Maciej Chalubinski

Claudin-1 expression in airway smooth muscle exacerbates airway remodeling in asthmatic subjects

BACKGROUND Increased airway smooth muscle (ASM) mass is an essential component of airway remodeling and asthma development, and there is no medication specifically against it. Tight junction (TJ) proteins, which are expressed in endothelial and epithelial cells and affect tissue integrity, might exist in other types of cells and display additional functions in the asthmatic lung. OBJECTIVE The aim of this study was to investigate the existence, regulation, and function of TJ proteins in ASM in asthmatic patients. METHODS The expression and function of TJ proteins in primary ASM cell lines, human bronchial biopsy specimens, and a murine model of asthma were analyzed by means of RT-PCR, multispectral imaging flow cytometry, immunohistochemistry, Western blotting, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester staining, tritiated thymidine incorporation, wound-healing assay, and luminometric bead array. RESULTS Increased claudin-1 expression was observed in ASM of asthmatic patients, as well as in a murine model of asthma-like airway inflammation. Whereas IL-1β and TNF-α upregulated claudin-1 expression, it was downregulated by the T(H)2 cytokines IL-4 and IL-13 in primary human ASM cells. Claudin-1 was localized to the nucleus and cytoplasm but not to the cell surface in ASM cells. Claudin-1 played a central role in ASM cell proliferation, as demonstrated by increased ASM cell proliferation seen with overexpression and decreased proliferation seen with small interfering RNA knockdown of claudin-1. Overexpression of claudin-1 induced vascular endothelial growth factor and downregulated IL-6, IL-8, and IFN-γ-induced protein 10 production by ASM cells. Claudin-1 upregulation by IL-1β or TNF-α was suppressed by dexamethasone but not by rapamycin, FK506, or salbutamol. CONCLUSION These results demonstrate that claudin-1 might play a role in airway remodeling in asthmatic patients by means of regulation of ASM cell proliferation, angiogenesis, and inflammation.

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.

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.

The effect of melatonin on circadian blood pressure in patients with type 2 diabetes and essential hypertension

Introduction The aim of this study was to evaluate the effect of melatonin on blood pressure in patients with essential hypertension receiving medical treatment and with type 2 diabetes in good metabolic control. Material and methods The study lasted 8 weeks. Patients were equipped with a 24-hour ambulatory blood pressure monitor and took melatonin (3 mg a day in the evening) for 4 weeks. The patients were divided into four groups: group 1 (n = 32) including dippers, group 2 (n = 34) non-dippers treated with melatonin; and two control groups: group 3 (n = 28) including dippers and group 4 (n = 30) non-dippers treated without melatonin. After 4 weeks patients took melatonin for the next 4 weeks (5 mg a day). In each visit were analyzed: systolic, diastolic and mean blood pressure in both day and night time. Results We observed that 29.5% non-dippers (n = 10) treated with melatonin in a dose of 3 mg/day achieved features of dippers compared to control group (p < 0.05). Five mg of melatonin per day restored normal diurnal blood pressure rhythm in 32.4% non-dippers (n = 11, p < 0.05). In non-dippers treated with melatonin significant decreases of diastolic, systolic and mean night blood pressure values (p < 0.05) were observed. Conclusions More than 30% of non-dippers with type 2 diabetes treated with melatonin were restored to the normal circadian rhythm of blood pressure. The effect of melatonin in both doses (3 mg and 5 mg) was significant for non-dippers only and included nocturnal systolic, diastolic and mean arterial pressure.

The effect of 7-ketocholesterol and 25-hydroxycholesterol on the integrity of the human aortic endothelial and intestinal epithelial barriers

Objective and designThe damage of barrtier tissues, such as the vascular endothelium and intestinal epithelium, may lead to disturbances of local immune homeostasis. The aim of the study was to assess and compare the effect of oxidized cholesterols (7-ketocholesterol and 25-hydroxycholesterol) on the barrier properties of human primary aortic endothelium (HAEC) and intestinal epithelium Caco-2 cells using a real-time cell electric impedance sensing system (RTCA-DP).Materials and methodsHAEC and Caco-2 cells were stimulated with 7-ketocholesterol and 25-hydroxycholesterol by the RTCA-DP system. Apoptosis was assessed by flow cytometry and cell monolayer morphology was assessed under a light microscope.Results7-ketocholesterol decreased impedance (nCI) in both the endothelium and epithelium. However, the decrease was more profound in the endothelium. Similarly, although 25-hydroxycholesterol decreased nCI in both the endothelium and epithelium, the effect was weaker than that of 7-ketocholesterol, which caused extensive damage to the endothelial monolayer, while 25-hydroxycholesterol caused partial damage and did not affect the epithelial monolayer. 7-ketocholesterol, but not 25-hydroxycholesterol, increased endothelial cell apoptosis and decreased the viability of endothelial cells. However, 7-ketocholesterol and 25-hydroxycholesterol decreased epithelial cell apoptosis and increased viability.ConclusionOxidized cholesterols destroy the HAEC, but not the Caco-2 epithelial barrier, via cell apoptosis dependent on the site of oxidation. Damage to the endothelium by oxidized cholesterol may disrupt local homeostasis and provide open access to inner parts of the vascular wall for lipids, other peripheral blood-derived agents, and immune cells, leading to inflammation and atherogenesis.

