Cytokines suppress the shear stress-stimulated release of vasoactive peptides from human endothelial cells

Endothelial cells from human umbilical vein perfused at 0.5 ml/min released vasopressin, endothelin, and substance P. Upon perfusion of the cells at 3.0 ml/min, the release of endothelia and vasopressin was significantly increased whereas the release of substance P was significantly decreased. Endothelial cells precultured for 24 h with interleukin-I (IL-1) and interferon-γ (IFN-γ) released more endothelin and less substance P at low flow and there was no further increase in release at high flow rate. These results suggest that cytokines suppress the normal responses of endothelial cells to increased fluid shear stress.     

Colocalization of vasoactive substances in the endothelial cells of human umbilical vessels

Human umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in local control and regulation of the blood flow. In the present study, we examined ultrathin sections of cultured human umbilical vein endothelial cells and tissue preparations of umbilical vein and artery, immunostained by the post-embedding colloidal gold double-labelling technique. We observed colocalization of atrial natriuretic peptide and neuropeptide Y, as well as colocalization of atrial natriuretic peptide and neuropeptide Y with other vasoactive substances, namely, vasoactive intestinal peptide, substance P, calcitonin gene-related peptide and arginine vasopressin. The functional significance of the colocalization of these vasoactive substances in the human umbilical vessel endothelial cells is discussed.     

Endothelin-1 induces prostacyclin release from bovine aortic endothelial cells

The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.     

Stimulation of growth and calcium influx in cultured, bovine, aortic endothelial cells by platelets and vasoactive substances.

Endothelial cells (EC) line the heart and blood vessels, and they are the principal cellular components of the microvasculature. The presence of circulating platelets is believed to be necessary to maintain the integrity of the capillary endothelium. Growing EC in culture provides an opportunity to simulate the in vivo situation and to study the response of these cells to platelets and platelet secretions. The addition of platelets at a concentration of 10⁴-10⁵/mm³ and substances which circulate in blood following injury (serotonin, thrombin, ADP, epinephrine, norepinephrine and histamine) stimulate endothelial proliferation from 150–1,000% of controls. That substances so diverse in form have similar effects suggests a common mode of action, such as mobilization of a second messenger. The influx of ⁴⁵calcium (⁴⁵Ca⁺⁺) in response to these agents was found to be 5 to 24 times that of controls. The stimulation of ⁴⁵Ca⁺⁺ influx appears to be dose-dependent, and it is inhibited by pre-incubation with lanthanum chloride and specific blocking agents. Calcium as a second messenger is implicated in a variety of cellular functions including division, secretion, motility and enzyme regulation. Thus, the theorized supportive role of platelets on endothelium may be dual and operate, at least at the initial level, by a common mechanism: to mobilize calcium for stimulus-division following injury and for stimulus-secretion in normal metabolic activities.     

Comparison of the release of vasoactive factors from venous and arterial bovine cultured endothelial cells.

The release of endothelium-derived relaxing factor (EDRF), prostacyclin (PGI2), and endothelin-1 (ET-1) was measured from endothelial cells (EC) cultured from either bovine vena cava (BVCEC) or bovine aorta (BAEC). EDRF release was determined by using the superfusion bioassay technique, whereas ET-1 and PGI2 were measured by specific radioimmunoassays. Bradykinin (BK) (0.05-30 pmol) given through columns of venous or arterial EC induced a dose-dependent release of EDRF. BK (0.05 pmol) evoked a release of EDRF from venous EC that was similar to the effect of a dose of 1 pmol from arterial EC. As with BAEC, infusions of NG-monomethyl-L-arginine (30 microM) caused an inhibition of EDRF release from BVCEC that was partially reversed by coinfusions of L-arginine (L-Arg; 100 microM). BK also induced a dose-dependent release of PGI2 from BVCEC. BVCEC and BAEC produced PGI2 in equivalent amounts when arachidonic acid (9.2 and 32 pmol) was added to the Krebs' solution perfusing the cells. BVCEC and BAEC released detectable amounts of ET-1 (0.4 +/- 0.1 and 0.9 +/- 0.3 ng/mL, respectively), over a 4-h period, and the release of ET-1 was increased approximately twofold by coincubations with thrombin (0.05-1 U/mL). These findings demonstrate that venous EC have a similar capacity to arterial EC to release vasoactive factors, thus supporting the hypothesis that veins have a functional endothelium that may modulate venous tone and platelet function.     

