HLA-DRB1*04 gene polymorphisms and expressions profiles of interleukin-18 and interleukin-18 binding protein following in vitro stimulation in human peripheral blood mononuclear cells of healthy individuals and patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic arthritic condition that can lead to deformities and disabilities. Interleukin-18 (IL-18) is a proinflammatory cytokine known to play a role in the acute and chronic inflammatory phases of RA. IL-18 binding protein is the natural antagonist of IL-18 protein. We aim to identify the effect of HLA-DRB1*04 gene polymorphisms on IL-18 and IL-18BP gene expressions profiles as well as the time-course profiles following in vitro stimulation with mitogens. Peripheral blood mononuclear cells from 16 RA patients and 21 healthy controls were cultured for 1, 4, 8, 12, 24, 48 and 72 h following stimulation with either LPS or PHA. mRNA expression of IL-18 and IL 18BP were determined by quantitative real-time PCR using a comparative Ct (threshold cycle) method. IL-18 levels in supernatants were measured by enzyme-linked immunosorbent assay. Basal mRNA (4.5-fold) and protein levels of IL-18 were increased and IL-18BP mRNA expression was decreased (8-fold) in RA patients when compared to controls. Similarly, increased IL-18 levels were observed in active RA patients, whereas IL-18BP expression was increased in inactive patients. There was an increase in mRNA and protein levels of IL-18 in RA patients that peaked at 4 h and 8 h respectively following LPS stimulation. A similar profile was observed for IL-18BP; however, the expression level was higher in controls than RA patients. Persistent high production of IL-18 in RA is associated with disease progression and IL-18 BP seems to inhibit this activity.     

Advances in understanding molecular regulation of innate immune memory

The epigenetic and functional reprogramming of immune genes during induction of trained immunity is accompanied by the metabolic rewiring of cellular state. This memory is induced in the hematopoietic niche and propagated to daughter cells, generating epigenetically and metabolically reprogrammed innate immune cells that are greatly enhanced in their capacity to resolve inflammation. In particular, these cells show accumulation of H3K4me3 and H3K27Ac epigenetic marks on multiple immune gene promoters and associated enhancers. However, the mechanism governing how these epigenetic marks accumulate at discrete immune gene loci has been poorly understood, until now. Here, we discuss some recent advances in the regulation of trained immunity, with a particular focus on the mechanistic role of a novel class of long non-coding RNAs in the establishment of epigenetic marks on trained immune gene promoters.     

Triacylglycerol-rich lipoproteins trigger the phosphorylation of extracellular-signal regulated kinases in vascular cells

Postprandial triacylglycerol-rich lipoproteins (TRL) have been implicated in the pathophysiology of atherosclerosis, but the intracellular processes by which TRL could affect vascular function are still unknown. Incubation of TRL obtained at 2 h postprandial period with vascular smooth muscle cells (VSMC) produced a tyrosine phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) that belong to the mitogen-activated protein kinase (MAPK) family. The activation of ERK1 and ERK2 had a maximum at 15 min, returned to baseline by 60 min, and was partially depleted after incubation of cells with a MAPKK inhibitor (PD 098059). In addition, postprandial TRL did competent VSMC for DNA replication through a MAPK pathway. These effects were dependent of the lipid composition of TRL. Our observations suggest that postprandial TRL can trigger activation of the MAPK pathway and induce a mitogenic response in VSMC in a lipid-dependent fashion.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

Mechanical regulation of epigenetics in vascular biology and pathobiology

Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to haemodynamic forces, including blood flow‐induced fluid shear stress and cyclic stretch from blood pressure. These forces modulate vascular cell gene expression and function and, therefore, influence vascular physiology and pathophysiology in health and disease. Epigenetics, including DNA methylation, histone modification/chromatin remodelling and RNA‐based machinery, refers to the study of heritable changes in gene expression that occur without changes in the DNA sequence. The role of haemodynamic force‐induced epigenetic modifications in the regulation of vascular gene expression and function has recently been elucidated. This review provides an introduction to the epigenetic concepts that relate to vascular physiology and pathophysiology. Through the studies of gene expression, cell proliferation, angiogenesis, migration and pathophysiological states, we present a conceptual framework for understanding how mechanical force‐induced epigenetic modifications work to control vascular gene expression and function and, hence, the development of vascular disorders. This research contributes to our knowledge of how the mechanical environment impacts the chromatin state of ECs and VSMCs and the consequent cellular behaviours.     

