Effect of hemopexin treatment on outcome after intracerebral hemorrhage in mice.

Heme release from hemoglobin may contribute to secondary injury after intracerebral hemorrhage (ICH). The primary endogenous defense against heme toxicity is hemopexin, a 57 kDa glycoprotein that is depleted in the CNS after hemorrhagic stroke. We hypothesized that systemic administration of exogenous hemopexin would reduce perihematomal injury and improve outcome after experimental ICH. Intraperitoneal treatment with purified human plasma hemopexin beginning 2 h after striatal ICH induction and repeated daily for the following two days reduced blood-brain barrier disruption and cell death at 3 days. However, it had no effect on neurological deficits at 4 or 7 days or striatal cell viability at 8 days. Continuous daily hemopexin administration had no effect on striatal heme content at 3 or 7 days, and did not attenuate neurological deficits, inflammatory cell infiltration, or perihematomal cell viability at 8 days. These results suggest that systemic hemopexin treatment reduces early injury after ICH, but this effect is not sustained, perhaps due to an imbalance between striatal tissue heme and hemopexin content at later time points. Future studies should investigate its effect when administered by methods that more efficiently target CNS delivery.

Platelets as drivers of ischemia/reperfusion injury after stroke.

Ischemic stroke is a leading cause of morbidity and mortality worldwide and, despite reperfusion either via thrombolysis or thrombectomy, stroke patients often suffer from lifelong disabilities. These persistent neurological deficits may be improved by treating the ischemia/reperfusion (I/R) injury that occurs following ischemic stroke. There are currently no approved therapies to treat I/R injury, and thus it is imperative to find new targets to decrease the burden of ischemic stroke and related diseases. Platelets, cell fragments from megakaryocytes, are primarily known for their role in hemostasis. More recently, investigators have studied the nonhemostatic role of platelets in inflammatory pathologies, such as I/R injury after ischemic stroke. In this review, we seek to provide an overview of how I/R can lead to platelet activation and how activated platelets, in turn, can exacerbate I/R injury after stroke. We will also discuss potential mechanisms by which platelets may ameliorate I/R injury.

CXCL12-CXCR4 Interplay Facilitates Palatal Osteogenesis in Mice.

Cranial neural crest cells (CNCCs), identified by expression of transcription factor Sox9, migrate to the first branchial arch and undergo proliferation and differentiation to form the cartilage and bone structures of the orofacial region, including the palatal bone. Sox9 promotes osteogenic differentiation and stimulates CXCL12-CXCR4 chemokine-receptor signaling, which elevates alkaline phosphatase (ALP)-activity in osteoblasts to initiate bone mineralization. Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion. Since we earlier demonstrated chemokine-receptor mediated signaling by the MES, we hypothesized that chemokine CXCL12 is expressed by the disintegrating MES to promote the formation of an osteogenic center by CXCR4-positive osteoblasts. Disturbed migration of CNCCs by excess oxidative and inflammatory stress is associated with increased risk of cleft lip and palate (CLP). The cytoprotective heme oxygenase (HO) enzymes are powerful guardians harnessing injurious oxidative and inflammatory stressors and enhances osteogenic ALP-activity. By contrast, abrogation of HO-1 or HO-2 expression promotes pregnancy pathologies. We postulate that Sox9, CXCR4, and HO-1 are expressed in the ALP-activity positive osteogenic regions within the CNCCs-derived palatal mesenchyme. To investigate these hypotheses, we studied expression of Sox9, CXCL12, CXCR4, and HO-1 in relation to palatal osteogenesis between E15 and E16 using (immuno)histochemical staining of coronal palatal sections in wild-type (wt) mice. In addition, the effects of abrogated HO-2 expression in HO-2 KO mice and inhibited HO-1 and HO-2 activity by administrating HO-enzyme activity inhibitor SnMP at E11 in wt mice were investigated at E15 or E16 following palatal fusion. Overexpression of Sox9, CXCL12, CXCR4, and HO-1 was detected in the ALP-activity positive osteogenic regions within the palatal mesenchyme. Overexpression of Sox9 and CXCL12 by the disintegrating MES was detected. Neither palatal fusion nor MES disintegration seemed affected by either HO-2 abrogation or inhibition of HO-activity. Sox9 progenitors seem important to maintain the CXCR4-positive osteoblast pool to drive osteogenesis. Sox9 expression may facilitate MES disintegration and palatal fusion by promoting epithelial-to-mesenchymal transformation (EMT). CXCL12 expression by the MES and the palatal mesenchyme may promote osteogenic differentiation to create osteogenic centers. This study provides novel evidence that CXCL12-CXCR4 interplay facilitates palatal osteogenesis and palatal fusion in mice.

