Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.     

Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients.     

Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha.

Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H₂O₂ production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H₂O₂ levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. phenotype; reactive oxygen species; inflammation; shear stress     

Synthesis and cardiovascular protective effects of quercetin 7‐O‐sialic acid

Oxidative stress and inflammation play important roles in the pathogenesis of cardiovascular disease (CVD). Oxidative stress‐induced desialylation is considered to be a primary step in atherogenic modification, and therefore, the attenuation of oxidative stress and/or inflammatory reactions may ameliorate CVD. In this study, quercetin 7‐O‐sialic acid (QA) was synthesized aiming to put together the cardiovascular protective effect of quercetin and the recently reported anti‐oxidant and anti‐atherosclerosis functions of N‐acetylneuraminic acid. The biological efficacy of QA was evaluated in vitro in various cellular models. The results demonstrated that 50 μM QA could effectively protect human umbilical vein endothelial cells (HUVEC, EA.hy926) against hydrogen peroxide‐ or oxidized low‐density lipoprotein‐induced oxidative damage by reducing the production of reactive oxygen species. QA attenuated hydrogen peroxide‐induced desialylation of HUVEC and lipoproteins. QA decreased lipopolysaccharide‐induced secretion of tumour necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1), and it significantly reduced the expression of intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, TNF‐α and MCP‐1. Furthermore, QA effectively promoted cholesterol efflux from Raw 264.7 macrophages to apolipoprotein A‐1 and high‐density lipoprotein by up‐regulating ATP‐binding cassette transporter A1 and G1, respectively. Results indicated that the novel compound QA exhibited a better capacity than quercetin for anti‐oxidation, anti‐inflammation, cholesterol efflux promotion and biomolecule protection against desialylation and therefore could be a candidate compound for the prevention or treatment of CVD.     

Contrasting effects of interferon γ and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor α

Tumour necrosis factor α (TNF-α) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelialcells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-α-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colon-stimulating factor (GM-CSF) from TNF-α-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-α-induced responses to those observed with endothelial cells of foetal origin. Additionaly, we report here that TNF-α and interferon γ (IFN-γ) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-α-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-γ plus TNF-α was markedly decreased when compared to the response by TNF-α alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NF-kappaB, and p38

Bile acids are synthesized in the liver, stored in gallbladder, and secreted into the intestine to aid in the absorption of lipid-soluble nutrients. In addition, bile acids also actively participate in regulation of gene expression through their ability to act as ligands for the nuclear receptor farnesoid X receptor or by activating kinase signaling pathways. Under cholestatic conditions, elevated levels of bile acids in the liver induce hepatic inflammation, and because bile acid levels are also elevated in the circulation, they might also induce vascular inflammation. To test this hypothesis, primary human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells were treated with bile acids, and the expression of ICAM-1, VCAM-1, and E-selectin were monitored. The three major bile acids found in the circulation, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, all strongly induced both the mRNA and protein expression of ICAM-1 and VCAM-1. To delineate the mechanism, the experiments were conducted in the presence of various kinase inhibitors. The results demonstrate that the bile acid-mediated induction of adhesion molecule expression occurs by stimulation of NF-kappaB and p38 MAPK signaling pathways through the elevation in reactive oxygen species. The bile acid-induced cell surface expression of ICAM-1 and VCAM-1 was sufficient to result in the increased adhesion of THP-1 monocytes to the HUVEC, suggesting that elevated levels of bile acids in the circulation may cause endothelium dysfunction and contribute to the initiation of early events associated with vascular lesion formation.     

Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation

Changes in endothelial cell (EC) phenotype are central to thefunction of endothelium in inflammation. Although these events mainlyoccur in the microvasculature, previous studies have predominantly usedlarge-vessel EC. Using enzyme-linked immunosorbent and flow cytometricassays, we compared the responses of human umbilical vein endothelialcells (HUVEC) and dermal microvascular endothelial cells (DMEC) to theactivation of protein kinase C (PKC). Stimulation with phorbol12,13-dibutyrate and more selective PKC agonists, including12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), inducedmorphological changes and proliferation in both EC types. PKCactivation induced a marked increase in Thy-1 expression on DMEC andonly a moderate rise on HUVEC. Furthermore, heterogeneity in theinduction of the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin between thetwo EC types following activation of PKC was demonstrated. Inparticular, E-selectin and VCAM-1 were significantly upregulated onHUVEC but not DMEC. The data indicate that the PKC pathway is unlikelyto be important for E-selectin and VCAM-1 expression in themicrovasculature but are consistent with a role for PKC inangiogenesis. This diversity in signaling in response to PKC activationmay depend on differential utilization of PKC isozymes and mayfacilitate specialized endothelial responses.

     

Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.     

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.     

Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-gamma-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against beta(1) and beta(2) integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-gamma, TNF-alpha, and IFN-gamma plus TNF-alpha-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4(-), but not CD4(+), T cell TEM, suggesting a role for tTG in recruitment of CD8(+) T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8(+) T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.     

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.     

Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2',5'-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a G(i) signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the G(s)/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the G(s)/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

Insulin potentiates cytokine-induced VCAM-1 expression in human endothelial cells

Hyperinsulinemia is an independent risk factor for cardiovascular events and may contribute to cardiovascular disease. Low-grade chronic inflammation has been implicated in the pathogenesis of atherosclerosis. We aimed at determining the impact of pathophysiologically high insulin concentrations on cytokine-induced endothelial activation in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with insulin (0-24 h)+/-tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS). At pathophysiological/pharmacological concentrations (10(-9)-10(-7) mol/L), insulin selectively induced VCAM-1 expression and potentiated the effects of TNF-alpha andLPS, effects reverted by the proteasome inhibitor lactacystin. Compared with TNF-alpha alone, insulin+TNF-alpha doubled U937 cell adhesion. Insulin markedly increased TNF-alpha-induced NF-kappaB activation and induced phosphorylated IkappaB-alpha accumulation. Therefore, hyperinsulinemia enhances cytokine-induced VCAM-1 expression in endothelial cells, thus potentially contributing to detrimental effects of other inflammatory stimuli on atherogenesis.     

Proinflammatory response of human endothelial cells to Brucella infection

Although vascular pathologies such as vasculitis, endocarditis and mycotic aneurysms have been described in brucellosis patients, the interaction of Brucella with the endothelium has not been characterized. In this study we show that Brucella abortus and Brucella suis can infect and replicate in primary human umbilical vein endothelial cells (HUVEC) and in the microvascular endothelial cell line HMEC-1. Infection led to an increased production of IL-8, MCP-1 and IL-6 in HUVEC and HMEC-1 cells, and an increased expression of adhesion molecules (CD54 in both cells, CD106 and CD62E in HUVEC). Experiments with purified antigens from the bacterial outer membrane revealed that lipoproteins (Omp19) but not lipopolysaccharide mediate these proinflammatory responses. Infection of polarized HMEC-1 cells resulted in an increased capacity of these cells to promote the transmigration of neutrophils from the apical to the basolateral side of the monolayer, and the same phenomenon was observed when the cells were stimulated with live bacteria from the basolateral side. Overall, these results suggest that Brucella spp. can infect and survive within endothelial cells, and can induce a proinflammatory response that might be involved in the vascular manifestations of brucellosis.