Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.
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Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD. (+info)
Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.
(10/5814)
The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion. (+info)
Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis.
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BACKGROUND: The pathogenesis of pancreatic fibrosis is unknown. In the liver, stellate cells play a major role in fibrogenesis by synthesising increased amounts of collagen and other extracellular matrix (ECM) proteins when activated by profibrogenic mediators such as cytokines and oxidant stress. AIMS: To determine whether cultured rat pancreatic stellate cells produce collagen and other ECM proteins, and exhibit signs of activation when exposed to the cytokines platelet derived growth factor (PDGF) or transforming growth factor beta (TGF-beta). METHODS: Cultured pancreatic stellate cells were immunostained for the ECM proteins procollagen III, collagen I, laminin, and fibronectin using specific polyclonal antibodies. For cytokine studies, triplicate wells of cells were incubated with increasing concentrations of PDGF or TGF-beta. RESULTS: Cultured pancreatic stellate cells stained strongly positive for all ECM proteins tested. Incubation of cells with 1, 5, and 10 ng/ml PDGF led to a significant dose related increase in cell counts as well as in the incorporation of 3H-thymidine into DNA. Stellate cells exposed to 0.25, 0.5, and 1 ng/ml TGF-beta showed a dose dependent increase in alpha smooth muscle actin expression and increased collagen synthesis. In addition, TGF-beta increased the expression of PDGF receptors on stellate cells. CONCLUSIONS: Pancreatic stellate cells produce collagen and other extracellular matrix proteins, and respond to the cytokines PDGF and TGF-beta by increased proliferation and increased collagen synthesis. These results suggest an important role for stellate cells in pancreatic fibrogenesis. (+info)
Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice.
(12/5814)
The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent. (+info)
Cell adhesion activity for murine carcinoma cells of a wheat germ 55-kDa protein with binding affinity for animal extracellular matrix proteins.
(13/5814)
A wheat germ 55-kDa protein was isolated by affinity chromatography with Matrigel immobilized on agarose, followed by preparative gel electrophoresis. This Matrigel-binding protein designated as WG-55 had an amino-terminal amino acid sequence which is identical to that of a putative mature form of wheat storage protein Gbl 1. WG-55 reacted with concanavalin A, indicating its glycoprotein nature as expected from the amino acid sequence of Gbl 1. As expected, similarly, WG-55 exhibited RGD-dependent cell adhesion activity for murine carcinoma cells. These data suggest that WG-55 or mature Gbl 1 protein may play a role in plant cell adhesion. (+info)
Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis.
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Ectopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21(WAF1/CIP1) (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) approximately 4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 approximately 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency. (+info)
Cortical bitufted, horizontal, and Martinotti cells preferentially express and secrete reelin into perineuronal nets, nonsynaptically modulating gene expression.
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Reelin (Reln) is a protein with some structural analogies with other extracellular matrix proteins that functions in the regulation of neuronal migration during the development of cortical laminated structures. In the cortex of adult animals, Reln is expressed primarily in gamma-aminobutyric acid (GABA)ergic neurons and is secreted into perineuronal nets. However, only 50-60% of GABAergic interneurons express Reln. We have characterized this subpopulation of cortical GABAergic neurons that expresses Reln by using two strategies: (i) a double immunolabeling procedure to determine the colocalization of Reln with neuropeptides and Ca2+-binding proteins and (ii) a combination of Golgi staining and Reln immunolabeling to determine the morphology of the rat cortical cells that store Reln. Many interneurons that express Neuropeptide Y (NPY) or somatostatin (but none of those that express parvalbumin) are Reln-immunopositive. A small population of calbindin-positive interneurons and very few calretinin-positive cells express Reln immunopositivity. Golgi staining revealed that layer I horizontal cells, layer II-V bitufted neurons, and some deep cortical layer Martinotti cells express Reln. Basket and chandelier cells are often immunopositive to parvalbumin, but never to Reln. Although Reln is secreted by GABAergic neurons, its target are not the GABA receptors, but rather may be extrasynaptically located in perineuronal nets and concerned with the modulation of neuronal plasticity. Dab1, the target adapter protein that presumably mediates transcription regulation via the extrasynaptic actions of Reln, is expressed predominantly in pyramidal neurons, but it can also be detected in a small population of GABAergic neurons that are neither horizontal nor bitufted neurons. (+info)
Fatty acids modulate the composition of extracellular matrix in cultured human arterial smooth muscle cells by altering the expression of genes for proteoglycan core proteins.
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In diabetes-associated microangiopathies and atherosclerosis, there are alterations of the extracellular matrix (ECM) in the intima of small and large arteries. High levels of circulating nonesterified fatty acids (NEFAs) are present in insulin resistance and type 2 diabetes. High concentrations of NEFAs might alter the basement membrane composition of endothelial cells. In arteries, smooth muscle cells (SMCs) are the major producers of proteoglycans and glycoproteins in the intima, and this is the site of lipoprotein deposition and modification, key events in atherogenesis. We found that exposure of human arterial SMCs to 100-300 micromol/albumin-bound linoleic acid lowered their proliferation rate and altered cell morphology. SMCs expressed 2-10 times more mRNA for the core proteins of the proteoglycans versican, decorin, and syndecan 4 compared with control cells. There was no change in expression of fibronectin and perlecan. The decorin glycosaminoglycan chains increased in size after exposure to linoleic acid. The ECM produced by cells grown in the presence of linoleic acid bound 125I-labeled LDL more tightly than that of control cells. Darglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, neutralized the NEFA-mediated induction of the decorin gene. This suggests that some of the NEFA effects are mediated by PPAR-gamma. These actions of NEFAs, if present in vivo, could contribute to changes of the matrix of the arterial intima associated with micro- and macroangiopathies. (+info)