Deferoxamine deconditioning increases neuronal vulnerability to hemoglobin.

Concomitant treatment with deferoxamine (DFO) protects neural cells from iron and heme-mediated oxidative injury, but also disrupts cell responses to iron loading that may be protective. We hypothesized that DFO treatment and withdrawal would subsequently increase neuronal vulnerability to hemoglobin. Pretreatment with DFO followed by its washout increased neuronal loss after subsequent hemoglobin exposure by 3-4-fold compared with control vehicle-pretreated cultures. This was associated with reduced ferritin induction by hemoglobin; expression of heme oxygenase-1, which catalyzes iron release from heme, was not altered. Increased neuronal loss was prevented by exogenous apoferritin or by continuing DFO or antioxidants throughout the experimental course. Cell nonheme iron levels after hemoglobin treatment were similar in DFO-pretreated and control cultures. These results indicate that DFO deconditions neurons and subsequently increases their vulnerability to heme-mediated injury. Its net effect after CNS hemorrhage may be highly dependent on the timing and duration of its administration. Withdrawal of DFO while heme or iron levels remain elevated may be deleterious, and may negate any benefit of prior concomitant therapy.

Antithrombotic effects of heme-degrading and heme-binding proteins.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2-/- mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin-/- mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs.NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

Rapid loss of perihematomal cell viability in the collagenase intracerebral hemorrhage model.

The effective time window of any therapeutic in an experimental stroke model is limited by the rate of injury progression. Intracerebral hemorrhage in rodents is commonly induced by striatal injection of either autologous blood or bacterial collagenase, which digests local blood vessels. During time window studies of the heme oxygenase-1 inducer hemin, which is protective when administered within 1-3 h in both models, the rate of perihematomal injury was directly compared after striatal blood or collagenase injection. Surprisingly, about 80% of the loss of perihematomal cell viability as measured by MTT reduction assay occurred within 6 h of collagenase injection. In contrast, significant viability loss was not observed at this time point after autologous blood injection, but rather it progressed over the subsequent four days to a level similar to that produced by collagenase. Consistent with these observations, systemic hemin therapy reduced blood-brain barrier disruption and perihematomal cell injury when initiated at 6 h after striatal injection of blood but not collagenase. These results indicate that the rate of early cell injury differs markedly in the collagenase and blood injection ICH models, which may contribute to inconsistent results in time window studies. The blood injection model may be more appropriate for prolonged time window studies of a neuroprotective agent.

Protective effect of vitreous against hemoglobin neurotoxicity.

Hemorrhage into the brain parenchyma or subarachnoid space is associated with edema and vascular injury that is likely mediated at least in part by the toxicity of hemoglobin. In contrast, extravascular blood appears to be less neurotoxic when localized to the retina or adjacent vitreous, the gel filling the posterior segment of the eye. In this study, the hypothesis that vitreous protects neurons from hemoglobin toxicity was investigated in a primary cortical cell culture model. Consistent with prior observations, hemoglobin exposure for 24 h resulted in death of most neurons without injury to co-cultured glia. Neuronal loss was reduced in a concentration-dependent fashion by bovine vitreous, with complete protection produced by 3% vitreous solutions. This effect was associated with a reduction in malondialdehyde but an increase in cell iron. At low vitreous concentrations, its ascorbate content was sufficient to account for most neuroprotection, as equivalent concentrations of ascorbate alone had a similar effect. However, other vitreous antioxidants provided significant protection when applied at concentrations present in undiluted vitreous, and prevented all neuronal loss when combined in the absence of ascorbate. These results indicate that vitreous is an antioxidant cocktail that robustly protects neurons from hemoglobin toxicity, and may contribute to the relative resistance of retinal neurons to hemorrhagic injury.

Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent.

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

Chemokine Signaling during Midline Epithelial Seam Disintegration Facilitates Palatal Fusion.

Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion, and failure results in cleft palate. Palatal fusion and wound repair share many common signaling pathways related to epithelial-mesenchymal cross-talk. We postulate that chemokine CXCL11, its receptor CXCR3, and the cytoprotective enzyme heme oxygenase (HO), which are crucial during wound repair, also play a decisive role in MES disintegration. Fetal growth restriction and craniofacial abnormalities were present in HO-2 knockout (KO) mice without effects on palatal fusion. CXCL11 and CXCR3 were highly expressed in the disintegrating MES in both wild-type and HO-2 KO animals. Multiple apoptotic DNA fragments were present within the disintegrating MES and phagocytized by recruited CXCR3-positive wt and HO-2 KO macrophages. Macrophages located near the MES were HO-1-positive, and more HO-1-positive cells were present in HO-2 KO mice compared to wild-type. This study of embryonic and palatal development provided evidence that supports the hypothesis that the MES itself plays a prominent role in palatal fusion by orchestrating epithelial apoptosis and macrophage recruitment via CXCL11-CXCR3 signaling.

