The outer arterial wall layers are primarily affected in spontaneous cervical artery dissection.

OBJECTIVE: To evaluate the macroscopic and microscopic phenotype of the distal superficial temporal artery (STA) in patients with spontaneous cervical artery dissection (sCAD, n = 14). Arteries of accident victims, free of clinically apparent vascular disease, served as reference samples (n = 9). METHODS: Specimens of distal STA branches were obtained by biopsy or at autopsy. Their fine and ultrafine structure was documented by close-up photography of native STA branches, light microscopy, and electron microscopy in a case-control study. RESULTS: STA specimens from patients with sCAD revealed pathologic changes mainly in the adventitial and medial layers. In these areas, vacuolar degeneration and fissuring were associated with neoangiogenesis of capillaries and microscopic erythrocyte extravasation into the connective tissue. In addition, some specimens showed overt microhematomas close to the medial/adventitial border visible at low magnification. The reference arteries showed virtually no pathologic changes in the outer arterial layers. CONCLUSION: Bearing in mind that the STA is only a surrogate for the cervical arteries affected by sCAD, we propose the following pathogenetic model. We hypothesize that sCAD affects primarily the outer arterial layers. The process starts with degenerative changes at the medial-adventitial border associated with neoangiogenesis of capillary vessels branching from vasa vasorum in the adventitia. Leakage of neoangiogenetic capillaries releases blood cells into the connective tissue and leads to formation of microhematomas along the medial/adventitial border, as well as disintegration of the medial and adventitial texture. Microhematomas might then cause successive rupture of multiple neoangiogenetic capillaries and vasa vasorum, ultimately resulting in dissection.

Isolation of phages infecting Actinoplanes SN223 and characterization of two of these viruses.

Phages infecting the industrially important Actinoplanes strain SN223 were isolated from soil samples collected at the shores of inland waters in Germany. The genome sizes range from 53 kb to 58 kb. Preliminary analyses revealed G+C contents comparable with the G/C bias of the host. Electron microscopy of three selected viruses displayed no obvious morphological differences, the phage heads being icosahedral and their tails non-contractible. Two of the phages (phiAsp2, phiAsp3.1) characterized in more detail are capable of provoking putative pseudolysogenic growth of the host bacterium. The carrier state for phiAsp2, in which cells are tightly packed with viruses, was demonstrated by electron microscopy. The latter phage is apparently widely distributed, as it was isolated from regions which are distantly located, i.e. more than 600 km apart from each other.

Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin, and lumican.

BACKGROUND: Among the members of the small leucine-rich proteoglycan family, decorin, biglycan, and fibromodulin have been proposed to be potent modulators of transforming growth factor-beta (TGF-beta) activity, thereby playing an important role in the pathogenesis of fibrotic kidney diseases. Furthermore, decorin expression influences the expression of p21WAF1/CIP1, which has been related to kidney hypertrophy and hyperplasia. However, none of the members of this proteoglycan family have been investigated in normal adult human kidney cortex, thus making it impossible to correlate disease-mediated alterations of their expression with the normal situation in vivo. METHODS: The chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, and the keratan sulfate proteoglycans, fibromodulin and lumican, were investigated in normal human adult renal cortex by immunohistochemistry on the light and electron microscopic level and by in situ hybridization. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) methods were used to get an estimate of their expression in isolated glomeruli. Decorin excretion with the urine was measured by Western blotting. RESULTS: Two bands of decorin and a single band of biglycan mRNA were identified in Northern blots of isolated glomeruli. Amplification by RT-PCR was required to detect the signals for fibromodulin and lumican. All four proteoglycans were preferentially expressed in the renal interstitium with accumulations around tubules. Weak expression was found in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, was synthesized and deposited in distal tubular cells and collecting ducts. Immunogold labeling indicated the presence of the proteoglycans in the glomerular basement membrane, which was interpreted as a result of glomerular filtration. Indirect evidence suggested tubular reuptake of decorin after glomerular filtration. CONCLUSION: The data indicate that the different cells of the adult human kidney are characterized by a distinct expression pattern of the four small proteoglycans. It is suggested that these proteoglycans may have distinct pathophysiological roles depending upon whether they are expressed by mesangial cells, endothelial cells, epithelial cells, or cells of the tubulointerstitium.

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.

Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen-expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.

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 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.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates the expression of type VIII collagen mRNA in vascular smooth muscle cells and both are codistributed during atherogenesis.

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.

