Examining and mitigating acellular hemoglobin vasoactivity

Abstract

Significance: There has been a striking advancement in our understanding of red cell substitutes over the past decade. Although regulatory oversight has influenced many aspects of product development in this period, those who have approached the demonstration of efficacy of red cell substitutes have failed to understand their implication at the level of the microcirculation, where blood interacts closely with tissue.

Recent advances: The understanding of the adverse effects of acellular hemoglobin (Hb)-based oxygen carriers (HBOCs) has fortunately expanded from Hb-induced renal toxicity to a more complete list of biochemical mechanism. In addition, various unexpected adverse reactions were seen in early clinical studies. The effects of the presence of acellular Hb in plasma are relatively unique because of the convergence of mechanical and biochemical natures.

Critical issues: Controlling the variables using genetic engineering and chemical modification to change specific characteristics of the Hb molecule may allow for solving the complex multivariate problems of acellular Hb vasoactivity. HBOCs may never be rendered free of negative effects; however, quantifying the nature and extent of microvascular complications establishes a platform for designing new ameliorative therapies.

Future directions: It is time to leave behind the study of vasoactivity and toxicity based on bench-top measurements of biochemical changes and those based solely on systemic parameters in vivo, and move to a more holistic analysis of the mechanisms creating the problems, complemented with meaningful studies of efficacy.

FIG. 1.

Mechanistic analysis of Hb based transfusion associate vascular dysfunction. In physiological condition (panel A), blood flow provides oxygen and mechanical signals (τ, wall shear stress) to the vasculature, which serve to self-regulate local blood flow. After severe blood losses, anemia or any condition that originates a need for blood transfusion (panel B), oxygenation and vascular shear stress are compromised, thus the local vascular regulatory processes become impaired. Consequently, when the blood transfusions are given or a HBOC is infused (panel C), they do not achieve their intended goal; because they only increase oxygen carrying capacity without restoring blood flow. Even following perfusion recovery, vascular regulatory processes are not fully functional, such as endothelial NO synthase, which becomes uncoupled and instead of generating NO produces superoxide, contributing to oxidative stress, and triggering an inflammatory cascade. The additive role of oxidative and nitrosative stress results in increased NO release from inducible NO synthase (iNOS) in perivascular tissue and from macrophages/monocytes. The NO generated by iNOS is metabolized to peroxynitrite, which is cytotoxic to tissues. Within panels A, B and C: The flow arrow shows blood flow direction. A, RBC column; B, cell-free layer (CFL) width; C, endothelial cells; and D, tissue space.

FIG. 2.

Hb structures, auto-oxidation products, and toxicity. Hb is safely contained within the RBCs, although it appears in the plasma, when the RBCs rupture (hemolysis), or when HBOCs are infused. All Hb chains contain an Fe-containing protoporphyrin IX group, which binds molecular oxygen. Cell-free Hb auto-oxidizes, leading to formation of reactive oxygen species (ROS) and degradation products as heme and methemoglobin (metHb). All of these molecules are highly reactive and have the ability to damage lipids, protein, and DNA, and are therefore proinflammatory, pro-oxidant, and toxic. Furthermore, Hb also binds and reacts with NO, thereby affecting the vasomotor tone. Thus, hemolysis and elevated extracellular Hb concentrations are associated with vasoconstriction and hypertension. Finally, the metabolites formed by the Hb degradation are hemolytic, and thus oxidative stress accelerates the hemolysis, in a positive feedback loop.

FIG. 3.

Molecular weight and hydrodynamic radius of HBOCs. Schematic representation of Hb, polyethylene glycol-conjugated Hbs (PEG-Hb), encapsulated Hb vesicles (HbV), and polymerized Hb (PolyHb, large, and small) relative to RBCs. The size/molecular mass of the HBOC molecules have a direct impact on their proximity to the vascular endothelium and their molecular diffusivity, which determine NO scavenging and vessel wall oxygen oversupply. In the absence of plasma Hb in the circulation, the plasma layer adjacent to the vessel wall limits the NO scavenging by Hb in the RBCs; however, after infusion of HBOC, the plasma Hb becomes a sink for NO. Large HBOCs have a high solution viscosity relative to smaller HBOCs; thus, they increased plasma viscosities when infused, leading to a hyperviscous oxygen carrier. An increase in plasma viscosity directly increases vessel WSS and endothelial cell mechanotransduction.

FIG. 4.

Schematic representation of haptoglobin (Hp)-Hb complex formation and endocytosis mediated by CD163 receptor. Hp is synthesized predominantly in the liver. If plasma Hb is released into the circulation, it binds immediately to Hp to form an Hp-Hb complex, which subsequently is removed by the CD163 receptor expressed mainly on monocytes, macrophages, and Kupffer cells in the liver. The binding between Hp and Hb is among the strongest noncovalent interactions known, with very slow dissociation rates. When hemolysis depletes Hp, Hb accumulates in the kidney and is secreted in the urine. Hp can offer an effective protection against kidney damage. The Hb-Hp complex is not filtrated by the kidney, and instead directs Hb to CD163 on macrophages for a process of endocytosis and final degradation. Within macrophages, the heme oxygenase enzyme breaks down the heme group of Hb into bilirubin and CO, which both have been shown to be an antioxidant and vasodilator.

FIG. 5.

Intravascular NO delivery based on nanoparticles (NO-releasing nanoparticles [NOnps]). NOnps are a hydrogel precursor in conjunction with a glass-forming (glass in the context of a solid comprised of an amorphous network of hydrogen-bonded elements) combination of chitosan and polyethylene glycol to form a fine powder (25). This material retains NO in a stable form when dry, and release NO upon exposure to moisture. NOnps have shown a high efficacy in relaxing blood vessel in vivo compared to NONOates (25). About 30 times less NO is required from NOnps compared to NONOates to produce similar dilation when applied intravascularly. In addition, the effects of NOnps were sustained longer compared to NONOates that only lasted minutes. The higher doses of NO required by NONOate significantly increase metHb levels and decrease the oxygen-carrying capacity (25).