In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1-2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-kappaB nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 microM), and by bilirubin (1 microM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium.

Heme oxygenase (HO) represents an intrinsic antiinflammatory system based on its ability to regulate leukocyte function and inhibit expression of proinflammatory cytokines. This anti-inflammatory function is linked to the inducible isoform HO-1; the role of the constitutive isoform HO-2 is unknown. The current study was undertaken to investigate the role of HO-2 in the regulation of the acute inflammatory and reparative response by using HO-2-null mice and well-established animal models of epithelial injury and antigen-induced peritonitis. Here we show that in vivo deletion of HO-2 disables execution of the acute inflammatory and reparative response after epithelial injury and leads to an exaggerated inflammatory response in antigen-induced peritonitis. HO-2 deletion was associated with impaired HO-1 induction, indicating that HO-2 is critical for HO-1 expression and that the subsequent failure to up-regulate the HO system may contribute to unresolved inflammation and the development of chronic inflammatory conditions. Indeed, supplementation with the HO bioactive product, biliverdin, rescued the acute inflammatory and reparative response in HO-2-null mice. Thus, HO-2 sets in place a basal tone of anti-inflammatory signals that may be a prerequisite for the ordered execution of an inflammatory and reparative response.

Hemin is released from hemoglobin after CNS hemorrhage and is present at high micromolar concentrations in intracranial hematomas. This highly reactive compound is potentially cytotoxic via a variety of oxidative and nonoxidative mechanisms. However, despite its clinical relevance, little is known of its effect on neuronal cells. In this study, we tested the hypotheses that hemin is toxic to human neurons at physiologically relevant concentrations and that its toxicity is iron dependent and oxidative. A homogeneous population of neuron-like cells was produced by sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor, using the protocol of Encinas et al. Hemin exposure for 24 hr resulted in cell death that progressively increased between 3 and 30 microM (EC(50) approximately 10 microM); protoporphyrin IX, the iron-free congener of hemin, was not toxic. Cell death commenced at 14 hr and was preceded by a marked increase in cellular reactive oxygen species (ROS). Most injury and ROS production were prevented by concomitant treatment with an equimolar concentration of the lipid-soluble iron chelator phenanthroline; the water-soluble chelator deferoxamine was also effective at concentrations of 0.1 mM or higher. Heme oxygenase-2 was constitutively expressed by these cells, and heme oxygenase-1 was induced by hemin. Heme oxygenase inhibition attenuated ROS generation and reduced injury by about one-third. Cell death was also prevented with the sulfhydryl reducing agents glutathione and mercaptoethanol. Nuclear morphology in the hours prior to cell lysis revealed a predominantly homogenous staining pattern; the percentage of fragmented nuclei was increased only at 4 hr and then accounted for only 1.45% +/- 0.25% of cells. The general caspase inhibitor zVAD-fmk had no effect on cell viability. These results suggest that hemin is toxic to human neuron-like cells at concentrations that are less than 3% of those observed in intracranial hematomas. In this model, its toxicity is iron dependent, oxidative, and predominantly necrotic.

Tumor necrosis factor-alpha (TNF-alpha) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF-alpha-induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF-alpha alone, or with 10 microg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-kappaB, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF-alpha did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF-alpha than did wild-type mice. TNF-alpha increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF-alpha response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-alpha. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF-alpha did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF-alpha-induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation.

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