Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.     

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1)

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Endothelin-1 is expressed and released by a human endothelial hybrid cell line (EA.hy 926).

A permanent vascular endothelial cell line, EA.hy 926, was shown to express endothelin-1 (ET-1) mRNA and to secrete big ET-1 and ET-1 into culture medium. The concentration of both big ET-1 and ET-1 was significantly increased in EA.hy 926 culture medium by phosphoramidon, a metalloproteinase inhibitor, suggesting that phosphoramidon sensitive protease(s) may be responsible for the degradation of ET-1 and big ET-1. EA.hy 926 cells responded to various regulators of ET-1 similarly as primary human vascular endothelial cells. The production of ET-1 was increased by thrombin and decreased by vasodilators such as atrial natriuretic peptide, brain natriuretic peptide and nitroprusside, and by 8-bromo cyclic GMP and papaverine. This continuous human endothelial hybrid cell line could facilitate studies of regulation of ET-1 production in human endothelial cells, which in primary cultures have limited replication potential.     

源自天蚕素A和爪蟾素2的杂合肽P18体外抑制内皮细胞血管生成

目的探讨杂合肽P18体外对内皮细胞EA.hy926血管生成的抑制作用.方法采用MTT法检测P18对EA.hy926细胞增殖的影响;应用Matrigel实验检测P18对内皮细胞形成管状结构的影响;利用流式细胞术分析P18对内皮细胞的损伤作用.结果 MTT结果显示P18可明显抑制EA.hy926细胞的增殖,且抑制率存在剂量依赖性;Matrigel实验表明P18具有抑制EA.hy926细胞体外分化成管状结构的作用;流式结果显示15 μM P18作用内皮细胞6 h后,所诱导的细胞坏死比例达到81.4%.结论体外实验结果表明,杂合肽P18具有体外抑制EA.hy926细胞血管生成的作用.     

Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates.

Human endothelial cells (EA.hy 926 line) were loaded with cationized low density lipoprotein (LDL) and subsequently incubated with fatty acid/bovine serum albumin complexes. The fatty acids were palmitic, oleic, linoleic, arachidonic, and eicosapentaenoic acids. The preincubations resulted in extensively modified fatty acid profiles in cell membrane phospholipids and in cellular cholesteryl esters. The cholesterol efflux from these fatty acid-modified cells was measured using 0.2 mg high density lipoprotein3 (HDL3)/ml medium. The efflux was significantly higher for the palmitic acid-treated cells, compared to all other fatty acid treatments. These differences in efflux rates were not caused by changes in the binding of HDL3 to high affinity receptors on the EA.hy 926 cells. Efflux mediated by dimethyl suberimidate-treated HDL3, which does not interact with high affinity HDL receptors, was similar to efflux induced by native HDL3 after all fatty acid treatments. Our results indicate that high affinity HDL receptors are not important for HDL-mediated efflux of cell cholesterol. The fatty acid composition of the cell membrane phospholipids may be an important determinant.     

松鼠胰腺型核糖核酸酶A基因的克隆与表达

核糖核酸酶A(ribonucleases A,RNases A)构成一个大家族,其成员表现出不同程度的水解RNA的酶活性。一些成员还表现出特殊的生物学活性。旨在克隆松鼠核糖核酸酶A的基因,利用原核细胞系统表达重组蛋白,并进行初步的酶学性质的研究。结果表明,所克隆的基因属于核糖核酸酶A家族,保留了核糖核酸酶A家族酶活性必要的保守序列。进化分析表明,该基因属于松鼠胰腺型核糖核酸酶基因,与其它啮齿类动物胰腺型酶一样,重组的核糖核酸酶具有酶活性,为进一步进行啮齿类动物核糖核酸酶的宿主防卫功能研究打下了基础。     

Effects of prolonged exposure to hypoxia on morphological changes of endothelial cells plated on fibrin gel

Since tissue oxygenation has a profound effect on capillary growth, the effect of pO2 on endothelial cell functions was studied. Under normoxic conditions, EA.hy926 endothelial cells and HUVEC plated onto fibrin gels in low-serum culture medium underwent rapid and profound morphological changes within 12 to 48 hours depending on the cell line used. Their characteristic cobblestone organisation was transformed into a network of cord-like or tube-like structures. We showed that when exposed to low oxygen concentrations for 3 days, HUVEC and EA.hy926 have their ability to rearrange reduced to around 50 %. With EA.hy926 this effect was amplified by 79% after 9 days of hypoxia. The altered behaviour of hypoxia-adapted cells was not caused by a loss in their fibrinolytic activity. In fact, the fibrin degradation rate and the generated fibrin fragments appeared identical in normoxia and hypoxia. Confocal microscopy and gel densitometry showed that in normoxia the remaining undegraded fibrin gel underwent a dynamic remodeling whereas in hypoxia it remained undisturbed. It is likely that hypoxia induces modification in the factors that integrate matrix information and cytoskeletal organisation in order to contract fibrin.     

