Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

High concentrations of magnesium modulate vascular endothelial cell behaviour in vitro

Magnesium supplementation has been reported to prevent cardiovascular diseases through the decrease of plasma lipids and to improve endothelial function in patients with coronary artery disease. In the present work, we evaluated whether high magnesium concentrations can directly affect the function of cultured endothelial cells, which play a crucial role in maintaining the functional integrity of the vascular wall. We cultured human umbilical vein endothelial cells for various times in media containing different concentration of magnesium (range 2 to 10 mM) and compared them to the corresponding controls (1 mM Mg). High Mg concentrations stimulated endothelial proliferation, enhanced the motogenic response to angiogenic factors and attenuated the response to lipopolysaccharide (LPS). In addition, we demonstrate that high concentrations of magnesium did not modulate the levels of plasminogen activator inhibitor-1, but enhanced the synthesis of nitric oxide, in part through the up-regulation of endothelial nitric oxide synthase. Our results demonstrate a direct role of magnesium in maintaining endothelial function. We therefore anticipate that magnesium may have a protective effect against atherosclerosis and could play a role in promoting the growth of collateral vessels in chronic ischemia. Moreover, because it induces the synthesis of nitric oxide, this cation could be a helpful tool in hypertension as well as in preventing thrombosis.     

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

Impact of modeled microgravity on microvascular endothelial cells

Microvascular endothelial cells are protagonists in inflammation and angiogenesis. They contribute to the integrity of microvasculature by synthesizing a large array of cytokines, growth factors and mediators active on the endothelium itself, on smooth muscle cells and circulating leukocytes. Because space flight (i) associates with vascular impairment and (ii) modulates the cytokine network, we evaluated the effect of modeled microgravity on microvascular 1G11 cells. We found that modeled microgravity reversibly inhibits endothelial growth and this correlates with an upregulation of p21, a cyclin-dependent kinases inhibitor. By protein array, we found that microgravity inhibits the synthesis of interleukin 6, an event that may contribute to growth retardation. We also detected increased amounts of nitric oxide, a mediator of inflammatory responses, a potent vasodilator and a player in angiogenesis. The increased synthesis of nitric oxide is due, at least in part, to an upregulation of endothelial nitric oxide synthase. Because low levels of IL-6 might contribute to endothelial growth retardation as well as to the enhancement of nitric oxide synthesis, we hypothesize a central role of IL-6 in modulating microvascular endothelial cell behaviour in modeled microgravity.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

Cytochrome P450 epoxygenases and vascular tone: novel role for HMG-CoA reductase inhibitors in the regulation of CYP 2C expression

Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI(2))-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI(2)-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.     

Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS-cGMP-PKG pathway in macrovascular endothelial cells

Angiogenesis is a physiological process involving the growth of blood vessel in response to specific stimuli. The present study shows that limited microgravity treatments induce angiogenesis by activating macrovascular endothelial cells. Inhibition of nitric oxide production using pharmacological inhibitors and inducible nitric oxide synthase (iNOS) small interfering ribo nucleic acid (siRNA) abrogated microgravity induced nitric oxide production in macrovascular cells. The study further delineates that iNOS acts as a molecular switch for the heterogeneous effects of microgravity on macrovascular, endocardial and microvascular endothelial cells. Further dissection of nitric oxide downstream signaling confirms that simulated microgravity induces angiogenesis via the cyclic guanosine monophosphate (cGMP)-PKG dependent pathway.     

Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine

Many cell types are known to synthesize nitric oxide (NO.) from L-arginine. There appear to be at least two forms of NO. synthase: an inducible, tetrahydrobiopterin- and flavin-dependent activity exemplified by the macrophage enzyme and a constitutive, Ca+(+)-dependent activity exemplified by the endothelial cell enzyme. L-NG-methylarginine inhibits NO. synthesis by both cell types. We now report that L-NG-aminoarginine and L-NG-nitroarginine are about 100-fold more potent than NG-methylarginine in blocking endothelial cell NO. synthesis. In contrast, NG-aminoarginine and NG-methylarginine are about equipotent with macrophages whereas NG-nitroarginine is much less potent. Since macrophage and endothelial cell NO. synthesis are differentially sensitive to the inhibitors, the panel of inhibitors can be used in complex biological systems to determine if macrophage-like or endothelial-like cells are the predominant source of NO.. Indeed, all three inhibitors elicit a strong pressor response in the anesthetized guinea pig, a result consistent with the view that endothelial cells continually produce vasodilatory NO(.).     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium

The inhibition of platelet adhesion by nitric oxide (NO) and prostacyclin and their mechanism of action was studied. Platelet adhesion to collagen fibrils and endothelial cell matrix was inhibited completely by NO but only partially by prostacyclin. Adhesion of platelets to endothelial cell monolayers was inhibited by bradykinin. This effect of bradykinin was unaffected by aspirin, and was accounted for by the amounts of NO released by the endothelial cells. Inhibition of platelet adhesion by NO and prostacyclin was potentiated by selective inhibitors of cGMP phosphodiesterase, but not of cAMP phosphodiesterase, indicating that elevation of cGMP regulates platelet adhesion.     

