Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

Exosomal Transfer of Vasorin Expressed in Hepatocellular Carcinoma Cells Promotes Migration of Human Umbilical Vein Endothelial Cells

Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.     

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.     

MicroRNA Expression Profiles of Human iPS Cells,Retinal Pigment Epithelium Derived From iPS,and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.     

Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.Age-related macular degeneration (AMD)¹ is one of the most common pathologies in the retina, consisting in a chronic degenerative disorder that constitutes the leading cause of blindness in the elderly, probably affecting 14 million people in the United States. AMD is a multifactorial disease in nature in which age is the predominant risk factor, although there are also environmental factors involved. In this regard, smoking is thought to be a major environmental risk factor as supported by extensive epidemiological evidence (1–5). AMD develops in two different stages: early AMD (also referred to as dry AMD) and the late stage of AMD known as wet AMD by virtue of the extensive neovascularization taking place in the retina choroid. Although there is a fair understanding of the mechanisms involved in wet AMD, little is known about dry AMD and its transition into the most severe stage of this disorder, i.e. wet AMD (6).Early AMD targets the retinal pigment epithelium (RPE) and the Bruch membrane (BrM) in the retina. The RPE constitutes a cell monolayer that is crucial to maintain a normal photoreceptor function. In fact, RPE participates in the cycling of the visual molecules, provides nutrients to rods and cones, and is responsible for withdrawing waste debris from the outer segments of photoreceptors (7). The early stage of AMD is characterized by initial deregulation of the normal extracellular matrix (ECM) turnover leading to thickening of the BrM, sub-RPE deposit accumulation, and drusen formation (8). As mentioned earlier, cumulative evidence suggests that smoking may constitute a major risk factor for early AMD. In fact, we and others have provided evidence that hydroquinone (HQ), a major component of cigarette smoke, has the ability to deregulate the ECM (9–12). Aside from cigarette smoke, HQ is a compound of environmental relevance because of its broad presence in plastics, foodstuff, and air pollution (13, 14).Mild injuries inflected to the retina elicit a cellular response in the RPE consisting in pinching off small areas of the plasma membrane, which renders small microvesicles called blebs (15). The reason(s) behind membrane blebbing remains unknown, although it has been postulated to be an attempt to discard damaged cellular constituents by the RPE cell (8). Under prolonged injury, blebs may accumulate between the RPE and the basal lamina underneath this cell monolayer. Based on this concept, a plausible role for blebs in the pathogenesis of dry AMD has been suggested as a likely contributor to build-up of the sub-RPE deposits, which are characteristic of the early stages of this disorder (8). To date, however, RPE bleb composition and potential functions remain largely unexplored.However, membrane bleb or microvesicle production stimulated by a variety of stress has been extensively described in many different cell types (16–23). To gain a better understanding of the functional relevance of blebs in general and the pathogenic mechanism(s) involved in early AMD in particular, we sought to investigate the identity of proteins carried by human RPE blebs. Previously microvesicles from lymphocytes have been subjected to analysis leading to the identification of a number of proteins (24). In our study, we show the proteomics characterization of stress-induced blebs in RPE cells from human retina. We report identification of several proteins, some of them potentially involved in matrix metalloproteinase (MMP) activation, membrane lipid raft formation, and immunogenic processes. Interestingly RPE blebs were found to carry basigin (including highly glycosylated species) and MMP-14, which are key proteins regulating the ECM turnover and remodeling. A previous proteomics study also has revealed the presence of basigin in the blebs from malignant lymphocytes (24). In the present study, we intended to gain some insight into the functional characterization of blebs to unravel some of the biological consequences of cell membrane blebbing in disease.     

人脐静脉内皮细胞的原代培养和鉴定

目的：探索建立稳定有效的脐静脉内皮细胞体外培养方法。方法：用0．1％II型胶原酶消化分离人脐静脉肉皮细胞,加入含内皮细胞生长因子的M199完全培养液中培养,用胰蛋白酶-EDTA进行消化传代培养,用光镜和免疫组化方法对培养的细胞进行形态观察和鉴定。结果：原代培养的内皮细胞在接种后24h后完全贴壁生长,第445天后融合呈铺路石样镶嵌排列,免疫组化可见胞浆中第Ⅷ因子相关抗原呈阳性反应,证实培养的细胞为内皮细胞。结论：脐静脉灌注II型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞,细胞可传代5—6次,但细胞产出量不高,不能传10代以上,5代以后细胞形态变化较大,对于复杂的基础研究应用受限。     

SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤

采用以下方法探讨SelS在内皮细胞中的表达和作用:将SelS基因克隆到真核表达载体pLNCX2,RT-PCR、XhoⅠ/ClaⅠ双酶切以及DNA序列分析验证目的基因;利用脂质体转染技术将pLNCX2-SelS或pLNCX2转染至人脐静脉内皮细胞(ECV304细胞),RT-PCR检测重组基因SelS的表达;MTT方法检测转染后过氧化氢(H2O2)对内皮细胞增殖能力的影响;硫代巴比妥酸法测定暴露于H2O2中不同转染组细胞脂质过氧化产物丙二醛含量.结果表明:成功构建真核表达载体pLNCX2-SelS;转染后重组SelS mRNA表达水平是内源性水平的1.76倍;H2O2对ECV304细胞损伤后,高表达SelS组细胞活性增强、H2O2诱导产生的丙二醛减少.上述结果表明,高表达SelS可保护内皮细胞免于H2O2诱导的细胞损伤,其作用机制与抗氧化有关.     

