antibodies to different lipoproteins and their apolipoprotein constituents, namely LDL, formaldehyde-fixed LDL, apolipoprotein B-100, HDL, and formaldehyde-fixed apolipoprotein A-I. We also applied antibodies to alpha-actin and cathepsin D to characterize the cells and organelles involved in lipoprotein uptake and metabolism. Semiquantitative evaluation was carried out for a detailed comparison of the results obtained. Electron microscopic examination revealed that the majority of HDL and LDL in the pathological tissue was localized intracellularly in macrophage-derived foam cells and smooth muscle cells, whereas only LDL was found in the extracellular matrix. In some cases, we observed an intracellular accumulation of lipoproteins in electron-dense vesicles, which appeared to be of lysosomal origin, as shown by double labeling with an antibody to cathepsin D. These vesicles were present only in macrophage-derived foam cells, which were localized in the necrotic cores of arteriosclerotic plaques, and could not be found in healthy tissue or in the early stages of arteriosclerotic disease.

Arteriosclerosis↗

PubMed1993

Aortic smooth muscle cells in a three-dimensional collagen lattice culture. Evidence for posttranslational regulation of collagen synthesis.

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.

Actins↗

PubMed1993

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.

Animals↗

PubMed1993

Genetic defects in proteoglycan biosynthesis.

An overview on the structure of proteoglycans and on genetic defects in proteoglycan biosynthesis is given. Several patients with progeroid-like symptoms have been shown to have abnormalities in the biosynthesis of chondroitin/dermatan sulfate proteoglycans. A partial inactivity of galactosyltransferase I which catalyzes the second glycosyl transfer reaction in the assembly of glycosaminoglycan chains has been shown to represent the primary defect in one of the patients. A diminished concentration of a collagen-associated proteoglycan is considered to play a pathogenetic role in the development of loose skin.

Carbohydrate Sequence↗

PubMed1992

Differential regulation of protein synthesis in smooth muscle cells from normotensive and spontaneously hypertensive rats by a three-dimensional matrix of type I collagen.

OBJECTIVE: The objective of this study was to gain insight into the metabolic and morphological properties of smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) when cultured in vivo under similar conditions. DESIGN: Three-dimensional cell-collagen systems represent living tissue equivalents in vitro and simulate natural conditions more closely than conventional monolayer cultures. METHODS: The effect of a three-dimensional matrix of type I collagen on ultrastructure, total protein and collagen synthesis and cell cycle distribution of SMC from SHR and normotensive Wistar-Kyoto (WKY) rats was studied. RESULTS: Collagen lattice-cultured SMC from SHR and WKY rats showed the synthetic phenotype, i.e. the cytoplasm was filled with organelles characteristic of secretory protein synthesis. There was a decrease in the percentage of cells in the 5 phase compared with monolayer cultures. Total protein synthesized by SMC from SHR and WKY rats in lattices was lowered compared with monolayer cultures. However, reduction of protein synthesis in SMC from SHR was less than in SMC from WKY rats. Differences in the proportion of collagen in SMC from SHR and WKY rats were not demonstrable in collagen lattice cultures. CONCLUSION: The present study suggests that a three-dimensional matrix of type I collagen may modulate total protein synthesis in SMC from SHR and WKY rats. However, cells from SHR react less to this matrix than those from WKY rats.

Animals↗

PubMed1992

Actions of lipoproteins in cultured human mesangial cells: modulation by mitogenic vasoconstrictors.

Recent studies have suggested that hypercholesterolemia may aggravate glomerulosclerosis. Mesangial cells actively participate in this process. To elucidate mechanisms by which lipids act on human mesangial cells (HMC), we measured the receptor-specific uptake of apolipoprotein (Apo) B- and Apo B- and E-containing lipoproteins in the presence and absence of growth factors and studied the growth-related mechanisms in HMC after exposure to low-density lipoprotein (LDL). Human LDL and very low density beta-lipoprotein (beta-VLDL) isolated from cholesterol-fed rabbits were bound, internalized, and degraded by a receptor-specific mechanism (dissociation constants for degradation LDL 30.0 and for beta-VLDL 4.1 micrograms protein/ml medium). Maximal capacities were 30-50% of those of human fibroblasts. Acetylated and copper-oxidized LDL were not taken up specifically, suggesting no active scavenger-receptor activity. Preexposure to endothelin-1 (5 x 10(-7) M) and platelet-derived growth factor (PDGF A, B, 83 x 10(-12) M) for 16 or 15 h, respectively, doubled the uptake of LDL by HMC. In addition, PDGF synergized with LDL in stimulating DNA synthesis. Exposure of HMC to LDL resulted in a transient elevation of mRNA that encodes c-fos and c-jun, with a maximal effect seen after 30-60 min. In addition, PDGF A- and B-chain mRNAs were transiently elevated, peaking at 3 h in response to LDL (25 micrograms protein/ml medium) and continued to increase in a concentration-dependent manner (25-75 micrograms protein/ml medium). These data demonstrate that HMC take up lipoproteins via a receptor-specific mechanism with a high affinity for Apo E-containing lipoproteins which are often found in plasma of patients with renal disease. Vasoconstrictor and mitogenic peptides enhance lipoprotein receptor activity and have a synergistic effect on the mitogenic effect of LDL. LDL stimulates a number of growth-related genes. These data suggest that lipoproteins may play a critical role in mediating mesangial cell hypertrophy or proliferation, events intimately involved in the development of glomerulosclerosis.

