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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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*.

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

The low density lipoprotein receptor-related protein (LRP) discovered in 1988 (1) was proposed as a candidate for the postulated apo E-binding chylomicron remnant receptor. Recent results suggest LRP to be a multifunctional cell surface receptor which might have a pivotal function in linkage of diverse metabolic pathways not previously considered to be related. Although biochemical evidence for lipoprotein binding to LRP has been presented (rev in 2), the need to add apo E to lipoproteins to demonstrate lipoprotein binding to LRP has raised doubts as to its lipoprotein receptor function. Therefore, it has yet to be established whether there is a physiologically relevant binding of naturally occurring apo E-containing lipoproteins to LRP. Here we describe cytochemical studies in which naturally occurring apo E-containing lipoproteins carefully isolated by gelfiltration chromatography were found to bind specifically to the cell surface of mouse peritoneal macrophages (MPM). These lipoproteins compete specifically with the binding of activated alpha 2-macroglobulin, which is a generally accepted ligand of LRP, to these cells without previous enrichment with exogenous apo E. We therefore conclude that LRP is indeed a physiologically relevant lipoprotein receptor.

Effects of calcium on the migration and recruitment of macrophages and macrophage-derived foam cells.

Calcium is thought to play an important role in the genesis of atherosclerotic lesions, but the precise mechanisms involved are unclear. In the present investigation, we have used in vitro systems to investigate the effects of calcium on one key aspect of lesion development: the migration of macrophages and macrophage/foam cells. Using agarose plate migration assays, the migratory characteristics of macrophages exposed to 1) no lipoprotein, 2) low density lipoprotein (LDL), 3) acetylated low density lipoprotein (acLDL), and ) oxidized low density lipoprotein were examined. The most marked stimulatory effect on macrophage mobility was observed when freshly isolated cells were exposed to acLDL during the migration assay. High levels of exogenous calcium were found to suppress the stimulatory effect of acLDL on migration. As the responses of macrophages exposed to a uniform concentration of agents in the surrounding medium may differ from chemotactic responses to a concentration gradient, the migration of macrophages, with and without preexposure to acLDL or LDL, was studied using microchemotaxis Boyden chambers. Under these conditions, calcium acted as a highly potent chemoattractant, especially to cells that had been preincubated with acLDL. These results suggest how elevated external calcium concentration leads initially to macrophage recruitment, and subsequently to foam cell aggregation and lipid core formation, in association with calcification, in the developing atherosclerotic plaque.

Repression of the macrophage scavenger receptor in macrophage-smooth muscle cell heterokaryons.

Macrophage scavenger receptors mediate the uptake of chemically modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid and thus a foamy cellular morphology. In atherosclerotic lesions, foam cells originate not only from macrophages but also from smooth muscle cells, yet smooth muscle cells do not normally express scavenger receptors, and when exposed to chemically modified LDL in vitro, lipid accumulation does not occur. The mechanism of conversion of smooth muscle cells into foam cells in the arterial wall is thus still under discussion. To investigate whether direct interaction between macrophages and smooth muscle cells may be involved and to explore the effects of components of the two cell types on the expression of scavenger receptors, we report here experiments using somatic cell hybrids formed by fusion of the two cell types. Immunofluorescent labeling and confocal microscopic techniques were applied to investigate and measure (1) lipid accumulation (using Nile Red staining), (2) the binding and uptake of acetylated LDL (using 1,1'-dioctadecyl-1-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated LDL), and (3) receptor expression (assessed using a specific anti-receptor antibody) in smooth muscle cell-macrophage heterokaryons, macrophage-macrophage homokaryons, smooth muscle cell-smooth muscle cell homokaryons, and unfused macrophages and smooth muscle cells. The results demonstrate that scavenger receptor expression becomes repressed in macrophage-smooth muscle cell heterokaryons but not in macrophage-macrophage homokaryons. One possible explanation for the observed repression would be the existence of a negative regulatory cytoplasmic factor produced by smooth muscle cells.

Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture.

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid alpha-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, beta-hexosaminidase (N-acetylglucosaminidase) and alpha-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; beta-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and beta-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.