瑞舒伐他汀对OX-LDL诱导的人脐静脉内皮细胞PPAR-gamma表达的影响

目的：探讨经氧化低密度脂蛋白（OX-LDL）刺激后,人脐静脉内皮细胞（PPAR-gamma）表达的变化,以及瑞舒伐他汀对动脉粥样硬 化的影响。方法：将实验标本随机分为2 组,分为（OX-LDL）刺激组、瑞舒伐他汀干预组。应用RT-PCR及Western blot 技术,观察 OX-LDL 诱导的人脐静脉内皮细胞PPAR-gamma表达情况及瑞舒伐他汀对人脐静脉内皮细胞PPAR-gamma及浓度依赖的方式降低了人脐静脉内皮细胞PPAR-gamma的表达;2）瑞舒伐他汀可以逆转OX-LDL 对人脐静脉内细胞的影响 并可能与甲羟戊酸有关。结论：OX-LDL可降低人脐静脉内皮细胞PPAR-gamma的表达。瑞舒伐他汀可以抑制OX-LDL诱导人脐静脉 内皮细胞PPAR-gamma表达的增强,从而可能抑制了OX-LDL信号通路介导的与炎症有关的血管损伤,延缓动脉粥样硬化的进程。     

Effective Human Herpesvirus 8 Infection of Human Umbilical Vein Endothelial Cells by Cell-Mediated Transmission

Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be difficult, if not impossible. The present experiments were conducted with the idea that cell-cell contact may produce much more effective transmission, so-called cell-mediated transmission. Primary human umbilical vein endothelial cells (HUVECs) were cocultured with an HHV-8-infected lymphoma cell line, BCBL-1 cells. When a ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BCBL-1 cells to HUVECs of 10:1 was used, more than 20% of HUVECs were found to express the HHV-8 latency-associated nuclear antigen (LANA) 48 h after the start of coculturing; this value increased to more than 30% after 72 h. HHV-8-encoded ORF26, K8, K8.1, K10, K11, ORF59, and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA stimulation. These results indicated a far more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo.     

Nonselective Cation Channels in Endothelial Cells Derived from Human Umbilical Vein

(i) We have used a combined patch-clamp and fura-2 fluorescence technique to characterize a nonselective cation channel (NSC) in Ea.hy926 (EA) cells, an endothelial cell line derived from human umbilical vein. (ii) Stimulation with ATP, histamine and bradykinin activated slowly and with a long delay after application of the agonist, a nonselective cation current (I _NSC) which is time- and voltage-independent. The permeability sequence for cations was P _Na > P _Cs >> P _NMDG , P _Ca . In the absence of external Ca²⁺ and at rather high concentrations, La³⁺ and Gd³⁺ blocked I _NSC . (iii) Single channel analysis revealed that ATP activates in the cell-attached configuration a nonselective cation channel with a conductance of approximately 24 pS and a permeation sequence identical to that of the macroscopic current. The channel activity disappeared after membrane excision. (iv) Activation of NSC required physiological intracellular Ca²⁺ levels (100 nm or higher). All agonists failed to activate NSC if cytosolic Ca²⁺ ([Ca²⁺] _i ) was lowered by 10 mm BAPTA. Clamping internal Ca²⁺ at 1 μm sometimes (8 out of 17 cells) spontaneously activated I _NSC in the absence of any additional stimulus. (v) Application of 2,5-di-tert-butylhydroquinone and internal perfusion of inositol 1,4,5-trisphosphate also activated I _NSC . The phospholipase C inhibitor, U-73122 inhibited I _NSC and the sustained Ca²⁺ plateau during agonist stimulation whereas the inactive analogue, U-73343 had no effect. (vi) These results indicate NSC may act as a Ca²⁺ entry pathway in endothelium. [Ca²⁺] _i and inositol 1,4,5-trisphosphate play a role in the activation cascade of NSC, and possibly also store depletion. Received: 13 October 1998/Revised: 28 January 1999     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的:研究胰蛋白酶对IL-8释放的影响。方法:分离、培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体-2(proteinase-activated receptor-2,PAR-2)表达,ELISA检测HUVECs培养上清中IL-8水平。结果:HUVECs表达内皮细胞标志和PAR-2。刺激16 h,1 g/ml胰蛋白酶和100M PAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论:胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的：研究胰蛋白酶对IL-8释放的影响。方法：分离、培养人脐静脉内皮细胞（human umbilical vein endothelialcells,HU-VECs）、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体．2（proteinase．activatedreceptor．2,PAR-2）表达,ELISA检测HUVECs培养上清中IL-8水平。结果：HUVECs表达内皮细胞标志和PAR-2。刺激16h,1g／ml胰蛋白酶和100MPAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论：胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。     

