Activation Of Vascular Endothelial Cells Research Articles

Regulation of NF-κB activation and nuclear translocation by exogenous nitric oxide (NO) donors in TNF-α activated vascular endothelial cells

Nitric oxide (NO) is a unique mediator which may promote or suppress inflammation. In this study, we examine the effect of exogenous NO on nuclear translocation of nuclear factor-kappa B (NF-κB) in quiescent human umbilical vein endothelial cells (HUVECs) subsequently activated by tumor necrosis factor-α (TNF-α), and in HUVECs previously activated by TNF-α, a model of vascular inflammation. Quiescent and activated HUVECs are exposed to exogenous NO donors of varying half-lives and the degree of NF-κB translocation into the nucleus determined by unique application of immunofluorescence image analysis in whole cells and correlative biochemical analysis of activated NF-κB proteins in the nucleus. NO donors with shorter half-lives are more effective in blocking the activation and translocation of NF-κB, when added to quiescent HUVECs prior to cellular activation by TNF-α. However, in previously activated HUVECs where NF-κB had relocated into the cytoplasm, addition of short half-life NO donors, but not TNF-α, induced re-translocation of NF-κB back into the nucleus sustaining the inflamed cell phenotype. These data suggest that NO as an inhibitor or activator of NF-κB may depend on the state of activation of vascular endothelial cells in which it contacts. Additionally, in activated cells, NO may modulate expression of NF-κB-dependent gene products, when cytokines are ineffective.

Expression of angiogenic and fibrogenic factors in proliferative vitreoretinal disorders

To investigate the expression of connective tissue growth factor (CTGF) in the retina of human subjects with diabetes mellitus, and CTGF, CD105, and gelatinase B in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Twelve donor eyes from six subjects with diabetes mellitus, 10 eyes from five nondiabetic subjects, 14 PDR membranes, and five PVR membranes were studied by immunohistochemical techniques. In situ zymography was used to examine gelatinolytic activity in four PDR membranes. In nondiabetic retinas, there was no immunoreactivity for CTGF. Diabetic retinas showed immunoreactivity for CTGF in ganglion cells and microglia. Vascular endothelial cells in PDR membranes expressed CTGF, CD105, and gelatinase B in 10 (71.4%), 11 (78.6%), and 5 (35.7%) membranes, respectively. Myofibroblasts in PDR membranes expressed CTGF, and gelatinase B in 14 (100%), and 6 (42.9%) membranes, respectively. There was a significant correlation between the number of blood vessels expressing the panendothelial marker CD34 and the number of blood vessels expressing CTGF (r = 0.7884; P = 0.0008), and CD105 (r = 0.6901; P = 0.0063), and the number of myofibroblasts expressing CTGF (r = 0.5922; P = 0.0257). There was a significant correlation between the number of myofibroblasts expressing alpha-smooth muscle actin and the number of myofibroblasts expressing CTGF (r = 0.8393; P = 0.0002). In situ zymography showed the presence of gelatinolytic activity in vascular endothelial cells in PDR membranes. Myofibroblasts in PVR membranes expressed CTGF, and gelatinase B. These results suggest a possible role of CTGF, CD105, and gelatinase B in the pathogenesis of proliferative vitreoretinal disorders.

Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non-small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non-small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.

Beneficial Effect of Zinc Supplementation on Oxidative Stress, Cytokines, and NF-κB DNA Binding in Sickle Cell Disease Patients.

