Heme oxygenase-2 protects against ischemic acute kidney injury: influence of age and sex.

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2+/+ and HO-2-/- mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2+/+ and HO-2-/- mice, between young female HO-2+/+ and HO-2-/- mice, or between aged female HO-2+/+ and HO-2-/- mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2-/- mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2-/- mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2-/- mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and ß-catenin was accentuated in aged male HO-2-/- mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

Deletion of the hemopexin or heme oxygenase-2 gene aggravates brain injury following stroma-free hemoglobin-induced intracerebral hemorrhage.

BACKGROUND: Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to heme and participates in its transport, while heme oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes. METHODS: Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induce ICH. Hpx knockout (Hpx(-/-)) or HO2 knockout (HO2(-/-)) mice were injected with 10 µL of SFHb in the striatum. After injection, behavioral/functional tests were performed, along with anatomical analyses. Iron deposition and neuronal degeneration were depicted by Perls' and Fluoro-Jade B staining, respectively. Immunohistochemistry with anti-ionized calcium-binding adapter protein 1 (Iba1) was used to estimate activated microglial cells around the injured site. RESULTS: This study shows that deleting Hpx or HO2 aggravated SFHb-induced brain injury. Compared to wild-type littermates, larger lesion volumes were observed in Hpx(-/-) and HO2(-/-) mice, which also bear more degenerating neurons in the peri-lesion area 24 h postinjection. Fewer Iba1-positive microglial cells were detected at the peri-lesion area in Hpx(-/-) and HO2(-/-) mice, interestingly, which is associated with markedly increased iron-positive microglial cells. Moreover, the Iba1-positive microglial cells increased from 24 to 72 h postinjection and were accompanied with improved neurologic deficits in Hpx(-/-) and HO2(-/-) mice. These results suggest that Iba1-positive microglial cells could engulf the extracellular SFHb and provide protective effects after ICH. We then treated cultured primary microglial cells with SFHb at low and high concentrations. The results show that microglial cells actively take up the extracellular SFHb. Of interest, we also found that iron overload in microglia significantly reduces the Iba1 expression level and resultantly inhibits microglial phagocytosis. CONCLUSIONS: This study suggests that microglial cells contribute to hemoglobin-heme clearance after ICH; however, the resultant iron overloads in microglia appear to decrease Iba1 expression and to further inhibit microglial phagocytosis.

Heme oxygenase-2 deletion impairs macrophage function: implication in wound healing.

Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.

Delayed cutaneous wound closure in HO-2 deficient mice despite normal HO-1 expression.

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO-1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO-2 deficient mice is impaired with exorbitant inflammation and absence of HO-1 expression. This study addresses the role of HO-2 in cutaneous excisional wound healing using HO-2 knockout (KO) mice. Here, we show that HO-2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO-2 KO mice compared to WT controls. Surprisingly, wound closure in HO-2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO-1 induction in HO-2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C-X-C) ligand-11 (CXCL-11) in wounds of HO-2 KO mice. Abnormal regulation of CXCL-11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL-11 expression in HO-2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Age sensitizes the kidney to heme protein-induced acute kidney injury.

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (~16 mo old), but not young mice (~6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2(+/+) and HO-2(-/-) mice: HO-2(-/-) mice, compared with HO-2(+/+) mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.

Coordinated expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 and heme oxygenase 2: evidence for a regulatory link between glycolysis and heme catabolism.

Heme is an essential requirement for cell survival. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and consists of two isozymes, HO-1 and HO-2. To identify the protein that regulates the expression or function of HO-1 or HO-2, we searched for proteins that interact with both isozymes, using protein microarrays. We thus identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) that synthesizes or degrades fructose-2,6-bisphosphate, a key activator of glycolysis, depending on cellular microenvironments. Importantly, HO-2 and PFKFB4 are predominantly expressed in haploid spermatids. Here, we show a drastic reduction in expression levels of PFKFB4 mRNA and protein and HO-2 mRNA in HepG2 human hepatoma cells in responses to glucose deprivation (≤ 2.5 mM), which occurred concurrently with remarkable induction of HO-1 mRNA and protein. Knockdown of HO-2 expression in HepG2 cells, using small interfering RNA, caused PFKFB4 mRNA levels to decrease with a concurrent increase in HO-1 expression. Thus, in HepG2 cells, HO-1 expression was increased, when expression levels of HO-2 and PFKFB4 mRNAs were decreased. Conversely, overexpression of HO-2 in HepG2 cells caused the level of co-expressed PFKFB4 protein to increase. These results suggest a potential regulatory role for HO-2 in ensuring PFKFB4 expression. Moreover, in D407 human retinal pigment epithelial cells, glucose deprivation decreased the expression levels of PFKFB4, HO-1, and HO-2 mRNAs. Thus, glucose deprivation consistently down-regulated the expression of PFKFB4 and HO-2 mRNAs in both HepG2 cells and RPE cells. We therefore postulate that PFKFB4 and HO-2 are expressed in a coordinated manner to maintain glucose homeostasis.

