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Objective— The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. Methods and Results— Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. Conclusion— The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

The organization of the brain extracellular matrix appears to be based on aggregates of hyaluronan and proteoglycans, connected by oligomeric glycoproteins. Mild phenotypical consequences were reported from several mouse strains lacking components of this matrix such as neurocan, brevican, tenascin-R, and tenascin-C. To further challenge the flexibility of the extracellular matrix network of the brain, mice lacking all four brain extracellular matrix molecules were generated, which were found to be viable and fertile. Analysis of the brains of 1-month-old quadruple KO mice revealed increased protein levels of fibulin-1 and fibulin-2. Histochemical analysis showed an unusual parenchymal deposition of these fibulins. The quadruple KO mice also displayed obvious changes in the pattern of deposition of hyaluronan. Further, an almost quadruple knockout like extracellular environment was noticed in the brains of triple knockout mice lacking both tenascins and brevican, since these brains had strongly reduced levels of neurocan.

Resident dendritic cells (DC) within the T cell area of the lymph node take up soluble antigens that enter via the afferent lymphatics before antigen carrying DC arrive from the periphery. The reticular network within the lymph node is a conduit system forming the infrastructure for the fast delivery of soluble substances from the afferent lymph to the lumen of high endothelial venules (HEVs). Using high-resolution light microscopy and 3D reconstruction, we show here that these conduits are unique basement membrane-like structures ensheathed by fibroblastic reticular cells with occasional resident DC embedded within this cell layer. Conduit-associated DC are capable of taking up and processing soluble antigens transported within the conduits, whereas immigrated mature DC occur remote from the reticular fibers. The conduit system is, therefore, not a closed compartment that shuttles substances through the lymph node but represents the morphological equivalent to the filtering function of the lymph node.

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.

Neurocan is one of the major chondroitin sulfate proteoglycans of perinatal rodent brain. HEK-293 cells producing neurocan recombinantly show changes in their behavior. The expression of full-length neurocan led to a detachment of the secreting cells and the formation of floating spheroids. This occurred in the continuous presence of 10% fetal bovine serum in the culture medium. Cells secreting fragments of neurocan-containing chondroitin sulfate chains and the C-terminal domain of the molecule showed a similar behavior, whereas cells expressing fragments of neurocan-containing chondroitin sulfate chains but lacking parts of the C-terminal domain did not show spheroid formation. Cells secreting the hyaluronan-binding N-terminal domain of neurocan showed an enhanced adhesiveness. When untransfected HEK-293 cells were plated on a surface conditioned by spheroid-forming cells, they also formed spheroids. This effect could be abolished by chondroitinase treatment of the conditioned surface. The observations indicate that the ability of the chondroitin sulfate proteoglycan neurocan to modulate the adhesive character of extracellular matrices is dependent on the structural integrity of the C-terminal domain of the core protein.

Background and Objectives 5-fluorouracil (5-FU) exerts its effects mainly by its incorporation into RNA and inhibition of DNA synthesis. Its toxicity may therefore be estimated by measuring its incorporation into RNA. Protein malnutrition has been considered to increase the toxicity of 5-FU. Methods Rats with a hepatoma implanted into the liver were fed on either a 25% or a 0% casein diet for 1 week. On the last day, they were infused via the hepatic artery with a therapeutic dose of 3H-5-FU. Its incorporation into RNA was measured in hepatoma and several normal tissues. Results Protein deprivation increased the incorporation of 5-FU into liver and intestinal RNA. Incorporation into hepatoma RNA did not increase significantly, but the ratio, liver/hepatoma RNA incorporation, remained unchanged. Conclusions Protein deprivation might increase the toxicity of 5-FU on liver and intestine. J. Surg. Oncol. 1997;65:155–158. © 1997 Wiley-Liss, Inc.

The cytotoxicity of 5-FUra has been related to its incorporation into RNA. In a model of secondary liver cancer in the rat, the incorporation of 5-FUra into the acid-soluble fraction, RNA and DNA of several normal tissues and an adenocarcinoma of the colon transplanted to the liver was determined. A therapeutic labelled dose of the drug was infused via the hepatic artery for 2 hr and the rats killed 1 hr later. Half of the rats were starved overnight before treatment. The incorporation of 5-FUra into liver and intestinal RNA increased at starvation. It was unchanged in kidney and bone marrow.The incorporation into tumor RNA decreased insignificantly. The incorporation into tumor RNA was significantly higher than in hepatic, intestinal, and renal RNA at ad libitum feeding. This difference disappeared at overnight starvation. © 1993 Wiley-Liss, Inc.