Isoflavone genistein protects human vascular endothelial cells against tumor necrosis factor-α-induced apoptosis through the p38β mitogen-activated protein kinase

Isoflavone genistein may have beneficial effects on vascular function, but the mechanism is unclear. Here, we investigated whether genistein protects vascular endothelial cells against apoptosis induced by tumor necrosis factor-α. We show that genistein significantly inhibited TNF-α-induced apoptosis in human aortic endothelial cells as determined by caspase-3 activation, 7-amino actinomycin D staining, in situ apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of Bcl-2 protein and its promoter activity. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptors had no effect on the cytoprotective effect of genistein. However, inhibition of p38 mitogen-activated protein kinase (p38) completely abolished this genistein effect. Accordingly, stimulation of HAECs with genistein resulted in rapid activation of p38β, but not p38α. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Preservation of the functional integrity of the endothelial monolayer may represent an important mechanism by which genistein exerts its vasculoprotective effect.     

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological properties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistein, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis.     

Positive and negative regulation of insulin action by genistein in the endothelium

Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.     

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 microM Hcy alone or in combination with 2.5 microM genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting. Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation; and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect on apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action.     

Kruppel-like factor 4 regulates endothelial inflammation

The vascular endothelium plays a critical role in vascular homeostasis. Inflammatory cytokines and non-laminar blood flow induce endothelial dysfunction and confer a pro-adhesive and pro-thrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factor 4 expression has been documented in endothelial cells, but a function has not been described. In this communication we describe the expression in vitro and in vivo of Kruppel-like factor 4 in human and mouse endothelial cells. Furthermore, we demonstrate that endothelial Kruppel-like factor 4 is induced by pro-inflammatory stimuli and shear stress. Overexpression of Kruppel-like factor 4 induces expression of multiple anti-inflammatory and anti-thrombotic factors including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of Kruppellike factor 4 leads to enhancement of tumor necrosis factor alpha-induced vascular cell adhesion molecule-1 and tissue factor expression. The functional importance of Kruppel-like factor 4 is verified by demonstrating that Kruppel-like factor 4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. Kruppel-like factor 4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. These data implicate Kruppel-like factor 4 as a novel regulator of endothelial activation in response to pro-inflammatory stimuli.     

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFP-paxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (<7.5 µm), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. mechanotransduction; endothelial cell; paxillin; viscoelastic model     

Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-alpha and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-alpha-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.     

Loss of Syndecan-1 Induces a Pro-inflammatory Phenotype in Endothelial Cells with a Dysregulated Response to Atheroprotective Flow

Fluid shear stresses are potent regulators of vascular homeostasis and powerful determinants of vascular disease progression. The glycocalyx is a layer of glycoaminoglycans, proteoglycans, and glycoproteins that lines the luminal surface of arteries. The glycocalyx interacts directly with hemodynamic forces from blood flow and, consequently, is a prime candidate for the mechanosensing of fluidic shear stresses. Here, we investigated the role of the glycocalyx component syndecan-1 (sdc-1) in controlling the shear stress-induced signaling and flow-mediated phenotypic modulation in endothelial cells. We found that knock-out of sdc-1 abolished several key early signaling events of endothelial cells in response to shear stress including the phosphorylation of Akt, the formation of a spatial gradient in paxillin phosphorylation, and the activation of RhoA. After exposure to atheroprotective flow, we found that sdc-1 knock-out endothelial cells had a phenotypic shift to an inflammatory/pro-atherosclerotic phenotype in contrast to the atheroprotective phenotype of wild type cells. Consistent with these findings, we found increased leukocyte adhesion to sdc-1 knock-out endothelial cells in vitro that was reduced by re-expression of sdc-1. In vivo, we found increased leukocyte recruitment and vascular permeability/inflammation in sdc-1 knock-out mice. Taken together, our studies support a key role for sdc-1 in endothelial mechanosensing and regulation of endothelial phenotype.     

Effects of protein and gene transfer of the angiopoietin-1 fibrinogen-like receptor-binding domain on endothelial and vessel organization

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e. dissociated from its state as an oligomer. A truncated monomeric Ang1 variant (i.e. DeltaAng1) containing the isolated fibrinogen-like receptor-binding domain of Ang1 was created and recombinantly produced in insect cells. DeltaAng1 ligated the Tie2 receptor without triggering its phosphorylation. Moreover, monomeric DeltaAng1 was observed to bind alpha(5)beta(1) integrin with similar affinity compared with Tie2. Unexpectedly, in vitro treatment of endothelial cells with DeltaAng1 showed some of the known effects of full-length Ang1, including inhibition of basal endothelial cell permeability and stimulation of cell adhesion as well as activation of MAPKs. Local treatment of the microvasculature of the developing chicken chorioallantoic membrane with the DeltaAng1 protein led to profound reduction of the mean vascular length density, thinning of vessels, and reduction of the number of vessel branching points. Similar effects were observed in side-by-side experiments with the recombinant full-length Ang1 protein. These effects of simplification of the vessel branching pattern were confirmed through local gene transfer with lentiviral particles encoding DeltaAng1 or full-length Ang1. Together, our findings suggest a potential use for exogenous Ang1 in reducing rather than increasing vascular density. Furthermore, we show that the isolated receptor-binding domain of Ang1 is capable of mediating some effects of full-length Ang1 independently of Tie2 phosphorylation, possibly through integrin ligation.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Reduction of ENU-induced transversion mutations by the isoflavone genistein in Escherichia coli

