参与脂蛋白代谢酶研究的进展

肝性脂酶(HL)、脂蛋白脂酶(LPL)和卵磷脂胆固醇酰基转移酶(LCAT)是血浆脂蛋白代谢中三个关键酶,与血浆脂质转运蛋白(LTP)一起调控脂蛋白代谢。几十年来它们一直受到人们关注。本文重点介绍近几年来有关这三个酶研究的进展。 1.HL 肝素化血浆中存在二种脂酶活性:HL和LPL。HL是肝实质细胞合成、转运到肝窦状隙内皮细胞表面发挥生理作用,故又称为肝内皮细胞脂酶(HEL)。它是一种糖蛋白,在肝细胞经过一系列糖基化加工。HL-Ⅰ(Mr.51000)和HL-Ⅱ(Mr.53000)代表细胞内加工过程两个不同阶段。在合成类固醇激素的细胞如肾上腺和卵巢细胞也发现HL存在。     

血浆脂蛋白对血小板功能的影响

脂蛋白和血小板同时与许多疾病的病理过程密切相关,包括动脉粥样硬化(冠心病)、血栓形成等。一般认为,高水的低密度脂蛋白(LDL)和低水平的高密度脂蛋白(HDL)以及血小板的高反应性是这些疾病发生发展的共同危险因素。对于以高水平LDL为特征的家族性Ⅱa型高脂血症患者的研究,更加明显地提示脂蛋白与血小板之间存在直接关系的可能性。来源于Ⅱa型高脂血症患者的血小板对许多生理刺激剂(包括肾上腺素、ADP、凝血酶、胶原等)诱导的聚集、释放以及花生四烯酸代谢的反应性均增高。能够降低血浆脂蛋白(LDL或极低密度脂蛋白VLDL)水平的烟酸和安妥明,同时也能降低Ⅱa型高脂蛋白血症患者的血小板高反应性。这些线索引起了人们对于LDL、HDL与血小板之间的直接关系进行深入探讨的兴趣。     

血浆脂蛋白研究的某些进展

1929年,Macheboeuf首先从马血清中分离出一种含有脂类和蛋白质的物质,命名为“酸沉淀性Coenapse。其后,这种物质被称为脂蛋白。但直到1948年,脂蛋白的分类未超出α与β两类。进入50年代后,将超速离心法和电泳法用于脂蛋白的分离获得成功,脂蛋白被分为CM(原点)、VLDL(前β脂蛋白)、LDL(β脂蛋白)及HDL(α脂蛋白)等四类。尤其在发现冠心病(CHD)人血浆LDL升高、HDL降低后,血浆脂蛋白的研究引起人们极大的关注和兴趣。开始,人们的注意力集中在LDL与CHD的关系,对     

小鼠空肠黏膜微血管内皮细胞糖萼的透射电镜观察

目的 建立可靠的小肠黏膜微血管内皮糖萼显示方法,为探讨其生物学功能提供基础。方法 取小鼠6只,分为对照组和心脏灌注组,每组3只。对小鼠行乙醚吸入麻醉,心脏灌注组使用梯度阿尔新蓝溶液心脏灌注固定后取空肠组织,对照组直接取空肠组织,然后按照常规方法对其黏膜层制备超薄切片,透射电镜观察。 结果 透射电镜观察显示,心脏灌注组小鼠肠黏膜微血管内皮细胞膜表面有明显的糖萼结构,呈不连续型和连续型两种形态,不连续性糖萼厚度为50~100nm,连续型糖萼厚度为20~30nm;而对照组未能观察到明显的糖萼结构。结论 常规透射电镜样品处理方法,不能显示肠黏膜微血管内皮细胞的糖萼,而心脏灌注阿尔新蓝溶液进行前固定,能够较好地保存该结构,是一种较为可靠的研究方法。     

脂蛋白（a）及低密度脂蛋白与成纤维细胞表面结合特性的比较

脂蛋白（a)[Lipoprotein(a),Lp(a)]的代谢途径尚有争议。本文比较研究了Lp(a)和低密度脂蛋白（Low Density Lipoprotein,LDL)与成纤维细胞表面的结合特性，结果显示，Lp(a)与成纤维细胞呈特异性，高亲和力，可饱和性结合，但是Lp(a)与细胞的亲和力，最大结合容量均比LDL低，竞争性结合试验显示LDL只能部分抑制Lp(a)与成纤维细胞表面特异性的结合；细胞表面LDL受体活性的下调，可使LDL与细胞的结合量下降58％，而Lp(a)的结合量仅下降12．3％，这些结果表明LDL受体只能参与部分Lp(a)与细胞表面的结合及代谢。     

