Venous and arterial endothelial proteomics: mining for markers and mechanisms of endothelial diversity

Endothelial cells (ECs) line the inside of arterial and venous blood vessels in a continuous monolayer and have the important function of responding to environmental cues to regulate vascular tone and new blood vessel formation. They also have well-defined roles in supporting tumorigenesis, and alterations in their function lead to cardiovascular disease. Consequently, ECs have been studied extensively as a cellular model of both normal and abnormal physiology. Despite their importance and the increased utility of proteomic tools in medical research, there are relatively few publications on the topic of vascular endothelial proteomics. A thorough search of the literature mined 52 publications focused exclusively on arterial and/or venous endothelial proteomics. These studies mostly relied upon examination of whole-cell lysates from cultured human umbilical vein ECs to investigate in vitro effects of various molecules, such as VEGF in the context of altering human umbilical vein EC functions related to angiogenesis. Only a few of these publications focused solely on a proteomic characterization of ECs and our analysis further revealed a lack of published studies incorporating proteomic analysis of freshly isolated ECs from tissues or in vitro conditions that mimic in vivo variables, such as oxygen tension and shear stress. It is the purpose of this article to account for the diversity of vascular EC proteomic investigations and comment on the issues that have been and should be addressed in future work.     

Suppression of Tie-1 in endothelial cells in vitro induces a change in the genome-wide expression profile reflecting an inflammatory function

Tie-1 is an endothelial specific receptor tyrosine kinase that is upregulated in diseases such as atherosclerosis and rheumatoid arthritis. We recently demonstrated that Tie-1 induced a proinflammatory response when overexpressed in endothelial cells. Here, we used a complementary approach and suppressed endogenous Tie-1 expression in endothelial cells to examine its function by microarray analysis. Tie-1 appeared to govern expression of many genes involved in inflammation. Expression knockdown of Tie-1 significantly reduced endothelial conditioned medium ability to stimulate MCP-1 production in U937 cells. Collectively, our results support the notion that Tie-1 has an inflammatory function in endothelial cells.     

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 μm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.     

原子力显微镜研究高乌甲素对人脐静脉内皮细胞形态的影响

在纳米量级上探测药物对人脐静脉内皮细胞(HUVEC)的抑制作用对于揭示药物的功效及肿瘤的有效治疗十分重要.通过高分辨率的原子力显微镜研究了不同浓度的高乌甲素培养的HUVEC的形貌特征,包括整个细胞的形貌和超微结构的表面膜的差异.从形貌学方面探讨了高乌甲素对HUVEC的抑制作用,可为临床应用高乌甲素提供依据.结果表明,我...     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

A PAUF-neutralizing antibody targets both carcinoma and endothelial cells to impede pancreatic tumor progression and metastasis

Pancreatic adenocarcinoma up-regulated factor (PAUF) is expressed in pancreatic ductal adenocarcinoma (PDAC) and plays an important role in tumor progression and metastasis. Here we evaluate the anti-tumor efficacy of a human monoclonal antibody against PAUF, PMAb83, to provide a therapeutic intervention to treat the disease. PMAb83 reduced tumor growth and distant metastasis in orthotopically xenografted mice of human PDAC cells. PMAb83 treatment retarded proliferation along with weakened aggressiveness traits of the carcinoma cells. AKT/β-catenin signaling played a role in the carcinoma cell proliferation and the treated xenograft tumors exhibited reduced levels of β-catenin and cyclin D1. Moreover PMAb83 abrogated the PAUF-induced angiogenic responses of endothelial cells, reducing the density of CD31⁺ vessels in the treated tumors. In combination with gemcitabine, PMAb83 conferred enhanced survival of xenografted mice by about twofold compared to gemcitabine alone. Taken together, our findings show that PMAb83 treatment decreases the aggressiveness of carcinoma cells and suppresses tumor vascularization, which culminates in mitigated tumor growth and metastasis with improved survival in PDAC mouse models.     

Influence of protein histidine phosphatase overexpression and down-regulation on human umbilical-vein endothelial cell viability

PHP (protein histidine phosphatase) is expressed by mammalian tissues, particularly in blood vessel walls. We investigated whether PHP plays a significant role in endothelial cells. By Western blot and immunofluorescence analysis PHP was found in HUVEC (human umbilical-vein endothelial cells). Overexpression of PHP by the use of a plasmid vector, pIRES2-AcGFP1-PHP, induced apoptosis in HUVEC. To exclude the possibility that increased cellular protein alone unspecifically caused cell damage, the inactive H53A mutant of PHP was also overexpressed as a control; it did not lead to apoptosis. Down-regulation of PHP by the RNAi (RNA interference) technique did not affect cell viability. In conclusion, HUVEC are damaged by overexpression, but not down-regulation, of PHP, suggesting a pronounced impact of the enzyme on the cells when its activity is increased.     

腺苷对人脐静脉内皮细胞三维管状结构形成的影响

目的:探讨腺苷对脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)三维管状结构形成的影响.方法:建立上、下两层内皮细胞,中间为胶原凝胶的三维培养方式.设对照和试验各3孔.对照孔不加腺苷,实验孔内加入10-4mol/L腺苷.观察并记录特定视野下芽生的管状结构数目.结果:HUVEC可以在Ⅰ型胶原凝胶中形成三维网状结构,腺苷实验组细胞生长快,出芽快,管状结构粗大,甚可形成贯穿胶原的三维网状结构.血管芽生数与对照组比较在24 h、48 h、72 h、96 h均有统计学差异(P＜0.05或P＜0.01).结论:腺苷对HUVEC三维网状结构的形成有促进作用.     