Zinc deficiency is common in adult sickle-cell disease (SCD) patients, due to continued hemolysis and hyperzincuria. Growth retardation, hypogonadism, and immune dysfunctions due to zinc deficiency have been described in SCD patients. Our studies show that zinc has not only anti-inflammatory functions, but is also an antioxidant. We have previously shown that zinc supplementation to adult SCD patients decreased the incidences of infection and hospital admissions. We hypothesize that zinc supplementation improves T-helper cell and vascular endothelial cell activation, and decreases oxidative stress and NF-κB activation in SCD patients. To test this hypothesis, we recruited 36 ambulatory SCD (homozygous) patients (ages 18–47 years, 11 males and 7 females in each group) and randomly divided these into 2 groups. One group (n=18) received 25 mg zinc as acetate orally thrice a day for 3 months. The other group (n=18) received placebo. All these patients were free of pain crisis for 3 months and were not receiving hydroxyurea. The results indicate that zinc supplemented group had decreased incidence of infection in comparison to the placebo group (Chi square analysis: p=0.017). After 3 months of zinc supplementation, the plasma zinc level increased. The anti-oxidant power increased and the plasma levels of NO, lipid peroxidation products (MDA+HAE), DNA oxidation product (8-OHdG), and sVCAM-1 decreased in the zinc supplemented group, compared to the placebo group (p<0.05), as shown in the Table.ParameterGroupPrePostp valueΔp valueZn (μM)Placebo88.8±9.989.3±8.50.8930.0001Zn Supp.85.6±9.3104.2±15.00.0005MDA+HAE (μM)Placebo1.62±0.311.75±0.270.9230.004Zn Supp.1.60±0.241.28±0.240.009NO (μM)Placebo78.5±12.676.7±11.80.2560.051Zn Supp.75.2±11.260.9±17.10.0218-OHdG (ng/ml)Plecebo0.75±0.230.79±0.350.4270.036Zn Supp.0.81±0.250.63±0.210.093Antioxidant power (u/ml)Placebo6.89±1.656.61±1.500.2780.002Zn Supp.7.36±7.210.52±2.330.001sVCAM-1 (ng/ml)Placebo1587±3591495±4540.2630.008Zn Supp.1434±4551251±3530.023p value: Paired T-test; Pre (baseline) vs Post (6 m of supplementation). Δp value: T-test of the differences (pre vs post) between placebo vs Zn group)Relative levels of TNF-α, IL-1β, VCAM-1 mRNAs by RT-PCR were reduced in LPS-stimulated MNC isolated from zinc supplemented group, compared to placebo. In vitro zinc addition to the MNC isolated from placebo decreased TNF-α and IL-1β mRNAs. Zinc supplementation decreased TNF-induced NF-κB DNA binding (a biomarker of oxidative stress) by EMSA in isolated MNC, compared to placebo. Zinc supplementation also increased the relative level of IL-2 mRNA in PHA-stimulated MNC, compared to placebo. Our results showed that zinc supplementation to SCD patients decreased incidence of infection, and decreased oxidative stress markers. The mRNAs of TNF-α, IL-1β, and sVCAM-1, as well as TNF-induced NF-κB activation were also decreased in the zinc-supplemented subjects, suggesting that zinc supplementation may be beneficial to in SCD patients.

Sequences in Intron 51 of the VWF Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice.

The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons. VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences −487 to +247 that functions as an endothelial specific promoter in vitro. Subsequently a number of transacting factors including, NF1, Oct 1, Ets, GATA6, NFY and Ebp4 that positively and negatively regulate the activity of this promoter were identified by us and others. However, in vivo analysis of the promoter demonstrated that this promoter fragment (sequences −487 to +247) targets activation of fused heterologous transgenes (LacZ and amyloid precursor proteins) specifically and exclusively to brain vascular endothelial cells of transgenic mice. A longer VWF promoter fragment, including 2182 bp of the 5′ flanking sequences, the first exon and the first intron was reported by Aird et al to activate LacZ transgene expression in endothelial cells of the heart and muscle as well as brain of transgenic mice. Considering that endogenous VWF expression is observed in almost all endothelial cells, these results suggested that additional VWF gene sequences were required for transcriptional activation of the VWF promoter in vascular endothelial cells of multiple other organs in vivo; and that distinct regions of the VWF gene are required to achieve promoter activity in endothelial cells of distinct organs. To identify additional cis acting elements within the VWF gene that may participate in its transcriptional regulation generally and/or in distinct organs, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells. This region was shown to interact with YY1 transcription factor in a manner that forms endothelial and non-endothelial specific complexes. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site significantly increased a heterologous SV40 promoter activity specifically in endothelial cell and that this increase was dependent on the presence of an intact YY1 binding site. In contrast, the HSS sequences significantly decreased the SV40 promoter activity in non-endothelial cells. These results suggested that the HSS sequences may participate in activation of gene expression in an YY1 dependent manner in endothelial cells, while repress gene expression in non-endothelial cells. Nevertheless the HSS sequences did not significantly affect the homologous VWF promoter activity that was analyzed by in vitro transfection analyses. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (−487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene participate in organ specific regulation of VWF gene expression, an observation that could not be determined by in vitro analysis. These analyses suggest that the HSS sequences contain cis-acting elements that are specifically necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