Lp(a)与LDL氧化修饰的比较

脂蛋白（a）［Lp（a）］是一种特殊的血浆脂蛋白,其结构、理化性质和脂质组成与低密度脂蛋白（LDL）极为相似。氧化低密度脂蛋白（Ox－LDL）在动脉粥样硬化（AS）的发生、发展中起重要作用,Lp（a）的氧化亦发生于体内动脉壁。在体外,Lp（a）能经铜离子（Cu2＋）介导发生氧化修饰,但与LDL相比,Lp（a）氧化的延迟时间是LDL的1．62倍,氧化速率和氧化程度均只有LDL的63％,故Lp（a）对氧化的敏感性和氧化程度均较LDL为低,其高度致AS作用,难以完全用较高的氧化敏感性来加以解释。     

脂蛋白α与动脉粥样硬化

脂蛋白a（Lipoprotein（a），Lp（a））是与低密度脂蛋白（low density lipoprotein，LDL）相似的一种脂蛋白，有足够证据表明，Lp（a）是动脉粥样硬化性疾病的一项独立危险因子，本文就Lp（a）的生物化学特性、代谢、可能的致动脉粥样硬化机制及与冠心病关系作一综述。     

离体培养动脉内皮细胞结合、内移和降解脂蛋白(α)作用的研究

本文研究离体培养动脉内皮细胞通过LDL受体结合、内移和降解脂蛋白(α)[lipopro-tein(a),Lp(α)]的作用.实验取第3～4代离体培养的动脉内皮细胞分为两组,对照组培养液中不加碘标的脂蛋白(a)[~(125)I—Lp(α)],实验组培养液中加~(125)I—Lp(α)最终浓度分别为0.25、0.5、1、10和50μg/ml.用γ计数器测定内皮细胞对Lp(α)的结合、内移和降解值,并测定内皮细胞的蛋白质量,以μg/mg细胞蛋白表示.结果随培养液中加进~(125)I—Lp(α)的浓度递增而增加,在10μg/ml浓度时最大的结合、内移和降解值分别是0.219、0.390和0.465μg/mg细胞蛋白;最大的结合率、内移率和降解率分别是2.19%、3.90%和4.65%.结果提示Lp(α)通过动脉内皮细胞LDL受体途径的摄取和降解作用是非特异性的.     

糖萼的作用及其在脓毒症中的研究进展

糖萼(glycocalyx)作为内皮细胞腔内侧的多糖结构,主要由蛋白聚糖、氨基葡聚糖(glycosaminoglycans,GAGs)、膜糖蛋白及血浆蛋白组成.糖萼对维持内皮细胞的通透性、形态及结构、血管内微环境有着重要作用,参与包括脓毒症在内多种疾病的生理病理过程.而在脓毒症时,各种炎症因子的作用损伤糖萼的完整性,从而导致糖萼的功能障碍.     

细胞表面硫酸肝素蛋白多糖在信号传导中的作用

硫酸肝素蛋白多糖由硫酸肝素和核心蛋白共价连接而成。细胞表面硫酸肝素蛋白多糖主要包括Syndecans和Glypicans两类 ,它们作为共受体调节许多配体的特异性受体的激活 ;此外 ,它们本身也具有信号传导功能 ,尤其是Syndecans这类跨膜分子 ,通过胞外区与配体结合 ,通过胞浆区与细胞骨架和下游信号传导分子相互作用。本文主要就细胞表面硫酸肝素蛋白多糖的结构、生物合成及其在信号传导中的作用作一综述     

Binding of human extracellular-superoxide dismutase C to cultured cell lines and to blood cells

