To date, accumulation of hydroxyethyl starch (HES) has been studied mainly in skin specimens, but there are no detailed reports available regarding starch accumulation in the endothelium. Because endothelial cells play an essential role during shock, we studied the accumulation of HES in human umbilical venous endothelial cells (HUVEC). HUVEC (n = 9) were incubated with a fluorescein-conjugated HES 200/0.5 (FITC-HES) at 0.5–20 mg/ml for 1–72 h. FITC-HES was internalized dose- and time-dependently by pinocytosis into secondary lysosomes. Asymptotic elimination curves showed that 50% of the formerly ingested molecules could not be eliminated. Despite accumulation, starch molecules did not attenuate the expression of E-selectin, ICAM-1 or VCAM-1 on TNF-α-activated HUVEC. However, apart from adhesion molecule expression, perfusion studies showed that HES reduced neutrophil adhesion by direct inhibition of integrin-mediated interactions.

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 degrees C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 degrees C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non-activated monocytes are needed for adhesion assays under flow.

Despite up-regulated CD11b, integrin-mediated adhesion is markedly impaired in salvaged blood. Therefore, the effect of blood salvage cannot be predicted from cell surface expression but rather from functional assays. The former hypothesis, that leukocytes from SWB aggravate leukocyte-mediated tissue injury by increased adhesion, may not be as great a concern as previously suggested.

For the first time, a functional protection of HUEC in a model simulating physiologic vascular conditions by using nonviral transfection of the cells in a setup with high relevance for clinical applicability was demonstrated. Therefore, siRNA transfection of bypass material may develop into a new therapeutic option to improve the quality of venous graft material in the future.

Monocyte adhesion contributes to perfusion abnormalities, tissue damage, and activation of the coagulation system seen during trauma, shock, or overwhelming inflammation. This study was performed to determine whether an intravenous fish oil emulsion used for parenteral nutrition attenuates monocyte-endothelial interactions under flow and reduces procoagulant activity, measured as tissue factor (TF) expression on adherent monocytes in vitro. Endothelial cell monolayers were incubated with either an intravenous fish oil emulsion or a conventional omega-6 lipid emulsion at 0.05 to 1 mg/ml for 24 h. Six hours following activation with TNFalpha (25 ng/ml), expression of endothelial cell adhesion molecules was measured by flow cytometry. Adhesion of isolated monocytes to pretreated endothelium was examined in a parallel plate flow chamber at a shear stress of 1.5 dynes/cm2. Following perfusion, the cells were cocultured for an additional 4 h and TF expression on monocytes was determined by flow cytometry. In contrast to omega-6 lipids, fish oil down-regulated E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in a dose-dependent manner. P-selectin, however, remained unchanged. In addition, firm adhesion was reduced to 54%, whereas rolling interactions remained unchanged. Fish oil exhibited no effect on the TF expression on cocultured monocytes. We conclude that intravenous fish oil emulsions reduce both endothelial cell adhesion molecule expression and monocyte adhesion. However, under postcapillary flow conditions, rolling interactions via P-selectin remain unaltered. The functional importance of this effect is illustrated by the corresponding upregulation of TF in response to residual monocyte-endothelial interactions.