Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice

Abstract

Transgenic sickle mice expressing betaS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-kappaB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation-induced stasis, leukocyte-endothelium interactions, and NF-kappaB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-kappaB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation-induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice.

Figure 1

HO-1 expression is elevated in the organs of sickle mice. Western blots for HO-1 were performed on organ homogenates (1 μg of organ DNA per lane) from lungs, livers, and spleens of untreated normal, S+S-Antilles, and BERK mice. (A) The 32-kDa HO-1 bands are shown for each organ and each mouse. (B) The mean HO-1 band intensities (n = 4) ± SD are expressed as a percentage of those in normal control mice. *P < 0.05, normal versus sickle.

Figure 2

Hemin increases HO-1 expression. (A) HO-1 expression can be further upregulated in the organs of sickle mice with hemin treatment. S+S-Antilles mice were either untreated or injected with hemin (40 μmol/kg/d, i.p.) for 3 days. Twenty-four hours after the third injection, the organs were harvested, and Western blots for HO-1 were performed on lung, liver, and spleen homogenates (1 μg of organ homogenate DNA per lane). The mean HO-1 band intensities (n = 3) ± SD are expressed as a percentage of those in untreated S+S-Antilles mice (100%), which represent the same untreated S+S-Antilles organs shown in Figure 1. Below each bar is a representative HO-1 band from the Western blot. *P < 0.05, untreated versus hemin. (B) HO-1 activity in normal and S+S-Antilles livers. HO-1 activity was measured in microsomes isolated from another group of normal and S+S-Antilles sickle mice as previously described (36). Mice were untreated, injected with hemin (40 μmol/kg/d, i.p.) for 3 days, or injected with hemin plus SnPP (40 μmol/kg/d of each porphyrin, i.p.) for 3 days. Twenty-four hours after the third injection, the livers were harvested, microsomes were isolated at 105,000 g, and HO-1 enzymatic activity was measured. The results in triplicate are expressed as mean ± SEM picomoles of bilirubin generated per milligram microsomal protein per hour. *P < 0.05, normal versus sickle.

Figure 3

Further upregulation of HO-1 by hemin inhibits stasis and HO-1 inhibition by SnPP exacerbates stasis in sickle mice. S+S-Antilles (A) and BERK (B) mice with an implanted DSFC were untreated, injected with hemin (40 μmol/kg/d, i.p.) for 3 days, or injected with SnPP (40 μmol/kg/d, i.p.) for 3 days. Twenty-four hours after the third injection, stasis was measured after 1 hour of hypoxia (7% O₂/93% N₂) and 1 hour and 4 hours of reoxygenation in room air. n = 3–10 mice and a minimum of 20 venules per mouse. *P < 0.05, untreated versus hemin or SnPP. The proportions of venules exhibiting stasis at each time point were compared using a z test.

Figure 4

CO and biliverdin inhibit stasis in sickle mice. S+S-Antilles mice with an implanted DSFC were either untreated or treated with inhaled CO (250 ppm CO in air for 1 hour per day) for 3 days or biliverdin injections (50 μmol/kg i.p. twice, at 16 hours and 2 hours, before hypoxia). Twenty-four hours after the third CO treatment or 2 hours after the second biliverdin injection, stasis was measured after 1 hour of hypoxia (7% O₂/93% N₂) and 1 hour of reoxygenation in room air. n = 3–10 mice and a minimum of 20 venules per mouse. *P < 0.05, untreated versus CO or biliverdin. The proportions of venules exhibiting stasis at each time point were compared using a z test.

Figure 5

Further upregulation of HO-1 by hemin inhibits hypoxia/reoxygenation–induced increases in leukocyte-endothelium interactions. S+S-Antilles mice with an implanted DSFC were treated with either placebo (saline) or hemin injections (40 μmol/kg/d, i.p.) for 3 days. Twenty-four hours after the third injection, leukocyte rolling and adhesion were measured in the subcutaneous venules at base line in ambient air and again in the same venules after exposure of the mice to 1 hour of hypoxia (7% O₂/93% N₂) and 1 hour of reoxygenation in room air. Results are mean ± SEM percentage change in leukocyte rolling and adhesion after hypoxia/reoxygenation. n = 2 mice and a minimum of 20 venules per group. *P < 0.05, placebo versus hemin.

Figure 6

Further upregulation of HO-1 by hemin inhibits NF-κB activation and VCAM-1 and ICAM-1 overexpression in the organs of sickle mice. S+S-Antilles mice were either untreated or injected with hemin (40 μmol/kg/d, i.p.) for 3 days. Twenty-four hours after the third injection, the organs were harvested from mice in ambient air. NF-κB activation was measured by EMSA, and VCAM-1 and ICAM-1 expression was measured by Western blotting in organ homogenates of the lungs, liver, and spleen of sickle mice. (A) The NF-κB, VCAM-1, and ICAM-1 bands are shown for each organ and each sickle mouse. (B) The bar graph shows the mean band intensity (n = 3 mice per group) ± SD for each organ treatment group. *P < 0.05, untreated versus hemin.

Figure 7

Biliverdin or CO treatment inhibits NF-κB activation in the lungs of sickle mice. S+S-Antilles mice were untreated, treated with biliverdin injections (50 μmol/kg i.p. twice, at 16 hours and 2 hours), or treated with inhaled CO (250 ppm in air for 1 hour per day for 3 days). Two hours after the second biliverdin injection or 24 hours after the third CO treatment, mice were exposed to 3 hours of hypoxia (7% O₂/93% N₂) and 2 hours of reoxygenation in room air. After 2 hours of reoxygenation, the lungs were harvested, and NF-κB activation was measured in organ homogenates by EMSA. n = 3 mice per group. Below each bar is a representative NF-κB band from the EMSA. *P < 0.05, untreated versus biliverdin or CO.

Figure 8

HO-1-ADV increases HO-1 expression and inhibits stasis. Local administration of HO-1-ADV increases HO-1 expression (A) and inhibits hypoxia/reoxygenation–induced stasis (B) in the skin. S+S-Antilles sickle mice with an implanted DSFC were treated with either a rat HO-1-ADV construct (n = 3 mice and 84 venules) or an empty Control-ADV construct (n = 4 mice and 64 venules). The adenovirus constructs (2 × 10⁷ MOI) in sterile saline were dripped onto the subcutaneous skin inside the DSFC. Forty-eight hours after adenovirus treatment, hypoxia/reoxygenation–induced stasis was measured (B). After measurement of stasis, the skin inside the DSFC window was harvested, and HO-1 expression was measured in the skin homogenates by Western blotting (A). Below each bar is a representative HO-1 band from the Western blot. *P < 0.05, Control-ADV versus HO-1-ADV.