Astrocyte heme oxygenase-1 reduces mortality and improves outcome after collagenase-induced intracerebral hemorrhage.

Pharmacotherapies that increase CNS expression of heme oxygenase-1 (HO-1) and other antioxidant proteins have improved outcome in experimental models of spontaneous intracerebral hemorrhage (ICH). In order to more specifically investigate the relationship between HO-1 and ICH outcome, mice expressing human HO-1 driven by the glial fibrillary acidic protein (GFAP) promoter (GFAP·HMOX1 mice) were tested in a model of in situ parenchymal hemorrhage. Injection of collagenase into the striata of wild-type (WT) mice resulted in a 26.3% mortality rate, with deaths equally distributed between males and females. Mortality was reduced to 4.48% in GFAP·HMOX1 mice. Cell viability in the injected striata of surviving WT mice was reduced by about half at one week and was significantly increased in transgenics; this benefit persisted over a 22day observation period. Cell counts guided by design-based stereology indicated loss of ~40% of neurons in WT hemorrhagic striata at one week, which was decreased by half in transgenics; no significant differences in microglia or astrocyte numbers were observed. Blood-brain barrier disruption and short-term neurological deficits were also mitigated in GFAP·HMOX1 mice, but long-term outcome did not differ from that of WT survivors. These results suggest that astrocyte HO-1 overexpression provides robust neuroprotection after acute intracerebral hemorrhage. Further investigation of drug or genetic therapies that selectively increase astrocyte HO-1 is warranted.

Targeting the Nrf2-Heme Oxygenase-1 Axis after Intracerebral Hemorrhage.

BACKGROUND: Injury to cells adjacent to an intracerebral hemorrhage (ICH) is likely mediated at least in part by toxins released from the hematoma that initiate complex and interacting injury cascades. Pharmacotherapies targeting a single toxin or pathway, even if consistently effective in controlled experimental models, have a high likelihood of failure in a variable clinical setting. Nuclear factor erythroid-2 related factor 2 (Nrf2) regulates the expression of heme oxygenase-1 (HO-1) and multiple other proteins with antioxidant and antiinflammatory effects, and may be a target of interest after ICH. METHODS: Studies that tested the effect of HO and Nrf2 in models relevant to ICH are summarized, with an effort to reconcile conflicting data by consideration of methodological limitations. RESULTS: In vitro studies demonstrated that Nrf2 activators rapidly increased HO-1 expression in astrocytes, and reduced their vulnerability to hemoglobin or hemin. Modulating HO-1 expression via genetic approaches yielded similar results. Systemic treatment with small molecule Nrf2 activators increased HO-1 expression in perivascular cells, particularly astrocytes. When tested in mouse or rat ICH models, Nrf2 activators were consistently protective, improving barrier function and attenuating edema, inflammation, neuronal loss and neurological deficits. These effects were mimicked by selective astrocyte HO-1 overexpression in transgenic mice. CONCLUSION: Systemic treatment with Nrf2 activators after ICH is protective in rodents. Two compounds, dimethyl fumarate and hemin, are currently approved for treatment of multiple sclerosis and acute porphyria, respectively, and have acceptable safety profiles over years of clinical use. Further development of these drugs as ICH therapeutics seems warranted.

Haptoglobin increases the vulnerability of CD163-expressing neurons to hemoglobin.

Haptoglobin (Hp) binds hemoglobin (Hb) with high affinity and provides the primary defense against its toxicity after intravascular hemolysis. Neurons are exposed to extracellular Hb after CNS hemorrhage, and a therapeutic effect of Hp via Hb sequestration has been hypothesized. In this study, we tested the hypothesis that Hp protects neurons from Hb in primary mixed cortical cell cultures. Treatment with low micromolar concentrations of human Hb for 24 h resulted in loss of 10-20% of neurons without injuring glia. Concomitant treatment with Hp surprisingly increased neuronal loss five-sevenfold, with similar results produced by Hp 1-1 and 2-2 phenotypes. Consistent with a recent in vivo observation, neurons expressed the CD163 receptor for Hb and the Hb-Hp complex in these cultures. Hp reduced overall Hb uptake, directed it away from the astrocyte-rich CD163-negative glial monolayer, and decreased induction of the iron-binding protein ferritin. Hb-Hp complex neuronal toxicity, like that of Hb per se, was iron-dependent and reduced by deferoxamine and 2,2' bipyridyl. These results suggest that Hp increases the vulnerability of CD163+ neurons to Hb by permitting Hb uptake while attenuating the protective response of ferritin induction by glial cells. Cover Image for this issue: doi: 10.1111/jnc.13342.