Differential expression of connexin43 and desmin defines two subpopulations of medial smooth muscle cells in the human internal mammary artery.

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Colony stimulating factors modulate the transcription of type VIII collagen in vascular smooth muscle cells.

Colony stimulating factors belong to a family of cytokines that regulate proliferation in macrophages and other vascular cell types. They have been implicated in the inflammatory-fibroproliferative response of atherosclerosis. The present study was undertaken to assess the effect of granulocyte-macrophage and macrophage colony stimulating factors on the transcription of type VIII collagen by vascular smooth muscle cells and their potential relevance for the expression of collagen in atherosclerotic lesions. The influence of colony stimulating factors was studied in relation to transforming growth factor beta1, the factor exhibiting the most potent effect on collagen metabolism. Northern blot experiments showed that treatment with both colony stimulating factors and transforming growth factor beta1 transiently stimulated the transcription of type VIII collagen mRNA. Maximal levels were reached after 2 h and 100 pg/ml granulocyte macrophage colony stimulating factor (4-fold), 1 U/ml macrophage colony stimulating factor (4.6-fold) and 1 ng/ml transforming growth factor beta1 (1.6-fold). While overnight treatment with colony stimulating factors stimulated the expression of transforming growth factor beta1 mRNA, short incubations did not influence or downregulate the transcription. In turn, treatment with transforming growth factor beta1 reduced the expression of granulocyte-macrophage and macrophage colony stimulating factor mRNA. The in vitro mRNA expression patterns were directly reflected in the distribution patterns found in intimal thickenings and advanced atherosclerotic lesions. This study demonstrates that colony stimulating factors and transforming growth factor beta1 modulate the transcription of type VIII collagen in vitro. Our data indicate a direct mechanism and exclude a pathway, which is mediated via the stimulation of transforming growth factor beta1 transcription. Our studies further support the hypothesis that colony stimulating factors in concert with transforming growth factor beta1 affect the collagenous composition of the extracellular vascular matrix.

Expression of type VIII collagen after cholesterol diet and injury in the rabbit model of atherosclerosis.

This study presents an analysis of the expression of type VIII collagen mRNA in response to cholesterol diet and balloon injury in the rabbit iliac artery. The design of the animal experiments was as follows: 28 male New Zealand White rabbits were divided into the 3 different treatment groups. Group 1 received regular chow; group 2 was fed with a 1% cholesterol diet for 6 weeks and normal chow for 5 weeks; and group 3 underwent balloon injury, then 6 weeks of a 1% cholesterol diet, which was followed by 5 weeks of normal chow. The expression pattern of type VIII collagen mRNA was compared with that of the fibrillar collagen types I and III, transforming growth factor-beta1, a factor known to exert the most potent stimulatory effect on collagen synthesis in vitro, and matrix metalloproteinase 1, a collagen-degrading enzyme. The cholesterol diet resulted in an upregulation of type VIII collagen, fibrillar collagens, transforming growth factor-beta1, and matrix metalloproteinase I in the adventitia. Although the number of type VIII collagen mRNA-expressing cells in the media increased, no significant difference in overall expression levels was detectable by northern blot analysis. The ratio of medial smooth muscle cells expressing type VIII collagen mRNA to those expressing type I and type III collagen mRNA (CVIII:CI:CIII) changed from 1:1.88:0.03 in the normal media to 1:0.78:0.29. When cholesterol feeding was preceded by balloon injury, type VIII collagen mRNA expression concomitant with the fibrillar collagens was further upregulated over and above that level reported after cholesterol diet alone. In general, low levels of transforming growth factor-beta1 mRNA correlated with high expression of matrix metalloproteinase I. Our study indicates that a cholesterol diet resulted in a balanced reorganization of the collagen composition but did not result in marked collagen accumulation. This may provide an extracellular environment that favors migration and proliferation processes during early atherogenesis. It also demonstrates that type VIII collagen is highly expressed and deposited at later stages, and this may be linked to processes such as tissue reorganization during vascular repair and plaque stabilization.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices.

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

Left-ventricular expression of interleukin-6 messenger-RNA higher in idiopathic dilated than in ischemic cardiomyopathy.

