Incubation Of Human Umbilical Vein Endothelial Cells Research Articles

Effect of aspirin on constitutive nitric oxide synthase and the biovailability of NO

Introduction: Aspirin decreases the activity of iNOS and the formation of prostanoids. Constitutive nitric oxide synthase (cNOS) is present in endothelial cells, platelets, leukocytes and neurons, yet no data are available on the effect of aspirin on cNOS and the bioavailability of NO produced by this enzyme. Materials and methods: Human umbilical vein endothelial cells (HUVECs), rat adrenal gland pheocromocytoma cells (PC-12) and human platelets were incubated with different aspirin concentrations. The kinetics of NO, O2− and ONOO− release were measured simultaneously in single cells or platelet suspensions using tandem electrochemical nanosensors. The NO, O2− and ONOO− release from cells and platelets was stimulated with calcium ionophore and collagen, respectively. cNOS expression was estimated by Western blot analysis. Results: Incubation of HUVECs and PC-12 with 10−5 mol/l of aspirin increased cNOS expression by 70±7% and 50±5, respectively. However, the NO concentration increased only by 33% in HUVECs incubated with the same aspirin concentration. Incubation of HUVECs with aspirin also increased the O2− and ONOO− production. Therefore the bioavailability of NO increased only slightly in endothelium and did not reflect the increase in eNOS. This was in contrast to platelets, where maximal NO bioavailability almost doubled after incubation with aspirin. Conclusions: Aspirin did not have a significant effect on the NO bioavailability in endothelial cells. However, aspirin highly improved the NO production in platelets. The high NO production in platelets may counteract the effect of thromboxane, inhibit platelet aggregation, and compensate for the reduction of prostacycline concentration by aspirin.

Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism.

GTP cyclohydrolase I (GTPCH I) catalyzes the de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor of NO-synthase. The enzyme underlies negative feedback regulation by the end product BH(4). This feedback inhibition is mediated through complex formation with the GTP cyclohydrolase I feedback regulatory protein (GFRP). To further classify the mechanism involved in the regulation of BH(4) synthesis, we measured expression of GTPCH I and GFRP in different human tissues. Furthermore, we looked for the influence of phenylalanine that is known to reverse BH(4)-mediated feedback inhibition of GTPCH I, and of immunostimulation with interferon gamma on the expression of GTPCH I and GFRP. Using RT-PCR and northern blot technique, coexpression of GFRP and GTPCH I could be demonstrated in a number of different tissues such as endothelial cells and peripheral blood cells. Following stimulation of human umbilical vein endothelial cells (HUVEC) with phenylalanine (1 mM), there was no change of GFRP mRNA. In contrast, the mRNA level of GTPCH I was significantly upregulated with a maximum after 6 hours (p = 0.04). Incubation of HUVEC with interferon-gamma (100 U/ml) showed an increase of GTPCH I mRNA and a significant downregulation of GFRP mRNA after 24 hours (p = 0.03). This study shows for the first time the expression of GFRP in different human tissues. The biosynthesis of BH(4) is not only regulated on the substrate level but also through transcription of the interacting proteins. Phenylalanine stimulates the biosynthesis of BH(4) not only by reversing the negative feedback inhibition of GTPCH I but also by increasing the mRNA level of GTPCH I. Immunostimulation alters protein expression of GTPCH I and GFRP in a way that favors BH(4) synthesis.

Paraoxonase-1 reduces monocyte chemotaxis and adhesion to endothelial cells due to oxidation of palmitoyl, linoleoyl glycerophosphorylcholine.