Relevance of disease- and organ-specific endothelial cells for in vitro research

The endothelium is a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic and immunological functions. Endothelial dysfunction has been implicated as a key factor in the development of organ-specific vascular diseases. This minireview gives a brief overview on EC (endothelial cell) biomarkers in arterial and venous endothelium and critically discusses the different sources of ECs that are most frequently applied in in vitro assays and research. The relevance of organ- and disease-specific endothelial cell cultures for studying cellular responses as a basis for improving therapeutic interventions is highlighted with particular emphasis on endothelial dysfunction in transplant-associated coronary artery disease, in atherosclerotic lesions and in response to diabetes mellitus.     

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty microg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB-treated group produced significantly more ultrafiltration than the non-treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with the non-treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB-treated endothelial cells, compared with the non-treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter.     

A novel caspase dependent pathway is involved in apoptosis of human endothelial cells by Shiga toxins

Shiga toxins have been shown to induce apoptosis on primary cultures, but not passaged ones, of human umbilical vein endothelial cells, independent of cytokine pre-treatment. Here, a peculiar pattern of caspase activation was observed; caspase-3 and -2, but not conventional upstream caspases, were activated at the initial phase of 6 hr, whereas a broad range inhibitor of caspases, VAD-fmk, but not mono-specific ones, suppressed DNA fragmentation and cell death. These results suggest additional analogous molecules, which have yet to be delineated, are involved. The requirement of retrograde uptake of toxins was also proved by the intervening effect of brefeldin A.     

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.     

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

脂多糖直接损伤血管内皮细胞后内皮细胞膜脂的修饰

我们前期的研究显示,脂多糖（lipopolysaccharide,LPS）直接损伤人脐静脉血管内皮细胞（human umbilical vein endothelial cell,HUVEC）后,HUVEC膜粘度随LPS浓度的增加而增大,这暗示LPS可能改变HUVEC的膜结构和组成。本文旨在研究LPS直接损伤HUVEC后内皮细胞膜脂的修饰。用高效毛细管电泳（high performance capillary electrophoresis,HPCE)污测定HUVEC膜磷脂组成,HUVEC用不同浓度（O、0.3125、0.625、1.25、2.5、5、7、8.5、9、10μg/ml）的LPS无血清培养液直接损伤3h;用含LPS（0.625μg/ml）的无血清培养液直接损伤1、3、6、12、24、48h。结果显示：在3h LPS的作用下,HUVEC的总磷脂含量随LPS浓度的增加而增加,在0.625μg/ml LPS的浓度条件下,HUVEC的总磷脂含量随LPS作用时间的延长而降低;LPS的作用浓度和作用时间对磷脂酰鞘磷脂（SM）、磷脂酰乙醇胺（PE）以及磷脂酰丝氨酸（PS）的含量影响不大。LPS激活膜磷脂代谢的反应特性表现出典型的酶促动力学特性。实验结果表明,LPS可直接激活血管内皮细胞膜磷脂代谢,诱导HUVEC膜磷脂的修饰,提示LPS直接损伤血管内皮细胞HUVEC的途径可能与膜结构组成及膜脂代谢有关。     

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

Background: To evaluate the influence of hypoxia and molecular events in endothelial and embryonic stem cells.Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantification of apoptotic cells by TUNEL assay measured by flowcytometry, and Western blot analysis for the molecular events of apoptosis were performed.Results: DNA fragmentation could be identified under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended.In situ internucleosomal DNA fragmentation-TUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions.Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression.     

Sperm proteomics: potential impact on male infertility treatment

Spermatozoa are unique cells that have highly compact DNA, motility (and hypermotility) patterns, a specific morphology, localized mitochondria and an apical acrosome. They are the end product of a dynamic process termed spermatogenesis. Sperm are therefore produced with specific proteins in order to effect different traits, such as the presence of cysteine-rich protamines in DNA, which effectively compacts DNA. Moreover, specific proteins are transferred during epididymal maturation and after ejaculation in order to render sperm capable of undergoing post-ejaculatory alterations, generally termed capacitation, which confers capacity to fertilize a mature oocyte. In addition, sperm exhibit several post-translational modifications, which are fundamental to their function, such as SUMOylation and ubiquitination. Discussed in this review is the current knowledge of the sperm proteome in terms of its composition and the function that these proteins determine, as well as their post-translational modifications and how these alter sperm functional integrity. Studies are emphasized that focus on shotgun proteomics – untargeted determination of the protein constituent of a cell in a given biological condition – and technologies currently applied toward that end are reviewed.     

睾酮对人血管内皮细胞产生NO、tPA和PAI-1的影响

目的:观察不同浓度睾酮对人血管内皮细胞生长、产生舒张因子及纤溶活性的影响.方法:体外培养人脐静脉内皮细胞(HUVEC),分为五个浓度睾酮组及单纯培养基对照组.做MTT实验观察睾酮对HUVEC生长的影响;还原酶法测定各组HUVEC释放NO量;ELISA法测定各组培养基中纤溶酶原激活物(tPA)及其抑制物(PAI-1)含量.结果:3×10-10mol/L-3×10-8mol/L睾酮组与对照组相比细胞生长良好,无明显差别;而大于生理剂量的两组(3×10-6～3×10-1mol/L)3 d后细胞生长明显受到抑制(P＜0.05).各浓度睾酮组产生NO量与对照组无明显区别.而3×10-10 mol/L～3×10-8 mol/L睾酮组tPA含量明显高于对照组(P＜0.01);大剂量组tPA产生明显减少(P＜0.01).所有实验组的PAI-1含量均明显降低.结论:生理及略低于生理剂量的睾酮对HUVEC生长及释放NO无不利影响,且增加纤溶活性.说明生理剂量睾酮对血管内皮功能、心血管系统有一定的保护作用,有利于防止动脉粥样硬化的发生、发展.