The high heparin-affinity subtype C of the secretory enzyme extracellular-superoxide dismutase (EC-SOD) was found to bind to cultured mammalian cells, forming an equilibrium between the cells and the medium. To anchorage-dependent cell lines, binding apparently occurred both to the glycocalyx of the cell surfaces and to the sub- and intercellular matrix produced by the cells. Heparan sulfate proteoglycan appeared to be the principal binding substance. The binding capacities of anchorage-dependent cultures were very high, and at maximal binding the amount of EC-SOD C activity associated with the exterior of the cells was several-fold higher than the endogenous intracellular SOD activity. Half-maximal binding occurred at about 8 micrograms/ml EC-SOD C. At low, nonsaturating, physiologic EC-SOD C concentrations, the enzyme concentration in the glycocalyx of cells may be several thousand times higher than in the medium. All 14 investigated anchorage-dependent cell lines, including endothelial cells, bound EC-SOD C avidly. The 10 suspension-growing cell lines were all weaker binders. Blood monomorphonuclear leukocytes and platelets bound little EC-SOD C, whereas no significant binding to neutrophil leukocytes, to erythrocytes and to E. coli could be demonstrated. The findings are compatible with the notion that EC-SOD C in the vasculature forms an equilibrium between plasma and heparan sulfate in the glycocalyx of the endothelium. Furthermore, tissue EC-SOD is probably distributed between heparan sulfate on the surface of most cell types in the organs and in the interstitial matrix. The binding pattern suggests that EC-SOD C has the potential to protect most normal cells in the body and the interstitial matrix, without protecting microorganisms lacking affinity, and without interfering with superoxide radicals produced at the surface of activated neutrophil leukocytes.     

Impaired glycocalyx barrier properties contribute to enhanced intimal low-density lipoprotein accumulation at the carotid artery bifurcation in mice

The glycocalyx contributes to the barrier properties of vascular endothelium, and recently, we reported using electron microscopy that glycocalyx is diminished at lesion prone sites in arterial bifurcations in mice. In the present study, we examined using confocal microscopy the dimension and composition of the endothelial glycocalyx at low- and high-risk atherogenic regions within the common carotid (common) and internal carotid branch (sinus) of C57BL/6J mice and compared dimensional variations with its ability to limit transendothelial leakage of low-density lipoprotein (LDL). Confocal laser scanning microscopy of arterial surfaces stained for heparan sulfate and hyaluronan revealed thinner glycocalyces at the sinus region (2.2 +/- 0.7 and 2.3 +/- 0.7 mum, respectively; P < 0.05) than the glycocalyx thickness at the common region (4.3 +/- 1.6 and 4.3 +/- 1.6 mum, respectively). This thinner glycocalyx was associated with impaired LDL retention by the glycocalyx resulting in a two to three times increase in intimal accumulation of LDL 15 min after i.v. bolus administration: 10.8 +/- 5.6 vs. 4.0 +/- 1.9 x 10,000 a.u. (sinus vs. common, P < 0.05). These results indicate that impaired glycocalyx barrier properties may contribute to transendothelial leakage of atherogenic LDL at lesion prone arterial sites.     

Proliferation, morphology, and low-density lipoprotein metabolism of arterial endothelial cells cultured from normal and diabetic minipigs

Aortic endothelial cells from control and streptozotocin diabetic minipigs were cultured. Both groups of cells exhibited the typical cobblestone-like appearance and gap junction formation. Endothelial cells derived from diabetic minipigs differed, however, from those from control animals by a higher rate of proliferation and a higher percentage of large and often multinucleated cells. In these cells the specific binding of low-density lipoproteins (LDL) to coated pits on the cell surface, the LDL uptake, and the intracellular transport of LDL to lysosomes were visualized by gold-labeled LDL complexes. The binding, internalization, and degradation of LDL by subconfluent, non-contact-inhibited endothelial cells was quantified using 125I-labeled LDL. The LDL metabolism of endothelial cells derived from diabetic animals was increased by about 40% compared to endothelial cells derived from nondiabetic animals.     

Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression,localization, and function via glycocalyx degradation

Background

Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization.

Methods

We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation.

Results

Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177.

Conclusions

This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

     

Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses

Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase-N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.     

Monoclonal antibody to heparan sulfate from autoimmune tight skin (TSK) mice binds to the endothelial cell surface

Heparan sulfate proteoglycans have pleiotropic functions in the normal vasculature. Autoimmunity to heparan sulfate may play a role in vascular injury. In this study, monoclonal antibody (mb) 28C3–1 to heparan sulfate derived from autoimmune Tight skin (TSK) mice was investigated for its reactivity with endothelial cells. Mb 28C3–1 was previously demonstrated to inhibit the heparin-accelerated formation of antithrombin III-thrombin complexes. In the current studies it is shown that mAb 28C3–1 bound to heparan sulfate proteoglycan with the highest affinity in direct binding solid phase radioimmunoassay. Binding to the heparan sulfate was stronger than binding to the protein core, indicating that the primary epitope of 28C3–1 is the polysaccharide component. Using confocal fluorescent microscopy, mAb 28C3–1 was demonstrated to bind to the endothelial cell surface. Furthermore, treatment of endothelial cells with heparitinase abolished mAb 28C3–1 binding. These studies support the hypothesis that naturally occurring anti-heparan sulfate autoantibodies from autoimmune mice may cause vascular injury by initial interaction with endothelial cell surface heparan sulfate.     

