Apohemoglobin‐haptoglobin (Apohb‐Hp) Complex as a Vasculo‐protective Agent in Hypoxia Induced Vaso‐occlusion in Sickle Cell Disease

Abstract

Sickle Cell Disease is associated with episodes of vaso‐occlusive crises (VOCs) caused by hemoglobin S (HbS) polymerization and resultant microvascular endothelial dysfunction due to hemolysis, inflammation, defects in nitric oxide metabolism and oxidative stress. In the acute setting, care for VOC involves supportive care including hydration and aggressive pain management. There are no specific therapies known to alter the course of the disease during an acute VOC. Apohemoglobin‐haptoglobin (Apohb‐Hp) complex is a two‐protein formulation that scavenges free hemoglobin (Hb) as well as free heme and has been shown to have a role in alleviating vasoconstriction and hypertension induced by cell free Hb1. The objective of this study was to evaluate microcirculatory effects of Apohb‐Hp during hypoxia induced vaso occlusion in sickle cell mice.We used intravital microscopy on the dorsal skin fold to observe the microcirculatory effects of Apo Hb‐Hp, haptoglobin (Hp), vehicle control in HbSS‐Townes mice (6‐8 week and ~25 g) and Hb AA mice. Each group consisted of 11 mice, they were initially kept in normal conditions (21% O2) for an hour followed by a hypoxic state (7% O2) for another hour and finally brought back to normal conditions (21% O2) for an hour before being sacrificed. In some cases, some mice were followed for 72 hours before being sacrificed. ApoHb‐Hp and Hp were injected intravenously at a concentration of 59 mg/mL and 86 mg/mL respectively at a single dosing quantity of 150 mg/kg. The Hb binding capacity of Apohb‐Hp was 15 mg/mL and the heme binding capacity was 0.5 mg/mL, the Hb binding capacity of Hp was 30 mg/mL with no heme binding capacity. During baseline, hypoxia and normoxia systemic parameters (mean arteriole pressure and heart rate), blood gases (pO2,A, pO2,v, sO2,A­, sO2,v), hematological properties (total Hb concentration, hematocrit, and concentration of lactate) and microcirculatory hemodynamics (velocity, diameter, flow and functional capillary density) were measured. After the mice were sacrificed a count of present macrophages, neutrophiles and lymphocytes were quantified in the plasmaThere was no notable changes in blood gas concentrations with any of the treatments. Apohb‐hp treatment treated animals showed an improvement in microcirculatory dynamics. Apohb‐Hp treated animals showed a statically higher diameter in arterioles (<40 um and >40 um) as well as venules with a diameter between 20‐60 um when compared to controls and hp treated mice, suggesting that treatment prevented vasoconstriction in arterioles. Apohb‐hp treatment also improved flow rates in arterioles and venules when compared to the control and hp treated mice. Furthermore, the functional capillary density of the mice followed for 72 hours after hypoxia showed that the mice treated with apohb‐hp had a greater number of functional capillaries compared to the control and hp treated group. Lastly, the amount macrophages, neutrophiles and lymphocytes decreased when treated with apohb‐hp illustrating another promising result as VOCs are generally associated with an active immune response.These results suggest a role for ApoHb‐Hp complex in alleviating microvascular changes occurring during hypoxia mediated acute vaso‐occlusion in SCD and merits further study for development as a potential therapeutic.