Knockdown of heme oxygenase-2 impairs corneal epithelial cell wound healing.

Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti-oxidative and anti-inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO-2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO-1 and HO-2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO-1 and HO-2 proteins. Injury elicited a rapid and transient increase in HO-1 and HO activity; HO-2 expression was unchanged. Treatment with biliverdin or CORM-A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM-A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO-2 expression, but not HO-1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO-2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin.

Haem oxygenase-1 prevents cell death by regulating cellular iron.

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.

Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2.

Nitric oxide (NO) is well established as a neurotransmitter in the central and peripheral nervous systems. More recently, another gas, carbon monoxide (CO) has also been implicated in neurotransmission. In the nervous system CO is formed by a subtype of heme oxygenase (HO) designated HO2. HO2 is localized to discrete neuronal populations in the brain resembling localizations of soluble guanylyl cyclase, which is activated by CO. CO may also function in the peripheral autonomic nervous system, in conjunction with NO. The majority of ganglia in the myenteric plexus possess both HO2 and neuronal NO synthase (NOS). Defects in myenteric plexus neurotransmission occur both in mice with targeted deletion of genes for HO2 and neuronal NOS. HO2 also occurs in other autonomic ganglia including the petrosal, superior cervical and nodose ganglia. Neuronal NOS is localized to neurons regulating male reproductive behavior, such as penile erection, and NOS inhibitors prevent erection. Because of the other parallels between NO and CO, we speculated that CO may play a role in male reproductive behavior. In the present study we describe HO2 localization in neuronal structures regulating copulatory reflexes. Reflex activity of the bulbospongiosus muscle, which mediates ejaculation and ejaculatory behavior, is markedly diminished in mice with targeted deletion of the gene for HO2 (HO2-).

Heme oxygenase-2 deletion causes endothelial cell activation marked by oxidative stress, inflammation, and angiogenesis.

In previous studies, we have shown that heme oxygenase (HO)-2 null [HO-2(-/-)] mice exhibit a faulty response to injury; chronic inflammation and massive neovascularization replaced resolution of inflammation and tissue repair. Endothelial cells play an active and essential role in the control of inflammation and the process of angiogenesis. We examined whether HO-2 deletion affects endothelial cell function. Under basal conditions, HO-2(-/-) aortic endothelial cells (mAEC) showed a 3-fold higher expression of vascular endothelial growth factor receptor 1 and a marked angiogenic response compared with wild-type (WT) cells. Compared with WT cells, HO-2(-/-) mAEC showed a 2-fold reduction in HO activity and marked increases in levels of gp91(phox)/NADPH oxidase isoform, superoxide, nuclear factor kappaB activation, and expression of inflammatory cytokines, including interleukin (IL)-1alpha and IL-6. HO-2 deletion transforms endothelial cells from a "normal" to an "activated" phenotype characterized by increases in inflammatory, oxidative, and angiogenic factors. This switch may be the result of reduced HO activity and the associated reduction in the cytoprotective HO products, carbon monoxide and biliverdin/bilirubin, because addition of biliverdin to HO-2(-/-) cells attenuated angiogenesis and reduced superoxide production. This transformation underscores the importance of HO-2 in the regulation of endothelial cell homeostasis.

Blood pressure and renal blow flow responses in heme oxygenase-2 knockout mice.