改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。     

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation

The histamine receptors (HRs) represent a subclass of G protein‐coupled receptors (GPCRs) and comprise four subtypes. Due to their numerous physiological and pathological effects, HRs are popular drug targets for the treatment of allergic reactions or the regulation of gastric acid secretion. Hence, an understanding of the functional selectivity of HR ligands has gained importance. These ligands can bind to specific GPCRs and selectively activate defined pathways. Supporting the activation of a therapeutically necessary pathway without the activation of other signaling cascades can result in drugs with more specific activity and fewer side effects. To evaluate the cellular consequences resulting from receptor binding, comprehensive analyses of cellular protein alterations upon incubation with ligands are required. For this purpose, endothelial cells are treated with histamine, as the endogenous ligand of HRs, to obtain a global overview of its cellular effects. Quantitative proteomics and pathway analyses of histamine‐treated and untreated cells reveal enrichment of the nuclear factor‐κB and tumor necrosis factor signaling pathways, cytokine?cytokine receptor interactions, complement and coagulation cascades, and acute inflammatory processes upon histamine treatment. This strategy offers the opportunity to monitor HR‐mediated signaling in a multidimensional manner.     

二苯乙烯苷通过上调XIAP抑制人脐静脉内皮细胞凋亡

目的:二苯乙烯苷(2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside,TSG)具有抗炎、抗氧化、抗动脉粥样硬化(atherosclerosis,As)等作用。课题组前期研究表明TSG对过氧化氢(H2O2)诱导损伤的内皮细胞具有保护作用,并抑制内皮细胞的凋亡,但机制尚未完全明确。本研究目的在于探讨TSG是否通过影响X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)、Caspase-9的表达来抑制细胞凋亡。方法:体外培养人脐静脉内皮细胞,实验分为正常对照组、模型组(300μmol·L-1H2O2)、TSG预处理组(10μmol·L-1TSG+300μmol·L-1H2O2)、Embelin与TSG联合处理组(30μmol·L-1Embelin+10μmol·L-1TSG+300μmol·L-1H2O2)、TSG单独处理组(10μmol·L-1TSG)、Embelin组(30μmol·L-1Embelin)。采用MTT法检测细胞增殖率,Hoechst 33258染色观察凋亡细胞核形态,RT-PCR和Western blot检测XIAP、Caspase-9的表达。结果:与空白对照组相比,H2O2组内皮细胞增殖率降低,核损伤明显,XIAP表达显著性下降,Caspase-9表达显著增加(P0.01);与H2O2组比较,经TSG预处理后,细胞增殖率增加,核损伤减轻,XIAP的表达上升,Caspase-9表达减少,差异均有显著性(P0.01)。与TSG预处理组比较,用XIAP阻断剂Embelin与TSG联合处理后,内皮细胞活力下降,XIAP表达显著降低,Caspase-9表达增加(P0.01)。结论:TSG具有抑制H2O2诱导的人脐静脉内皮细胞凋亡作用,其机制与增加XIAP的表达,抑制Caspase-9的表达有关。     

胞浆型磷脂酶A2介导弱氧化修饰低密度脂蛋白诱导的人脐静脉内皮细胞凋亡

探讨弱氧化修饰低密度脂蛋白(MM-LDL)能否诱导人脐静脉内皮细胞(HUVECs)凋亡以及胞浆型磷脂酶A2(cPLA2)在此过程中的作用.MTT法测定细胞存活率;相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡;3H-花生四烯酸(3H-AA)预标法测定PLA2活性;蛋白质印迹检测cPLA2磷酸化;激光共聚焦显微镜检测单个细胞内钙离子浓度的变化.结果表明,MM-LDL(100～300 mg/L)作用后的HUVECs呈现凋亡典型的形态特征,凋亡率随MM-LDL浓度的增加而上升.MM-LDL能引起胞内钙离子浓度增加,cPLA2的活化及磷酸化.15 μmol/L AACOCF3和5 mmol/L EGTA在抑制cPLA2活性的同时,部分抑制MM-LDL诱导的HUVECs凋亡.加入外源性AA(50 μmol/L)能逆转AACOCF3引起的凋亡抑制.结果提示,cPLA2参与了MM-LDL诱导HUVECs凋亡的信号传递.     

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．