High-density lipoprotein (HDL) is postulated to protect against the development of atherosclerosis, in part, by inhibiting the oxidation of low density lipoprotein (LDL) in the sub-endothelial space and thus inhibiting activation of the endothelium. The HDL-associated enzyme, paraoxonase-1, is proposed to be a major protective factor. However, HDL is also prone to oxidation when exposed to peroxynitrite and may therefore, once oxidized, have properties similar to oxidized LDL. We exposed human HDL to the peroxynitrite donor 3-morpholinosydnonimine and incubated oxidized HDL with human umbilical vein endothelial cells (HUVECs). Oxidized HDL increased monocyte binding (P<0.001) and enhanced chemotaxis (P<0.001). The major oxidized phospholipids were 1-palmitoyl (stearoyl)-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, derived from linoleate-containing phosphatidylcholines, and 1-palmitoyl(stearoyl)-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine, derived from arachidonate-containing phosphatidylcholines. Incubation of HUVECs with synthetically prepared 1-palmitoyl-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, or 1-palmitoyl-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine increased binding of monocytes (P<0.001) and chemotaxis (P<0.001). Purified paraoxonase-1 reduced monocyte adhesion and chemotaxis (P<0.001). (i) HDL can be a source of oxidatively-derived bioactive phospholipids; (ii) the fragmented phospholipids with a 9-carbon aldehyde or acid are as effective as a 5-carbon aldehyde or acid at inducing monocyte adhesion and chemotaxis; and (iii) paraoxonase-1 is effective at reducing the activity of these phospholipid oxidation products.

Inhibitory effects of catechins on neutrophil-dependent gastric inflammation

The inhibitory effects of tea against carcinogenesis have been attributed to the biological activity of the polyphenol fraction of tea. However, the molecular mechanisms of these effects are not completely understood. Chronic inflammation induced by Helicobacter pylori has been proposed to be a causative pathway in the carcinogenesis of stomach cancer. Therefore, an agent possessing anti-inflammatory properties may be chemopreventative against stomach cancer. In the present study, we have investigated the anti-inflammatory effects of tea catechins. After addition of IL-1β to MKN45 cells, a gastric cancer cell line, or human umbilical vein endothelial cells (HUVECs), IL-8 production was detected in supernatants. This IL-8 production was inhibited by catechins. Incubation of HUVECs or polymorphonuclear leukocytes (PMNs) with IL-1β or IL-8, respectively, resulted in an increased surface expression of adhesion molecules. Catechins also inhibited this expression of adhesion molecules on HUVECs and PMNs. Of these major effects, the strongest effect of catechins was to reduce expression of the adhesion molecules CD11b and CD18 on PMNs. These results suggest that tea may inhibit carcinogenesis partly through the anti-inflammatory effects of tea catechins on PMN-dependent gastric mucosal inflammation.

Dose-dependent Activation of Antiapoptotic and Proapoptotic Pathways by Ethanol Treatment in Human Vascular Endothelial Cells: DIFFERENTIAL INVOLVEMENT OF ADENOSINE

Moderate but not heavy drinking has been found to have a protective effect against cardiovascular morbidity. We investigated the effects of ethanol (EtOH) treatment on the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cultured human umbilical vein endothelial cells (HUVEC). Exposure of cells to 2-20 mm EtOH resulted in rapid (<10 min) induction of Akt phosphorylation that could be prevented by pertussis toxin or the PI3K inhibitors wortmannin and LY294002. Among the downstream effectors of PI3K/Akt, p70S6 kinase, glycogen synthase kinase 3alpha/beta, and IkappaB-alpha were phosphorylated, the latter resulting in 3-fold activation of NF-kappaB. EtOH also activated p44/42 mitogen-activated protein kinase in a PI3K-dependent manner. Low concentrations of EtOH increased endothelial nitric-oxide synthase activity, which could be blocked by transfection of HUVEC with dominant-negative Akt, implicating the PI3K/Akt pathway in this effect. The adenosine A1 receptor antagonist 1,3-dipopylcyclopentylxanthine prevented the phosphorylation of Akt observed in the presence of EtOH, adenosine, or the A1 agonist N(6)-cyclopentyladenosine. Incubation of HUVEC with 50-100 mm EtOH resulted in mitochondrial permeability transition and caspase-3 activation followed by apoptosis, as documented by DNA fragmentation and TUNEL assays. EtOH-induced apoptosis was unaffected by DPCPX and was potentiated by wortmannin or LY294002. We conclude that treatment with low concentrations of EtOH activates the cell survival promoting PI3K/Akt pathway in endothelial cells by an adenosine receptor-dependent mechanism and activation of the proapoptotic caspase pathway by higher concentrations of EtOH via an adenosine-independent mechanism can mask or counteract such effects.

