Endothelial integrity may be regulated by a specific antigen via an IgE-mediated mechanism.

BACKGROUND: Human vascular endothelial function and integrity may be regulated by many non-specific factors. However, the potential influence of specific antigens via an IgE-mediated mechanism remains unknown. The aim of the study was to determine the expression of the IgE receptors FcεRI and FcεRII in the human vascular endothelium and to assess their relevance in the IgE-mediated regulation of endothelial integrity. MATERIAL/METHODS: FcεRI and FcεRII expression in human umbilical vein endothelial cells (HUVEC) was genetically assessed by PCR with respective primers and sequencing. HUVEC were cultured with IL-4, and changes in FcεRI and FcεRII mRNA expression were analyzed by real-time PCR. Changes in the integrity of endothelium pre-treated with anti-BSA-DNP IgE following exposure to the specific BSA-DNP antigen was assessed using the Real-time Cell Electric Impedance Sensing system (RTCA-DP). RESULTS: PCR and sequencing revealed the expression of FcεRI and FcεRII receptors in the human vascular endothelium. IL-4 caused respective 2- and 3-fold increases in FcεRI and FcεRII mRNA expression. Exposure of endothelium pre-treated with anti-BSA-DNP IgE to specific BSA-DNP antigen led to a 20% increase of endothelial integrity (p<0.05) after 24 hours, but only in cells pre-incubated with IL-4. CONCLUSIONS: The presence of FcεRI and FcεRII may allow the human vascular endothelium to respond to a specific antigen by increasing its integrity via an IgE-mediated mechanism.

[The effect of 7-ketocholesterol on surface ICAM-1 and PECAM-1 expression in human aortic endothelial cells]. / Ocena wplywu 7-ketocholesterolu na powierzchniowa ekspresje molekul adhezyjnych ICAM-1 i PECAM-1 w ludzkich komórkach sródblonka aortalnego.

UNLABELLED: Proatherogenic factors lead to activation of endothelial cells, which symptom is an increased expression of surface adhesion molecules enabling the initiation of a local inflammatory response. 7-ketocholesterol (7-KCH) is a product of oxidation of cholesterol with proven pro-apoptotic effect on the cells of the vessel wall. So far, however, the impact of 7-KCH on surface expression of adhesion molecules has not been assessed. The aim of this study was to evaluate the influence of 7-KCH on the surface expression of adhesion molecules--intracellular adhesion molecule 1 (ICAM-1, CD 54) and .platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31) on human aortic endothelial cells (HAECs). MATERIAL AND METHODS: After treatment with 7-KCH surface expression of adhesion molecules on HAEC was measured with antihuman CD31 and anti-human CD54 antibodies using flow cytometer. RESULTS: 7-KCH significantly increases percentages of CD 54 on viable HEAC, but does not affect expression of CD 31. CONCLUSION: 7-KCH may enhance the initiation of a local inflammatory response in atherosclerosis by increasing the expression of ICAM-1.

Escherichia coli lipopolysaccharide may affect the endothelial barrier and IL-10 expression of apolipoprotein B100-pulsed dendritic cells.

Atherogenesis is associated with chronic gut infections; however, the mechanisms are not clear. The aim of the study was to determine whether lipopolysaccharide of E. coli (E. coli LPS) may affect endothelial barrier and modify IL-10 expression in dendritic cells (DCs). Human umbilical vein endothelial cells (HUVECs) and monocyte-derived DCs were treated with E. coli LPS, apolipoprotein B100 (ApoB100) and 7-ketocholesterol (7-kCH) - harmful oxidized form of cholesterol. The effect of E. coli LPS, 7-kCH and ApoB100 on the barrier functions of HUVECs in real-time cell electric impedance sensing system (RTCA-DP) was assessed. Furthermore, the effect of 7-kCH and ApoB100 on barrier functions of HUVECs co-cultured with DCs previously treated with LPS was analyzed. Both E. coli LPS and 7-kCH decreased barrier functions of HUVECs and reduced tight junction protein mRNA expression, whereas ApoB100 increased endothelial barrier. In DCs, ApoB100 and E. coli LPS decreased IL-10 mRNA expression. In HUVECs co-cultured with DCs treated with LPS and subsequently pulsed with ApoB100 or 7-kCH, IL-10 mRNA expression was lower. E. coli LPS-exposed DCs diminished the protective effect of ApoB100 on endothelial integrity and led to the decrease in occludin mRNA expression. LPS potentially derived from gut microflora may destabilize endothelial barrier together with oxidized cholesterol and intensify the immunogenicity of ApoB100.

