Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin

Abstract

The heme oxygenase (HO) enzymes catalyze the rate-limiting step of heme breakdown. Prior studies have demonstrated that the vulnerability of neurons and astrocytes to hemoglobin is modified in cells lacking HO-2, the constitutive isoform. The present study assessed the effect of the inducible isoform, HO-1. Wild-type astrocytes treated for 3-5 days with 3-30 microM hemoglobin sustained no loss of viability, as quantified by LDH and MTT assays. The same treatment resulted in death of 25-50% of HO-1 knockout astrocytes, and a 4-fold increase in protein oxidation. Cell injury was attenuated by transfer of the HO-1 gene, but not by bilirubin, the antioxidant heme breakdown product. Conversely, neuronal protein oxidation and cell death after hemoglobin exposure were similar in wild-type and HO-1 knockout cultures. These results suggest that HO-1 induction protects astrocytes from the oxidative toxicity of Hb, but has no effect on neuronal injury.

Fig. 1

Effect of HO-1 gene deletion on vulnerability of astrocytes and neurons to Hb. A) Mean medium LDH (± S.E.M., 14–20/condition) in astrocyte cultures treated for 75–120 h with indicated concentrations of Hb. LDH values were scaled to those in sister cultures treated with 0.3% Triton X-100 (= 100), which produces 100% cell lysis. B) Mixed neuron/astrocyte cultures (27–31/condition) were treated with indicated Hb concentrations for 24h. LDH values were scaled to those in sister cultures treated with 300 μM NMDA, which releases all neuronal LDH without injuring astrocytes. *** P < 0.001 versus signal in corresponding WT culture, Bonferroni multiple comparisons test.

Fig. 2

Hb-induced protein oxidation in HO-1 knockout astrocytes and neurons. Bars represent mean carbonyl signal intensities (±S.E.M., 4/condition) from cultures treated with Hb 10 μM for 75h (astrocytes) or 24h (mixed), or subjected to media exchange only (wash). *** P < 0.001 versus signal in HO-1 knockout cultures treated with Hb.

Fig. 3

HO-1 gene transfer protects HO-1 knockout astrocytes from Hb. Cultures (12/condition) were pretreated for 24h with 100 MOI of adenovirus encoding the human HO-1 gene (HO-1-Hb), control virus (Null-Hb), or culture medium only, then incubated with 10 μM Hb for 75h and 120h. LDH values were scaled to those in sister cultures treated with 0.3% Triton X-100 for 1 h (=100). ***P<0.001 versus cultures exposed to Hb with control virus pretreatment or without any virus treatment.