Leptin induces endothelin-1 in endothelial cells in vitro.

Leptin, a protein encoded by the obese gene, is produced by adipocytes and released into the bloodstream. In obese humans, serum leptin levels are increased and correlate with the individual's body mass index and blood pressure. Elevated serum concentrations of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, were also observed in obese subjects. The pathomechanisms underlying this ET-1 increase in obesity are poorly understood. In the present study, we investigated the influence of the ob gene product leptin on the expression of ET-1 in human umbilical vein endothelial cells (HUVECs). Binding studies using (125)I-radiolabeled leptin revealed high- and low-affinity leptin binding sites on HUVECs (Kd1=13.1+/-3.1 nmol/L and Kd2=1390+/-198 nmol/L, respectively), mediating a time- and dose-dependent increase of ET-1 mRNA expression and protein secretion after incubation of HUVECs with leptin. This leptin-induced ET-1 expression was inhibited by preincubation of HUVECs with 0.75 micromol/L antisense phosphorothioate oligonucleotides directed against the leptin receptor Ob-Rb. Furthermore, after incubation with leptin, increased nuclear staining of c-fos and c-jun, the major components of the transcription factor AP-1, and increased AP-1 DNA binding were observed. Transient transfection studies with ET-1 promoter constructs showed that leptin-induced promoter activity was abolished in the absence of AP-1 binding sites or by cotransfection with a plasmid overexpressing a mutated jun, which is able to bind c-fos but not DNA. Thus, leptin upregulates ET-1 production in HUVECs via a mechanism potentially involving jun binding members of the bZIP family.

Effect of cytokines on adhesion molecules expression in cell culture of blood vessel endothelium

