A need exists for an alternative oxygen carrier to blood for transfusion. Whole blood has the disadvantage that transfusion may facilitate transmission of infectious agents such as HIV and hepatitis viruses. Although the development of stable hemoglobin (Hb)-based oxygen carriers has progressed, unexpected toxicities have arisen in clinical trials. These effects are believed to stem from the interactions of the carrier molecules with vascular endothelium and their subsequent extravasation into the surrounding tissue. If the events leading to extravasation could be inhibited, then the occurrence of toxic side-effects would be greatly reduced. The long-term aim of the proposed research is to identify a Hb-based blood substitute which remains in the circulation for an appropriate time period and does not alter capillary permeability. If exchange is altered by Hb- based oxygen carriers, not only will passage of nutrients, drugs, hormones and enzymes be compromised, but the blood substitute itself may extravasate, enter the interstitium and produce toxic effects. The immediate goal of this proposal is to examine the effects of blood substitutes on microvascular permeability, through intravital microscopy of an in situ preparation of rat intestinal mucosa. The intestine is one of the principal sites of transvascular exchange in the body. Human stroma-free Hb (SFHb) and polyethylene glycol (PEG)-conjugated bovine Hb (Enzon, Inc.) will be used, both in free form and encapsulated into artificial vesicles. The kinetics of extravasation of SFHb and PEG-Hb will be determined and their effects on transcapillary passage of bovine serum albumin (BSA) will be measured using epifluorescence microscopy and computer analysis of video images. In addition, the predominant transendothelial pathways taken by SFHb, PEG-Hb and BSA will be determined using immunogold labelling and electron microscopy. The following hypothesis will be tested: Hemoglobin and PEG-Hb interact with intestinal capillary endothelium to disrupt endothelial actin cytoskeleton, increase the density of fenestrae (diaphragm-covered, 70 nm pores through endothelium) and alter the electrostatic charge on the endothelial surface, leading to increased permeability. Endothelial cell actin fibers will be stained for confocal microscopy using fluorescent phalloidin. Fluorescent and electron-dense anionic and cationic tracers will be used to evaluate changes in positive and negative surface charge densities. One possible way of inhibiting Hb-induced changes in endothelial permeability will be pursued. Hemoglobin has a strong affinity for nitric oxide, which affects endothelial permeability. Nitric oxide donors will therefore be included in perfusates containing Hb-based carriers to determine whether capillary permeability is reduced.
