Native LDL-Cholesterol Mediated Monocyte Adhesion Molecule Overexpression is Blocked by Simvastatin

Aim of the study This study sought to evaluate the effect of nLDL concentrations on monocyte adhesion molecule expression in hypercholesterolemic patients with stable corollary artery disease (CAD) and to determine whether lipid-lowering therapy with simvastatin Would change this effect. Methods Blood samples from patients with hypercholesterolemia (mean LDL 152 mg/dL) and CAD (HC, n = 23) were collected before and after a 12-week treatment with 40 mg of simvastatin. Healthy individuals (mean LDL 111 mg/dL) were used as controls (CT, n = 15). Isolated nLDL, at a fixed concentration of 100 mg/dL, was added to monocyte suspensions obtained before and after the simvastatin treatment. Monocyte activation was determined by changes in cellular adhesion molecule expression. Results In response to nLDL, CD11b and CD14 adhesion molecule expression was higher in HC patients than in CT patients before treatment (174.2+/-8.4 vs 102.2+/-6.3, P<0.03 and 140.4+/-5.0 vs 90.4+/-6.7, P<0.04). After simvastatin treatment, CD11b expression decreased to 116.9+/-12.5 (P< 0.03) and CD14 expression to 107.5+/-6.2 (P<0.04). Alternatively, L-selectin expression was lower in HC patients than in CT patients before therapy (46.0+/-3.5 vs 62.1+/-5.5, P<0.04), and it increased markedly after lipid reduction to 58.7+/-5.0 (P<0.04 vs baseline). After simvastatin treatment, LDL was reduced to mean 101.5 mg/dL. Conclusions These data demonstrate that monocytes from HC patients are more prone to marked nLDL-mediated changes of adhesion molecule expression than monocytes from controls. Simvastatin is capable of inhibiting such nLDL effects. This proinflammatory response to nLDL may have a role in the early onset of atherosclerosis.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Connexin37 protects against atherosclerosis by regulating monocyte adhesion

A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 μg/ml for 0-60 min at 37°C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 μg/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.

Effects of oral vitamin C on monocyte:Endothelial cell adhesion in healthy subjects

Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean = 67μM, n = 20, mean = 32μM, n = 20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P < 0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation. © 2002 Elsevier Science (USA). All rights reserved.

α-Tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (α-tocopherol). We have tested the hypothesis that α-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 μM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received α-tocopherol supplements (400 IU RRR-α-tocopherol /day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM- 1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-κB in isolated resting monocytes, nor any effect of α-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and α-tocopherol concentration. In conclusion, α-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration. © W. S. Maney & Son Ltd.

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.

Dietary fats induce human monocyte activation in vitro

In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased, due partly to de novo expression or activation of monocyte adhesion molecules. Although there is increasing evidence that CMRs (chylomicron remnants) are strongly atherogenic, the outcomes of interactions between blood monocytes and circulating CMRs are not known. Here, we have studied the effects of CRLPs (CMR-like particles) on THP-1 human monocyte oxidative burst. The particles induced a significant increase in reactive oxygen species within 1 h, which persisted for 24 h. We suggest that monocyte–CMR interactions may be important in early atherosclerosis when many activated monocytes are found in susceptible areas of the artery wall.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR

Aims We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. Methods and results Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-beta-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. Conclusion We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Molecular role of Cx37 in advanced atherosclerosis: a micro-array study.

Recently, we showed that connexin37 (Cx37) protects against early atherosclerotic lesion development by regulating monocyte adhesion. The expression of this gap junction protein is altered in mouse and human atherosclerotic lesions; it is increased in macrophages newly recruited to the lesions and disappears from the endothelium of advanced plaques. To obtain more insight into the molecular role of Cx37 in advanced atherosclerosis, we used micro-array analysis for gene expression profiling in aortas of ApoE(-/-) and Cx37(-/-)ApoE(-/-) mice before and after 18 weeks of cholesterol-rich diet. Out of >15,000 genes, 106 genes were significantly differentially expressed in young mice before diet (P-value of <0.05, fold change of >0.7 or <-0.7, and intensity value >2.2 times background). Ingenuity pathway analysis (IPA) revealed differences in genes involved in cell-to-cell signaling and interaction, cellular compromise and nutritional disease. In addition, we identified 100 genes that were significantly perturbed after the cholesterol-rich diet. Similar to the analysis on 10-week-old mice, IPA revealed differences in genes involved in cell-to-cell signaling and interaction as well as to immuno-inflammatory disease. Furthermore, we found important changes in genes involved in vascular calcification and matrix degradation, some of which were confirmed at protein level by (immuno-)histochemistry. In conclusion, we suggest that Cx37 deficiency alters the global differential gene expression profiles in young mice towards a pro-inflammatory phenotype, which are then further influenced in advanced atherosclerosis. The results provide new insights into the significance of Cx37 in plaque calcification.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.