The interaction between leukocytes and endothelial cells plays the essential role in inflammation. Endothelial cells express a variety of adhesive receptors that regulate their adhesion to leukocytes and also to the extracellular matrix. These interactions are complex phenomena that require multiple recognition mechanisms, and include the first rolling and later the stationary adhesion and transmigration of leukocytes. It is known that cytokines have regulatory effects on cell adhesion molecules expression. In the present study we investigated the influence of cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8 IL-10, TNF-a, IFN-g) and the combined use of IL-2, IL-4, IL-6, IL-8 and IL-10 with IL-1b, TNF-b or IFN-g on the expression of adhesion molecules of cultured human umbilical vein endothelial cells (HUVECs) after stimulation for 16 hours. Likewise, in vitro model described herein is designed to mimic the activation of endothelial cells by cytokines as seen during inflammatory processes. This process is mediated by specific cell adhesion molecules being crucial for the generation of immune and inflammatory responses. Therefore, HUVECs are treated with two different cytokine combinations consisting of either IL-2, IL-6, IL-8 IFN-g and TNF-a or IL-1b, IL-2, IL-4, IL-6, IL-10, IFN-g and TNF-a. Endothelial cells were collected from hunam umbilical vain using collagense type II, and cell cultures in complete medium were kept in the incubator (37.4?C, 5% CO2). After stimulation cells were prepared for analysis using tripsinisation procedure. The surface expression of the following adhesion molecules was determined in cultured human umbilical vein endothelial cells (HUVECs) by means of flow-cytometric analysis: CD 62P (P-selectin), CD 62E (E-selectin, ELAM-1) CD 106CD 34 (L-selectin ligand). The highest CD 62E expression on the surface of HUVECs was found when endothelial cells were stimulated with TNF-a alone. Also they were increased after stimulation with IL-1b, while IL-4 led to down-regulation of CD 62E. Incubation of HUVEC monolayers with IL-1b, IL-4 as well as TNF-a and IFN-g, statistically significant, reduced the surface expression of CD 34 while other cytokines did not affect CD 34 expression. Incubation of HUVECs with a single cytokine caused no statistically significant changes in CD 62P expression compared to controls. The most potent effect on CD 54 expression was found under TNF-a stimulation; IL-1b and IFN-g had also amplifying effects, while all other tested cytokines caused no significant changes in surface molecule expression. Surface expression of CD 106 was amplified during incubation with IL-1b, IL-4, TNF-a and IFN-g. Single stimulation of tested cytokines did not significantly alter the cell surface expression of CD 31. Concomitant stimulation with IL-2, IL-4, IL-6, IL-8 or IL-10 with IL-1b, TNF-a or IFN-g led to different effects compared with effects of single cytokine stimulation: CD 62E were up-regulated under co-stimulation with combination of IL-1b and IFN-g, IL-6 and IL-1b, and also in all combinations with TNF-a. Statistically significant differences were found in CD 62P surface expression after concomitant stimulation with IL-1b and IFN-g, and in combinations with TNF-a. Co-stimulation with IL-10 and IL- 1b, TNF-a or IFN-g, or IL-8 with IL-1b or IFNg, IL-6 with IFN-g, IL-4 with TNF-a or IFN-g and IL-2 with IFN-g significantly decreased the level of CD 34 surface expression. (VCAM-1), CD 54 (ICAM-1), CD 31 (PECAM-1) and CD 54 expression was up-regulated after stimulation with IL-1b and IFN-g, and under concomitant stumulation with TNF-a. Surface expression of molecule CD 106 was higher after co-stimulation of cytokines with TNF-a, and IL-4 or IL-10 with IL-1b. These effects indicate modulation of single cytokine effects. Intracellular mechanisms included in those effects need to be investigated. Also there were found modulatory effects of cytokine combinations. Some effects of cytokine combinations were different in comparison to single cytokine effect. This finding indicates that intracellular mechanisms are present and responsible for signal modulation of single cytokine. The application of these two cytokine combinations mimicing inflammation reactions results in effects of comparable dimensions significantly increasing the mean fluorescence intensity of E-selectin, VCAM-1 and ICAM-1 surface expression accompanied by the induction of P-selectin expression. The experiments reveal a strong up-regulation of these cell surface antigens under conditions mimicing inflammation. This is an essential finding stressing the importance of endothelial cells during inflammatory processes.

Influence of hepatocyte growth factor, epidermal growth factor, and mycophenolic acid on endothelin-1 synthesis in human endothelial cells.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells. ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction. HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1+/-4.3% of control, n=6, mean+/-SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 microg/ml) for 3-5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1-50 microg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.

Cell-derived Apolipoprotein E (ApoE) Particles Inhibit Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression in Human Endothelial Cells

Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.

Hydrocortisone modulates the effect of estradiol on endothelial nitric oxide synthase expression in human endothelial cells

The interaction between hydrocortisone and estradiol on the regulation of endothelial nitric oxide synthase (eNOS) expression was investigated in human umbilical vein endothelial cells (HUVECs). Following incubation in medium containing dextran-coated-charcoal-stripped serum (DCC-stripped medium) for 4 days, incubation of HUVECs with 0.1 nM estradiol for 24 hr in the absence of hydrocortisone increased levels of eNOS mRNA measured by ribonuclease protection assay above control (0 nM estradiol). 2 μM hydrocortisone applied for 24 hr preceding and during estradiol application inhibited the estradiol-elicited increase in eNOS mRNA levels, reducing mRNA levels from 134% ± 14% of control to 85% ± 5% of control. Significant (ANOVA, p<0.01) reductions of estradiol-mediated increases of mRNA levels occurred over a range of hydrocortisone concentrations (10 nM, p<0.05; 2 μM, p<0.05; n=3–12). In the presence of 2 μM hydrocortisone, 10 nM estradiol significantly reduced eNOS mRNA levels to 59% ± 3% of control. The ability of hydrocortisone to block or reverse the estradiol-mediated increase in eNOS mRNA levels may provide a link between elevated hydrocortisone levels and decreased NO production, potentially contributing to the development of hypertension and cardiovascular disease in vivo and antagonizing cardioprotective effects of estrogens.