The influence of statin monotherapy and statin-ezetimibe combined therapy on FoxP3 and IL 10 mRNA expression in patients with coronary artery disease.

BACKGROUND: FoxP3 is a marker of human T regulatory cells (Tregs), which are supposed to play an important role in the pathophysiology of atherosclerosis. Interleukin 10 (IL-10) is a cytokine with pleiotropic, immunoregulatory properties, produced mostly by Tregs and B regulatory cells. Due to their anti-inflammatory action, both Tregs and IL-10 are believed to inhibit plaque development and decrease atherosclerosis progression. The effect of hypolipidemic drugs - statins or ezetimibe - on FoxP3-positive Tregs and anti-inflammatory cytokines, such as IL-10, is still unclear. OBJECTIVES: The objective of the study was to investigate the effects of 3 different therapies of equivalent hypolipidemic activity: atorvastatin, rosuvastatin, and combination therapy of atorvastatin and ezetimibe on FoxP3-Tregs transcription factor and IL-10 mRNA expression in peripheral blood mononuclear cells (PBMCs) from patients with stable coronary artery disease (CAD). MATERIAL AND METHODS: Sixty-five patients with diagnosed CAD participated in the study. They were randomly assigned to 3 therapeutic groups: atorvastatin at a dose of 40 mg/day (A40 group); rosuvastatin 20 mg/day (R20 group); and atorvastatin 10 mg/day combined with ezetimibe 10 mg/day (A10+E10 group). After 1 month and 6 months of therapy, the mRNA expression for FoxP3 and IL-10 in PBMCs was evaluated using real-time polymerase chain reaction (RT-PCR) and lipid parameters. RESULTS: An improvement in lipid parameters was observed in each of the groups studied; however, hypolipidemic treatment did not induce any change in FoxP3 and IL-10 mRNA expression. After 6 months, an increase in FoxP3 mRNA expression was noted in A40 group as compared to R20 group. CONCLUSIONS: None of the therapies of equal hypolipidemic efficacy affected FoxP3 and IL-10 mRNA expression in patients with stable CAD.

Troglitazone, a PPAR-γ agonist, decreases LTC4 concentration in mononuclear cells in patients with asthma.

BACKGROUND: Asthma is an inflammatory disorder with multiple mediators involved in the inflammatory response. Despite several attempts, no new anti-inflammatory drugs have been registered for asthma treatment for several years. However, thiazolidinediones, peroxisome proliferator-activated receptor agonists, have demonstrated some anti-inflammatory properties in various experimental settings. The aim of this study was to assess the influence of troglitazone on LTC4 and 15-HETE concentrations. It also evaluates TNF-induced eotaxin synthesis in peripheral blood mononuclear cells from 14 patients with mild asthma and 13 healthy controls. METHODS: PBMCs were isolated from the whole blood of the asthmatics and healthy subjects and pretreated with 0.1, 1 or 10µM of Troglitazone. The cells were then exposed to 10-6M calcium jonophore or 10ng/ml TNF. The production and release of LTC4, 15-HETE and eotaxin were then assessed. RESULTS: Troglitazone caused a dose-dependent inhibition in LTC4 synthesis in both asthmatics and healthy subjects. Troglitazone did not influence 15-HETE or eotaxin production in either asthmatic patients or in healthy individuals. CONCLUSION: Due to its inhibition of LTC4 synthesis, troglitazone therapy is an interesting potential therapeutic approach in asthma and other LTC4 related inflammatory disorders.

