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 proper-ties. 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 genistem, 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. (C) 2004 Elsevier B.V All rights reserved.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis. (c) 2006 Elsevier Inc. All rights reserved.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

A role for Notch signaling in human corneal epithelial cell differentiation and proliferation

PURPOSE. To identify the role of Notch signaling in the human corneal epithelium. METHODS. Localization of Notch1, Notch2, Delta1, and Jagged1 in the human corneal epithelium was observed with the use of indirect immunofluorescence microscopy. Gene and protein expression of Notch receptors and ligands in human corneal epithelial cells was determined by RT-PCR and Western blot analysis, respectively. The effects of Notch inhibition (by {gamma}-secretase inhibition) and activation (by recombinant Jagged1) on epithelial cell proliferation (Ki67) and differentiation (CK3) were analyzed after Western blotting and immunocytochemistry. RESULTS. Immunofluorescent labeling localized Notch1 and Notch2 to suprabasal epithelial cell layers, whereas Delta1 and Jagged1 were observed throughout the corneal epithelium. Notch1, Notch2, Delta1, and Jagged1 genes and proteins were expressed in human corneal epithelial cells. {gamma}-Secretase inhibition resulted in decreased Notch1 and Notch2 expression, with an accompanying decrease in Ki67 and increased CK3 expression. The activation of Notch by Jagged1 resulted in the upregulation of active forms of Notch1 and 2 proteins (P < 0.05), with a concurrent increase in Ki67 (P < 0.05) and a decrease in CK3 (P < 0.05) expression. Interestingly, {gamma}-secretase inhibition in a three-dimensional, stratified corneal epithelium equivalent had no effect on Ki67 or CK3 expression. In contrast, Jagged1 activation resulted in decreased CK3 expression (P < 0.05), though neither Notch activation nor inhibition affected cell proliferation in the 3D tissue equivalent. CONCLUSIONS. Notch family members and ligands are expressed in the human corneal epithelium and appear to play pivotal roles in corneal epithelial cell differentiation.

Nox2 regulates endothelial cell cycle arrest and apoptosis via p21 (cip1) and p53

Endothelial cells (EC) express constitutively two major isofonns (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 similar to 1:13), but was upregulated 24 It after starvation and increased to 8 +/- 3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28 +/- 0.18-fold increase in O-2(-); production, a dramatic induction of p21(cip1) and p53, cell cycle arrest, and the onset of apoptosis (all p < 0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8 +/- 0.5fold increase in Nox4 mRNA expression after 36 h of starvation (p < 0.01), neither production nor the p21(cip1) or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O-2(-), through the modulation of p21(cip1) and p53 expression, participates in endothelial cell cycle regulation and apoptosis. (c) 2007 Elsevier Inc. All rights reserved.

Photochemical cross-linking of plastically compressed collagen gel produces an optimal scaffold for corneal tissue engineering

The experiments were designed to use photochemically cross-linked plastically compressed collagen (PCPCC) gel to support corneal epithelial cells. A plastically compressed collagen (PCC) scaffold was photo cross-linked by UVA in the presence of riboflavin to form a biomaterial with optimal mechanical properties. The breaking force, rheology, surgical suture strength, transparency, ultrastructure, and cell-based biocompatibility were compared between PCPCC and PCC gels. The breaking force increased proportionally with an increased concentration of riboflavin. The stress required to reach breaking point of the PCPCC scaffolds was over two times higher compared to the stress necessary to break PCC scaffolds in the presence of 0.1% riboflavin. Rheology results indicated that the structural properties of PCC remain unaltered after UVA cross-linking. The PCC gels were more easily broken than PCPCC gels when sutured on to bovine corneas. The optical density values of PCPCC and PCC showed no significant differences (p > 0.05). SEM analyses showed that the collagen fibres within the PCPCC gels were similar in morphology to PCC gels. No difference in cell-based biocompatibility was seen between the PCPCC and PCC scaffolds in terms of their ability to support the ex vivo expansion of corneal epithelial cells or their subsequent differentiation evidenced by similar levels of cytokeratin 14. In conclusion, PCPCC scaffold is an optimal biomaterial for use in therapeutic tissue engineering of the cornea.

Influence of substrate on corneal epithelial cell viability within ocular surface models

Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modeling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.