Electron-microscopic immunolabelling of vasoactive substances in human umbilical endothelial cells and their actions in early and late pregnancy

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of feto-placental blood flow. The pharmacological effects of 5-hydroxytryptamine, histamine and endothelin were examined in umbilical arteries and veins from legal terminations (gestational age 8–17 weeks, n=12) and normal term vaginal deliveries (gestational age 38–41, n=12). Immunocytochemistry of human unbilical vessels indicated that 5-hydroxytryptamine, histamine and endothelin were localised in subpopulations of endothelial cells of both artery and vein in late, but not early, pregnancy. 5-Hydroxytryptamine (10 nM–30 μM) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy. Histamine (0.1 μM–30 mM) also caused sustained contractions in all vessels from late pregnancy but only 27% of arteries and 41% of veins from early pregnancy responded. Endothelin (10 pM–30 nM) caused slow long-lasting contractions in all vessels from early and late pregnancy. Atrial natriuretic peptide and neuropeptide Y did not alter vascular tone. The endothelium may thus play an autocrine/paracrine role, by synthesizing and releasing the above reactive substances in late pregnancy to influence feto-placental blood flow. Received: 23 May 1995 / Accepted: 13 October 1995     

Bradykinin induces superoxide anion release from human endothelial cells

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2-200 microM) stimulated the synthesis of prostacyclin (PGI2), as reflected by the release of 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha), in endothelial cells cultured from bovine aorta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF1 alpha triggered by ADP was rapid and onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI2. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

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.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2−200 μN) stimulated the synthesis of prostacyclin (PGI₂), as reflected by the release of 6-keto-prostaglandin F_1α (6-K-PGF_1α), in endothelial cells cultured from bovine orta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF_1α triggered by ADP was rapid in onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could be thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI₂. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates

Soy-derived proteins (soy protein isolate, glycinin, and β-conglycinin) and bovine whey-derived proteins (whey protein isolate, -lactalbumin, β-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates obtained was studied as a function of pH. At neutral pH, all soy-derived protein hydrolysates, particularly those from glycinin, obtained by hydrolysis with subtilisin Carlsberg, chymotrypsin, bromelain, and papain showed a stronger aggregation compared to the non-hydrolyzed ones. This increase in aggregation was not observed upon hydrolysis by trypsin. None of the whey-derived protein hydrolysates exhibited an increase in aggregation at neutral pH. The high abundance of theoretical cleavage sites in the hydrophobic regions of glycinin probably explains the stronger exposure of hydrophobic groups than for the other proteins, which is suggested to be the driving force in the aggregate formation.     

Modulation of secretion of vasoactive materials from human and bovine endothelial cells by cyclic strain

The effects of cyclical expansion and elaxation of the vessel wall on endothelial cell metabolism have been modeled using a uniaxial strain device and cultured endothelial cell monolayers. Also, the effects of stopping and then restarting cyclic strain on metabolite secreation rates were determined. Secretion rates of prostacyclin (PGI(2)), endothelin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-type 1 (PaI-1) by endothelial cells were constant over24-h periods The secreation of both PGI(2) and endothelin was enhanced in cells exposed to high physiological levels of cyclical strain (10% at 1Hz) compared with controls, while tPA production was unaltered. These results were true for both human and bovine endothelial cells. Characterization of the response of human endothelial cells to cyclical strain made evaluation of stretch effects on PAl-1 secretion possible. A nearly twofold increase in PAl-1 secretion by cells exposed to arterial levels of strain was observed. Endothelin secretion remained elevated even after strain was stopped for 12 h, while PGl(2) secretion returned to control values upon cessation of cyclic stretch. These results indicate that physiological levels of cyclic mechanical strain ca significantly modulate secretion of vasoactive metabolited form endothelial cells. The changes sen secretion are, in some cases, quite different from those caused by arterial levels of fluid shear stress exposure. (c) 1994 John Wiley & Sons, Inc.     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

The release of platelet-activating factor from human endothelial cells in culture

The release of platelet-activating factor (PAF) from stimulated human endothelial cells (HEC) cultured from normal term, umbilical cord veins is described. HEC in primary cultures released PAF after challenge with A23187, rabbit anti-human factor VIII (RaHu/FVIII), angiotensin II, and vasopressin. HEC subcultures maintained the ability to release PAF in the presence of A23187 and RaHu/FVIII, whereas the release of PAF in response to angiotensin II and vasopressin was not constant and was reduced. Control cultured, smooth muscle cells derived from umbilical cord veins, previously depleted of endothelial cells, did not release PAF under the above-mentioned stimulation. Plastic-adherent or cultured monocytes released PAF with A23187, but not with RaHu/FVIII, angiotensin II, and vasopressin. The release of PAF from HEC in primary cultures required the presence of extracellular cations and the activation of membrane phospholipase A2. PAF release induced by A23187, RaHu/FVIII, angiotensin II, and vasopressin was unaffected by indomethacin, an inhibitor of cyclooxygenase, which, however, favored the release of PAF from HEC stimulated with thrombin, a stimulus that did not affect HEC in the absence of indomethacin. PGI2 inhibited PAF release from stimulated HEC. The relevance of an acetylation process in the biosynthesis of PAF and HEC was supported by the following evidence: 1) the increase in PAF yield in the presence of sodium acetate and, particularly, of acetyl-CoA; 2) the incorporation of [14C]acetate into PAF molecules; 3) the loss of radioactivity and of biologic activity after treatment with phospholipase A2. These results indicate that HEC in culture are able to release PAF and that metabolic pathways similar to those described for leukocytes are involved.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.