Molecular mechanisms involved in anti-inflammatory effects of proton pump inhibitors

Interleukin (IL)-8 has been reported to participate in neutrophil infiltration in Helicobacter pylori (H. pylori)-induced gastritis in humans. In this study, we investigated the anti-inflammatory actions beyond the suppression of acid secretion by proton pump inhibitors (PPI), such as omeprazole and lansoprazole, on IL-8 production by gastric epithelial cells (MKN45) and human umbilical vein endothelial cells (HUVEC) and on the transendothelial migration of polymorphonuclear neutrophils (PMN). MKN45 and HUVEC were stimulated with H. pylori water extract (HPE) and IL-1beta, respectively, and nuclear factor kappa B (NFkappaB) activation and subsequent IL-8 production was assessed in the absence or presence of PPI. We also assessed the effect of PPI on IL-8-induced PMN transendothelial migration and on the alteration of cytoplasmic calcium concentration in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN. HPE and IL-1beta induced a significant increase in IL-8 production by MKN45 and HUVEC, respectively, along with NFkappaB activation, which was significantly inhibited by PPI. PPI also inhibited the IL-8-induced transendothelial migration of PMN and the fMLP-induced cytosolic calcium increase in PMN. PPI attenuate PMN-dependent gastric mucosal inflammation partly by interfering with NFkappaB activation in vascular endothelial cells and gastric epithelial cells, and partly by modulating the calcium concentration of PMN.

Synthesis of estradiol-pheophorbide a conjugates: evidence of nuclear targeting, DNA damage and improved photodynamic activity in human breast cancer and vascular endothelial cells

The synthesis, physico-chemical properties, cellular localization and photocytotoxicity of estradiol-pheophorbide a conjugates in estrogen-dependent cancer and vascular endothelial cells are described with the aim of increasing the photodynamic activity by targeting the nucleus of both tumor and blood vessel cells.

Apolipoprotein CIII Induces Expression of Vascular Cell Adhesion Molecule-1 in Vascular Endothelial Cells and Increases Adhesion of Monocytic Cells

Activation of vascular endothelial cells (ECs) plays an important role in atherogenesis and plaque instability. Lipoproteins containing apolipoprotein CIII (apoCIII) predict coronary heart disease (CHD). We recently reported that apoCIII has a proinflammatory effect on human monocytes. In this study, we looked for a direct effect of apoCIII on EC expression of adhesion molecules, leading to monocytic cell adhesion. Treatment of ECs with apoCIII or apoCIII-rich VLDL caused human monocytic THP-1 cells to adhere to them under static condition or under laminar sheer stress (1.0 dyne/cm2). ApoCIII increased EC expression of vascular cell adhesion molecule-1 (VCAM-1) protein and intercellular cell adhesion molecule-1 (ICAM-1) protein (4.9 +/- 1.5-fold and 1.4 +/- 0.5-fold versus control, respectively). Furthermore, apoCIII remarkably increased membrane-bound protein kinase C (PKC) beta in ECs, indicating activation. A selective inhibitor of PKCbeta prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, exposure of ECs to apoCIII induced nuclear factor-kappaB (NF-kappaB) activation. PKCbeta inhibition abolished apoCIII-induced NF-kappaB activation, and NF-kappaB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. ApoCIII-rich VLDL also activated PKCbeta and NF-kappaB in ECs and increased expression of VCAM-1. Pretreatment of ApoCIII-rich VLDL with anti-apoCIII neutralizing antibody abolished its effect on PKCbeta activation. Our findings provide the first evidence that apoCIII increases VCAM-1 and ICAM-1 expression in ECs by activating PKCbeta and NF-kappaB, suggesting a novel mechanism for EC activation induced by dyslipidemia. Therefore, apoCIII-rich VLDL may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