Aldosterone and the vascular system

Aldosterone can act in different tissues exerting physiological and pathological effects. At the vascular level, aldosterone affects endothelial function since administration of aldosterone impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorate relaxation to acetylcholine in models of both hypertension and atherosclerosis and in patients with heart failure. A reduction in nitric oxide levels seems to be the main mechanism underlying this effect due to a reduction in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone is a pro-inflammatory factor that can participate in the vascular inflammatory process associated with different pathologies including hypertension through activation of the NFkappaB system, which mediates the vascular production of different cytokines. This mineralocorticoid also participates in the vascular remodeling observed in hypertensive rats since the administration of eplerenone improved the media-to-lumen ratio in these animals. This effect seems to be due to an increase in extracellular matrix. In summary, aldosterone through mineralocorticoid receptors can participate in the vascular damage associated with different pathologies including hypertension through its prooxidant, pro-inflammatory and profibrotic effects that triggered endothelial dysfunction, an inflammatory process and vascular remodeling.     

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

The prothrombotic mediator thromboxane A₂ is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.     

Interdigitating cells in the thymus of the turtle Mauremys caspica

Summary Interdigitating cells are non-lymphoid elements in the thymus and peripheral, secondary lymphoid organs of higher vertebrates. Their origin and functional significance are a matter of controversy. In the present investigation we analyze, for the first time, the nature of presumptive interdigitating cells of the thymus of an ectothermic vertebrate, the turtle Mauremys caspica. This model is specially useful because of the seasonal variations that affect the reptilian lymphoid organs. Immature pro-interdigitating cells and phagocytosing mature interdigitating cells are described with special emphasis on their ultrastructural characteristics and possible relationships with monocytes and macrophages.     

Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion

Short interfering RNAs (siRNAs) are powerful sequence-specific reagents that suppress gene expression in mammalian cells. We report for the first time that gene silencing of endothelial E-selectin by siRNAs leads to successful inhibition of leukocyte-endothelial interaction under flow. siRNAs designed to target human E-selectin were tranfected into human umbilical vein endothelial cells (HUVEC). Western blotting analysis revealed that transfection of these siRNAs, but not the scrambled control siRNA (100nM each), attenuated E-selectin expression in HUVEC activated with TNF-alpha (10ng/ml, 4h) without affecting expression of ICAM-1. Moreover, a leukocyte adhesion assay under flow (shear stress=1.0dyne/cm(2)) demonstrated that HUVEC transfected with a siRNA against E-selectin (siE-01) supported significantly less HL60 adhesion as compared to those transfected with the control siRNA (scE-01) after activation (p<0.03). This technique provides a powerful strategy to dissect a specific function of a given molecule in leukocyte-endothelial interaction.     

Unfolding the relationship between secreted molecular chaperones and macrophage activation states

Over the last 20 years, it has emerged that many molecular chaperones and protein-folding catalysts are secreted from cells and function, somewhat in the manner of cytokines, as pleiotropic signals for a variety of cells, with much attention being focused on the macrophage. During the last decade, it has become clear that macrophages respond to bacterial, protozoal, parasitic and host signals to generate phenotypically distinct states of activation. These activation states have been termed ‘classical’ and ‘alternative’ and represent not a simple bifurcation in response to external signals but a range of cellular phenotypes. From an examination of the literature, the hypothesis is propounded that mammalian molecular chaperones are able to induce a wide variety of alternative macrophage activation states, and this may be a system for relating cellular or tissue stress to appropriate macrophage responses to restore homeostatic equilibrium.     

The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

Summary The peroxidatic (PO) activity of monocytes differentiating into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas was investigated with three different media for the demonstration of PO activity. Irrespective of the stage of differentiation, these cells did not show PO activity in the rough endoplasmic reticulum (RER) or nuclear envelope. In addition, it was found that the morphologically characteristic types of granule of the various cells of the monocyte line (the primary granules and secondary granules of monocytes, the macrophage granules, and the epithelioid cell granules), all have distinct cytochemical characteristics.Monocytes lose their primary and secondary granules during differentiation into mature macrophages. Simultaneously, the granules of both types become elongated and the secondary granules lose their halo. In contrast to monocytes, mature macrophages may contain a few microperoxisomes. During the differentiation of macrophages into epithelioid cells or multinucleated giant cells there is an increase in the number of microperoxisomes.     

Cellular, molecular and clinical aspects of vitamin E on atherosclerosis prevention

Randomised clinical trials and epidemiologic studies addressing the preventive effects of vitamin E supplementation against cardiovascular disease reported both positive and negative effects, and recent meta-analyses of the clinical studies were rather disappointing. In contrast to that, many animal studies clearly show a preventive action of vitamin E in several experimental settings, which can be explained by the molecular and cellular effects of vitamin E observed in cell cultures. This review is focusing on the molecular effects of vitamin E on the cells playing a role during atherosclerosis, in particular on the endothelial cells, vascular smooth muscle cells, monocytes/macrophages, T cells, and mast cells. Vitamin E may act by normalizing aberrant signal transduction and gene expression in antioxidant and non-antioxidant manners; in particular, over-expression of scavenger receptors and consequent foam cell formation can be prevented by vitamin E. In addition to that, the cellular effects of -tocopheryl phosphate and of EPC-K1, a composite molecule between -tocopheryl phosphate and l-ascorbic acid, are summarized.     

