Proteoglycans Released from Cultured Bovine Aortic Endothelial Cell Layers by Sodium Spirulan Are Both Perlecan and Biglycan

Abstract

Proteoglycans (PGs) are macromolecules that are composed of a core protein and one or more glycosaminoglycan (GAGs) side chains in a common feature. 1) In the vascular tissue, PGs are involved in the regulation of vascular properties such as permeability, lipid metabolism, hemostasis, thrombosis, and extracellular matrix assembly. [2][3][4][5] In addition, PGs influence the regulation of vascular endothelial and smooth muscle cell function mediated by growth factors 6) and cytokines. 7) There are two types of PGs classified by the structure of GAG chains: one is heparan sulfate PGs (HSPGs) and the other is chondroitin/dermatan sulfate PGs (CS/DS PGs). Vascular endothelial cells predominantly synthesize and secrete a large HSPG, perlecan,8) and a small CS/DS PG, biglycan. 9)Perlecan exhibits antithrombin activity through the activation of antithrombin III 10) by heparin-like sequences in the heparan sulfate chains.11) On the other hand, the antithrombin activity of biglycan is achieved by the activation of heparin cofactor II by the dermatan sulfate chains.12) It is therefore suggested that PGs synthesized and secreted by vascular endothelial cells contribute to local anticoagulant activity on the endothelium.Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated as an antiviral agent from a hot water extract of the blue-green alga Spirulina platensis. 13,14) On the other hand, Na-SP exhibits antithrombin activity in vitro in a fashion different from heparin, 15) suggesting that Na-SP may be an agent that can control anticoagulant activity in the blood. In addition, we found that Na-SP enhances the fibrinolytic activity of the cells through inhibition of plasminogen activator inhibitor-1 secretion.16) This indicates that Na-SP can modulate the anticoagulant and fibrinolytic properties of vascular endothelial cells. We hypothesized that Na-SP may influence the metabolism of endothelial PGs, since heparin has a stimulatory effect on the release of PGs from vascular endothelial cells.17) It was shown that Na-SP stimulates the endothelial PG release as does heparin 18) ; the stimulatory effect requires both the sulfate group and a sodium ion. 19) However, the type(s) of endothelial PGs whose release is stimulated by Na-SP has not been identified yet. In the present study, we suggest that Na-SP-releasable PGs in vascular endothelial cells are both perlecan and biglycan with the intact structure, although the core proteins may be partially degraded. MATERIALS AND METHODSMaterials Vascular endothelial cells derived from bovine aorta were purchased from Dainippon Pharmaceutical (Osaka, Japan). Na-SP was purified as described previously.1,2) Dulbecco's modified Eagle's medium and fetal bovine serum were from Nissui Pharmaceutical (Tokyo, Japan) and Equitech-Bio (Kerrville, TX, U.S.A.), respectively. ASF 301 medium was from Ajinomoto (Tokyo, Japan); tissue culture dishes and plates were from Asahi Technoglass (Chiba, Japan Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spiruli...

Proteoglycans Predominantly Synthesized by Human Brain Microvascular Endothelial Cells in Culture are Perlecan and Biglycan

“…Nolan and Shaikh 25) suggested that the initial effect of acute cadmium administration is on the integrity and permeability of the vascular endothelium and other necrotic changes in the underlying tissue occur secondarily. We have shown that cadmium, 26,27) lead, [27][28][29] arsenite, 30) and the biologically active sulfated polysaccharide sodium spirulan [31][32][33] influence the functions, including proteoglycan metabolism, of vascular endothelial cells. Proteoglycans are involved in the regulation of vascular endothelial cell functions; however, excluding a few exceptions such as the inhibition of vascular endothelial repair by lead, the toxicity or biological activity that mediate altered proteoglycan metabolism has not been completely understood.…”

Extracellular matrix remodelling properties of human fibrocytes

“…The extracts were concentrated with DEAE‐Sephacel minicolumns and precipitated with 1.3% potassium acetate in 95% ethanol. The precipitated PGs were separated on 0.75% agarose gel or by SDS‐PAGE (with a 4–12% gradient slab gel and a 3% stacking gel), using equal fractions of radio‐labelled material from fibrocytes and fibroblasts, with or without prior digestion of the GAG chains with both heparinase/heparitinase I (27.6 U/ml) and heparinase/heparitinase II (13.8 U/ml) (Sigma‐Aldrich) or with chondroitinase ABC (1.67 U/ml, Seikagaku, Tokyo, Japan) [27]. The radio‐labelled proteins were visualized by autoradiography.…”

“…For metabolic labelling of newly synthesized proteins, cells were incubated for 24 hrs with 50 μCi/ml Tran 35 S‐Label (ICN Biomedicals, Irvine, CA, USA) in methionine and cysteine‐free DMEM medium. Cell samples were then processed for analysis of the PG core proteins as previously described [26, 27]. Briefly, radio‐labelled PGs were extracted from the conditioned medium and the cell layers.…”

Inhibition of Cultured Bovine Aortic Smooth Muscle Cell Proliferation by Colominic Acid

“…11) In vascular endothelial cells, Na-SP elevates the fibrinolytic activity of vascular endothelial cells through inhibition of the secretion of plasminogen activator inhibitor type 1. 12) In addition, Na-SP promotes the secretion of anticoagulant proteoglycans 13) such as perlecan and biglycan, 14) as well as exhibiting an antithrombin activity through activation of heparin cofactor II. 15,16) We hypothesized that CA or SCA may inhibits the proliferation of vascular smooth muscle cell proliferation, as does natural or semi-synthetic polysaccharides, including Na-SP, heparin, 17) pentosan polysulfate 18) and fucoidan.…”