Toxins of Bacteroides fragilis and Bacteroides thetaiotaomicron rods as stimulators of adhesion molecule expression on the surface of vascular endothelial cells

Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF), and from three B. thetaiotaomicron strains were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the procedure of van Tassell et al. (1992). The influence of the examined toxins on the expression of adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human dermal microvascular endothelial cells) was assayed in ELISA test with monoclonal antibodies. Four concentrations of toxins were applied: 0.01, 0.1, 1.0 and 10.0 (micrograms/ml). Endothelial cells were activated for 24 hours (ICAM-1 and VCAM-1 expression) and for 4 hours (E-selectin expression). The coloured product of immunoenzymatic reaction was measured by reading the absorbance at wavelength 492 nm. Two controls were performed in each experiment: with resting HMEC-1 and E. coli O55:B5 LPS (Sigma, USA). Bacteroides fragilis and B. thetaiotaomicron lipopolysaccharides stimulated three adhesion molecules under investigation. Their activity was comparable, but weaker than the activity of E. coli O55:B5 LPS. ICAM-1 was the most stimulated molecule. B. fragilis enterotoxin induced two adhesion molecules: VCAM-1 and E-selectin demonstrating weaker stimulatory activity than E. coli LPS. Stimulation of adhesion molecules on vascular endothelial cells should be considered to be a biological activity of B. fragilis and B. thetaiotaomicron endotoxins and B. fragilis enterotoxin.     

The effect of metronidazole on stimulation of adhesion molecule expression on the surface of vascular endothelial cells by Bacteroides fragilis endotoxins and enterotoxin]

The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 micrograms/ml in the presence of metronidazole at the concentration of 4 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.     

Influence of clindamycin, metronidazole and polymyxin B on the expression of cell adhesion molecules stimulated by endotoxin and enterotoxin of Bacteroides fragilis

The aim of this study was to compare the influence of antimicrobials (clindamycin, metronidazole and polymyxin B) on the expression of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) on the HMEC-1 cell line stimulated by LPS and enterotoxin of B. fragilis. LPS was extracted from two reference: ATCC 43858 and NCTC 11295 and one isolated in our laboratory (W2) enterotoxigenic strains, and one nonenterotoxigenic reference strain--IPL E 323. Enterotoxin preparations (Tox 1 and Tox 2) were isolated from supematant of B. fragilis ATCC 43858 culture and purified. HMEC-1 cell line was stimulated with bacterial preparations at concentration of 10 mg/ml. For measuring the expression of adhesion molecules we used ELISA test. Clindamycin, metronidazole and polymyxin B supressed the ICAM-1 expression when endothelium was stimulated with B. fragilis LPS and augmented ICAM-1 expression by Tox 1 and Tox 2. The expression of VCAM-1 was augmented by antimicrobials when endothelium was stimulated with LPS or enterotoxin preparations. The expression of E-selectin was differentiated.     

Stimulation of adhesion molecule expression in human vascular endothelium by Bacteroides fragilis toxins--influence of polymyxin B

The aim of this study was to examine the influence of polymyxin B on the level of expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 on human vascular endothelium activated with B. fragilis endotoxins or enterotoxin. Lipopolysaccharides were extracted by phenol-water method from one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) B. fragilis strains. LPS preparations were purified with nucleolytic enzymes and ultracentrifugation. Enteotoxin (BFT) was prepared from the supernatant of reference B. fragilis ATCC 43858 culture by precipitation with ammonium sulphate. BFT preparations were purified with the application of ion-exchange chromatography and hydrophobic chromatography. Adhesion molecule expression on the surface of human vascular endothelial cells (HMEC-1 cell line) was determined after simultaneous stimulation with bacterial compounds at the concentration of 10 micrograms/ml and polymyxin B at the concentration of 20 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) or for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with the use of mouse, monoclonal antibodies against human ICAM-1, VCAM-1, and E-selectin. The results of performed experiments suggest, that polymyxin B changes the level of adhesion molecule expression on human vascular endothelium. This antibiotic causes changes in the expression of endothelial ICAM-1, VCAM-1, and E-selectin during simultaneous stimulation of endothelium with B. fragilis endotoxins or enterotoxin. In the majority of cases the addition of polymyxin B leads to the up-regulation of examined adhesion molecules.     

