Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h  following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.     

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.     

Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.     

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb3, and Gb4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB3H4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were GM3 and IV3NeuAcnLc4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.     

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.     

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.     

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.     

Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells

The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C. Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet. Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total. Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM). Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide. In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not. Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain. The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells. Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.     

Isolation of human umbilical vein endothelial cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.     

Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells

The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells.     

Antiapoptotic effect of ouabain on human umbilical vein endothelial cells

The present study investigates the effect of ouabain on caspase-3 activation in human umbilical vein endothelial cells (HUVEC). Ouabain (EC(50) 20 nM) reduced caspase-3 activity in HUVEC treated for 24h in a medium deprived of fibroblast growth factor (FGF). Incubation for 5h in the absence of both FGF and serum produced an increase in caspase-3 activity that was completely abolished by 100 nM ouabain. Pretreatment with the phosphatidylinositol 3 kinase (PI-3K) inhibitor, wortmannin, prevented the protective effect of ouabain against serum deprivation. Furthermore, Western blotting analysis revealed an increase in phosphorylation of extracellular signal-regulated kinases (ERK-1 and ERK-2) induced by 100nM ouabain in serum-deprived cells. In accord, pretreatment of HUVEC with PD98059, inhibitor of the ERK pathway, abrogated the effect of ouabain. Our results show that ouabain has an antiapoptotic effect on HUVEC through the activation of PI-3K and ERK dependent pathways.     

Proteomic study of human umbilical vein endothelial cells in culture

The endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (~ 30 nug), the association of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, liquid chromatography-tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F-actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione-S-transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2-D maps of HUVECs will be soon available on the web at http://www. huvec.com.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.