Interaction of biglycan with type I collagen.

The small proteoglycan decorin is known to interact with type I collagen fibrils, thereby influencing the kinetics of fibril formation and the distance between adjacent collagen fibrils. The structurally related proteoglycan biglycan has been proposed not to bind to fibrillar collagens. However, when osteosarcoma cells were cultured on reconstituted type I collagen fibrils, both decorin and biglycan were retained by the matrix. Immunogold labeling at the electron microscopic level showed that both proteoglycans were distributed along collagen fibrils not only in osteosarcoma cell-populated collagen lattices but also in human skin. Reconstituted type I collagen fibrils were able to bind in vitro native and N-glycan-free biglycan as well as recombinant biglycan core protein. From Scatchard plots dissociation, constants were obtained that were higher for glycanated biglycan (8.7 x 10(-8) mol/liter) than for glycanated decorin (7 x 10(-10) mol/liter and 3 x 10(-9) mol/liter, respectively). A similar number of binding sites for either proteoglycan was calculated. Recombinant biglycan and decorin were characterized by lower dissociation constants compared with the glycanated forms. Glycanated as well as recombinant decorin competed with glycanated biglycan for collagen binding, suggesting that identical or adjacent binding sites on the fibril are used by both proteoglycans. These data suggest that, because of its trivalency, biglycan could have a special organizing function on the assembly of the extracellular matrix.

Abnormal processing of Golgi elements and lysosomes in Tangier disease.

It has been demonstrated that the cellular defect in Tangier disease is associated with morphological abnormalities in the lysosomal compartment and in the Golgi apparatus of mononuclear phagocytes (MNPs) after lipid loading by exposure to acetylated low density lipoproteins (acetyl-LDLs). On exposure to acetyl-LDL, Tangier MNPs accumulate two unusual types of vacuoles that were not observed in control MNPs. The electron-lucent type I vacuoles are round or ovoid, form aggregates, and are filled with fine flocculent or fibrillar material. Type II vacuoles are filled with more electron-dense material, are larger, and contain scrolls and lamellae with varying degrees of electron opacity. By immunoelectron microscopy both types of vacuoles could be identified as lysosomes. This abnormality of the lysosomal compartment in Tangier MNPs was not observed in Tangier fibroblasts, where the few lysosomes detected were found to be similar to those of controls. Morphological analysis of the Golgi apparatus in Tangier MNPs revealed numerous dilated Golgi cisternae, which were distributed more widely throughout the cell. However, both Tangier MNPs and especially Tangier fibroblasts showed a marked hyperplasia of the Golgi complex. Specific staining of the Golgi apparatus with the fluorescent ceramide analogue N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproylsphingosine (C6-NBD-ceramide) showed that Tangier fibroblasts incorporated significantly higher amounts of C6-NBD-ceramide than did control fibroblasts. Moreover, the Tangier fibroblasts that appear generally larger than normal fibroblasts also show significant staining along the cytoskeleton, which may indicate the association of fluorescent ceramide and sphingomyelin to cytoskeletal elements. Phospholipid synthesis and catabolism were studied in Tangier fibroblasts from five patients with the use of tritiated choline as a tracer. A significantly higher incorporation rate was measured for sphingomyelin (176 +/- 18% of control) and phosphatidylcholine (144 +/- 12% of control). A moderate increase in catabolism was found for sphingomyelin (20 +/- 8% of control) and phosphatidylcholine (11 +/- 4% of control). These data are principally in agreement with our former studies of the abnormalities of phospholipid metabolism in Tangier MNPs (G. Schmitz et al, Arteriosclerosis 1990; 10:1010-1019). It is concluded that the defect in Tangier disease that we have recently described as a "disorder of intracellular traffic" (G. Schmitz et al, Proc Natl Acad Sci U S A 1985;82:6305-6309) is also expressed in Tangier fibroblasts. Tangier disease seems to be associated with morphological abnormalities of intracellular organelles, thus strongly indicating that the translocation disorder in this disease affects the Golgi apparatus and the regular processing of lysosomes and shows abnormalities in cellular lipid metabolism.