Hemoglobin-induced lung vascular oxidation, inflammation, and remodeling contribute to the progression of hypoxic pulmonary hypertension and is attenuated in rats with repeated-dose haptoglobin administration

Abstract

Haptoglobin (Hp) is an approved treatment in Japan for trauma, burns, and massive transfusion-related hemolysis. Additional case reports suggest uses in other acute hemolytic events that lead to acute kidney injury. However, Hp's protective effects on the pulmonary vasculature have not been evaluated within the context of mitigating the consequences of chronic hemoglobin (Hb) exposure in the progression of pulmonary hypertension (PH) secondary to hemolytic diseases. This study was performed to assess the utility of chronic Hp therapy in a preclinical model of Hb and hypoxia-mediated PH. Rats were simultaneously exposed to chronic Hb infusion (35 mg per day) and hypobaric hypoxia for 5 weeks in the presence or absence of Hp treatment (90 mg/kg twice a week). Hp inhibited the Hb plus hypoxia-mediated nonheme iron accumulation in lung and heart tissue, pulmonary vascular inflammation and resistance, and right-ventricular hypertrophy, which suggests a positive impact on impeding the progression of PH. In addition, Hp therapy was associated with a reduction in critical mediators of PH, including lung adventitial macrophage population and endothelial ICAM-1 expression. By preventing Hb-mediated pathology, Hp infusions: (1) demonstrate a critical role for Hb in vascular remodeling associated with hypoxia and (2) suggest a novel therapy for chronic hemolysis-associated PH.

Keywords: Free radicals; Heme; Hemoglobin; Hemolysis; Iron; Lipid peroxidation; Sickle cell disease; Thalassemia.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1. Infusion pump, plasma analysis of Hb-Hp complex

(A) Percentages of free and Hp bound Hb over 34 days of Hb infusion. (B) Chromatography of plasma from representative Hb exposed and Hp administered animals demonstrated a free Hb peak at an elution time of 21 minutes. This was confirmed by the chromatographic profile of Hb standard solution shown as a dotted red trace. An elution time shift to a broad band of peaks eluting over 13-17.5 minutes was observed as extra-cellular plasma Hb became bound to Hp. Hb- hemoglobin, Hp-haptoglobin.

Figure 2. Lung iron accumulation

Localization of non-heme iron in the lungs of either normoxic control animals or animals exposed to free Hb infusion plus hypoxia in the presence of absence of Hp. * p

Figure 3. Quantification and localization of HO-1 lung expressionan

(A) Western Blot analyses of lung HO-1 from densitometry analysis. (B) qRT-PCR analysis of whole lung CD163 mRNA . (C) Representative microphotographs of stained lung sections for HO-1 localization. 40X are high magnification of white boxed areas.HO-1 expression- (red), CD163 (green) and DAPI-. L- vessel lumen. White arrows show areas of HO-1 and CD163 co-expression. * p< 0.04 Hb+HX vs. all other groups. NX- normoxia. HX-hypoxia. Hb- hemoglobin. Hp- haptoglobin.

Figure 4. Lung vascular 4-hydroxynonenal (4-HNE)

Microphotographs of stained lung sections of 4-hydroxynonenal localization. Pictures captured at 40X are high magnification of the vessels in the white box captured at 10x. M- media. L-lumen. i- Intima. SMA- smooth muscle actin. 4-HNE- 4-hydroxynonenal. NX- Normoxia. HX-Hypoxia. Hp- Haptoglobin. Hb-Hemoglobin

Figure 5. Hb induced lipid peroxidation in rat pulmonary artery endothelial cells exposed to hypoxia

(A) photomicrographs that show areas of lipid perioxidation. (B) Image J quantification of lipid peroxidation. Green lipid peroxidation- (White arrows). HX-Hypoxia. Hb-Hemoglobin.Hp-Haptoglobin. * p

Figure 6. Quantification and localization of ICAM-1 lung expressionan and plasma IL-6 concentration

(A) Whole lung ICAM-1 protein expression. (B) Plasma IL-6 concentration. (C and D) Representative microphotographs of stained lung sections showing ICAM expression in the endothelium and extravascular cells respectively. White boxes show the regions that 40X microphotographs were captured. ICAM expression- (red), Factor VIII (green) and DAPI.White arrows show regions of ICAM expression. * p< 0.04 Hb+HX vs. HX and Hp+HX groups. NX- normoxia. HX- hypoxia. Hb- hemoglobin. Hp- haptoglobin.

Figure 7. Hb mediated pulmonary vascular remodeling is attenuated with Hp therapy

(A) Quantification of muscularization of small arteries. (B) Quantification of medial hypertrophy of pulmonary arteries. (C) Microphotographs of lungs stained with -smooth muscle actin or hematoxylin and eosin (H&E) to visualize small arterial muscularization, medial hypertrophy and accumulation of cells in the adventitia compartment. White arrows show muscularized small arteries, black arrows show the accumulation of cells in the advanticia surrounding the vessel. * p < 0.044 Hb+HX vs. all other groups (one tailed test). ** p < 0.04 Hb+HX vs. all other groups. NX- normoxia. HX-hypoxia. Hb- hemoglobin. Hp- haptoglobin.

Figure 8. Hemodynamic characterization of pulmonary arterial hypertension

(A) Cardiac output. (B) Pulmonary arterial pressures. (C) Pulmonary vascular resistance. (E) Fulton index of right ventricular hypertrophy. * p

Figure 9. Heart iron accumulation

Localization of non-heme iron in the cardiac tissue of either NX control animals or animals exposed to free Hb infusion plus HX in the presence of absence of Hp. HX-hypoxia, Hb- hemolglobin, Hp- haptoglobin.