Dysregulation of the haem-haemopexin axis is associated with severe malaria in a case-control study of Ugandan children.

BACKGROUND: Malaria is associated with haemolysis and the release of plasma haem. Plasma haem can cause endothelial injury and organ dysfunction, and is normally scavenged by haemopexin to limit toxicity. It was hypothesized that dysregulation of the haem-haemopexin pathway contributes to severe and fatal malaria infections. METHODS: Plasma levels of haemin (oxidized haem), haemopexin, haptoglobin, and haemoglobin were quantified in a case-control study of Ugandan children with Plasmodium falciparum malaria. Levels at presentation were compared in children with uncomplicated malaria (UM; n = 29), severe malarial anaemia (SMA; n = 27) or cerebral malaria (CM; n = 31), and evaluated for utility in predicting fatal (n = 19) vs non-fatal (n = 39) outcomes in severe disease. A causal role for haemopexin was assessed in a pre-clinical model of experimental cerebral malaria (ECM), following disruption of mouse haemopexin gene (hpx). Analysis was done using Kruskall Wallis tests, Mann-Whitney tests, log-rank tests for survival, and repeated measures ANOVA. RESULTS: In Ugandan children presenting with P. falciparum malaria, haemin levels were higher and haemopexin levels were lower in SMA and CM compared to children with UM (haemin, p < 0.01; haemopexin, p < 0.0001). Among all cases of severe malaria, elevated levels of haemin and cell-free haemoglobin at presentation were associated with subsequent mortality (p < 0.05). Compared to ECM-resistant BALB/c mice, susceptible C57BL/6 mice had lower circulating levels of haemopexin (p < 0.01), and targeted deletion of the haemopexin gene, hpx, resulted in increased mortality compared to their wild type littermates (p < 0.05). CONCLUSIONS: These data indicate that plasma levels of haemin and haemopexin measured at presentation correlate with malaria severity and levels of haemin and cell-free haemoglobin predict outcome in paediatric severe malaria. Mechanistic studies in the ECM model support a causal role for the haem-haemopexin axis in ECM pathobiology.

Astrocyte overexpression of heme oxygenase-1 improves outcome after intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Heme oxygenase-1 (HO-1) catalyzes the rate-limiting reaction of heme breakdown and may have both antioxidant and pro-oxidant effects. In previous studies, HO-1 overexpression protected astrocytes from heme-mediated injury in vitro. In the present study, we tested the hypothesis that selective astrocyte overexpression of HO-1 improves outcome after intracerebral hemorrhage. METHODS: Male and female transgenic mice overexpressing human HO-1 driven by the GFAP promoter (GFAP.HMOX1) and wild-type controls received striatal injections of autologous blood (25 µL). Blood-brain barrier disruption was assessed by Evans blue assay and striatal cell viability by methylthiazolyldiphenyl-tetrazolium bromide assay. Neurological deficits were quantified by digital analysis of spontaneous cage activity, adhesive removal, and elevated body swing tests. RESULTS: Mortality rate for wild-type mice was 34.8% and was similar for males and females; all GFAP.HMOX1 mice survived. Striatal Evans blue leakage at 24 hours was 23.4±3.2 ng in surviving wild-type mice, compared with 10.9±1.8 ng in transgenics. Perihematomal cell viability was reduced to 61±4% of contralateral at 3 days in wild-type mice, versus 80±4% in transgenics. Focal neurological deficits were significantly reduced and spontaneous cage activity was increased in GFAP.HMOX1 mice. CONCLUSIONS: Selective HO-1 overexpression in astrocytes reduces mortality, blood-brain barrier disruption, perihematomal cell injury, and neurological deficits in an autologous blood injection intracerebral hemorrhage model. Genetic or pharmacological therapies that acutely increase astrocyte HO-1 may be beneficial after intracerebral hemorrhage.

Targeting heme oxygenase after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the primary event in approximately 10% of strokes, and has higher rates of morbidity and mortality than ischemic stroke. Experimental evidence suggests that the toxicity of hemoglobin and its degradation products contributes to secondary injury that may be amenable to therapeutic intervention. Hemin, the oxidized form of heme, accumulates in intracranial hematomas to cytotoxic levels. The rate limiting step of its breakdown is catalyzed by the heme oxygenase (HO) enzymes, which consist of inducible HO-1 and constitutively-expressed HO-2. The effect of these enzymes on perihematomal injury and neurological outcome has been investigated in ICH models using both genetic and pharmacological approaches to alter their expression, with variable results reported. These findings are summarized and reconciled in this review; therapeutic strategies that may optimize HO expression and activity after ICH are described.