Effect of clindamycin on stimulation of cell adhesion molecules by endotoxins and enterotoxin of Bacteroides fragilis strains]

The influence of clindamycin on expression of B. fragilis endotoxins (LPS) and enterotoxin stimulated cell adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human microvascular endothelial cell line) was tested. Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the method described by van Tassel et al. (1992). All bacterial preparations were used for stimulation at concentration 10 micrograms/ml. Clindamycin was used in concentration of 2 micrograms/ml. The influence of antimicrobial agent on the endotoxins and enterotoxin stimulation and expression of adhesion molecules was tested by ELISA, using monoclonal mouse anti-human antibodies (Genzyme, USA). Peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO A/S Denmark) and OPD (Sigma USA) were used. The coloured reaction product was measured by reading the absorbance at 492 nm in SPECTRA II reader (SLT, Austria). It was observed that clindamycin influenced the expression of cell adhesion molecules on resting cell line. HMEC-1 cells stimulated with Bacteroides fragilis LPS preparations have suppressive effect on ICAM-1 expression. ICAM-1 expression was augmented when stimulated with Tox 1 and Tox 2 preparations. Clindamycin augmented the VCAM-1 expression in tests with all bacterial preparations. All used bacterial preparations of Bacteroides fragilis LPS and enterotoxin enhanced the expression of E-selectin with exception of LPS of NTBF strain.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.     

Cryptotanshinone inhibits oxidized LDL-induced adhesion molecule expression via ROS dependent NF-κB pathways

Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, play important roles in the initial stage of atherosclerosis. Cryptotanshinone (CPT), a natural compound isolated from Salvia miltiorrhiza Bunge, exhibits anti-atherosclerotic activity although the underlying mechanisms remain elusive. In this study, the protective effect of CPT against oxidized low-density lipoprotein (ox-LDL)-induced adhesion molecule expression was investigated in human umbilical vein endothelial cells. Ox-LDL significantly induced ICAM-1, VCAM-1, and E-selectin expression at the mRNA and protein levels but reduced eNOS phosphorylation and NO generation, which were reversed by CPT pretreatment. Sodium nitroprusside, a NO donor, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF-κB inhibitor, inhibited ox-LDL-induced ICAM-1, VCAM-1, and E-selectin expression. Ox-LDL-induced ROS production was significantly inhibited by CPT and NAC. Furthermore, ox-LDL activated the NF-κB signaling pathway by inducing phosphorylation of IKKβ and IκBα, promoting the interaction of IKKβ and IκBα, and increasing p65 nuclear translocation, which were significantly inhibited by CPT. In addition, CPT, NAC, and BAY117082 inhibited ox-LDL-induced membrane expression of ICAM-1, VCAM-1, E-selectin, and endothelial–monocyte adhesion and restored eNOS phosphorylation and NO generation. Results suggested that CPT inhibited ox-LDL-induced adhesion molecule expression by decreasing ROS and inhibiting the NF-κB pathways, which provides new insight into the anti-atherosclerotic mechanism of CPT.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin,P-selectin,ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

A functional role of I kappa B-epsilon in endothelial cell activation

The NF-kappa B inhibitor I kappa B-epsilon is a new member of the I kappa B protein family, but its functional role in regulating NF-kappa B-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, I kappa B-epsilon associates predominantly with the NF-kappa B subunit Rel A and to a lesser extent with c-Rel, whereas I kappa B-alpha and I kappa B-beta associate with Rel A only. Following stimulation with TNF-alpha, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented I kappa B kinase-induced I kappa B-alpha, but not I kappa B-beta or I kappa B-epsilon phosphorylation and degradation. Since the activation of NF-kappa B is required for the induction of adhesion molecule expression, we examined the role of I kappa B-epsilon in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of kappa B cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 kappa B promoter activity. By electrophoretic mobility shift assay, NF-kappa B heterodimers containing c-Rel specifically bind to the kappa B motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 kappa B promoter activity to a greater extent than that of E-selectin and overexpression of I kappa B-epsilon inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated I kappa B-epsilon is involved in the induction of ICAM-1 expression.     

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells

Cell adhesion molecules expressed on endothelial cells in inflamed skin appear to be controlled by the actions of cytokines and reactive oxygen species. However, molecular mechanisms of the expression of adhesion molecules during skin inflammation are currently not well understood. To evaluate the role of antioxidants and nitric oxide in modulating inflammatory processes in the skin, we examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human dermal microvascular endothelial cells (HDMEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). The up-regulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were significantly decreased by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had no significant inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. These findings indicate that both cell surface and mRNA expression of adhesion molecules in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. These results suggest a potential therapeutic approach using antioxidant agents or nitric oxide pathway modulators in the treatment of inflammatory skin diseases.     

