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Upon induction of experimental hyperglycemia (i.e. diabetes) pathological modifications are early detected (approximately 7 days) at the level of the cardiac valves leading rapidly to the development of valvular atheroma. Monocyte adhesion to the vascular endothelium is one of the initial event at the onset of atherosclerosis. We questioned whether high glucose enhances monocyte adhesion to the valvular endothelial cells (VEC) so as to explain, in part, the accelerated atheroma formation that occur in diabetic conditions. To this purpose we compared the adhesion of monocytes to VEC cultured in 5.5 mM (normal) glucose (NG) or in 33 mM (high) glucose (HG) or in high mannitol (HM) (27.5 mM mannitol plus 5.5 mM glucose), a concentration known to simulate the hyperosmolar effect of high glucose. After incubation for 30 min at 37 degrees C, the adhesion of monocyte cell line (U937 cells) to VEC was quantitated by a fluorimetric assay or by direct counting. Statistical data showed a significant increased adhesion of monocytes to VEC grown in HG (up to 4 fold) or in HM (up to 2.7) when compared to normal conditions. Using a battery of specific monoclonal antibodies molecules it was found that the increased adhesion of monocytes to VEC grown in high glucose was specifically inhibited (p < 0.05) by anti-ICAM-1, anti-VCAM-1 and anti-CD18 monoclonal antibodies. Together, the results indicate that high glucose induces enhanced monocyte adhesion to VEC via a mechanism involving in part an osmotic effect and mainly the cell adhesion molecules: ICAM-1, VCAM-1 and CD18.

RATIONALE AND OBJECTIVES For assessment of the tissue blood pool and overall vascularity, macromolecular contrast media have significant advantages over low molecular weight contrast agents. The authors evaluated the vascular effect and transport of a new macromolecular contrast media(MMCM), an iodinated dextran polymer of 32 kDa. METHODS The new MMCM was obtained from dextran activated by carboxy methylation, followed by linkage with triiodinated aminophtalamid conjugates. To detect whether the tracer induces vascular leakage, MMCM (350 mg I/kg) was administered intravenously in 10 mice, or applied on the cremaster muscle of 26 mice previously injected with carbon particles; after 30 or 45 minutes, the cremaster was fixed and examined by optical microscopy. For investigation of the vascular transport 3, 5, and 15 minutes after MMCM administration, various tissue fragments were processed and examined by electron microscopy. RESULTS In all vascular beds examined, MMCM does not induce plasma extravasation and the probe was detected mostly within the vascular lumen. At the ultrastructural level, a small fraction of MMCM was found in endothelial plasmalemmal vesicles (endosome-like structures) and, in time, transcytosed to the subendothelial space. No intercellular junctions were permeated by MMCM. CONCLUSIONS The MMCM induces no vascular leakage and it is retained mainly in the plasma. Transport of MMCM is restricted to endothelial vesicles, which may explain, in part, its prolonged vascular space retention.

Aim Resistin is an adipokine with potent proinflammatory properties, acting as a potential regulator of inflammation. Several studies proposed resistin to be an important link between obesity and insulin resistance. The obesity-related inflammatory state is linked to the increased risk of developing cardiovascular diseases and type 2 diabetes mellitus. The aim of this study is to determine whether resistin, a cytokine with an important role in the inflammatory process associated with atherosclerosis, induces the expression of fractalkine (CX3CL1) and CX3CR1 in human smooth muscle cells (SMC) and to investigate the potential signaling pathways involved. Methods and Results Cultured human aortic SMC were stimulated with 100ng/ml resistin for 4, 6, 12, and 24h and then the CX3CL1 and CX3CR1 expression was assessed by quantitative RT-PCR and Western-blot. We found that resistin up-regulated CX3CL1 and CX3CR1 in SMC and induced the phosphorylation of p38MAPK and STAT3. Inhibitors of p38MAPK, JAK-STAT and NF-kB and AP-1reduced significantly the CX3CL1 and CX3CR1 expression. Knockdown of STAT1 and STAT3 with decoy oligodeoxinucleotides and silencing p65 and cjun with siRNA down-regulated the CX3CL1 and CX3CR1 expression. Anti-TLR4 antibody and pertussis toxin reduced CX3CL1 and CX3CR1 protein expression. The xCELLigence experiments revealed that resistin may use Gi-proteins for its effect on SMC. The resistin-induced CX3CL1 had chemotactic effect on monocytes transmigration. Conclusions Resistin contributes to the pro-inflammatory state of SMC by up-regulation of CX3CL1 and CX3CR1 expression via a mechanism involving NF-kB, AP-1 and STAT1/3 transcription factors. Resistin employs TLR 4 and Gi proteins signalling for its effect on SMC. Resistin-induce CX3CL1 is functional in monocytes chemotaxis. The data reveal new mechanisms by which resistin contributes to the development of atherosclerosis and indicate this cytokine as a novel therapeutic target.