Solid microparticles based on chitosan or methyl-ß-cyclodextrin: a first formulative approach to increase the nose-to-brain transport of deferoxamine mesylate.

We propose the formulation and characterization of solid microparticles as nasal drug delivery systems able to increase the nose-to-brain transport of deferoxamine mesylate (DFO), a neuroprotector unable to cross the blood brain barrier and inducing negative peripheral impacts. Spherical chitosan chloride and methyl-ß-cyclodextrin microparticles loaded with DFO (DCH and MCD, respectively) were obtained by spray drying. Their volume-surface diameters ranged from 1.77 ± 0.06 µm (DCH) to 3.47 ± 0.05 µm (MCD); the aerodynamic diameters were about 1.1 µm and their drug content was about 30%. In comparison with DCH, MCD enhanced the in vitro DFO permeation across lipophilic membranes, similarly as shown by ex vivo permeation studies across porcine nasal mucosa. Moreover, MCD were able to promote the DFO permeation across monolayers of PC 12 cells (neuron-like), but like DCH, it did not modify the DFO permeation pattern across Caco-2 monolayers (epithelial-like). Nasal administration to rats of 200 µg DFO encapsulated in the microparticles resulted in its uptake into the cerebrospinal fluid (CSF) with peak values ranging from 3.83 ± 0.68 µg/mL (DCH) to 14.37 ± 1.69 µg/mL (MCD) 30 min after insufflation of microparticles. No drug CSF uptake was detected after nasal administration of a DFO water solution. The DFO systemic absolute bioavailabilities obtained by DCH and MCD nasal administration were 6% and 15%, respectively. Chitosan chloride and methyl-ß-cyclodextrins appear therefore suitable to formulate solid microparticles able to promote the nose to brain uptake of DFO and to limit its systemic exposure.

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.

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.

Neuroprotective effect of heme oxygenase-2 knockout in the blood injection model of intracerebral hemorrhage.

BACKGROUND: The toxicity of heme breakdown products may contribute to the pathogenesis of intracerebral hemorrhage (ICH). Heme catabolism is catalyzed by the heme oxygenase enzymes. We have previously reported that heme oxygenase-2 (HO-2), the constitutive isoform, protects neurons from hemin in vitro and reduces oxidative stress after striatal blood injection. In order to further evaluate HO-2 as a therapeutic target, we tested the hypothesis that HO-2 gene deletion protects neurons and attenuates behavioral deficits after ICH. FINDINGS: Injection of 20 µl blood into the right striatum of HO-2 wild-type mice resulted in loss of approximately one third of striatal neurons 4-8 days later. Neuronal survival was significantly increased in HO-2 knockout mice at both time points. This was associated with reduced motor deficit as detected by the corner test; however, no differences were detected in spontaneous activity or the adhesive removal or elevated body swing tests. CONCLUSION: HO-2 knockout attenuates perihematomal neuron loss in the blood injection ICH model, but has a weak and variable effect on neurological outcome.

Systemic hemin therapy attenuates blood-brain barrier disruption after intracerebral hemorrhage.

Injury to the blood-brain barrier (BBB) is a key feature of intracerebral hemorrhage (ICH) and may contribute to perihematomal cell injury. Pretreatment with the heme oxygenase (HO)-1 inducer hemin improves barrier function and neurological outcome in experimental models of traumatic and ischemic CNS injury. Since hemin is already in clinical use to treat acute porphyrias, this translational study was designed to test its effect on BBB function when initiated after ICH in two mouse models. At a dose similar to those used in most preconditioning studies (26mg/kg i.p.), post-hemorrhage treatment with hemin reduced parenchymal extravasation of Evans blue by about three-quarters in both the blood injection and collagenase ICH models. Similar efficacy was observed when treatment was begun at 1 or 3h. At the lower dose that is currently in clinical use (4mg/kg beginning at 3h), hemin also improved barrier function in both models, as assessed by both Evans blue and FITC-dextran leakage; however, it was somewhat less potent, reducing Evans blue leakage by about half. This dose was nevertheless sufficient to attenuate striatal cell loss and accelerate neurological recovery. Consistent with prior observations, striatal HO-1 expression was increased by hemin, and was localized to perivascular cells. These results suggest that hemin may be an effective therapy for ICH with a clinically relevant time window. Further study of the repurposing of this old drug seems warranted.