During end-stage heart failure, plasma levels of interleukin-6 (IL6) are elevated. This cytokine exerts a negative inotropic influence on the myocardium. The production site of IL6 is unclear. We examined the hypothesis that IL6 in end-stage heart-failure patients is produced in the myocardium itself and is differentially regulated according to etiology. Cardiac tissue was obtained from 27 patients (idiopathic dilated cardiomyopathy, (DCM) 9/6 m/f, age 46 +/- 14 y; ischemic cardiomyopathy (ICM), 11/1 m/f, age 55 +/- 8 y) at the time of transplantation. The tissue was subjected to IL6 Northern-blot analysis. Signals were quantified by densitometric scanning after normalization to G3 PDH mRNA. Data were compared by Mann-Whitney test between DCM and ICM patients, divided by chamber origin. IL6 transcripts were found in all patients. In DCM, left-ventricular IL6 mRNA expression was higher than in ICM (p = 0.006). Median right-ventricular as well as left- and right-atrial IL6 mRNA expression was not significantly different in both groups. In summary, in end-stage heart failure, IL6 mRNA is consistently expressed in the myocardium. Left-ventricular expression is higher in DCM than in ICM. These data support the concept of a potentially reversible inflammatory component in the etiology of DCM which is more pronounced than in patients with ICM of comparable clinical severity.

Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.

Monocytes/macrophages in atherosclerosis.

The realization that the monocyte/macrophage is central to both atherogenesis and the progression of the atherosclerotic plaque has arisen only recently. In this chapter the role of the monocyte/macrophage in the genesis of the atherosclerotic plaque will be discussed. As will be demonstrated, the pivotal role of the macrophage in atherosclerosis depends not only on its ability to handle lipids but also on its physical and secretory functions and its role as a mediator of inflammation.

Smooth muscle cells express granulocyte-macrophage colony-stimulating factor in the undiseased and atherosclerotic human coronary artery.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of a family of cytokines that regulate proliferation in macrophages and other types of cells, has been implicated in the inflammatory-fibroproliferative response of atherosclerosis. However, previous studies have been restricted to cultured cells and animal models. In the present study, we investigated GM-CSF expression in undiseased and atherosclerotic human coronary arteries at both the mRNA and protein levels. Dual in situ hybridization/cell-marking experiments demonstrated that subpopulations of intimal smooth muscle cells (SMCs) and endothelial cells express the cytokine in the histologically normal human coronary artery and that augmented expression occurs at these sites, and in macrophage accumulations and medial SMCs, in the atherosclerotic vessel. Corresponding data were obtained by in situ hybridization and reverse transcription-polymerase chain reaction and Northern analyses of cultured cells. Cultured human coronary arterial SMCs showed constitutive expression of GM-CSF in cells that had adopted an activated synthetic phenotype. Electron microscope immunocytochemistry revealed that GM-CSF is a protein localized in the cytoplasmic matrix of SMCs of both the undiseased and atherosclerotic vessel wall; extracellular matrix was largely unlabeled, with only occasional small patches of amorphous immunopositive material. The expression of GM-CSF by subpopulations of intimal SMCs in the undiseased artery and the marked upregulation of GM-CSF apparent in atherosclerotic lesions suggest roles for the cytokine in the cellular events underlying initiation and progression of the human atherosclerotic lesion.

Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol.

Apart from dipalmitoyl phosphatidylcholine, cholesterol is the most abundant surfactant lipid. About 90 to 99% of cholesterol of the alveolar surfactant is derived from serum lipoproteins. The aim of this study was to identify the lipoprotein which preferentially supplements type II pneumocytes with cholesterol destined for surfactant production. Ultrastructural investigations revealed that type II pneumocytes bind and take up HDL, LDL and VLDL. Binding and uptake of VLDL occurred even in the presence of excess LDL indicating that, besides LDL receptors, type II pneumocytes express additional binding sites for VLDL. Type II pneumocytes in primary culture are able to take up cholesterol added in the form of HDL, LDL and VLDL. Cholesterol uptake was lowest from HDL and highest from VLDL. The maximal velocity of cholesterol uptake from VLDL was more than three times that of cholesterol uptake from LDL. The half-maximal saturation of cholesterol uptake from VLDL was nearly half that of LDL. From these kinetic data and the distribution of free cholesterol among the serum lipoproteins, we calculated that the cholesterol uptake from VLDL is more than three times that of cholesterol uptake from LDL. In double-labeling experiments type II pneumocytes secreted palmitic acid-labeled phospholipids together with labeled free cholesterol taken up from lipoproteins. The secretion rates of both phospholipids and free cholesterol were stimulated to nearly the same extent by isoproterenol. From our results we conclude that type II pneumocytes interact specifically with HDL, LDL and VLDL. Cholesterol taken up in the form of the individual lipoproteins shows no difference in its availability for the formation of cholesterol ester and surfactant by type II pneumocytes in vitro. Based on the kinetic studies, it appears that VLDL is the major gateway through which cholesterol is provided to satisfy the cholesterol requirements of type II pneumocytes for the synthesis of surfactant.

