Lipoprotein lipase enhances the binding of native and oxidized low density lipoproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells.
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Retention of low density lipoproteins (LDL) by vascular proteoglycans and their subsequent oxidation are important in atherogenesis. Lipoprotein lipase (LPL) can bind LDL and proteoglycans, although the effect of different proteoglycans to influence the ability of LPL to act as a bridge in the formation of LDL-proteoglycan complexes is unknown. Using an electrophoretic gel mobility shift assay, [(35)S]SO(4)-labeled versican and biglycan, two extracellular proteoglycans secreted by vascular cells, bound native LDL in a saturable fashion. The addition of bovine milk LPL dose-dependently increased the binding of native LDL to both versican and biglycan, approaching saturation at 30-40 microgram/ml LPL for versican and 20 microgram/ml LPL for biglycan. LDL was oxidized by several methods, including copper, 2, 2-azo-bis(2-amidinopropane)-2HCl and hypochlorite. Extensively copper- and hypochlorite-oxidized LDL bound poorly to versican and biglycan. Proteoglycan binding to LDL was correlated inversely with the extent of LDL; however, the addition of LPL to oxidized LDL together with biglycan or versican allowed the oxidized LDL to bind the proteoglycans in an LPL dose-dependent manner. Addition of LPL had a greater relative effect on the binding of extensively oxidized LDL to proteoglycans compared with native LDL. LPL had a slightly greater effect on increasing the binding of native and oxidized LDL to biglycan than versican. Thus, LPL in the artery wall might increase the atherogenicity of oxidized LDL, since it enables its binding to vascular biglycan and versican. (+info)
Acute glomerular upregulation of ornithine decarboxylase is not essential for mesangial cell proliferation and matrix expansion in anti-Thy-1-nephritis.
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BACKGROUND: Pathways of L-arginine metabolism including nitric oxide, agmatine and polyamine synthesis are upregulated during glomerular inflammation in experimental glomerulonephritis. In anti-Thy-1-glomerulonephritis L-arginine-deficient diets ameliorate the disease course in this model. However, it is unclear which metabolic pathway is affected by this substrate depletion. Since polyamines are important proproliferative molecules, we studied the effect of specific polyamine synthesis blockade in vivo on mesangial cell proliferation and glomerular fibrosis in this model. METHODS: Anti-Thy-1-glomerulonephritis was induced in male Sprague-Dawley rats by single-bolus injection of monoclonal ER4-antibodies. Rats were treated with difluoromethylornithine (0.5-2% in the drinking water), a selective inhibitor of the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC). Mesangial cell proliferation and matrix expansion were evaluated in PAS-stained kidney tissues. Glomerular TGF-beta and biglycan-mRNA-expression were determined by Northern blot analysis and albuminuria was measured using a competitive ELISA. Data were compared to untreated controls. RESULTS: Though complete inhibition of ODC activity was achieved at any time point, difluoromethlornithine treatment had no significant effect on albuminuria, glomerular matrix protein expression and mesangial cell count in this model. CONCLUSIONS: The acute upregulation of glomerular ODC activity above baseline in anti-Thy1-glomerulonephritis is not pathophysiologically important for disease development however, biological effects of available polyamine pools cannot be excluded by our study. (+info)
Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease.
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BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases. (+info)
The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle.
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The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy. (+info)
Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein.
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Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation. (+info)
Altered dermatan sulfate structure and reduced heparin cofactor II-stimulating activity of biglycan and decorin from human atherosclerotic plaque.
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Biglycan and decorin are small dermatan sulfate-containing proteoglycans in the extracellular matrix of the artery wall. The dermatan sulfate chains are known to stimulate thrombin inhibition by heparin cofactor II (HCII), a plasma proteinase inhibitor that has been detected within the artery wall. The purpose of this study was to analyze the HCII-stimulatory activity of biglycan and decorin isolated from normal human aorta and atherosclerotic lesions type II through VI and to correlate activity with dermatan sulfate chain composition and structure. Biglycan and decorin from plaque exhibited a 24-75% and 38-79% loss of activity, respectively, in thrombin-HCII inhibition assays relative to proteoglycan from normal aorta. A significant negative linear relationship was observed between lesion severity and HCII stimulatory activity (r = 0.79, biglycan; r = 0.63, decorin; p < 0.05). Biglycan, but not decorin, from atherosclerotic plaque contained significantly reduced amounts of iduronic acid and disulfated disaccharides DeltaDi-2,4S and DeltaDi-4,6S relative to proteoglycan from normal artery. Affinity coelectrophoresis analysis of a subset of samples demonstrated that increased interaction of proteoglycan with HCII in agarose gels paralleled increased activity in thrombin-HCII inhibition assays. In conclusion, both biglycan and decorin from atherosclerotic plaque possessed reduced activity with HCII, but only biglycan demonstrated a correlation between activity and specific glycosaminoglycan structural features. Loss of the ability of biglycan and decorin in atherosclerotic lesions to regulate thrombin activity through HCII may be critical in the progression of the disease. (+info)
Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan.
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Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs. (+info)
Proteoglycan composition in the human sclera during growth and aging.
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PURPOSE: Scleral proteoglycans were characterized from human donor eyes aged 2 months to 94 years to identify age-related changes in the synthesis and/or accumulation of these extracellular matrix components. METHODS: Newly synthesized proteoglycans (previously radiolabeled with 35SO4) and total accumulated scleral proteoglycans were extracted with 4 M guanidine hydrochloride and separated by molecular sieve chromatography on a Sepharose CL-4B column. The elution positions of newly synthesized and total accumulated proteoglycans were determined by assaying each fraction for radioactivity and glycosaminoglycans, respectively. Regression analyses were performed on the three major proteoglycan peaks to identify age-related changes in scleral proteoglycan composition. Scleral proteoglycans were further purified by anion-exchange chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses. RESULTS: Human scleral proteoglycans were apparent as three major peaks after chromatography on Sepharose CL-4B. The two faster eluting peaks contained alternative forms of the cartilage proteoglycan, aggrecan, whereas the third peak contained the small proteoglycans biglycan and decorin. The relative percentage of newly synthesized and total accumulated aggrecan increased approximately two- to sixfold from infancy to 94 years. In contrast, the relative percentage of newly synthesized and total accumulated biglycan and decorin decreased by approximately 25%. Chromatography and Western blot results indicated that the absolute amounts of all three proteoglycans significantly increased in concentration within the sclera from birth to the fourth decade. Beyond the fourth decade, decorin and biglycan decreased in all scleral regions and were present in lowest concentrations by the ninth decade. In contrast, aggrecan, which was present in highest concentration in the posterior sclera, was not significantly reduced with increasing age. CONCLUSIONS: The age-related changes in scleral proteoglycan composition observed in the present study are likely to contribute to the regional alterations in biomechanical properties of the sclera associated with growth and aging. (+info)