Comprehensive insight into immune regulatory mechanisms and vascular wall determinants of atherogenesis – emerging perspectives of immunomodulation

For many years atherosclerosis was believed to be the passive accumulation of cholesterol in vessel walls. Today the picture is more complex, as immune processes occur in atherogenesis. Considerable attention is focused on the particular role of adaptive immune responses orchestrated by T cell subsets. Since the role of Th1/Th2 balance and Th1 cell domination in atherogenesis is already known, the involvement of regulatory T lymphocytes and recently described Th17 cells raises new concerns. On one hand, each of these cells may specifically drive responses of vascular wall tissues and immune cells; however, they are subject to the control of a plethora of tissue- and pathogen-derived agents. Due to ineffective tissue regeneration, remodeling of the vascular wall occurs. The understanding of the immune regulatory network gives perspectives of innovative immunomodulatory therapies of atherosclerosis and the prevention of its complications, such as coronary artery disease.

The effect of oxidized cholesterol on barrier functions and IL-10 mRNA expression in human intestinal epithelium co-cultured with dendritic cells in the transwell system.

The intestinal epithelium is exposed to oxygenated cholesterol products present in foodstuffs. In vitro studies demonstrate the effect of oxysterols on cytokine release by intestinal cells cultured alone. However, physiologically, the response of the intestinal epithelium to external agents occurs in the presence of dendritic cells (DCs). The aim of the study was to analyze the effect of 7-ketocholesterol on the barrier functions and IL-10 mRNA expression of Caco-2 cells in the presence of DCs, and secondly, on IL-10 mRNA expression in DCs. Caco-2 cells were co-cultured with monocyte-derived dendritic cells and induced with 7-ketocholesterol in a transwell system. DCs did not affect the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer, but increased IL-10 mRNA expression in Caco-2 cells. 7-ketocholesterol decreased the TER of Caco-2 cells co-cultured with DCs and diminished IL-10 mRNA expression in Caco-2 cells induced by the presence of DCs. IL-10 mRNA expression fell in DCs co-cultured with Caco-2 cells after treatment with 7-ketocholesterol. Oxidized cholesterols present in gut mucosa may contribute to the decrease of epithelial barrier functions and the inappropriate development of an inflammatory response to food compounds.

Effect of statins on platelet function in patients with hyperlipidemia

Introduction It is generally assumed that cholesterol reduction by statins is the predominant therapeutic result underlying their beneficial effects in cardiovascular disease. However, the action of statins may be partially independent of their effects on plasma cholesterol levels, as they combine lipid lowering with positive effects on hemorheological conditions and endothelial function. We evaluated the impact of statin treatment on platelet adhesion to fibrinogen (spontaneous and ADP-activated), along with ADP, collagen or ristocetin-induced aggregation in type II hyperlipidemic patients. Material and methods The study group included 70 persons: 50 patients affected by type II hyperlipidemia without concomitant diseases and 20 healthy volunteers. The effects of 8-week statin treatment (atorvastatin 10 mg/day, simvastatin 20 mg/day, or pravastatin 20 mg/day) on platelet activation were evaluated. Results Regardless of the type of statin, a significant decrease in ADP-induced platelet aggregation was observed: for atorvastatin 50.6 ±12.8% vs. 41.1 ±15.8% (p < 0.05), for simvastatin 57.2 ±18.0% vs. 44.7 ±22.1% (p = 0.05), and for pravastatin 55.8 ±19.5% vs. 38.8 ±23.3% (p < 0.05). There was no significant effect of statins on collagen or ristocetin-induced platelet aggregation and adhesion. Conclusions Therapy with statins beneficially modifies ADP-induced platelet aggregation in patients with hyperlipidemia and does not affect spontaneous or ADP-induced platelet adhesion to fibrinogen and platelet aggregation induced by collagen or ristocetin.

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.

Putative consequences of exposure to Helicobacter pylori infection in patients with coronary heart disease in terms of humoral immune response and inflammation

Introduction Pathogens, including Helicobacter pylori (Hp), have been suggested to contribute to the development of coronary heart disease (CHD), although the evidence still remains insufficient. The study was focused on the exposure of CHD patients to Hp and resulting anti-Hp heat shock protein B HspB antibody production in relation to the level of serum lipopolysaccharide binding protein (LBP) as a marker of inflammation. Material and methods One hundred seventy CHD patients and 58 non-CHD individuals participated in this study. Coronary angiography confirmed the atheromatic background of CHD. The panel of classical risk factors included: arterial hypertension, diabetes, total cholesterol, low-density lipoprotein (LDL)/high-density lipoprotein (HDL) cholesterol, triglycerides, obesity and nicotinism. The Hp status was estimated by 13C urea breath test and serology. Immunoblot and ELISA were used for screening the sera samples for anti-Hp HspB immunoglobulins (Igs) and LBP. Results Coronary heart disease patients were exposed to Hp more frequently than non-CHD individuals. This was associated with increased levels of specific anti-Hp IgG2 and IgA as well as total IgA. Hp infected CHD and non-CHD donors produced anti-Hp HspB IgG cross-reacting with human Hsp 60. In CHD patients the LBP level was significantly higher in comparison to non-CHD donors. This was related to the severity of the disease. Type I Hp strains stimulated higher LBP levels than less pathogenic type II isolates. Conclusions Lipopolysaccharide binding protein secreted in excess together with anti-Hp HspB, cross-reacting with human Hsp60, may increase the risk of vascular pathologies in Hp-exposed CHD patients.