Apolipoprotein E (apoE) isoforms differentially induce nitric oxide production in endothelial cells

Although apolipoprotein E3 (apoE3) is atheroprotective, two common isoforms, apoE2 and apoE4, produce recessive and dominant hyperlipidaemias, respectively. Using a fluorescent assay, we report herein that apoE3 particles secreted from recombinant cells stimulate more nitric oxide release in cultured human EA.hy926 endothelial cells than apoE2 or apoE4 (141% more than controls vs. 61 or 11%). Phosphatidylinositol (PI) 3-kinase inhibitors suppressed the apoE effect, while apoE receptor 2 (apoER2) was tyrosine phosphorylated. We conclude that apoE stimulates endothelial nitric oxide release in an isoform-dependent manner, and propose that tyrosine phosphorylation of apoER2 initiates PI3-kinase signalling and activation of nitric oxide synthase.     

Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H2O2 is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.Key words: Rac1, angiogenesis, endothelial, motility, lamellipodia, nitric oxide, nitric oxide synthase     

Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)

Endothelial cells synthesize and release various factors that regulate angiogenesis, inflammatory responses, hemostasis, as well as vascular tone and permeability. Endothelial dysfunction has been associated with a number of pathophysiological processes. Oxidative stress appears to be a common denominator underlying endothelial dysfunction in cardiovascular diseases. However, depending on the pathology, the vascular bed studied, the stimulant, and additional factors such as age, sex, salt intake, cholesterolemia, glycemia, and hyperhomocysteinemia, the mechanisms underlying the endothelial dysfunction can be markedly different. A reduced bioavailability of nitric oxide (NO), an alteration in the production of prostanoids, including prostacyclin, thromboxane A2, and/or isoprostanes, an impairment of endothelium-dependent hyperpolarization, as well as an increased release of endothelin-1, can individually or in association contribute to endothelial dysfunction. Therapeutic interventions do not necessarily restore a proper endothelial function and, when they do, may improve only part of these variables.     

A protective role of ciglitazone in ox‐LDL‐induced rat microvascular endothelial cells via modulating PPARγ‐dependent AMPK/eNOS pathway

Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox‐LDL receptor‐1 (LOX‐1) plays a central role in ox‐LDL‐mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox‐LDL through regulating eNOS activity and LOX‐1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox‐LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox‐LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre‐treatment. Meanwhile, phosphorylation of AMP‐activated protein kinase (AMPK) was suppressed by ox‐LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone‐mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone‐mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX‐1 protein expression in RMVECs was suppressed by ciglitazone, but re‐enhanced by blocking PPARγ or AMPK. Ox‐LDL‐induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX‐1. Collectively, these data demonstrate that ciglitazone‐mediated PPARγ activation suppresses LOX‐1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.     

Possible Role of Nitric Oxide in Catecholamine Secretion by Chromaffin Cells in the Presence and Absence of Cultured Endothelial Cells

Abstract: We studied the effect of cultured endothelial cells on the secretion of catecholamines by cultured bovine chromaffin cells. Chromaffin cell catecholamine secretion was stimulated by either boluses of potassium (K⁺) or the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Endothelial cells inhibited the catecholamine release and stimulatory effects of K⁺ and DMPP. This inhibition increased with time, and in 25 min the initial stimulated secretory response (100%) to 30 m M K⁺ or 25 μ M DMPP dropped to 45 ± 3% and 53.5 ± 2.3%, respectively. This endothelial cell-induced inhibition was blocked by the nitric oxide synthase inhibitors N -nitro- l -arginine methyl ester ( l -NAME) and N -monoethyl- l -arginine ( l -NMMA), and by the guanylate cyclase inhibitor methylene blue, indicating that the l -arginine/nitric oxide/ cyclic GMP pathway is involved in this endothelial cell-chromaffin cell interaction. In the absence of endothelial cells, incubation of chromaffin cells with l -NAME, l -NMMA, or methylene blue also augmented the secretagogue-induced catecholamine secretion, indicating that nitric oxide from chromaffin cells could be implicated in an autoinhibitory process of catecholamine release. These results provide indirect evidence for the presence of nitric oxide synthase in bovine adrenomedullary chromaffin cells. Our results show that there is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that an additional level of inhibition is observed when cultured vascular endothelial cells are present. These two inhibitory processes may have different origins, but they appear to converge into a common pathway, the l -arginine/nitric oxide synthase/guanylate cyclase pathway.     

