MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice

Abstract

Transgenic sickle mice expressing β(S) hemoglobin have activated vascular endothelium in multiple organs that exhibits enhanced expression of NF-ĸB and adhesion molecules and promotes microvascular stasis in sickle, but not normal, mice in response to hypoxia/reoxygenation (H/R), or heme. Induction of heme oxygenase-1 (HO-1) or administration of its products, carbon monoxide (CO) or biliverdin, inhibits microvascular stasis in sickle mice. Infusion of human hemoglobin conjugated with polyethylene glycol and saturated with CO (MP4CO) markedly induced hepatic HO-1 activity and inhibited NF-ĸB activation and H/R-induced microvascular stasis in sickle mice. These effects were mediated by CO; saline or MP4 saturated with O2 (MP4OX) had little to no effect on H/R-induced stasis, though unmodified oxyhemoglobin exacerbated stasis. The HO-1 inhibitor, tin protoporphyrin, blocked MP4CO protection, consistent with HO-1 involvement in the protection afforded by MP4CO. MP4CO also induced nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), an important transcriptional regulator of HO-1 and other antioxidant genes. In a heterozygous (hemoglobin-AS) sickle mouse model, intravenous hemin induced cardiovascular collapse and mortality within 120 minutes, which was significantly reduced by MP4CO, but not MP4OX. These data demonstrate that MP4CO induces cytoprotective Nrf2 and HO-1 and decreases NF-ĸB activation, microvascular stasis, and mortality in transgenic sickle mouse models.

Figures

Figure 1

Exhaled CO from NY1DD mice 24 hours prior to administration (baseline) and for 2 3-hour periods following intravenous MP4OX or MP4CO (12 mL/kg). Values are means + standard deviation (SD). N = 4 mice per treatment with the same mice used at each of the 3 time points. *P < .025 for MP4CO vs baseline.

Figure 2

Effect of treatment pre-H/R. LRS, MP4CO, MP4OX, or Hb (8 mL/kg) was infused into NY1DD sickle mice with implanted DSFCs 24 hours before H/R. Twenty-four hours after infusion, flowing venules in the subcutaneous skin were selected and mapped. Mice were then exposed to 1 hour of hypoxia (7% O2/93% N2) and returned to room air. After 1 hour of reoxygenation, the same mapped venules were re-examined for blood flow, and the percentage of static (no flow) venules was calculated. Hemin pretreatment (40 µmols/day IP × 3 days) is a positive control known to prevent stasis by induction of HO-1. Values are means + SD. N = 4 mice per treatment except hemin (n = 6). *P < .017 vs LRS.

Figure 3

Effect of treatment post-H/R. LRS, MP4CO, MP4OX, or Hb (8 mL/kg) was infused into NY1DD sickle mice with implanted DSFCs 30 minutes after H/R (1 hour hypoxia at 7% O2/93% N2 followed by 30 minutes in room air) during the reoxygenation phase. Microvascular stasis was measured after an additional 30 minutes in room air (1 hour reoxygenation total) as described in Figure 2. The effect of pre-H/R + post-H/R treatment is shown on the right (×2 IV) where MP4CO or MP4OX was infused 24 hours before H/R and 30 minutes after H/R. Values are means + SD. N = 4 mice per treatment. *P < .017 vs LRS; #P < .026 MP4CO vs MP4OX.

Figure 4

NY1DD sickle mice with implanted DSFCs were infused with LRS, MP4CO, MP4OX, or Hb (8 mL/kg) 24 hours prehypoxia or 30 minutes post-H/R. Livers were removed in all cases after 1 hour of hypoxia and 4 hours of reoxygenation. Thus, livers were removed 29 hours and 3.5 hours postinfusion as indicated. (A) Western blot of liver nuclear NF-ĸB phospho-p65 29 hours postinfusion. (B) Western blot of liver nuclear Nrf2 expression 29 hours postinfusion. (C) Western blot of liver microsomal HO-1 protein expression 3.5 hours and 29 hours postinfusion. (D) Liver microsomal HO activity 3.5 hours and 29 hours postinfusion and after treatment with both MP4CO (29 hours postinfusion) and pretreatment with HO inhibitor SnPP (40 µmols/kg/d × 3 days, IP). N = 4 mice/group. *P < .025. (E) Microvascular stasis after 1 hour of hypoxia and 1 hour of reoxygenation in mice treated with LRS (IP × 3 days) + LRS (IV, 8 mL/kg, 24 hours prehypoxia), LRS (IP × 3 days) + MP4CO (IV, 8 mL/kg, 24 hours prehypoxia), or SnPP (IP, 40 µmols/kg/d × 3 days) + MP4CO (IV, 8 mL/kg, 24 hours prehypoxia). N = 3 mice/group. *P < .025 LRS (IP) + MP4CO (IV) vs LRS (IP) + LRS (IV).

Figure 5

Effect of treatment on MAP and survival after hemin infusion into HbAS mice. (A) MAP in heterozygous HbAS mice infused with hemin followed by saline (n = 12, solid squares), MP4CO (n = 6, gray circles), or MP4OX (n = 6, open triangles). Values are means ± SD. *P < .016 vs saline. (B) Survival plot of HbAS mice infused with hemin followed by an 8 mL/kg infusion of saline (solid squares), MP4CO (gray circles), or MP4OX (open triangles). Arrows indicate the point of hemin administration and the duration of infusion of saline, MP4CO, or MP4OX. Values are fraction alive at indicated time points. *P < .016 vs saline.