Background and aims Atherosclerosis is characterized by an inflammatory process and a specific repertoire of chemokines and cell adhesion molecules expressed by endothelial cells (EC) that attract leukocytes to the developing atheroma. The inhibition of chemokine/chemokine receptor interactions and prevention of accumulation of pro-inflammatory cells in the vascular wall may constitute an important therapeutic option. Our aim was to follow in real-time the effect of chemokine receptor antagonists (CRA) on monocyte adhesion and transmigration through endothelial cell monolayer. Materials and Methods We used a method based on changes in electrical impedance to study in real-time the monocytes infiltration through endothelial cell monolayer. The endothelial cells (line EA.hy926) were cultured in 16-well e-plates covered with gold microelectrode that monitors and quantifies the infiltration of human monocytes (U937 cell line) in the presence/absence of CRA specific to chemokine receptors CCR2 (Teijin1 coumpound 1 and BMS CCR2) and CCR5 (maraviroc) and a broad-range CRA (pertussis toxin). Results The formation of EC monolayer was documented by the increase in electrical impedance in the first 24 hours of the experiments. The infiltration of monocytes, characterized by the opening of endothelial cell junctions, retraction of endothelial monolayer and replacement by monocytes determined a significant decrease in impedance. In contrast, the incubation of EC with monocytes in the presence of chemokine receptors antagonists lead to increases in impedance values indicating that monocytes infiltration was inhibited in the presence of CRA. The percentage of increases in impedance were calculated at different time points over three days and at the end of experiment the values were as follows: for Teijin compound 1 by ∼ 17%, for BMS CCR2 an maraviroc by ∼30% and for PTX by ∼ 20% Conclusion This novel technique monitors in real-time the effect of CRA on adhesion and transmigration of monocytes through endothelial monolayer. The advantages of this method in comparison with other methods (Boyden chamber, matrigel assay) are: 1) the endothelial cell-monocyte interaction mimics more closely the in vivo process, and 2) the data are obtained in real-time and are more reliable quantify as opposed to end-point analysis for other methods.

To study the roles played by cardiac valvular endothelium in normal and pathologic conditions, we have established and characterized a system of bovine valvular endothelial cells (VEC) in culture. Viable VEC from calf atrioventricular valves were obtained by a non-enzymatic procedure using 3 m m ethylenediamine-tetraacetic acid (EDTA) as dissociating agent. The cells grown in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, vitamins and 20% fetal calf serum, developed as monolayers of closely apposed polygonal cells which were subcultured for up to seven passages. VEC maintained in culture the general ultrastructure displayed in vivo , expressed von Willebrand factor, presented angiotensin converting enzyme activity and synthesized a rich extracellular matrix. VEC preserved the cell surface anionic sites (detected with cationized ferritin, pI 8.4) and cationic sites (visualized with haemeundecapeptide pI 4.85), and took up, especially by adsorptive endocytosis, albumin-gold conjugate. The cells were coupled by functional communicating (gap) junctions, as demonstrated by microinjection of 6-carboxyfluorescein. VEC in culture produced fibronectin, prostacyclin, hyaluronic acid and heparin-like glycosaminoglycans (identified by electrophoresis, enzyme digestion, and deaminative cleavage of molecules). These properties render cultured VEC a suitable model for investigating their functions and involvement in normal and pathologic heart valves.