In vitro endothelial cell activation and inflammatory responses in end-stage heart failure

vascular endothelial cell activation and dysfunction are observed in patients with severe heart failure and may contribute to systemic manifestations of this syndrome. It remains unknown whether inflammatory activation of these cells occurs in these patients because of increased circulating proinflammatory mediators. to determine whether the serum from patients with heart failure possesses a net proinflammatory bioactivity to active proinflammatory pathways in cultured endothelial cells. serum was obtained from stable patients with end-stage heart failure undergoing elective cardiac transplantation (Tx) and severely decompensated patients with heart failure requiring emergency left ventricular assist device (LVAD) implantation. Net proinflammatory bioactivity of serum was investigated by monitoring IkappaBalpha degradation and E-selectin expression in cultured human pulmonary artery endothelial cells (HPAEC) following incubation with serum samples. Serum cytokine concentrations were measured by ELISA and neutralizing antibodies were used to determine the role of specific factors in the observed bioactivity. serum from both patient groups induced HPAEC IkappaBalpha degradation. Low basal HPAEC E-selectin expression significantly increased following treatment with Tx but not LVAD serum. Serum tumor necrosis factor-alpha (TNF-alpha) and IL-10 concentrations were higher in patients with LVAD than those with Tx, and soluble TNF-alpha receptor expression was high in both groups. Neither TNF-alpha nor IL-10 blocking experiments altered either bioassay result. activation of a specific profile of pro- and anti-inflammatory mediators is associated with heart failure resulting in HPAEC nuclear factor (NF)-kappaB activation. However, E-selectin expression is further regulated by unidentified factors. TNF-alpha is upregulated but appears to play no part in NFkappaB activation in these patients. These findings could have important therapeutic implications.

Dual Role of VEGF-Induced Heme-Oxygenase-1 in Angiogenesis

Heme-oxygenase-1 (HO-1) is an inducible cytoprotective molecule that displays antioxidant, antiapoptotic, and antiinflammatory effects. In addition, HO-1 appears to have a complex role in angiogenesis. Recent in vivo studies report that vascular endothelial growth factor (VEGF) regulates HO-1 expression and activity in vascular endothelial cells (ECs) and that inhibition of HO-1 abrogates VEGF-induced endothelial activation and subsequent angiogenesis, while promoting VEGF-induced monocyte recruitment and inflammatory angiogenesis. HO-1 may also regulate the synthesis and activity of VEGF, resulting in a positive-feedback loop. In contrast, HO-1 activity has the opposite effect on lipopolysaccharide-driven inflammatory angiogenesis, inhibiting leukocyte invasion and preventing subsequent angiogenesis. In this review, we summarize the current understanding of the role of HO-1 in angiogenesis. We conclude that further investigation, using targeted molecular approaches specifically to alter HO-1 activity, are required to develop our understanding of the role of HO-1 and its products, carbon monoxide, biliverdin, bilirubin, and free iron in angiogenesis. We propose that during chronic inflammation, HO-1 has two roles, first an antiinflammatory action inhibiting leukocyte infiltration, and second, promotion of VEGF-driven noninflammatory angiogenesis, which facilitates tissue repair. Additional studies will help determine whether modulating the activity of HO-1 and/or its products has therapeutic potential in chronic inflammatory dise.

Ectoadenylate Kinase and Plasma Membrane ATP Synthase Activities of Human Vascular Endothelial Cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.