Endotoxin and TNF alpha directly stimulate nitric oxide formation in cultured rat hepatocytes from chronically endotoxemic rats.

We examined the effects of endotoxin on nitric oxide formation in isolated rat hepatocytes in primary culture. Endotoxin was administered either in vivo, by continuous infusion for 30 or 3 h, or in vitro, on cultured cells. The spontaneous production of nitrites in hepatocytes from in vivo ET-infused rats was lower than equivalent saline controls in the absence of added stimuli. However in vitro addition of endotoxin in culture to hepatocytes from 30 h ET-infused rats greatly enhanced production relative to saline controls. This effect was mimicked by TNF alpha, and activators of protein kinase C (PMA and Ca2+ ionophore A23187). The effects of ET were blocked by NMMA, dexamethasone and protein synthesis inhibitors Actinomycin D and cycloheximide. No in vitro effect of ET was observed in the 3 h infusion model. The results show that chronic exposure to sub-lethal levels of ET primes liver parenchymal cells for the production of nitric oxide, when exposed in vitro to ET or TNF alpha.     

PARP inhibitor Veliparib (ABT-888) enhances the anti-angiogenic potentiality of Curcumin through deregulation of NECTIN-4 in oral cancer: Role of nitric oxide (NO)

Concurrent use of DNA damaging agents with PARP inhibitors contribute to the effectiveness of the anticancer therapy. But there is a dearth of reports on the antiangiogenic effects of PARP inhibitors and the suppression of angiogenesis by this drug combination is not yet reported. For the successful development of cancer therapeutics, anti-cancer drugs ought to have anti-angiogenic potentiality along with their DNA damaging abilities. In this current piece of work, we investigated the in vitro and in ovo anti-angiogenic effect of Curcumin and Veliparib (a PARP inhibitor) in oral cancer. Recent evidences suggest an involvement of the NECTIN-4 in cancer angiogenesis and the exact molecular pathway of this involvement remains to be delineated. We observed that the soluble NECTIN-4 secreted from H357 oral cancer cells enhanced the angiogenesis of endothelial cells (HUVECs) and this was inhibited by Curcumin-Veliparib combination. NECTIN-4 enhanced vascularization, induced vasodilation and triggered the angiogenic sprouting via endothelial tip cell filopodia. Data indicated that NECTIN-4 mediated angiogenesis is associated with PI3K-AKT-mediated nitric oxide (NO) formation. A noticeable increase in the NO enhanced epithelial NO level through HIF-1α mediated iNOS activation. We observed that increased NO enhanced the NECTIN-4 mediated eNOS expression and thereby elicited further angiogenesis. Curcumin antagonised the NECTIN-4-induced angiogenesis through inhibition of PI3K-AKT mediated eNOS pathway and Veliparib synergized the effect of Curcumin. Our observations indicate that NO is cardinal in inducing NECTIN-4 mediated angiogenesis in H357 cells. Thus, Curcumin-Veliparib combination suppresses angiogenesis through deregulation of the PI3K-AKT-eNOS pathway downstream to the NECTIN-4.     

eNOS, COX-2, and prostacyclin production are impaired in endothelial cells from diabetics

The vascular endothelium is a well-recognized target of damage for factors leading to increased cardiovascular risk. Among the agents playing an important role in cardiovascular homeostasis, nitric oxide and prostacyclin represent key markers of endothelial integrity. In the present work, we report for the first time the reduced expression of both endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) proteins, as well as decreased prostacyclin production, in unstimulated human endothelial cells from insulin-dependent diabetic mothers when compared to cells from non-diabetic, control subjects. According to a major role of COX-2 as a source of prostacyclin production even in unstimulated endothelial cells, prostacyclin production was concentration-dependently inhibited by the selective COX-2 inhibitor SC236. Overall, our results suggest a possible link between reduced endothelial COX-2 and NO-synthase expression and the increased risk of cardiovascular diseases affecting diabetic patients, and point to the use of endothelial cells from diabetic patients as a tool for investigating early dysfunction in pathological endothelium.     

Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells

We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm(2) using a cone-plate device, and the concentrations of 6-keto-PGF(1alpha) and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.