Oxidative DNA damage induced by high glucose and its suppression in human umbilical vein endothelial cells

In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45 mM) compared with normal glucose (5 mM) medium ( P<0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P<0.05, 5 days: P<0.001), whereas l-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites ( P<0.05). Flow cytometric analysis using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with l-glucose produced more reactive oxygen species than incubation with d-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 μM) or pyridoxamine (1 mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites ( P<0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species. In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 μg/ml induced FPG-sensitive sites ( P<0.001, P<0.01, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species.

Induction of cyclo-oxygenase-2 in human endothelial cells by SIN-1 in the absence of prostaglandin production.

Nitric oxide (NO) regulates cyclo-oxygenase (COX) activity in various cell systems and reports conflict in regard to its stimulatory versus inhibitory role. Incubation of human umbilical vein endothelial cells (HUVEC) with SIN-1 (3-morpholinosydnonimine), a donor of NO, resulted in a rapid and dose-dependent increase in the expression of COX-2 as analysed by Western and Northern blotting. Incubation of HUVEC with SIN-1 and interleukine (IL)-1alpha resulted in increased induction of COX-2 compared with IL-1alpha alone and corresponded to an additive effect. The COX-2 induction was dependent on a de novo synthesis since cycloheximide, an inhibitor of protein synthesis, blocked the enzyme expression. The increase in COX-2 expression was not accompanied by a corresponding change in prostaglandin (PG) production. However, the COX activity was partially recovered when immunoprecipitated COX-2 was incubated with arachidonic acid and haematin. Peroxynitrite, a highly reactive nitrogen molecule derived from the interaction of NO and superoxide anion, significantly increased COX-2 expression. Under these conditions and within the limit of detection of the antibody, selective antibody for nitrotyrosine failed to detect nitrated COX-2 in immunoprecipitated COX-2 when cells where incubated with SIN-1 or SIN-1+IL-1alpha. Ro 31-8220, a specific inhibitor of protein kinase (PK) C, blocked the induction of COX-2. Also, SB203580, the selective inhibitor of p38 MAP kinase, strongly blocked the induction of COX-2 by SIN-1 in the presence or absence of IL-1alpha, whereas the MEK-1 inhibitor, PD 98059, affected it to a lesser extent. These data demonstrate that SIN-1 induces COX-2 in HUVEC in the absence of PG formation and suggest a complex regulation of COX-2 expression and PG formation by NO in endothelial cells.

Acute regulation of fatty acid oxidation and amp-activated protein kinase in human umbilical vein endothelial cells.

It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of (3)H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with (3)H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

Proteinase 3 enhances endothelial monocyte chemoattractant protein-1 production and induces increased adhesion of neutrophils to endothelial cells by upregulating intercellular cell adhesion molecule-1.

Wegener's granulomatosis is an autoimmune disease that is characterized by systemic vasculitis and granuloma formation. Early influx of polymorphonuclear neutrophils (PMN), followed at a later stage by mononuclear cells, contributes to the granulomatous inflammation. Previous studies have shown that proteinase 3 (PR3), the major autoantigen in Wegener's granulomatosis, specifically binds to endothelial cells and plays a possible role in activation of these cells by enhancing interleukin-8 production, thus providing a chemotactic and activating stimulus for PMN. The present study demonstrated that PR3 enhances the production of monocyte chemoattractant protein-1 (MCP-1) by human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. The PR3-induced increase in MCP-1 production was demonstrated at both the protein and the mRNA levels and was chemotactic for monocytes. In addition, it was demonstrated that PR3 induces a dose- and time-dependent increase in the expression of intercellular adhesion molecule-1 (ICAM-1) as determined by fluorescence-activated cell sorter analysis. The PR3-induced increase in expression of ICAM-1 was also demonstrated at the mRNA level. PR3 induced a slight increase in vascular cell adhesion molecule-1 expression and had no effect on the expression of both P- and E-selectin. Incubation of HUVEC for 24 h in the presence of PR3 resulted in a significant increase in adhesion of PMN, which was reduced to baseline levels in the presence of blocking monoclonal antibody anti-ICAM-1 or anti-CD18 or a combination of both. Monocytes showed a slight but statistically not significant increase in adhesion. Incubation of HUVEC with PR3 for 4 h did not result in enhanced adhesion of either PMN or monocytes. It was hypothesized that PR3, which may be released locally at inflammatory sites after activation of cytokine primed PMN, plays a role in endothelial cell activation by enhancing both interleukin-8 and MCP-1 production, thus providing a chemotactic and activating stimulus for both PMN and monocytes. In addition, PR3 may contribute to the ongoing inflammation by enhancing the adhesion of PMN to endothelial cells by upregulating ICAM-1 expression.