Roles of heterogenous hepatic macrophages in the progression of liver diseases

Hepatic macrophages are key immune cells associated with the broad ranges of liver diseases including steatosis, inflammation and fibrosis. Hepatic macrophages interact with other immune cells and orchestrate hepatic immune circumstances. Recently, the heterogenous populations of hepatic macrophages have been discovered termed residential Kupffer cells and monocyte-derived macrophages, and identified their distinct population dynamics during the progression of various liver diseases. Liver injury lead to Kupffer cells activation with induction of inflammatory cytokines and chemokines, which triggers recruitment of inflammatory monocyte-derived macrophages. To understand liver pathology, the functions of different subtypes of liver macrophages should be regarded with different perspectives. In this review, we summarize recent advances in the roles of hepatic macrophages under liver damages and suggest hepatic macrophages as promising therapeutic targets for treating liver diseases.     

Interaction of monocytes with vascular endothelial cells synergistically induces interferon gamma-inducible protein 10 expression through activation of specific cell surface molecules and cytokines

To further understand the regulatory mechanisms involved in the process of angiogenesis, the present study was designed to determine the expression and regulation of interferon gamma-inducible protein 10 (IP-10) in peripheral blood monocytes and human umbilical vein endothelial cells (HUVECs). We found that the interaction of monocytes with HUVECs resulted in synergistic increases in IP-10 expression and secretion, which consequently inhibited endothelial tube formation in vitro. Induction of IP-10 was mediated via specific cell surface molecules, as indicated by the finding that IP-10 secretion was significantly inhibited by anti-CD40 ligand antibody, and to a lesser extent by anti-CD40 antibody. Furthermore, we examined the effects of soluble mediators, such as inflammatory and immune cytokines on IP-10 secretion. Addition of interleukin (IL)-1, as well as interferon gamma, induced a marked augmentation of IP-10 secretion by unstimulated monocytes, unstimulated HUVECs, and co-cultures of the two cell types. In contrast, IL-10, recognized as an anti-inflammatory cytokine, significantly inhibited IP-10 secretion by co-cultures. Our results suggest that the interaction of monocytes with endothelial cells results in synergistic increases in IP-10 expression and secretion, which contribute to the regulation of angiogenesis and initiation of inflammatory vascular diseases.     

Simultaneous determination of molecular species of monoacylglycerols,diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography

The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.     

Application of high-fat cell model in steady-state regulation of vascular function

In order to effectively apply the high-fat cell model to the regulation of vascular homeostasis and the repair of vascular endothelial cell injury, and to provide a new theoretical basis for the treatment of vascular homeostasis imbalance in the future, in this study, the mouse thoracic aorta tissue is extracted by using mouse endothelial cells. Western blotting and immunofluorescence resonance energy transfer (Immuno-FRET) are then used to verify the distribution and physical coupling properties of TRPV4 and Nox2 in cells. Finally, mouse mesenteric endothelial cells are isolated and cultured to induce FFA high-fat cell model. The results show that the nucleic acid expression levels of TRPV4 and Nox2 in RNA are significantly different from those of TRPV4 and Nox2 in protein. The relative values of TRPV4 and Nox2 in the control group are relatively low (0.8 ± 0.11). However, the relative values of TRPV4 and Nox2 are higher in the FFA high-fat cell model induced by the experimental group, and the values are (1.7 ± 0.8). Obviously, the relative values of TRPV4 and Nox2 in the experimental group are higher than those in the control group. The expression of reactive oxygen species (ROS) in vascular endothelial cells of control group is (1.0 ± 0.16), and that in FFA group is (2.5 ± 0.46). The expression of ROS in FFA cell model with HC067047A inhibitor is (1.5 ± 0.38). In the FFA cell model with apo inhibitor, ROS expression is (1.2 ± 0.23). Thus, in the FFA high-fat cell model induced successfully, the physical coupling of TRPV4 and Nox2 increases in primary endothelial cells, and the increase of physical coupling of TRPV4 and Nox2 results in the increase of ROS expression, which also means the imbalance of ROS homeostasis in vascular endothelial cells and the change of vascular endothelial cell permeability. The expression levels of TRPV4 and Nox2 are used as indicators of whether the vascular function is stable or unbalanced, thus providing a new theoretical basis for the treatment of cardiovascular diseases.