Liposome encapsulation attenuates hemoglobin-induced vasoconstriction in rabbit arterial segments

Free hemoglobin (Hb) induces a potent vasoconstrictor response that may limit its therapeutic application as a red blood cell replacement. We have investigated whether encapsulation of stroma-free Hb (SFHb) or cross-linked Hb (alpha alpha-Hb) in liposomes modulates Hb vasoactivity in isolated blood vessels. Relaxation of rabbit thoracic vessels was measured before and after exposure to acellular SFHb, alpha alpha-Hb, and liposome-encapsulated SFHb or alpha alpha-Hb. SFHb and alpha alpha-Hb caused significant inhibition of carbachol-induced relaxation at 0.5 mg/dl, whereas encapsulation inhibited vessel relaxation at 30- to 60-fold higher Hb concentrations. The contractile response of rabbit ear arterial segments to electrical stimulation in the presence of acellular alpha alpha-Hb resulted in a 150% increase (EC150) in contractile amplitude at 0.23 mg/dl, whereas the EC150 for encapsulated alpha alpha-Hb was 13.7 mg/dl. Mechanistic studies of the vasoconstrictor activity of Hb demonstrated that acellular alpha alpha-Hb had no effect on norepinephrine release in the rabbit ear artery. In addition, neither acellular nor encapsulated alpha alpha-Hb preparations inhibited endothelial nitric oxide (NO) synthase activity isolated from bovine pulmonary artery. However, inhibition of vessel relaxation by acellular or encapsulated alpha alpha-Hb was reversed by the NO donor S-nitrosylpenacillamine, implicating Hb-NO binding as a possible mechanism for the vasoconstrictor response. In vitro stopped-flow kinetic studies of Hb-NO binding showed similar rates of reaction for conversion of oxyhemoglobin to methemoglobin (metHb; < 2 ms), followed by rapid conversion of metHb to NO-Hb (300 ms) for both acellular and encapsulated alpha alpha-Hb, demonstrating that liposome encapsulation does not retard NO-Hb binding. The attenuated vasoactivity of encapsulated Hb may, therefore, result from the limited access of encapsulated Hb to NO imposed by the physical size of the liposome and reduced penetration of Hb across the vascular endothelium.