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.     

一种大容量细胞灌流液中外泌体的提取方法及鉴定*

目的: 建立一种从大容量细胞灌流液中提取外泌体的方法,并进行外泌体的鉴定。方法: 人脐静脉内皮细胞株(EA.HY926)是人脐静脉内皮细胞和人肺腺癌细胞株A549杂交成的永生化细胞株,因其具有血管内皮细胞的特性,广泛用于内皮细胞相关研究。本研究采用含10%胎牛血清的M199培养基培养,利用Flexcell STR-4000平行板流室系统对EA.HY926施以振荡剪切应力。收集流体剪切应力处理后的细胞灌流液,去除细胞碎片后冻成干粉,脱盐、提取纯化外泌体。电镜观察外泌体形态、纳米粒径电位分析仪检测外泌体大小、PKH26染色检测外泌体膜性结构、BCA蛋白定量法检测外泌体的蛋白浓度、Western blot检测外泌体特异性蛋白CD9和CD81的表达,荧光定量RT-PCR检测内皮细胞相关基因的表达。结果: 该方法提取的外泌体,大小均一,结构完整,呈典型囊泡样结构;粒径集中在30~150 nm,多数粒径为97.63 nm;表达外泌体特异性蛋白CD9和CD81;PKH26染色阳性,并可被细胞摄取;EA.HY926分泌的外泌体表达内皮细胞相关的CD31、vWF等mRNA,以及miR-126、miR-21、miR-155等microRNA分子。结论: 本方法能够有效从大容量细胞灌流液中提取到结构完整的、高浓度、高质量的外泌体,为开展以流体力学干预细胞为基础的外泌体相关研究提供技术方法。     

Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.

Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.     

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.     

Purification and characterization of human myometrial smooth muscle collagenase

Collagenase has been purified from the culture medium of a human myometrial smooth muscle cell line, and the properties of the pure enzyme compared to those of collagenase from another human mesenchymal cell, the fibroblast. The smooth muscle collagenase was purified using a new, rapid, and convenient three-step purification procedure consisting of chromatography on iminodiacetate-agarose chelated with zinc and on Cibacron Blue-agarose followed by gel filtration on Ultrogel AcA-44. The resultant pure collagenase is secreted as a zymogen indistinguishable from that of the fibroblast enzyme in molecular weight, amino acid composition, and in the nature of its conversion to active enzyme by trypsin. The amino acid sequence of the two enzymes at the trypsin cleavage site is the same. The two collagenases are also indistinguishable immunologically and display essentially identical kinetic behavior on a variety of collagen substrates. Although the two collagenases appear to be identical proteins, the mechanisms which regulate their production appear to be very different. Glucocorticosteroids, which inhibit collagenase production in human skin fibroblasts are without effect in the uterine smooth muscle cell. In contrast, the smooth muscle cell appears to require a component present in fetal bovine serum in order to produce the enzyme.     

Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9-6. 2)% to 91.4(83.0-96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-alpha/IL-1beta. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.     

Distribution of a Kidney Acid-ribonuclease-like Enzyme and the Other Ribonucleases in Bovine Organs and Body Fluids

To determine the distribution of a kidney acid RNase (RNase K₂) and other RNases, the levels of RNase K₂, RNase A, and seminal RNase (RNase Vs₁ in bovine tissues and body fluids were measured by enzyme immunoassay. The crude extracts of several tissues and body fluids were fractionated by phospho-cellulose column chromatography. The enzymatic activities at pH 7.5 and 6.0 and enzyme contents of each tube were measured by enzyme assay and enzyme immunoassay, respectively. In the pancreas, parotid gland, and heart, most RNase activity was due to a single peak of RNase A, but a small amount of RNase K₂ was always observed. In the kidney, there was about 5 times as much RNase K₂ as RNase A. In the lung, although RNase K₂ and RNase A were the major components, there are another two alkaline RNase peaks. In the spleen and liver, there are four RNases, two acid RNases, one of which is RNase K₂, and two alkaline RNases including RNase A. A new acid RNase (non RNase K₂-acid RNase) from both organs was immunologically the same. In serum, there are at least four RNases. By partial purification of serum RNases by phosphocellulose and heparin-Sepharose column chromatographies, at least 4 RNases, RNase A, RNase K₂ and the other two alkaline RNases, one of which is immunologically indistinguishable from liver alkaline RNase, were confirmed. The other serum alkaline RNase was immunologically related to lung and spleen alkaline RNases. In conclusion, in bovine tissues and body fluids there are at least 7 types of pyrimidine-base-specific RNases: brain RNase, seminal RNase, RNase A, RNase K₂, an acid RNase (RNase BSPJ, an alkaline RNase (RNase BL₄), and another alkaline RNase in serum.     

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.     

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.