Reduction in serum levels of adhesion molecules, interleukin-6 and C-reactive protein following short-term low-dose atorvastatin treatment in patients with non-familial hypercholesterolemia.

Hypercholesterolemia causes endothelial dysfunction, an early feature of atherosclerosis, leading to increased production of adhesion molecules and cytokines. The aim of this study was to investigate the effects of three months of treatment with low dose atorvastatin on serum levels of adhesion molecules, interleukin-6 (IL-6) and highly sensitive C-reactive protein (hs-CRP) in patients with non-familial hypercholesterolemia. Fifty-five patients with non-familial hypercholesterolemia were randomized to treatment with atorvastatin 10 mg/day or placebo for 3 months. Soluble intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, IL-6 and hs-CRP levels were measured to assess the inflammatory activity of the endothelium. There was a significant reduction in ICAM-1 at 2 weeks (p<0.0001) with further reduction at 3 months (p<0.0001). At 3 months, there were significant reductions in VCAM-1 (p<0.02), IL-6 (p<0.0001) and hs-CRP (p<0.01), but an increase in E-selectin levels (p<0.002). Treatment with statin was an independent determinant of change in ICAM-1 (p<0.05) and IL-6 levels (p<0.05) after correcting for anthropometric indices, blood pressure and lipid profile. Low-dose atorvastatin treatment leads to reduction in proinflammatory markers of endothelial function, suggesting an attenuation of endothelial activation and improvement in endothelial function, independent of lipid lowering. This may lead to a reduction in the progression of atherosclerosis.     

Insulin administration in the mild hyperglycaemia changes expression of proinflammatory adhesion molecules on human aortic endothelial cells

An overexpression of cell adhesion molecules (CAMs) on the surface of endothelial cells is one of the first steps in a high glucose-mediated endothelial dysfunction in diabetic patients. The effect of insulin administration in the condition of elevated glucose concentration on the E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) expression on human aortic endothelial cells (HAEC) was investigated. Cells were cultured for 4 h in a medium supplemented with homocysteine (7 pM) and different concentration of glucose (5.5, 8.0, 12.0 and 16.5 mM respectively) with or without insulin (1 mlU/mL) addition. Expression of CAMs was analysed by flow-cytometry using monoclonal antibodies. Controls were CAMs expression in the medium with a corresponding glucose concentration. Obtained results show that short-term exposure of HAECs to moderate high glucose concentrations results in increased expression of E-selectin (2-fold), VCAM-1 (3-fold) and ICAM-1 (47%). At the same time, HAEC grown with 12 mM glucose expressed lesser E-selectin and, more ICAM-1 (for 64%) and VCAM-1 (41%) molecules. 16.5 mM glucose decreased expression of all investigated adhesion molecules. Addition of insulin was not changed expression of CAMs in a medium with 5.5 mM glucose. In conditions of elevated glucose concentration (12 mM), addition of insulin significantly dropped E-selectin (27%) and increased VCAM-1 (23%) expression. In conclusion, moderate elevated glucose concentration increased expression of cell adhesion molecules on HAEC. Insulin administration in the mild hyperglycaemia reduces an expression of the proinflammatory adhesion molecule E-selectin which could contribute in deceleration of macrovascular complications development in diabetic patients.     

Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells

Chlamydophila pneumoniae is a common respiratory pathogen that has been shown to be associated with coronary artery disease. Recent studies have shown that one of the possible mechanisms of the atherogenicity of C. pneumoniae is overexpression of cell adhesion molecules (CAMs) in infected endothelial cells. We investigated whether exposure of C. pneumoniae-infected endothelial cells to oxidized LDL (oxLDL) leads to further upregulation of CAMs. Flow cytometry and immunoblot analysis of human aortic endothelial cells (HAECs) was performed for intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ICAM-1 was expressed in 78.7% of C. pneumoniae-infected HAECs. The addition of oxLDL (100 microg/ml) to infected HAECs increased the proportion of ICAM-1-positive cells to 92%. VCAM-1 was only observed in 9.3% of infected HAECs, and the addition of oxLDL had no further effect on the surface expression of VCAM-1. C. pneumoniae also upregulated the surface expression of E-selectin on 52.2% of the cells, and incubation with oxLDL further increased the proportion of positive cells to 63.64%. In conclusion, C. pneumoniae upregulated the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin on HAECs. The addition of oxLDL to the infected cells further enhanced the surface expression of ICAM-1 and E-selectin.