Heme oxygenase (HO) is the enzyme responsible for the breakdown of heme-generating carbon monoxide (CO) and biliverdin in this process. HO-2 is the constitutively expressed isoform in most tissues, such as the kidney and vasculature. CO generated by HO is believed to be an important vasodilator in the renal circulation along with another gas, nitric oxide (NO). To determine the importance of HO-2 in the regulation of blood pressure and renal blood flow (RBF), we treated HO-2 knockout (KO) mice chronically with either ANG II or N(G)-nitroarginine methyl ester (l-NAME). Basal blood pressures were not different between wild-type (WT), heterozygous (HET), or KO mice and averaged 113 +/- 3 vs. 115 +/- 2 vs. 116 +/- 2 mmHg. Similar increases in blood pressure to chronic ANG II as well as l-NAME treatment were observed in all groups with blood pressures increasing an average of 30 mmHg in response to ANG II and 15 mmHg in response to l-NAME. Basal RBFs were not different between the groups averaging 6.0 +/- 0.5 (n = 6) vs. 4.8 +/- 0.6 (n = 10) vs. 5.8 +/- 0.7 (n = 6) ml*min(-1)*g(-1) kidney weight in WT, HET, and KO mice. HO-2 KO and HET mice exhibited an attenuated decrease in RBF in response to acute administration of ANG II, while no differences were observed with l-NAME. Our data indicate that blood pressure and RBF responses to increased ANG II or inhibition of nitric oxide are not significantly enhanced in HO-2 KO mice.

Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice.

We have generated mice deficient in HO-2, the major cerebral isoform of heme oxygenase, in order to assess the potential role of carbon monoxide as a retrograde messenger in hippocampal LTP. Cerebral HO catalytic activity was markedly reduced in the HO-2 mutant mice, yet no differences were found between wild types and mutants in gross neuroanatomical structure, in basal hippocampal synaptic transmission, or in the amount of potentiation produced by various LTP induction protocols. Furthermore, zinc protoporphyrin IX, an inhibitor of HO, had nearly identical inhibitory effects on LTP in wild-type and HO-2 mutant hippocampal slices. Our data indicate that carbon monoxide produced endogenously by HO is unlikely to be a neuromodulator required for LTP in the hippocampus.

Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) remains a major medical problem and currently has no effective treatment. Hemorrhaged blood is highly toxic to the brain, and catabolism of the pro-oxidant heme, mainly released from hemoglobin, is critical for the resolution of hematoma after ICH. The degradation of the pro-oxidant heme is controlled by heme oxygenase (HO). We have previously reported a neuroprotective role for HO2 in early brain injury after ICH; however, in vivo data that specifically address the role of HO2 in brain edema and neuroinflammation after ICH are absent. Here, we tested the hypothesis that HO2 deletion would exacerbate ICH-induced brain edema, neuroinflammation, and oxidative damage. We subjected wild-type (WT) and HO2 knockout ((-/-)) mice to the collagenase-induced ICH model. Interestingly, HO2(-/-) mice had enhanced brain swelling and neuronal death, although HO2 deletion did not increase collagenase-induced bleeding; the exacerbation of brain injury in HO2(-/-) mice was also associated with increases in neutrophil infiltration, microglial/macrophage and astrocyte activation, DNA damage, peroxynitrite production, and cytochrome c immunoreactivity. In addition, we found that hemispheric enlargement was more sensitive than brain water content in the detection of subtle changes in brain edema formation in this model. Combined, these novel findings extend our previous observations and demonstrate that HO2 deficiency increases brain swelling, neuroinflammation, and oxidative damage. The results provide additional evidence that HO2 plays a critical protective role against ICH-induced early brain injury.

Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin.

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.

Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing.

In the immune-privileged cornea, epithelial wounds heal rapidly with almost no scarring and, unlike in most other tissues, acute inflammation in the absence of infection is beneficial to healing. Molecular mechanisms, which account for this striking property, remain to be clearly defined, but they likely include autacoids that control leukocyte activation. Two prominent enzymes, 12/15-lipoxygenase (LOX), which generates antiinflammatory lipid autacoids, and heme-oxygenase (HO), which generates antioxidants and carbon monoxide, are highly expressed in human and mouse corneas. LXA4, an endogenous 12/15-LOX product, proved to be a potent inhibitor of exacerbated inflammation and significantly increased re-epithelialization in corneal wounds. In vivo deletion of 12/15-LOX correlated with exacerbated inflammation and impaired wound healing in 12/15-LOX(-/-) mice, a phenotype that was rescued by treatment with LXA4. More importantly, 12/15-LOX(-/-) mice demonstrated impaired induction of HO-1 in both acute and exacerbated inflammation. Topical LXA4 restored HO-1 expression in 12/15-LOX(-/-) mice and amplified HO-1 gene expression in human corneal epithelial cells. HO-2(-/-) mice, which fail to induce HO-1, also demonstrated exacerbated inflammation in response to injury, a phenotype that, notably, correlated with a 50% reduction in endogenous LXA4 formation. Collectively, results demonstrate a critical role for LXA4 in inflammatory/reparative responses and provide the first evidence that 12/15-LOX and HO systems function in concert to control inflammation.

Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency.

The first known human case of heme oxygenase-1 (HO-1) deficiency is presented in this report. The patient is a six-year-old boy with severe growth retardation. He has been suffering from persistent hemolytic anemia characterized by marked erythrocyte fragmentation and intravascular hemolysis, with paradoxical increase of serum haptoglobin and low bilirubin. An abnormal coagulation/fibrinolysis system, associated with elevated thrombomodulin and von Willebrand factor, indicated the presence of severe, persistent endothelial damage. Electron microscopy of renal glomeruli revealed detachment of endothelium, with subendothelial deposition of an unidentified material. Iron deposition was noted in renal and hepatic tissue. Immunohistochemistry of hepatic tissue and immunoblotting of a cadmium-stimulated Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) revealed complete absence of HO-1 production. An LCL derived from the patient was extremely sensitive to hemin-induced cell injury. Sequence analysis of the patient's HO-1 gene revealed complete loss of exon-2 of the maternal allele and a two-nucleotide deletion within exon3 of the paternal allele. Growth retardation, anemia, iron deposition, and vulnerability to stressful injury are all characteristics observed in recently described HO-1 targeted mice. This study presents not only the first human case of HO-1 deficiency but may also provide clues to the key roles played by this important enzyme in vivo.

Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice.

Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.

Heme oxygenase-2 protects against lipid peroxidation-mediated cell loss and impaired motor recovery after traumatic brain injury.

After traumatic brain injury (TBI), substantial extracellular heme is released from hemoproteins during hemorrhage and cell injury. Heme oxygenase (HO) isozymes are thought to detoxify the pro-oxidant heme to the potent antioxidant, bilirubin. HO-1, the inducible isozyme, is expressed in glial populations after injury and may play a protective role. However, the role of HO-2, the predominant and constitutively expressed isozyme in the brain, remains unclear after TBI. We used a controlled cortical impact injury model to determine the extent and mechanism of damage between HO-2 knock-out (KO) (-/-) and wild-type (WT) (+/+) mice. The specific cellular and temporal expressions of HO-2 and HO-1 were characterized by immunocytochemistry and Western blots. HO-2 was immunolocalized in neurons both before and after TBI, whereas HO-1 was highly upregulated in glia only after TBI. HO activity determined by gas chromatography using brain sonicates from injured HO-2 KO mice was significantly less than that of HO-2 wild types, despite the induction of HO-1 expression after TBI. Cell loss was significantly greater in KO mice in areas including the cortex, the CA3 region of hippocampus, and the lateral dorsal thalamus. Furthermore, motor recovery after injury, as measured by the rotarod assay and an inclined beam-walking task, was compromised in the KO mice. Finally, brain tissue from injured HO-2 KO mice exhibited decreased ability to reduce oxidative stress, as measured with an Fe(2+)/ascorbic acid-mediated carbon monoxide generation assay for lipid peroxidation susceptibility. These findings demonstrate that HO-2 expression protects neurons against TBI by reducing lipid peroxidation via the catabolism of free heme.

Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice.

BACKGROUND: Heme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice. METHODS AND RESULTS: To determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (+/-), or homozygous null (-/-) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1(+/+) and HO-1(+/-) mice, whereas HO-1(-/-) mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1(-/-) mice (121+/-5 mm Hg) after 24 hours, compared with HO-1(+/+) (96+/-7 mm Hg) and HO-1(+/-) (89+/-13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1(-/-) mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1(+/+) and HO-1(+/-) mice. The end-organ damage and death in HO-1(-/-) mice was related to increased oxidative stress. CONCLUSIONS: These data suggest that the increased mortality during endotoxemia in HO-1(-/-) mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.