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 mu M Hcy alone or in combination with 2.5 mu M 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 oil 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

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

Background Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. Aim of the study To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells ( HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). Methods HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 muM) or by pharmacological intervention ( 25 muM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. Results Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. Conclusions Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Genistein affects the expression of genes involved in blood pressure regulation and angiogenesis in primary human endothelial cells

Background: Several lines of evidence suggest that the dietary isoflavone genistein (Gen) has beneficial effects with regard to cardiovascular disease and in particular on aspects related to blood pressure and angiogenesis. The biological action of Gen may be, at Least in part, attributed to its ability to affect cell signalling and response. However, so far, most of the molecular mechanisms underlying the activity of Gen in the endothelium are unknown. Methods and results: To examine the transcriptional response to 2.5 mu M Gen on primary human endothelial cells (HUVEC), we applied cDNA array technology both under baseline condition and after treatment with the pro-atherogenic stimulus, copper-oxidized LDL. The alteration of the expression patterns of individual transcripts was substantiated using either RT-PCR or Northern blotting. Gen significantly affected the expression of genes encoding for proteins centrally involved in the vascular tone such as endothelin-converting enzyme-1, endothetin-2, estrogen related receptor a and atria[ natriuretic peptide receptor A precursor. Furthermore, Gen countered the effect of oxLDL on mRNA levels encoding for vascular endothelial growth factor receptor 165, types 1 and 2. Conclusions: Our data indicate that physiologically achievable levels of Gen change the expression of mRNA encoding for proteins involved in the control of blood pressure under baseline conditions and reduce the angiogenic response to oxLDL in the endothelium. (c) 2005 Elsevier B.V. All rights reserved.

Targeting stem cell niches and trafficking for cardiovascular therapy.

Regenerative cardiovascular medicine is the frontline of 21st-century health care. Cell therapy trials using bone marrow progenitor cells documented that the approach is feasible, safe and potentially beneficial in patients with ischemic disease. However, cardiovascular prevention and rehabilitation strategies should aim to conserve the pristine healing capacity of a healthy organism as well as reactivate it under disease conditions. This requires an increased understanding of stem cell microenvironment and trafficking mechanisms. Engagement and disengagement of stem cells of the osteoblastic niche is a dynamic process, finely tuned to allow low amounts of cells move out of the bone marrow and into the circulation on a regular basis. The balance is altered under stress situations, like tissue injury or ischemia, leading to remarkably increased cell egression. Individual populations of circulating progenitor cells could give rise to mature tissue cells (e.g. endothelial cells or cardiomyocytes), while the majority may differentiate to leukocytes, affecting the environment of homing sites in a paracrine way, e.g. promoting endothelial survival, proliferation and function, as well as attenuating or enhancing inflammation. This review focuses on the dynamics of the stem cell niche in healthy and disease conditions and on therapeutic means to direct stem cell/progenitor cell mobilization and recruitment into improved tissue repair.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

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.)

Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days. and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB(4). TXB(2), IL-1 beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung Vitamins C, E and apocynin reduced the levels of LTB(4) in BAL-cultured cells of the FA and FA/OVA groups, but Increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1) In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Comparison of three vascular endothelial markers in the evaluation of microvessel density in breast cancer

Purpose: To evaluate the microvessel density by comparing the performance of anti-factor VIII-related antigen, anti-CD31 and, anti-CD34 monoclonal antibodies in breast cancer. Methods: Twenty-three postmenopausal women diagnosed with Stage II breast cancer submitted to definitive surgical treatment were evaluated. The monoclonal antibodies used were anti-factor VIII, anti-CD31 and anti-CD34. Microvessels were counted in the areas of highest microvessel density in ten random fields (200 x). The data were analyzed using the Kruskal-Wallis nonparametric test (p < 0.05). Results: Mean microvessel densities with anti-factor VIII, anti-CD31 and anti-CD34 were 4.16 +/- 0.38, 4.09 +/- 0.23 and 6.59 +/- 0.42, respectively. Microvessel density as assessed by anti-CD34 was significantly greater than that detected by anti-CD31 or anti-factor VIII (p < 0.0001). There was no statistically significant difference between anti-CD31 and anti-factor VIII (p = 0.4889). Conclusion: The density of stained microvessels was greater and staining was more intense with anti-CD34 compared to anti-CD31 and anti-factor VII-related antigen.