Endocytosis and retrograde transport of pertussis toxin to the Golgi complex as a prerequisite for cellular intoxication.

The uptake mechanism of pertussis toxin (PT) in CHO and insulin-producing HIT-T15 cells was studied. By electron microscopy after direct labeling of the toxin with gold particles, PT was found to be taken up by receptor-mediated endocytosis. The presence of active pertussis toxin in the Golgi complex was shown by subcellular fractionation. The importance of the Golgi localization of pertussis toxin for the S1-dependent ADP-ribosylation of G-proteins was investigated employing Brefeldin A (BFA) treatment to disrupt Golgi structures. Treatment with Brefeldin A completely blocked the pertussis toxin mediated ADP-ribosylation of cellular G-proteins in CHO and HIT-T15 cells, whereas the BFA-resistant MDCK cells were not protected. A mutant CHO cell line (V24.1) exhibiting a temperature-sensitive Golgi complex could be protected when grown at restrictive conditions. These results strongly indicate that retrograde transport to the Golgi network is a necessary prerequisite for pertussis toxin mediated ADP-ribosylation of G-proteins and thus also for cellular intoxication.

Copper-induced inflammatory reactions of rat carotid arteries mimic restenosis/arteriosclerosis-like neointima formation.

The pathogenesis of arteriosclerosis and of restenosis after angioplasty is linked with an inflammatory and fibroproliferative response of the arterial tissue. We have induced a non-infectious inflammation by implanting a silicon-copper cuff around rat carotid arteries. The copper ions released from the oxidized copper initiate and mimic all morphological features of post-angioplasty restenotic and arteriosclerotic lesions. The copper-induced lesions were analyzed by electron and light microscopy, immunohistochemical methods and quantified by morphometry. During the first phase of copper-induced tissue reaction (3 days), macrophages and polymorphonuclear leucocytes invaded through the endothelium, accumulated in the subendothelial space and triggered the proliferation of smooth muscle cells which then migrated from the tunica media through the lamina elastica interna into the intima. Within 3 weeks, the accumulated smooth muscle cells, macrophages, leucocytes and newly synthesized extracellular matrix formed a circular mostly eccentric fibrotic thickening that narrows the vessel lumen by 30-40%. The accompanying structural disorganization of the medial layer led to focal rupture and aneurysm-like dilatation of the vessel wall in 3 of 11 animals between day 20 and 43. The neointima progressively increased in thickness over time leading to corresponding reduction of the vessel lumen. The carotid arteries of control animals and animals treated with copper-free silicon cuffs showed no abnormal pathological appearance. Our results show that inflammation-inducing agents can contribute to and simulate restenosis- and arteriosclerosis-like lesions and that the copper-cuff model may be useful in the exploration of new approaches to intervention.

Interaction of apo E-containing lipoproteins with the LDL receptor-related protein LRP.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts with apolipoprotein E (apo E)-rich lipoproteins, and alpha2-macroglobulin (alpha2-M) in the activated state (alpha2-M*). Whether LRP is a physiologically relevant lipoprotein receptor for naturally occurring apo E-rich lipoproteins, however, is still under discussion. To address this question, we isolated beta-migrating very low density lipoprotein (beta-VLDL) from rabbits by using gel filtration chromatography. Biochemical analysis of beta-VLDL subfractions demonstrated that we isolated apo E- and cholesterol-rich triglycerides with differences in composition and size. Binding and uptake characteristics of beta-VLDL subfractions and alpha2-M* on mouse peritoneal macrophages (MPM) and Hep G2 cells were examined by electron microscopy. One of the beta-VLDL subfractions, beta-VLDL(II), bound specifically to LRP on MPM and Hep G2. beta-VLDL(II) competed with the binding of alpha2-M* without addition of exogenous apo E. Furthermore, binding and uptake of beta-VLDL(II) and alpha2-M* were not affected by either lactoferrin or Ca2+-free medium. The results indicate that naturally occurring apo E-rich lipoproteins do exist and that they very likely interact with LRP via the same binding site as alpha2-M*.