Cell Division↗

PubMed1992

Endocytosis of oxidized LDL and reversibility of migration inhibition in macrophage-derived foam cells in vitro. A mechanism for atherosclerosis regression?

The ability of macrophage-derived foam cells to migrate from atherosclerotic lesions represents one potential mechanism for the regression of atherosclerosis. It is, however, generally recognized that the transformation of macrophages into foam cells results in greatly reduced migrational ability. In the present study, we set out to investigate the factors affecting migratory capability in foam cell-like cells with the use of an in vitro assay. Foam cell-like cells were prepared by incubating macrophages in the presence of oxidized low density lipoprotein (LDL). The transformation to a typical foam cell morphology was demonstrated by Nile red staining (light microscopy), and the mechanisms of binding, uptake, and intracellular processing of oxidized LDL were established by colloidal gold labeling on the ultrastructural level. With the in vitro assay, the migration of these foam cell-like cells was found to be markedly inhibited compared with untreated, control macrophages. However, zymosan-activated mouse serum restored migration in oxidized LDL-treated cells to levels similar to those of control cells. Restoration of migratory capacity was accompanied by alterations in the cytoskeleton system, especially in actin arrangement.

Animals↗

PubMed1991

Distribution patterns of apolipoproteins A1, A2, and B in the wall of atherosclerotic vessels.

Atherosclerotic vessels were analysed histochemically for distribution, quantity, and composition of apolipoprotein (Apo) types in the vascular wall. The specimens comprised all stages of atherosclerosis, from very discrete intimal changes to complicated lesions. The vessel specimens were marked with antibodies against human Apo A1, A2, and B. Apo A1 can be demonstrated in even the earliest stage of atherosclerosis, and increases with the progression of the disease. In the initial stage, Apo A1 is found first in lumen-adjacent layers of the intima, and is evident in deeper layers of the wall as the disease progresses. Arteries of muscular type show accumulation of Apo in an earlier stage (or in greater quantity at the same stage) than arteries of elastic type. At all stages, the amount of Apo A1 always exceeds that of A2 and B. In the intima, Apo B is higher than Apo A2, the media contains hardly any Apo B, and the adventitia has less B than A2. Within the intimal layer, Apo A1 and A2 are found in an intracellular (mainly in foam cells) or in an extracellular location, according to the stage of atherosclerosis. Apo B is almost exclusively extracellular; only cases of advanced atherosclerosis show some intracellular localization (mostly in foam cells), visualized as electron dense lamellar organelles, probably of lysosomal origin. In the media, Apo A1 and A2 are accumulated in intracellular deposits, whereas the extracellular storage of Apo A1, A2 and B is observed only in cases with the most severe damage. Our investigations suggest that the accumulation of apolipoproteins in the vascular wall is affected not only by insudation from the plasma, but also by neosynthesis and/or metabolism by locally derived cells or cells immigrating in the process of atherosclerosis. The presence of Apo A1 and A2 in the vessel wall is now documented, and their role at this site apparently differs from that in the plasma.

Aorta↗

PubMed1991

Immunohistochemical double labeling of macrophages, smooth muscle cells, and apolipoprotein E in the atherosclerotic plaque.

To find out which cell types in the atherosclerotic plaque are actively involved in apolipoprotein-secretion, we stained tissue samples from the ascending aorta of patients undergoing coronary bypass surgery. We used monoclonal antibodies against macrophages, smooth muscle cells and apolipoprotein E. The present in situ-study of the atherosclerotic plaque has shown that apolipoprotein synthesis is predominantly secreted by macrophages transformed into foam cells. Besides this, there are obviously some smooth muscle cells equally transformed to foam cells, which also carry apolipoprotein E in their cytoplasm. Our data suggest that both cell types in the atherosclerotic plaque are actively involved in apolipoprotein secretion.