Oncostatin M is a novel glucocorticoid-dependent neuroinflammatory factor that enhances oligodendrocyte precursor cell activity in demyelinated sites

The innate immune reaction to tissue injury is a natural process, which can have detrimental effects in the absence of negative feedbacks by glucocorticoids (GCs). Although acute lipopolysaccharide (LPS) challenge is relatively harmless to the brain parenchyma of adult animals, the endotoxin is highly neurotoxic in animals that are treated with the GC receptor antagonist RU486. This study investigated the role of cytokines of the gp130-related family in these effects, because they are essential components of the inflammatory process that provide survival signals to neurons. Intracerebral LPS injection stimulated expression of several members of this family of cytokines, but oncostatin M (Osm) was the unique ligand to be completely inhibited by the RU486 treatment. OSM receptor (Osmr) is expressed mainly in astrocytes and endothelial cells following LPS administration and GCs are directly responsible for its transcriptional activation in the presence of the endotoxin. In a mouse model of demyelination, exogenous OSM significantly modulated the expression of genes involved in the mobilization of oligodendrocyte precursor cells (OPCs), differentiation of oligodendrocyte, and production of myelin. In conclusion, the activation of OSM signaling is a mechanism activated by TLR4 in the presence of negative feedback by GCs on the innate immune system of the brain. OSM absence is associated with detrimental effects of LPS, whereas exogenous OSM favors repair response to demyelinated regions. (C) 2010 Elsevier Inc. All rights reserved.

A pseudosymmetric cell adhesion regulatory domain in the β7 tail of the integrin α4β7 that interacts with focal adhesion kinase and src

The β7 integrins α4β7 and Eβ7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The α4β7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of α4β7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735-740 of the cytoplasmic tail of the β7 subunit inhibit the adhesion of T cells to β7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of α4β7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr⁷³⁵Phe and Tyr⁷⁴⁰Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of 4β7. The quasi-palindromic sequence YDRREY within the β7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of β7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease.

Endothelialisation and cell retention on gelation-chitosan coated electrospun polyurethane, poly (lactide-co-glycolide) and collagen-coated pericardium

Arterial bypass and heart valve replacements are two of the most common surgical treatments in cardiovascular surgery today. Currently, artificial materials are used as substitute for these cardiac tissues. However, these foreign materials do not have the ability to grow, repair or remodel and are thrombogenic, leading to stenosis. With the aid of tissue engineering, it is possible to develop functional identical copies of healthy heart valves and arteries, which are biocompatible. Although much effort has been made into this area, there are still inconsistencies with respect to
endothelialisation and cell retention on synthetic biological grafts. These variations may be attributed to differences in factors such as cell seeding density, incubation periods and effects of shear stress. In this study, we have compared the endothelialisation and cell retention between gelain chitosan-coated electrospun polyurethane (PU), poly (lactide co-glycolide) (PGA/PLA) and collagen-coated pericardium. Endothelial cells adhered to all of the materials as early as 1–day post seeding. After 7-day of seeding, the coverage on PU was almost 45% and that on PGA/PLA was about 25% and the least was on collagen-coated pericardium of approximately 15%. It was observed that the PU showed superior cell coverage and cell retention in comparison to the PGA/PLA and collagen-coated pericardium.

Polyethyleneterephthalate provides superior retention of endothelial cells during shear stress compared to polytetrafluoroethylene and pericardium

Background

Polyethyleneterephthalate (PET) and polytetrafluoroethylene (PTFE) are polymers successfully used as large diameter arterial grafts for peripheral vascular surgery. However, these prosthetic grafts are rarely used for coronary bypass surgery because of their low patency rates. Endothelialisation of the lumenal surface of these materials may improve their patency. This study aimed to compare the endothelialisation of PET, PTFE and pericardium by examining their seeding efficiency over time and the effect of various shear stresses on retention of endothelial cells.

Methods


Ovine endothelial cells at 4 × 105 cells/cm2 were seeded onto PET, PTFE and pericardium, and cultured for 1–168 hours. Cell coverage was determined via en face immunocytochemistry and cell retention was quantified after being subjected to shear stresses ranging from 0.018 to 0.037 N/m2 for 15, 30 and 60 minutes.

Results

Endothelial cells adhered to all of the materials one hour post-seeding. PET exhibited better cell retention rate, ranging from 66.9 ± 5.6% at 0.018 N/m2 for 15 min to 44.7 ± 1.9% at 0.037 N/m2 for 60 minutes, when compared to PTFE and pericardium (p < 0.0001, three-way ANOVA).

Conclusion

PET shows superior retention of endothelial cells during shear stress compare to PTFE and pericardium.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:  The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.

Methods:  Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay.

Results:  Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata.

Conclusions:  An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.