IL-22 modulates inflammatory properties of human primary aortic smooth muscle cells.

BACKGROUND: IL-22 is expressed at barrier surfaces, which suggests its critical role in the maintenance of normal barrier homeostasis and tissue repair. IL-22 can both promote pathological inflammation and prevent the destruction of tissues. The functional outcomes of IL-22 on vascular smooth muscle cells, which are shown to regulate immune processes within the vascular wall and which are involved in certain pathologies, have not been analyzed. OBJECTIVES: The effect of IL-22 on the expression of novel antiand pro-inflammatory and barrier disrupting cytokines, apoptosis and the expression of adhesive molecules in human primary aortic smooth muscle cells (AoSMC) was investigated. MATERIAL AND METHODS: Human AoSMC were induced with IL-22 for 24 h in vitro. The influence of IL-22 on IL-35 subunits EBI3 and p35, IL-33, IFN-γ and VEGF mRNA expression in Ao-SMC were assessed using real-time PCR. Additionally, the surface expression of ICAM-1 and apoptosis of AoSMC were analyzed in the flow cytometer. RESULTS: IL-22 caused a 2- and 3-fold increase of mRNA expression of the EBI3 and p35 IL-35 subunits, and a 40% decrease of IL-33 mRNA expression in AoSMC. Additionally, IL-22 decreased ICAM-1 expression on the surface of AoSMC by 30%. However, IL-22 affected neither IFN-γ and VEGF mRNA expression in AoSMC nor their apoptosis and viability. CONCLUSIONS: Our data suggest that IL-22, which is released by Th22 and NK cells, may be an agent affecting the inflammatory response of AoSMC, and thus it may regulate immune homeostasis of the vascular wall.

Increased plasma concentrations of interleukin 35 in patients with coronary artery disease.

INTRODUCTION: Atherosclerosis leading to coronary artery disease (CAD) is a chronic inflammatory condition. Interleukin 35 (IL-35) released by regulatory T cells (Tregs) has been found to be associated with CAD in the Chinese population. However, nothing is known about the relation between IL-35 concentrations and cholesterol levels. The aim of the study was to assess the levels of IL-35 in CAD patients and healthy subjects from a Caucasian population, and to analyze the relationship between IL-35 and the levels of total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, left ventricular ejection fraction (LVEF), sex and postmenopausal status. MATERIAL AND METHODS: Thirty-one patients with CAD and 30 healthy controls were included in the study. Levels of plasma IL-35 were analyzed by ELISA. The LVEF was assessed by transthoracic echocardiographic examination. Plasma levels of cholesterol fractions and C-reactive protein (CRP) were assessed by immunoenzymatic methods. RESULTS: The CAD patients had higher levels of IL-35 as compared to healthy controls (58.1 ±16.6 pg/ml vs. 5.35 ±3.35 pg/ml; p < 0.001). IL-35 levels negatively correlated with total and LDL cholesterol concentrations (R = -0.31, p < 0.01) and positively correlated with HDL cholesterol in men (R = 0.53, p < 0.01). In women, IL-35 levels negatively correlated with LVEF (R = -0.29, p < 0.05) and positively with the duration of postmenopausal status (R = 0.55, p < 0.01). CONCLUSIONS: These results suggest a possible association between high levels of IL-35 and CAD.

Innate lymphoid cells type 2 - emerging immune regulators of obesity and atherosclerosis.

