Background: The impact on the endothelial glycocalyx for the extravasation of colloidal infusion solutions has not been investigated sufficiently. Methods: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer in a Langendorff mode. Solutions of 0.9% saline, 5% albumin (70 kd), or 6% hydroxyethyl starch (200 kd) were infused into the coronary system for 20 min at a rate of one third of the coronary flow, also during reperfusion after 15 min of ischemia, and after enzymatic digestion of the endothelial glycocalyx by heparinase. Net coronary fluid filtration was assessed directly by measuring the formation of transudate on the epicardial surface, and solute extravasation was assessed by measuring albumin and hydroxyethyl starch in the coronary effluent and transudate. Hearts were perfusion fixed to visualize the endothelial glycocalyx using transmission electron microscopy. Results: Only infusion of hydroxyethyl starch, not infusion of albumin, significantly decreased net coronary fluid filtration. Heparinase application without ischemia increased coronary leak by 25% but did not accelerate the passage of colloids. Ischemia alone did not alter permeability. However, there was a large (approximately +200%), transient (approximately 4 min) increase in permeability for water, albumin, and hydroxyethyl starch after ischemia with heparinase application. Also, histamine (10 m) only increased permeability after pretreatment of the hearts with heparinase. The thickness of the glycocalyx after colloid administration was 0.2-0.3 microm. No glycocalyx could be detected after application of heparinase. Conclusion: The endothelial glycocalyx acts as a competent barrier for water and colloids. Only after its destruction do changes in endothelial morphology (postischemic reperfusion or histamine application) become effective determinants of coronary extravasation.

Platelets, although not phagocytotic, have been suggested to release O$2−$⁠. Since O$2−$-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O$2−$⁠. We further studied which agonists cause platelet O$2−$release and whether platelet-derived O$2−$ influences thrombus formation in vitro. Collagen, but not adenosine 5′-diphosphate (ADP) or thrombin, increased O$2−$ formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)–dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47^phox and p67^phoxand inhibition of platelet O$2−$ formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O$2−$ did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O$2−$increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase–dependent O$2−$ release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.

Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the method's efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.