人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑机细胞合成蛋白聚糖

从人正常胸主动脉分离硫酸乙肝素蛋白聚糖,观察其对体外培养的人主动脉平滑肌细胞合成ＰＧ的影响,ＨＡＳＭＣ在不加或加ＨＳＰＧ的＾３５Ｓ－硫酸钠培养液中培养,以标记ＰＧ继之,培养液及细胞层的４ｍｏｌ／Ｌ盐酸胍提取液是ＰＧｓ经离子交换及凝胶过滤柱层析分离,发现加ＨＳＰＧ后,培养液中的ＨＳＰＧ,硫酸软骨素ＰＧ及硫酸皮肤素－硫酸软骨素ＰＧ（ＤＳＣＳＰＧ）均明显增高,而细胞层中仅ＨＳＰＧ和ＣＳＰＧ增高,且加ＨＳ     

硫酸乙酰肝素蛋白聚糖对培养人脐静脉内皮细胞合成蛋白聚糖的…

以「＾３５Ｓ」－Ｎａ２ＳＯ４为示踪物,观察人正常主动脉中的硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对培养的第一代人脐静脉内皮细胞（ｈＵＶＥＣ）合成蛋白聚糖（ＰＧ）的影响,用解聚提取法及离子交换柱层析分离人主动脉ＨＳＰＧ,＾３５Ｓ－ＰＧｓ的混合物用离子交换及凝胶过滤柱层析法分离＾３５Ｓ－ＨＳＰＧ,＾３５Ｓ－硫酸软骨素－硫酸皮肤素ＰＧ（＾３５Ｓ－ＣＳＤＳＰＧ）及＾３５Ｓ－硫酸皮肤素ＰＧ（＾３５Ｓ－ＤＳＰＧ）,     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用＾３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣ ＤＮＡ合成的作用及对血小板源生长因子（ＰＧＤＦ）、ＰＧＤＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑肌细胞生长的影响

从动脉粥样硬化（ＡＳ）高（北京）、低（南宁）发区人正常胸主动脉内－中膜分离ＨＳＰＧ,观察其对体外培养的ＨＡＳＭＣ生长的影响,细胞计数、~３Ｈ－ＴｄＲ参入及形态观察均表明ＡＳ高、低发区人主动脉ＨＳＰＧ都能剂量依赖性地抑制ＨＡＳＭＣ增殖,但抑制百分数未见显著差异,结果提示,人动脉壁中ＨＳＰＧ的含量可能与ＡＳ发病有关．     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

人主动脉硫酸乙酰肝素蛋白聚糖对培养人的脐静脉内皮细胞生长的影响

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及离子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜、细胞计数、及￣３Ｎ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＦＣ）生长的影响。结果发现：（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ已糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）．（２）随着培养时间增加（２４,４８及７２ｈ）．根据细胞计数计算出同一剂量的ＨＳＰＧ（１７．０μｇ已糖醛酸／ｍｌ）对ｈＵＶＥＣ的促增殖％增高（分别为１４％,３０％及３７％）。（３）随着加入ＨＳＰＧ浓度的升高（４．３,８．５及１７．０μｇ已糖醛酸／ｍｌ．培养７２ｈ）．根据￣３Ｈ－ＴｄＲ参入计算出ＨＳＰＧ对ｈＵＶＥＣ的促增殖％亦增高（分别为４９％,７１％及９８％）。故人主动脉ＨＳＰＧ对培养的人脐静脉内皮细胞有促增殖作用。     

人主动脉硫酸乙酰肝系蛋白聚糖对培养人的脐静脉内皮细胞生长…

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及墩子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜,细胞计数,及＾３Ｈ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＥＣ）生长的影响,结果表明,（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ己糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）（２）随着培养时间增加（２４,４８及７２ｈ）根据细胞计数计算     

硫酸乙酰肝素蛋白聚糖对培养人脐静脉内皮细胞合成蛋白聚糖的影响

以[35S」-Na2SO4为示踪物,观察人正常主动脉中的硫酸乙酸肝素蛋白聚糖（HSPG）对培养的第一代人脐静脉内皮细胞（hUVEC）合成蛋白聚糖（PG）的影响．用解聚提取法及离子交换柱层析分离人主动脉HSPG．35S-PGs的混合物用离子交换及凝胶过滤柱层析法分离35S-HSPG,35S-硫酸软骨素-硫酸皮肤素PG（35S-CSDSPG）及35S-硫酸皮肤素PG（35S-DSPG）．结果发现实验组（加HSPG）与对照组（未加HSPG）相比,hU-VEC的35S-PGs总量（培养液+细胞层）无差别,但实验组培养液中35S-PGs总量升高、35S-DSPG、35S-CSDSPG及其相对百分含量均升高,而35S-HSPG及其百分含量降低．细胞层的35S-PGs,35S-HSPG及其相对百分含量降低,35S-DSPG及其相对百分含量升高,而CSDSPG未见差别．     

硒对培养人胚肝细胞Ⅲ型前胶原,羟脯氨酸合成的影响

原代培养人胚肝细胞经１．１５６×１０￣(－７）ｍｏｌ／Ｌ硒预处理４ｈ,加入２０ｍｍｏｌ／Ｌ四氟化碳作用２０ｈ,观察硒对其Ⅲ型前胶原（ＰＣⅢ）和羟脯氨酸（Ｈｙｐ）生成的影响。结果培养液中ＰＣⅢ水平、细胞内Ｈｙｐ含量及细胞内外丙二醛（ＭＤＡ）水平均降低,与未加硒对照组比较差别有显著性（Ｐ＜０．０１）。而硒谷腕甘肽过氧化物酶（Ｓｅ－ＧＳＨ－ＰＸ）活性则较对照组显著增高（Ｐ＜０．００１）,且ＰＣⅢ水平与Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值呈负相关（ｒ＝－０．９１５６,Ｐ＜０．０１）。提示硒可提高Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值,抑制脂质过氧化激发的肝细胞胶原合成。     

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.     

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.     

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.     

Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells. An underlying mechanism for the modulation of smooth muscle cell growth?

Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 microg/ml) increased (35)SO(4) incorporation into PG in media by 25-30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [(3)H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of (35)SO(4) to [(3)H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.     

The expression of specific surface antigen of smooth muscle cells is related to proliferation

Monoclonal antibody L1 has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) orginally isolated from rat aortic media. Antibody L1 recognizes only the surface antigen of cultured SMC and does not react with other cultured rat cell types. It has been shown that in primary culture of SMC the L1-positive cells appear on the 2nd to 3rd day and their proportion increases up to the 7th day up to 40% in DMEM supplemented with 10% of fetal calf serum (FCS), up to 25% in DMEM with 5% of rat whole-blood serum, but up to only 5% in DMEM with 5% rat plasma-derived serum. These results are in agreement with data on [¹⁴C]thymidine incorporation and on flow cytometry. Using FACS II, the SMC were sorted into subpopulations on the 4th and 8th days of primary culture according to the intensity of their specific immunofluorescence. It has been found that the DNA profile in intensively labelled cells corresponds to that in an intensively proliferating population of cells. These findings suggest that antigen L1 appears to be the specific marker of modulated SMC entering the cell cycle.     

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles: EVIDENCE FOR HEPARAN SULFATE EPITOPE SPECIFICITY AND ROLE OF BOTH SYNDECAN AND GLYPICAN*

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (αHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [³⁵S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.