Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction

Hemoglobin (Hb)-based O₂ carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (P_DH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing α-human and β-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)_n (molecular mass: ∼1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)_n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 × 10^-7 vs. 2.47 × 10^-6 cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of P_DH is also related to Hb size. When P_DH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.