A Radical View of Mitochondrial Function

Endogenous nitric oxide (NO) acts on cytochrome c oxidase, the terminal enzyme in the mitochondrial electron transport chain, inhibiting mitochondrial oxygen consumption and promoting the release of free radicals. Quintero et al. investigated the consequences of NO regulation of mitochondrial activity in vascular endothelial cells, which are highly glycolytic and have abundant NO. Analysis of the effects of inhibitors of mitochondrial electron transport or glycolysis at various oxygen concentrations indicated that ATP production in human umbilical vein endothelial cells (HUVECs) depended more on glycolysis than it did on mitochondrial respiration. Exposure to a low concentration of oxygen (1.5%) that promoted the accumulation of hypoxia-inducible factor 1α (HIF-1α) in human smooth muscle and epithelial kidney cells had little effect on HIF-1α abundance in human vascular endothelial cells unless the production of NO was inhibited. The ability of NO to inhibit HIF-1α accumulation in HUVECs depended on mitochondrial respiration. The production of reactive oxygen species (ROS) in HUVECs depended on oxygen concentration, being greater at 3% oxygen than at 0.5% or 21% oxygen, and, at 3% oxygen, depended on the production of NO. Activation of AMP-activated protein kinase (AMPK) also peaked at 3% oxygen, and this did not depend on the ratio of AMP to ATP or on the abundance of AMP. Rather, the activation of AMPK at 3% oxygen was stimulated by ROS and was inhibited by antioxidants. Thus, the authors came to the intriguing conclusion that, in vascular endothelial cells, mitochondria may function primarily as NO-regulated organelles that signal through oxygen or ROS rather than as the "powerhouse" that provides cells with ATP. M. Quintero, S. L. Colombo, A. Godfrey, S. Moncada, Mitochondria as signaling organelles in the vascular endothelium. Proc. Natl. Acad. Sci. U.S.A. 103 , 5379-5384 (2006). [Abstract] [Full Text]

Fabrication of endothelialized tube in collagen gel as starting point for self‐developing capillary‐like network to construct three‐dimensional organs in vitro

A possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro with similar volumes to the primary organs is to develop a capillary network throughout the constructs to provide sufficient oxygenation and nutrition to the cells composing them. Here, we propose a novel approach for the creation of a capillary-like network in vitro, based on the spontaneous tube-forming activity of vascular endothelial cells (ECs) in collagen gel. We fabricated a linear tube of 500 microm in diameter, the inner surface of which was filled with bovine carotid artery vascular endothelial cells (BECs), in type I collagen gel as a starting point for the formation of a capillary-like network. The BECs exposed to a medium containing vascular endothelial growth factor (VEGF) migrated into the ambient gel around the tube. After 2 weeks of VEGF exposure, the distance of the migration into the ambient gel in the radial direction of the tube reached approximately 800 microm. Cross-sections of capillary-like structures composed of the migrating BECs, with a lumen-like interior space, were observed in slices of the gel around the tube stained with hematoxylin-eosin (H&E). These results demonstrate that this approach using a pre-established tube, which is composed of ECs, as a starting point for a self-developing capillary-like network is potentially useful for constructing 3D organs in vitro.

Linoleic acid induces proinflammatory events in vascular endothelial cells via activation of PI3K/Akt and ERK1/2 signaling

Linoleic acid (18:2n-6), is a major unsaturated fatty acid in the American diet. Linoleic acid is considered to be atherogenic because of its pro-oxidative and proinflammatory properties. There is substantial evidence that linoleic acid (LA) can activate vascular endothelial cells and contribute to an inflammatory response. To explore the mechanisms of LA-induced proinflammatory signaling pathways, the present study addresses the role of the phosphatidylinositol 3-kinase/amino kinase terminal (PI3K/Akt), extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways during vascular endothelial cell activation. After a 3- to 6-h exposure, LA significantly activated both Akt and ERK in endothelial cells, as assessed by western blot and immunofluorescence. In contrast, LA activated p38 MAPK already at 10 min, suggesting that p38 MAPK signaling occurred upstream of the ERK1/2 pathway. Furthermore, inhibition of ERK activity by PD98059 and PI3K/Akt activity by LY294002 or wortmannin significantly reduced the LA-induced activation of nuclear factor kappa B (NF-κB). These results suggest a contribution of both the ERK1/2 and PI3K/Akt pathways to the effect of LA on NF-κB-dependent transcription. Indeed, LA-mediated gene expression of the vascular cell adhesion molecule 1 was suppressed by PD98059, wortmannin and LY294002. These data indicate that both PI3K/Akt- and ERK1/2-mediated proinflammatory signaling events are critical in LA-induced endothelial cell activation and vascular inflammation.