The modulation of heparin-like activity of endothelial cells in experimental systems

Anticoagulantly active heparin-like glycosaminoglycans are apparently present on the vascular surface. We have tried to modulate heparin-like substances on endothelial cells using an experimental cell culture system. Perturbation of the endothelial proteoglycan metabolism by beta-D-xyloside resulted in a reduced biosynthesis of cell surface heparan sulfate, and impaired antithrombin III binding to endothelial cells in parallel with an inhibition of endothelial cell heparin-like activity. In a separate series of experiments, treatments of endothelial cells with interleukin 1 beta and tumor necrosis factor alpha, physiological mediators of immunologic and inflammatory responses, were shown to cause an inhibition of the synthesis of endothelial cell surface heparan sulfate. The endothelial heparin-like activity was partially diminished by these cytokines, suggesting that cytokine-mediated suppression of heparin-like substance on endothelial cells is another cytokine-inducible endothelial effect affecting coagulation. The modulation of endothelial heparin-like activity by these pharmacological and physiological agents may have pathophysiological implications in thrombosis.     

Real-time analysis of cell-surface adhesive interactions using thickness shear mode resonator

The cell adhesion process and the molecular interactions that determine its kinetics were investigated using a thickness shear mode (TSM) sensor. The goal of this study was to correlate sensor readings with the progression of cell adhesion. In particular, the specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell-surface attachment and steady-state adhesion of endothelial cells were investigated. We found a strong correlation between resistance changes (DeltaR) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady-state adhesion strength. Coating the sensor surface with the positively charged poly-d-lysine (PDL) enhanced the initial interaction with substratum. These data confirm our current understanding of the contribution of these three phenomena to the adhesion process. The real-time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. This technique has the potential to facilitate the evaluation of biomaterials and surface treatments used for implants and tissue-engineering scaffolds for their bioactive effects on the cell adhesion process.     

A potential role of the heparan sulfate in the hepatitis C virus attachment

Several putative cell surface receptors have been identified in the Hepatitis C virus (HCV) infection including CD81, low-density lipoprotein (LDL) receptor, scavenger receptor class B type I (SR-BI), and highly sulfated heparan sulfate (HS). In this study, we showed that the binding of soluble heparin to the envelope glycoprotein E2 (E2) of HCV was dependent on the dose and conformation of E2. On the other hand, the binding of E2 to the cell surface after treatment with soluble heparin was not markedly inhibited, what was different from other viruses utilizing cellular HS as the primary receptor. However, the enzymatic removal of HS from the cell surface led to a significant reduction in the binding of E2 to the cells. These facts imply that E2 was bound to cellular HS but might also have another route for cell attachment. Monoclonal antibodies with neutralizing activity against E2 did not completely block the binding of E2 to the cell surface, but their neutralization activity was greatly enhanced in the presence of soluble heparin. Taken together, the cellular HS could act as an alternative receptor for HCV and the interaction of E2 with HS could play a distinct role in escaping of HCV from the humoral immunity. Key words: Hepatitis C virus; E2 glycoprotein; heparan sulfate; receptor; humoral immune response.     

血凝素样氧化低密度脂蛋白受体-1与动脉粥样硬化的研究进展

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1 } is a type-Ⅱ membrane protein belonging to the C-type lectin family molecules, which acts as a cell surface endocytosis receptor for atherogenic oxidized LDL (Ox-LDL）. LOX-1 supports the binding internalization and proteolytic degradation of oxidized LDL, but not of significant amounts of acetylated LDL. LOX-1 is initially synthesized as a 40 kD precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 48-kD mature form. In vivo, endothelial cells that cover early therosclerotic lesions, intimal macrophages and smooth muscle cells in advanced atherosclerotic plaques express LOX-1. LOX-1 is cleaved at membrane proximal extracellular domain and released from the cell surface. Measurement of soluble LOX-1 in vivo may provide novel diagnostic strategy for the evaluation and prediction of atherosclerosis and vascular diseases.