Wnt-1 signaling inhibits human umbilical vein endothelial cell proliferation and alters cell morphology

Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. There are many factors which can mediate this interaction including Wnt-signaling-related molecules. Wnt signaling is involved in many developmental processes and cellular functions. There is increasing evidence suggesting that Wnt signaling has a role in regulating endothelial cell growth although the precise mechanism is unclear. In this study, we established a coculture system to examine how Wnt-1 signaling regulates human umbilical vein endothelial cell (HUVEC) growth and behavior. We found that Wnt-1 signals inhibited BrdU incorporation in HUVECs and the number of labeled cells also decreased in proportion to the number of Wnt-1-expressing cells present (P < 0.05). Moreover, HUVECs cocultured with Wnt-1-expressing C57MG cells clumped together rather than remaining scattered throughout the culture. These effects were dependent on cell contact. Treatment of HUVEC with LiCl, which inhibits the activity of GSK-3β and mimicked Wnt-1 signaling, also inhibited the BrdU incorporation in endothelial cells. Our results suggest that Wnt signaling has a role in endothelial cell growth control and this is mediated through cell–cell contact. They also suggest that Wnt signaling might participate in angiogenesis by regulating endothelial cell growth and function.     

MiR-152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF-α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR-152. We found that miR-152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR-152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR-152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR-152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.     

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me₂SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me₂SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.     

Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells

Lipopolysaccharide (LPS), as an important proinflammatory agent, targets the endothelium. However, almost all in vitro experiments of the effect of LPS on vascular endothelial cells (VECs) were performed under an artificially decreased concentration of serum that was not enough to maintain the cell growth for a long time. The mechanism underlying LPS action on VECs cultured in a nutrient‐rich condition is not clear. To address this question and mimic the in vivo condition, we investigated the effect of LPS on VEC autophagy, which is involved in numerous physiological processes. The effect of LPS on microtubule‐associated protein 1 light chain 3 (LC3) distribution, LC3‐II accumulation and p62 degradation showed that LPS effectively induced autophagy in VECs cultured in the presence of 20% serum. To understand the mechanism by which LPS triggers the cell autophagy, we first investigated the effects of LPS on the expression of BIRC2 (cIAP1), a well‐known apoptosis inhibitor, and on the kinase activity of mammalian target of rapamycin (mTOR) and nuclear translocation of p53. LPS increased BIRC2 expression in a dose‐ and time‐dependent manner and elevated the intranuclear level of p53 but had no effect on the mTOR pathway when it triggered VEC autophagy. Furthermore, knockdown of BIRC2 by RNA interference inhibited the autophagy and the translocation of p53 to nuclei induced by LPS. These data suggest a novel role for BIRC2 in LPS‐induced autophagy in VECs. J. Cell. Physiol. 225: 174–179, 2010. © 2010 Wiley‐Liss, Inc.     

Proteoglycans from human umbilical vein endothelial cells

Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum).     

Organizational behavior of human umbilical vein endothelial cells

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.     

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Surface glycoproteins of cultured human umbilical vein endothelial cells

Radioactive surface-specific and metabolic labeling techniques were used to characterize the surface glycoprotein pattern of cultured human endothelial cells. Electrophoretic analysis of whole cells, surface labeled either by the galactose oxidase/sodium borotritide or the periodate/sodium borotritide method, revealed several major polypeptides in the Mr region of ca 40-220. During primary culture, the surface labeling pattern showed no changes related to cell density or to the establishment of confluence. A slightly different polypeptide profile was, however, seen when primary culture cells were labeled as an intact monolayer and not in suspension. On the other hand, in cells from later passages, when compared to their parental cells of early passages, there was a distinct intensification of polypeptides with Mr 155 and 90.     

CD151蛋白促内皮细胞增殖和eNOS蛋白表达

目的研究CD151及其突变体CD151-ARSA245-248对人脐静脉内皮细胞(HUVEC)增殖及eNOS表达的影响,探讨CD151促血管生成的机制。方法构建pAAV-CD151及其突变体CD151-ARSA245-248(囊泡运输缺陷突变体),并转染HU-VEC。CCK-8法测定HUVEC增殖的能力,Western Blot检测CD151及eNOS蛋白的表达。结果 pAAV-CD151组及pAAV-CD151-ARSA245-248组CD151蛋白表达均增加,显著高于正常对照组和pAAV-GFP组(P<0.05),但pAAV-CD151组及pAAV-CD151-ARSA245-248组之间CD151蛋白表达没有统计学意义(P>0.05)。CCK-8法测定HUVEC增殖能力亦无统计学意义(P>0.05)。正常对照组,pAAV-GFP组,pAAV-CD151组及pAAV-CD151-ARSA245-248突变体组的OD值分别为1.393?.685、1.498?.746、2.346?.52和1.71?.863,pAAV-CD151组较pAAV-GFP组和正常对照组细胞增殖能力明显增强(P<0.01),pAAV-CD151-ARSA245-248组较pAAV-CD151组细胞增殖能力减弱(P<0.05)。此外,pAAV-CD151组eNOS蛋白表达较pAAV-GFP组和正常对照组明显增加(P<0.01),pAAV-CD151-ARSA245-248组较pAAV-CD151组eNOS蛋白表达降低(P<0.05)。结论 CD151是促细胞增殖的重要蛋白质,CD151影响eNOS信号通路的激活。上述机制可能为CD151促血管生成的重要机制之一。     

肿瘤条件培养基对人脐静脉内皮细胞增殖、黏附、迁移能力的调节

本文旨在研究肿瘤条件培养基(tumor conditioned medium,TCM)对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)增殖、黏附和迁移能力的影响.采用MTT法测定TCM作用24 h后内皮细胞的增殖水平,实验设对照组、TCM原液(TCM stoc...     

High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.     

Donepezil attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells through SIRT1 activation

Cellular senescence of endothelial cells is a damage and stress response which induces pro-inflammatory, pro-atherosclerotic, and pro-thrombotic phenotypes. Donepezil is a drug used for the treatment of mild to moderate dementia of the Alzheimer’s disease (AD). The aim of the present study was to investigate the attenuation of endothelial cell senescence by donepezil and to explore the mechanisms underlying the anti-aging effects of donepezil. Our results indicated that high glucose (HG) markedly decreased cell viability of human umbilical vein endothelial cells (HUVECs), and this phenomenon was reversed by treatment with donepezil. Importantly, our results displayed that the frequency of senescent (SA-ß-gal-positive) cells and the expression level of senescence genes (PAI-1 and p21) were significantly higher in the HG group compared with the normal glucose (NG) group, and these changes were blocked by treatment with donepezil. Also, our results showed that donepezil inhibits the generation of reactive oxygen species (ROS), which promotes cellular senescence. Pretreatment with nicotinamide (NAM), a sirtuin 1 (SIRT1) inhibitor, inhibited the reduction in senescence associated with donepezil. Indeed, our results indicated that donepezil increased the SIRT1 enzyme activity. Therefore, these results show that donepezil delays cellular senescence that is promoted under HG condition via activation of SIRT1.     

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway

The biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.