Knockdown of CD146 reduces the migration and proliferation of human endothelial cells

Our previous study has demonstrated that CD146 molecule is a biomarker on vascular endothelium, which is involved in angiogenesis and tumor growth. However the mechanism behind is not clear. Here we have for the first time developed a novel CD146 blockade system using CD146 siRNA to study its function on endothelial cells. Our data showed that CD146 siRNA specifically blocked the expression of CD146 on both mRNA and protein levels, leading to the significant suppression of HUVEC proliferation, adhesion and migration. These results demonstrate that CD146 plays a key role in vascular endothelial cell activity and angiogenesis, and CD146 siRNA can be used as a new inhibitor for anti-angiogenesis therapy.

Antagonism of Sphingosine-1-Phosphate Receptors by FTY720 Inhibits Angiogenesis and Tumor Vascularization

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

Methods for Identifying Cardiovascular Agents: A Review

Basic and clinical investigation into many of the diverse aspects of cardiovascular drug discovery employs varied approaches aimed at determining physiologic and pathophysiologic efficacy of candidate agents for therapeutic utility with the ultimate hope of identifying those agents capable of exerting salutary influence upon cardiac and vascular tissues. Promising compounds may then be used for prophylactic cardiovascular protection and for the treatment of various disorders including hypertension, cardiomyopathy, occlusive vascular disease, and heart failure. The invention disclosed in Methods for identifying cardiovascular agents [1] provides screening methods which can be used to identify certain suspected cardiovascular agents that inhibit vascular smooth muscle cell (VSMC) activation and/or proliferation, functional adaptations inherent in the responses to disease or injury, or those that enhance vascular endothelial cell (VEC) activation and/or proliferation, processes thought to provide protection to jeopardized blood vessels. Additionally, these screening assays include agents that activate estrogen responsive genes in vascular cells, considering that estrogen signaling is generally suggested to serve pivotal functions in preventing many of the pathologic mechanisms contributing to occlusive vascular complications. The findings of this primary patent are directly relevant for discoveries in related inventions that disclose various provisions for cardiovascular drug discovery. This review will provide detailed synopses of these function-based screening methods capable of identifying cardiovascular protective agents for use in basic science research and clinical drug discovery.

Angiogenesis and Osteoclastogenesis Are Mutually Stimulated in Myeloma: A Role for VEGF and Osteopontin.