Regulation of Endothelial Nitric Oxide Synthase and Endothelin-1 Expression by Fluvastatin in Human Vascular Endothelial Cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on endothelial vasoactive substances using human umbilical vein endothelial cells (HUVECs). Incubation of HUVECs with fluvastatin for 12 h increased endothelial nitric oxide synthase (eNOS) mRNA expression in a concentration-dependent manner (peak, 276 ± 38%, mean ± S.D., of the control, at 1.0 μM fluvastatin, P0.01). In addition, fluvastatin increased eNOS protein production (245 ± 51% of the control level, P0.05) as well as nitrite production (165 ± 35% of the control level, P0.01). In contrast, incubation of HUVECs with 1.0 μM fluvastatin for 12 h significantly reduced the production of endothelin-1 (ET-1) and preproET-1 mRNA expression in HUVECs (28 ± 1% and 39 ± 1% of the control level, respectively, P0.01). Our results suggest that fluvastatin might be involved in improvement of endothelial function and prevention of the progression of atherosclerosis.

Localization of β2-Glycoprotein 1 in Late Endosomes of Human Endothelial Cells

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

Oxidized lipoprotein(a) enhanced the expression of P-selectin in cultured human umbilical vein endothelial cells

Lipoprotein(a) [Lp(a)] has been demonstrated to be an independent risk factor for coronary heart disease. However, the precise mechanism by which it contributes to the development of atherosclerosis remains unclear. P-selectin is one of the major adhesion molecules to mediate the interaction between monocytes, platelets and endothelial cells. Increased expression of P-selectin has been frequently found in atherosclerotic lesions. The present study assessed the effects of native and oxidized LDL (n-LDL and ox-LDL) and Lp(a) (n-Lp(a) and ox-Lp(a)) on the expression of P-selectin in cultured human umbilical vein endothelial cells (HUVECs). Cell ELISA was used to measure the expression of P-selectin in HUVECs and monocyte adhesion assay was used to detect whether the P-selectin expressed in HUVECs was functional. P-selectin mRNA expression in HUVECs was determined by Northern blot. Results showed that P-selectin protein expression was not influenced by n-LDL, but was moderately increased by ox-LDL and n-Lp(a). ox-Lp(a) was the most potent stimulus for P-selectin expression, increasing it in HUVECs by 144±10% at 10 nmol/l and 202±22% at 20 nmol/l. In addition, it showed that dose dependency induced monocyte adherence to endothelial cells after incubation of HUVECs with ox-Lp(a). Northern blot analysis demonstrated that the amount of P-selectin mRNA was markedly increased after treatment with ox-Lp(a) but not with n-LDL, ox-LDL and n-Lp(a). These results demonstrate that ox-Lp(a) can induce P-selectin expression in HUVECs, which may thereby influence the pathogenesis of athersclerosis.

Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis

Arsenic trioxide (As2O3) has recently been used successfully in the treatment of acute promyelocytic leukemia and has been shown to induce partial differentiation and apoptosis of leukemic cells in vitro. However, the mechanism by which As2O3 exerts its antileukemic effect remains uncertain. Emerging data suggest that the endothelium and angiogenesis play a seminal role in the proliferation of liquid tumors, such as leukemia. We have shown that activated endothelial cells release cytokines that may stimulate leukemic cell growth. Leukemic cells, in turn, can release endothelial growth factors, such as vascular endothelial growth factor (VEGF). On the basis of these observations, we hypothesized that As2O3 may interrupt a reciprocal loop between leukemic cells and the endothelium by direct action on both cell types. We have shown that treatment of proliferating layers of human umbilical vein endothelial cells (HUVECs) with a variety of concentrations of As2O3results in a reproducible dose- and time-dependent sequence of events marked by change to an activated morphology, up-regulation of endothelial cell adhesion markers, and apoptosis. Also, treatment with As2O3 caused inhibition of VEGF production in the leukemic cell line HEL. Finally, incubation of HUVECs with As2O3 prevented capillary tubule and branch formation in an in vitro endothelial cell–differentiation assay. In conclusion, we believe that As2O3 interrupts a reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of both cell types and by inhibiting leukemic cell VEGF production.

Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis

AbstractArsenic trioxide (As2O3) has recently been used successfully in the treatment of acute promyelocytic leukemia and has been shown to induce partial differentiation and apoptosis of leukemic cells in vitro. However, the mechanism by which As2O3 exerts its antileukemic effect remains uncertain. Emerging data suggest that the endothelium and angiogenesis play a seminal role in the proliferation of liquid tumors, such as leukemia. We have shown that activated endothelial cells release cytokines that may stimulate leukemic cell growth. Leukemic cells, in turn, can release endothelial growth factors, such as vascular endothelial growth factor (VEGF). On the basis of these observations, we hypothesized that As2O3 may interrupt a reciprocal loop between leukemic cells and the endothelium by direct action on both cell types. We have shown that treatment of proliferating layers of human umbilical vein endothelial cells (HUVECs) with a variety of concentrations of As2O3results in a reproducible dose- and time-dependent sequence of events marked by change to an activated morphology, up-regulation of endothelial cell adhesion markers, and apoptosis. Also, treatment with As2O3 caused inhibition of VEGF production in the leukemic cell line HEL. Finally, incubation of HUVECs with As2O3 prevented capillary tubule and branch formation in an in vitro endothelial cell–differentiation assay. In conclusion, we believe that As2O3 interrupts a reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of both cell types and by inhibiting leukemic cell VEGF production.

17Beta-estradiol modulates endothelin-1 expression and release in human endothelial cells.

In this study the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) mRNA expression and secretion was investigated in cultured human umbilical vein endothelial cells (HUVECs). Endothelial cells were either deprived of or treated with 17beta-estradiol (10(-9), 10(-7) M) for 48 h. After the incubation, the effect of E2 on ET-1 gene expression was evaluated by Northern blot analysis. ET-1 release into the media was measured by radioimmunoassay after 6 h of incubation under basal conditions and upon stimulation with thrombin (4 U/ml). In addition, the cyclic guanosine 5'-monophosphate (cGMP) content of cells was assayed by immunoassay. In order to exclude the role of nitric oxide (NO) in E2-induced effects on endothelin-1 gene expression and secretion, nitric oxide synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (1 mM) (L-NAME) was added to the media of some cultures. Incubation of HUVECs with 10(-9) and 10(-7) M E2 for 48 h resulted in a 30 and 47% inhibition of ET-1 mRNA expression, respectively. Incubation with E2 also decreased the basal and thrombin-stimulated ET-1 release while increasing the cGMP content of cells significantly. NOS inhibitor L-NAME increased the release of ET-1 from E2-incubated cells but did not alter the ET-1 release from hormone-deprived cells. However, ET-1 secretion of E2-treated cells were significantly less than the deprived ones. Northern blot analyses also demonstrated that inhibition of NOS only partly attenuated the effect of E2 on ET-1 gene expression. In the presence of L-NAME, treatment with 10(-7) M E2 caused a 12% decrease in ET-1 gene expression. The results demonstrate that E2 may play both direct and indirect role in regulation of ET-1 gene expression and production in human endothelial cells. E2-induced increase in NO but decrease in ET-1 production may partly explain the mechanism of the protective effects of the hormone on the cardiovascular system.