In studies of mutagenesis induced by the carcinogen N-ethyl-N-nitrosourea (ENU) in the bacterium Escherichia coli FX-11, it was observed that G:C to A:T transitions did not require the inducible umuDC gene products, while a portion of the A:T to G:C transitions and all transversion mutations were dependent on a functional umuC gene. This observation suggested that the different base substitutions may result from differential processing of specific DNA adducts produced by ENU. To further understand these processes, we have investigated the effect of the soybean isoflavone genistein on the production of ENU-induced mutations. This compound, in particular, has been shown to exhibit numerous effects including the inhibition of the growth or proliferation of a variety of cancers, inhibition of angiogenesis, inhibition of tyrosine protein kinases and anti-oxidant properties. In our experiments, tyrosine defective (TyrA(-)) E. coli were exposed to ENU and a portion of the ENU-treated cells were exposed to genistein. The results showed a three-fold reduction in the overall mutation frequency when cells were treated with genistein subsequent to ENU-exposure and this anti-mutagenic effect was dependent on the dose of genistein employed. However, only certain types of base substitution mutagenesis were affected. In particular, transversion mutations were reduced an average of about 8.5-fold, while transitions were not greatly affected. In addition, UV-mutagenesis was reduced about three-fold and induction of the SOS response (as monitored with a sulA-lacZ fusion) was decreased. These results suggest that genistein may interfere with expression of the SOS response, including the UmuC-mediated lesion bypass mechanism that is necessary for UV-mutagenesis and the generation of transversions by ENU in E. coli.     

Actinomyosin Contraction,Phosphorylation of VE-Cadherin,and Actin Remodeling Enable Melanoma-Induced Endothelial Cell-Cell Junction Disassembly

During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation.     

PPARalpha activators upregulate eNOS activity and inhibit cytokine-induced NF-kappaB activation through AMP-activated protein kinase activation

Endothelium-derived NO is an important mediator of vascular protection and adhesion molecule expression on the endothelial cell surface is critical for leukocyte recruitment to atherosclerotic lesions. We hypothesized that AMP-activated protein kinase (AMPK) activity is a down-stream mediator of the beneficial effects of PPARalpha activators on vascular endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with fenofibrate or WY14643 resulted in transient activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase. Fenofibrate caused phosphorylation of Akt and eNOS, leading to increased production of NO, and also caused inhibition of cytokine-induced NF-kappaB activation, leading to suppression of expression of adhesion molecule genes. Significant decreases in eNOS activity and NO production in response to fenofibrate were observed in cells treated with AMPK siRNA or with AraA, a pharmacological inhibitor of AMPK. The attenuation of fenofibrate-induced inhibition of NF-kappaB activation was observed in mouse endothelial (SVEC4) cells treated with AMPK siRNA or with AraA. We demonstrated that TNFalpha stimulates IkappaB-alpha phosphorylation through induction of IKK activity, and that fenofibrate inhibits IKK activity and TNFalpha-induced IkappaB-alpha phosphorylation. Our findings suggest that the beneficial effects of PPARalpha activators on endothelial cells such as inhibition of diabetic microangiopathy might be attributed to the induction of AMPK activation beyond its lipid-lowering actions.     

Individual and combined effects of shear stress magnitude and spatial gradient on endothelial cell gene expression

The apparent tendency of atherosclerotic lesions to form in complex blood flow environments has led to many theories regarding the importance of hemodynamic forces in endothelium-mediated atherosusceptibility. The effects of shear stress magnitude and spatial shear stress gradient on endothelial cell gene expression in vitro were examined in this study. Converging-width flow channels were designed to impose physiological ranges of shear stress gradient and magnitude on porcine aortic endothelial cells, and real-time quantitative PCR was performed to evaluate their expression of five genes of interest. Although vascular cell adhesion molecule-1 expression was insensitive to either variable, each of the remaining genes exhibited a unique dependence on shear stress magnitude and gradient. Endothelial nitric oxide synthase showed a strong positive dependence on magnitude but was insensitive to gradient. The expression of c-jun was weakly correlated with magnitude and gradient, without an interaction effect. Monocyte chemoattractant protein-1 expression varied inversely with gradient and also depended on the interaction of gradient with magnitude. Intercellular adhesion molecule-1 expression also exhibited an interaction effect, and increased with shear magnitude. These results support the notion that vascular endothelial cells are able to sense shear gradient and magnitude independently.     

Endothelial cells in culture: An experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [³H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor- and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selectin And vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also rn rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.Abbreviations AGEs advanced glycated end-products - ICAM intracellular adhesion molecule - IL-1 interleukin-1 - IFN- interferon- - MECIF monocyte-derived endothelial cell inhibitory factor - NO nitric oxide - PECAM-1 platelet-endothelial cell adhesion molecule-1 - RAGE receptor for advanced glycated end-products - RBCs red blood cells - TNF- tumor necrosis factor- - VCAM vascular adhesion molecule