The low-grade inflammation present in obese visceral adipose tissue impairs glucose metabolism, and contributes to the development of insulin resistance and weight gain. Immune processes occurring in response to the deposition of cholesterol within the vascular walls support atherosclerotic plaque growth and contribute to the cardiovascular complications. In both the obese adipose tissue and the atherosclerotic plaque, the Th1-type immune environment dominates over the Th2/Treg-type due to the overproduction of pro-inflammatory cytokines (IFN-γ, IL-6, TNF-α) and the deficiency of Th2-type processes and interleukins (IL-4, IL-5, IL-10, IL-13). So far, Th2 cells and eosinophils have been considered as the main providers of Th2-type mediators and the basis of Th2-type immunity in tissues. However, recently discovered innate lymphoid type 2 cells (ILC2s), which infiltrate lean visceral adipose tissue and the vascular wall, are believed to orchestrate local Th2-like immune responses. Upon activation by tissue-derived IL-33 and IL-25, ILCs2 secrete mostly IL-4, IL-5, IL-9 and IL-13: cytokines responsible for the accumulation of eosinophils and polarization of alternatively-activated macrophages, which altogether create the beneficial anti-inflammatory and metabo-regulatory environment in the adipose tissue and the vascular wall. Consequently, ILC2s-orchestrated immune environment seems to prevent obesity and atherosclerosis. Thus, ILCs2 appear to be the emerging immune regulators of immune and metabolic homeostasis in both adipose tissue and the vascular wall.

IL-33 and IL-4 impair barrier functions of human vascular endothelium via different mechanisms.

The vascular endothelium forms a barrier that controls flow of solutes and proteins and the entry of leukocytes into tissue. Injured tissue releases IL-33, which then alarms the immune system and attracts Th2 cells, thus increasing local concentration of IL-4. The aim of the study was to assess the influence of IL-33 and IL-4 on barrier functions of the human endothelium, expression of tight and adherent junction proteins, apoptosis and adhesive molecule surface expression in human endothelium in order to describe the mechanism of this effect. IL-33 and IL-4 decreased endothelial integrity and increased permeability. When added together, both cytokines lowered the endothelial integrity twice as much as used alone. This effect was accompanied by the down-regulation of occludin and VE-cadherin mRNA expression. Additionally, IL-4, but not IL-33, induced cell apoptosis. Both IL-33 and IL-4 showed the additive potency to down-regulate VE-cadherin mRNA expression. IL-33, unlike IL-4, increased the surface expression of ICAM-1, but not PECAM-1 in endothelial cells. Our results indicate that IL-33 may reversibly destabilize the endothelial barrier, thus accelerating the supply with immunomodulators and assisting leukocytes to reach wounded tissue. However, extended and less-controlled down-regulation of endothelial barrier, which may be a consequence of IL-33-initiated, but in fact IL-4-induced apoptosis of endothelial cells, may be deleterious and may eventually lead to the aggravation of inflammatory processes and the prolongation of tissue dysfunction.

Studies towards biocompatibility of PAMAM dendrimers--overall hemostasis potential and integrity of the human aortic endothelial barrier.

UNLABELLED: The last decade has brought many examples of utilization of dendrimers as drug delivery systems; however, their possible application is limited because of inherent toxicity associated with them. This study discusses the influence of G1-G4 PAMAM-NH2 dendrimers on the process of hemostasis and integrity of endothelial monolayer. The global assay of coagulation and fibrinolysis was investigated spectrophotometrically by means of CL-test at 405 nm. Thrombin (0.5 I U/mL) and t-PA (240 ng/mL) were used to obtain clotting and lysis curve. The activity of thrombin was determined by means of chromogenic substrate S-2238. The influence of PAMAM dendrimers on the barrier properties of human primary aortal endothelium was assessed by means of method based on the measurements of the impedance changes of the cells. Observed multidirectional impact of dendrimers, without affecting the thrombin activity, on clot formation, its stabilization and fibrinolysis could be regarded as important when trying to use them clinically. It is crucial that examined PAMAM dendrimers did not lead to spontaneous aggregation of fibrinogen. Importantly, examined polymers have concentration- and generation-dependent adverse effect towards the endothelial monolayer. RESULT: of described studies provide additional insight into PAMAM dendrimers toxicity associated with systemic administration and underscore the necessity for further research.