Background and Purpose-Many processes involved in the pathogenesis of atherosclerosis result in modifications of the extracellular matrix. These changes not only determine the mechanical stability of atherosclerotic lesions but can directly or indirectly influence further development of the lesions. The purpose of the present study was to compare the matrix composition of human carotid plaques from symptomatic patients with those obtained from patients without symptoms. Furthermore, matrix changes related to age were studied. Methods-Thirty atherosclerotic carotid plaques were removed by endarterectomy from 27 patients and divided into 2 groups on the basis of the presence of ipsilateral symptoms. The plaques were homogenized, and the total levels of the major components of the extracellular matrix were determined. Results-Plaques associated with symptoms were characterized by increased levels of elastin (1.58Ϯ0.46 versus 1.24Ϯ0.40 mg/g wet wt; Pϭ0.03) and decreased levels of hydroxyapatite (45.1Ϯ46.3 versus 131.4Ϯ111.7 mg/g wet wt; Pϭ0.02) compared with asymptomatic plaques. The increase in elastin in plaques from symptomatic patients was due to elevated levels of an intermediate-size fraction, as determined by liquid chromatography. Collagen and sulfated glycosaminoglycans were present in equal amounts in both groups. Elastin content in carotid plaques decreased with age. Conclusions-Carotid plaques from symptomatic patients have lower levels of hydroxyapatite than those from asymptomatic patients. The present study also raises the possibility that non-cross-linked forms of elastin, increased in plaques associated with symptoms, could be a marker of plaque vulnerability and/or directly induce harmful cellular activities or increase lipoprotein retention in the vascular wall.

This article describes the possible role of the endothelial cell-surface coat, containing proteoglycans (PGs) with connected glycosaminoglycans (GAGs), in maintaining glomerular permselectivity. Primary human glomerular endothelial cells (HGEC) in culture were treated with the nephrosis-inducing agent puromycin aminonucleoside (PAN). Analysis was made by TaqMan real-time PCR, Western blot analysis, and by metabolic labeling with [(35)S]sulfate. The HGECs express several PGs: syndecan, versican, glypican, perlecan, decorin, and biglycan, which may contribute to the glomerular charge barrier. PAN treatment downregulated both the protein expression (by 25%) and the mRNA expression (by 37 +/- 6%, P < 0.001, n = 8) of versican compared with control. Transferases important for chondroitin and heparan sulfate biosynthesis were also significantly downregulated by PAN, resulting in less sulfate groups, shorter GAG chains, and reduced PG net-negative charge. Moreover, analysis of the cell media after PAN treatment revealed a reduced content of [(35)S]sulfate-labeled PGs (40% of control). We conclude that PAN may cause proteinuria by affecting the endothelial cell-surface layer and not only by disrupting the foot process arrangement of the podocytes. Thus the endothelium may be a more important component of the glomerular barrier than hitherto acknowledged.

Phosphorylation of decorin was investigated by incubating a rat fibroblast cell line with radiolabelled phosphate and carbohydrate precursors. There was a transient phosphorylation of the linkage-region saccharides in intracellular decorin prior to assembly of the galactosaminoglycan chain. Phosphorylation gradually increased from xylosylated, galactosyl-xylosylated to galactosyl-galactosyl-xylosylated core protein where all trisaccharide stubs were phosphorylated. Addition of the first glucuronate residue was accompanied by rapid dephosphorylation. Brefeldin A treatment resulted in segregation of galactosaminoglycan synthesis and dephosphorylation. Enzymatic degradation of brefeldip-A-arrested immature proteo8lycan with incomplete galactosaminoglycan chain [Moses, J., Oldberg, A.,Biochem., in the press] by using chondroitin AC lyase and chondro-glycuronidase, followed by P-galactosidase treatment, demonstrated the sequence galactosyl-galactosyl-phosphoxylose. The xylose was resistant to direct periodate oxidation, but sensitive after treatment with alkaline phosphatase, showing that the phosphate was located at C2 of xylose. The transient 2-phosphorylation of xylose may be involved in intracellular transport and/or in the control of modifications of the glycan chain.

Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

Background and Purpose-Echolucent carotid plaques have been associated with increased risk for stroke. Histological studies suggested that echolucent plaques are hemorrhage-and lipid-rich, whereas echogenic plaques are characterized by fibrosis and calcification. This is the first study to relate echogenicity to plaque composition analyzed biochemically. Methods-Echogenicity of human carotid plaques was analyzed by standardized high-definition ultrasound and classified into echolucent, with gray-scale median (GSM) Ͻ32 and echogenic with GSM Ն32. The biochemical composition of the plaques was assessed by fast-performance liquid chromotography and high-performance thin-layer chromotography. Results-As

from eDNA indicates that decorin is synthesized as a precurbe due to a lower affinity between the core protein and the sor with a 14 amino acid residue long propeptide (Fig. 1).glycosyltransferases synthesizing the linkage region. Alternatively, the deletions may affect the intracellular transport of Previous investigations have suggested that this propeptide is removed intracellularly, before secretion of decorin [17].decorin. An antiserum prepared against the N-terminal propeptide immunoprecipitated decorin secreted by cultured cells, In contrast, it has been shown that biglycan, which is strucshowing that decorin is exported with the N-terminal region rurally related to decorin and also synthesized as a precursor, intact, is secreted from cultured smooth muscle cells with the propeptide intact [18].

No abstract

Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radiolabelled protein and galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulsekhase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Most connective tissues contain small leucine-rich proteoglycans substituted with dermatan sulphate or chondroitin sulfate depending on the source (Ciister and Fransson, 1981 ; Coster et al., 1984;Heineglrd et al., 1985; Heinegird and Oldberg, 1993;Cheng et al., 1994). One of these proteoglycans is named decorin because it decorates collagen fibrils in a characteristic manner (Krusius and Ruoslahti, 1986; Scott et al., 1981). The single galactosaminoglycan chain is attached to a serine residue located to the extreme N-terminal portion. In decorin isolated from extracellular matrices the N-terminal sequence is DEASGIlP in the rat (Kokenyesi and Woessner, 1990; Abramson and Woessner, 1992), and DEASGIGP in bovine and human decorin (Brennan et al., 1984), both of which conform with the proposed recognition consensus sequence for glycosaminoglycan attachment (Bourdon et al., 1987). Comparisons between the N-terminal sequence of extracellular decorin and the sequences deduced from cDNA indicate that decorin is synthesized as a precursor with a 14-amino-acid-long propeptide. We have studied expression and secretion of decorin in COS and CHO cells transformed with vectors encoding both wild-type rat decorin and variants with deletions in the N-terminal region (Oldberg et al., 1996). Cells expressing deletion variants synthesized proteoglycans with a shorter galactosaminoglycan chain compared with those derived from wild-type decorin with an intact N-terminal domain.