Apolipoproteins E↗

PubMed1991

Immunocytochemical localization of apolipoprotein A-I using polyclonal antibodies raised against the formaldehyde-modified antigen.

The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labelling intensity. To overcome the difficulties associated with fixation-induced changes in antigenicity, we produced antibodies against pre-fixed human apolipoprotein (apo) A-I. Purified apo A-I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein-A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulphone-activated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells. When HepG2 cells were exposed to anti-apo A-I antibodies followed by a secondary FITC-labelled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A-I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein-A-gold technique on ultrathin cryosections. The findings demonstrate the potential of using antibodies to fixed antigens as a strategy to overcome impaired localization due to fixation in cytochemistry at the light microscopic and electron microscopic levels.

Animals↗

PubMed1991

[Ultrastructural and proliferation studies of human, catheter atherectomy extracted plaque material].

Atherectomy with a Simpson catheter enables a detailed characterization of percutaneously removed human atheromatous plaque material. Cell biological investigations (flow-cytometric analyses, cell type-specific antibodies, immunohistological labelling methods of selected extracellular matrix proteins) are aimed at a comparative characterization of excised material from primary lesions and restenoses to yield further insight into the mechanisms of plaque formation. First results on 17 specimens would like to contribute to a better understanding of the structure and composition of the extracellular matrix and their interaction with cells in atherosclerotic plaques.

Aged↗

PubMed1991

Endocytosis of human IgG:Fc receptor complexes by transfected BHK cells.

We have analyzed the mode of uptake of human beta FcRII molecules expressed in BHK cells (clone 2/14). When challenged with aggregated human IgG (ahIgG), these cells bind the ligand at 4 degrees C and endocytose the IgG: receptor complexes rapidly upon warming to 37 degrees C, as seen by fluorescence microscopy with antibodies directed against human IgG. Using 125I-labeled ahIgG, we found that 40% of the bound ligand was internalized within 15 min, and approximately 60% within 2 h. Surface replication and thin sectioning combined with immunogold labeling revealed that the ligand was taken up by coated vesicles and was transferred to the endosomal/lysosomal compartment. This was confirmed by confocal laser microscopy of cells double labeled for clathrin and ahIgG. After modulation of the coated vesicle pattern by hypertonic medium, ahIgG transport was impaired. These data show that a single isoform of human FcRII, expressed in an animal cell negative for Fc receptors, can use the coated vesicle based endocytic pathway of the host cell. Reincubation of cycloheximide-treated cells with a second batch of ligand showed that approximately 20% of the beta FcRII was recycled. This finding is in apparent contrast to the fate of the endogenous Fc receptors expressed on mouse macrophages.

Animals↗

PubMed1991

Aortic smooth muscle cells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis.

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.

Animals↗

PubMed1990

Shedding of hyaluronate synthase from streptococci.

Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state.

Blotting, Western↗

PubMed1990

Organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen.

We investigated the organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen. The influence of these collagens on cell morphology and the distribution pattern of actin, vimentin, talin, and vinculin was analyzed by light microscopy, conventional electron microscopy, immunofluorescence, and immunogold labeling after lysis-squirting. Whereas the morphology of the endothelial cells is not markedly influenced, the structure of the cytoskeleton and the focal contacts of the cells are altered by the different collagen types. Stress fibers are more distinct in cells grown on type I collagen; cells on type III collagen show a more diffuse distribution of actin molecules. Intermediate filaments seem not to be affected by the collagens. The areas of focal contacts are larger in cells on type I collagen. Additionally, the labeling pattern of talin and vinculin is denser in focal contacts of cells grown on type I collagen. These results suggest an important role of the type of collagen in mediation of the organization of the microfilament system and the adhesion structures of bovine aortic endothelial cells in culture.

Actins↗

PubMed1990

Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein-mediated cholesterol efflux in macrophages.