Angiogenesis and osteoclastogenesis occur in the bone marrow as myeloma (MM) progresses. Suppression of osteoclastogenesis by bisphosphonates reduced tumor burden as well as vascular density in the BM in myeloma animal models(Croucher et al. 2003). These evidences suggest a link between angiogenesis, osteoclastogenesis and MM tumor expansion which orchestrates a network of co-operative cellular interactions. We have previously demonstrated that a cell-cell interaction between osteoclasts (OCs) and MM cells potently enhanced MM cell growth and survival as well as angiogenesis. Such angiogenic effects were almost completely abrogated by antibodies against VEGF and osteopontin (OPN) in combination, suggesting VEGF and OPN as predominant angiogenic factors produced in MM bone marrow microenvironment. Angiogenesis is a multi-step process including migration, proliferation and sprouting in response to angiogenic stimuli. The impact of these factors on these angiogenic processes as well as on osteoclastogenesis by vascular cells is largely unknown. We therefore investigated roles for OPN and VEGF in combination in different steps in angiogenesis and in induction of osteoclastogenic activity by vascular endothelial cells. Conditioned media (CM) from co-cultures of peripheral blood mononuclear cell-derived OCs and RPMI8226 cells at 10 % enhanced survival of HUVEC under factor-deprived conditions 3-fold compared to control culture media, and transmigration of HUVEC 1.5-fold. Antibodies against VEGF and OPN each alone partially and both in combination mostly abrogated these effects of the CM. Consistently, VEGF and OPN co-operatively promoted survival and transmigration of HUVEC as well as angiogenesis in vitro. In order to further clarify a role for VEGF and OPN in a link between angiogenesis and osteoclastogenesis, we next examined osteoclastogenic activity produced by HUVEC upon VEGF and OPN stimulation using rabbit bone cell cultures on dentine slices. CM were collected from HUVEC cultured on either OPN- or type 1 collagen-coated wells in the presence or absence of VEGF. CM from OPN-coated wells with VEGF enhanced OC formation most potently(2.8-fold) compared to control culture media, while CM from OPN-coated wells without VEGF, type 1 collagen-coated wells with VEGF and type 1 collagen-coated wells without VEGF enhanced 2-, 1.8- and 1.2-fold, respectively, indicating co-operative actions of VEGF and OPN. Furthermore, such VEGF and OPN treatment was found to induce osteoclastogenic IL-8 by HUVEC most apparently among 125 different cytokines determined by cytokine antibody arrays. These results suggest that VEGF and OPN derived from MM cells and OCs cause angiogenesis as well as osteoclastogenesis at least in part through the enhancement of migration and survival and the induction of osteoclastogenic activity of vascular endothelial cells, thereby mutually stimulating angiogenesis and osteoclastogenesis. Therefore, VEGF and OPN may be a therapeutic target against MM bone marrow environment to ameliorate bone destruction along with MM expansion.

Differential Regulation of bFGF- and VEGF-Induced Angiogenesis in Mice Lacking Junctional Adhesion Molecule-A.

Angiogenesis, the formation of new blood vessels from preexisting vessels, is a complex process involving vascular endothelial cell activation, proliferation, migration, and tube formation. Several growth factors have been shown to induce this process. Among them vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are most widely studied. These two growth factors are believed to induce angiogenesis through distinct signaling pathways. Recently, we have shown that signaling through junctional adhesion molecule-A (JAM-A) is required for bFGF-induced angiogenesis. Here, we present evidence that JAM-A differentially regulates both bFGF-induced and VEGF-induced angiogenic responses. Using human umbilical cord vein endothelial cells (HUVEC) we found that bFGF-induced but not VEGF-induced migration was inhibited by function blocking anti-JAM-A antibody. We also found that mutant JAM-A blocked bFGF-induced but not VEGF-induced MAP kinase activation. To conclusively determine the role of JAM-A in angiogenic signaling, we generated JAM-A null mice. Endothelial cells isolated from JAM-A null mice failed to migrate in response to bFGF stimulation, but showed enhanced cell migration in response to VEGF. Further, using aortic ring and Matrigel® plug assays, we found that bFGF failed to induce blood vessel formation in JAM-A null mice. Interestingly, an increase in the number of blood vessels was observed in response to VEGF in these mice suggesting that in the absence of JAM-A bFGF-induced angiogenic pathway is impaired, but VEGF-induced pathway is upregulated to compensate. We next determined the effect of increased angiogenesis on tumor growth. We found that B16F0 melanoma growth was enhanced at least by two-fold in JAM-A null mice compared to wild-type mice. To understand the mechanism of hypersensitivity of JAM-A null mice to VEGF, we determined the expression of VEGF receptors on endothelial cells. We found that surface expression of VEGFR-2 is upregulated in endothelial cells isolated from JAM-A null mice. These results suggest that signaling through JAM-A is essential for the bFGF-induced pathway and when this pathway is impaired, the VEGF-induced pathway is upregulated which leads to enhanced angiogenesis and tumor growth.