We have studied the rate of phospholipid synthesis and turnover in mouse peritoneal macrophages in reaction to cholesterol influx and high density lipoprotein (HDL)-mediated cholesterol efflux, using three different radioactive precursors, 32PO4(3-), [3H]choline, and [14C]oleic acid. The cells were loaded with cholesterol for up to 18 h with acetyl-low density lipoprotein (LDL), and phospholipid synthesis was measured at various time intervals and compared with nonloaded macrophages. In the first 2 h of cholesterol loading, a twofold increase in the rate of synthesis for sphingomyelin, phosphatidylcholine, phosphatidylserine-inositol, and phosphatidylethanolamine was observed. After this initial up-regulation, the rate of phospholipid synthesis continuously declined upon further cholesterol loading, while the turnover rate of cellular phospholipids was not affected under the same conditions. The lysosomal inhibitor chloroquine abolished the down-regulation, revealing a strong correlation between phospholipid synthesis and lysosomal enzyme activity which was presumably dependent on the release of cholesterol from the lysosome. The reduction in phospholipid synthesis induced by cholesterol loading is reversible by the addition of HDL3 to the cells. When HDL3 was added to the culture medium, a two- to threefold increase in phosphatidylcholine synthesis and a twofold increase in sphingomyelin formation was observed after 3 h. Ca2+ antagonists of the dihydropyridine type, which down-regulate HDL-receptor activity and promote the formation and cellular release of lamellar bodies derived from the lysosomal compartment (Schmitz, G., et al. 1988. Arteriosclerosis. 8: 46-56, and Robenek, H., and G. Schmitz. 1988. Arteriosclerosis. 8: 57-67), specifically enhance the synthesis of sphingomyelin in cholesterol-loaded macrophages. Inhibitors of acyl-CoA:cholesterol acyltransferase (Octimibate, progesterone) increase both the synthesis of sphingomyelin and phosphatidylcholine, and enhance HDL-receptor activity. The results indicate that cholesterol and phospholipid metabolism are coordinately regulated in macrophages. Moreover, the formation of phosphatidylcholine and sphingomyelin seems to be an important factor for the promotion of HDL-receptor-mediated cellular cholesterol efflux.

Animals↗

PubMed1990

Heterogeneity of human lipoprotein Lp[a]: cytochemical and biochemical studies on the interaction of two Lp[a] species with the LDL receptor.

Human Lp[a] can be fractionated into two species with different affinities for lysine-Sepharose. Forty to 81% of the total Lp[a] in the density fraction 1.055-1.15 g/ml from five individuals was retained by this affinity column. The remaining unretained Lp[a] species with no apparently functional lysine binding site was similar to the retained species in its electrophoretic mobility, lipid, protein, and apolipoprotein composition, and the heterogeneity was not related to apo[a] size polymorphism. Interaction of the two species with the low density lipoprotein (LDL) receptor was studied in human fibroblasts. Using gold-labeled lipoproteins and an immunochemical procedure, both Lp[a] species could be located in clusters on the cell surface, but the extent of labeling was far lower than that seen with LDL. Both Lp[a] variants were less effective than LDL in 1) down-regulation of LDL-receptor activity; 2) suppression of cellular sterol synthesis; and 3) stimulation of cholesteryl ester formation in human fibroblasts. Although degradation of both species of Lp[a] by the perfused rat liver was stimulated after estrogen induction of hepatic LDL-receptor activity, the stimulation amounted to only a quarter of that seen with LDL. The heterogeneity of Lp[a] with respect to the ability to bind epsilon-aminocarboxylic acid will need to be considered in studying the physiological role of this lipoprotein. Both Lp[a] species exhibited a similar interaction with the LDL-receptor in vitro, and confirmed previous investigations that Lp[a] is only a poor ligand for the LDL-receptor.

Affinity Labels↗

PubMed1989

Subcellular distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay.

Several types of the 14-15 kDa fatty acid-binding proteins (FABPs) are known to occur in the cytosol of mammalian cells. With antibodies raised against the cardiac-type protein from bovine heart, immunoblots indicated a more widespread distribution of the cardiac FABP in subcellular fractions, such as mitochondria and nuclei. A detailed view was obtained when the post-embedding protein A-gold labeling method was applied to cross-sections of heart cells and isolated subcellular fractions. Cardiac FABP in myocytes was associated with myofibrils and localized within mitochondria and nuclei. After subfractionation of mitochondria, the binding protein was recovered with matrix proteins only. A non-competitive enzyme-linked immunosorbent assay (ELISA) of the direct type was developed specifically for bovine cardiac FABP. This assay was sensitive in the range of 0.05 to 1 ng, and concentrations of cardiac FABP per mg protein were found for cytosol, matrix and nuclei to be around 3.18, 0.18 and 0.03 micrograms, respectively. The newly found compartmentation of cardiac FABP in the heart cell must be considered when the true functions of the protein, yet to be defined, are studied.

Animals↗