Heme Oxygenase-1 Knockout Mice Research Articles

Inhibition of heme oxygenase 1 alleviates thoracic aortic aneurysm via restoration of extracellular matrix

BackgroundThoracic aortic aneurysm (TAA) is a silent but life-threatening cardiovascular disease. Heme oxygenase 1 (HO-1) plays an important role in the cardiovascular diseases but is poorly understood in TAA. This study aims at investigating the role of HO-1 in TAA. MethodsSingle-cell RNA sequencing, Western blot and histological assay were performed to identify specific cellular expression of HO-1 in both human and β-aminopropionitrile (BAPN)-induced mice TAA. Zinc protoporphyrin (ZnPP), a pharmacological inhibitor of HO-1, was used to investigate whether inhibition of HO-1 could attenuate BAPN-induced TAA in rodent model. Histological assay, Western blot assay, and mRNA sequencing were further performed to explore the underlying mechanisms. ResultsSingle-cell transcriptomic analyses of 113,800 thoracic aortic cells identified an increase of HO-1(+) macrophage in aneurysmal thoracic aorta from BAPN-induced TAA mice and TAA patients. Histological assay verified HO-1 overexpression in clinical TAA specimens, which was co-localized with CD68(+) macrophage. HO-1(+) macrophage was closely associated with pro-inflammatory response and immune activation. Inhibition of HO-1 through ZnPP significantly alleviated BAPN-induced TAA in mice and restored extracellular matrix (ECM) in vivo. Further experiments showed that ZnPP treatment suppressed the expression of matrix metalloproteinases (MMPs) in aneurysmal thoracic aortic tissues from BAPN-induced TAA mice, including MMP2 and MMP9. Macrophages from myeloid specific HO-1 knockout mice displayed weakened pro-inflammatory activity and ECM degradation capability. ConclusionHO-1(+) macrophage subgroup is a typical hallmark of TAA. Inhibition of HO-1 through ZnPP alleviates BAPN-induced TAA in mice, which might work through restoration of ECM via suppressing MMP2 and MMP9 expression.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through modulating mitochondrial quality control.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD) and lacks effective treatment options. Heme oxygenase-1 (HO-1) is a critical defense against oxidative stress and inflammation in the liver injury. This study aims to investigate the protective role and underlying mechanisms of HO-1 in NASH pathogenesis. The hepatocyte-specific HO-1 knockout (HO-1HEPKO ) mice on a C57BL/6J background (HO-1fl/fl /Alb-Cre) were generated and fed a high-fat/western-style diet (HFD) or methionine-choline-deficient diet (MCD). Changes in mitochondrial ultrastructure were observed by transmission electron microscopy and confocal microscopy. A mitochondrial PCR array was used to identify the crucial genes associated with mitochondrial dysfunction. Hepatocyte-specific HO-1HEPKO mice developed steatohepatitis with severe steatosis, ballooning, and necroinflammation. Dysregulated hepatic expression of mitochondria-related proteins, including DRP1, Tomm20, MFN1 and MFN2 were detected in NASH animals. Ultrastructural mitochondrial damage was observed in HO-1HEPKO mice. Mitochondrial dysfunction was recapitulated in HO-1-knockdown cells in vitro, as evidenced by decreased membrane potential, reduced ATP content, and mtDNA damage. Conversely, HO-1 overexpression restored these changes in vitro. Mechanistically, HO-1 deficiency reduced the inhibitory effect on Tomm20, leading to mitochondrial dysfunction, and thereby causing steatohepatitis. HO-1 attenuates diet-induced steatohepatitis by preventing mitochondrial dysfunction, indicating that HO-1 may constitute a potential therapeutic target for NASH.

Role of Mast Cells in HIV-associated Chronic Widespread Pain

Chronic widespread pain (CWP) in people with HIV-1 (PWH) is associated with a high rate of disability and low quality of life, with prevalence estimates ranging from 25%-85%. However, the specific mechanisms that contribute to CWP in HIV are not understood. Previously we demonstrated that PWH with CWP have increased hemolysis, elevated plasma levels of cell-free heme, and attenuated levels of the heme degrading enzyme, heme oxygenase 1 (HO-1). We also found that high cell-free heme and low HO-1 were associated with hypersensitivity in animals. In this study, we hypothesized that high heme and low HO-1 activates mast cells leading to degranulation and release of pain mediators like, histamine and bradykinin in HIV. PWH who self-report CWP were recruited from the University of Alabama at Birmingham HIV clinic. Animal models used in the study included, C57BL/6 mice subjected to hemolysis by intraperitoneal injection of phenylhydrazine hydrochloride (PHZ), HO-1 knockout mice, and HIV-1 transgenic rats overexpressing HIV-1 proteins. Cellular studies were performed in MC/9 mast cells. Human findings show that PWH with CWP have elevated plasma levels of histamine and bradykinin. HIV-1 transgenic rats also have elevated plasma levels of histamine and bradykinin and are hypersensitive as estimated by a decrease in withdrawal threshold to von Frey stimulation. In addition, data in mice with elevated plasma levels of free-heme, or decreased expression of HO-1, show significantly elevated levels of the pain mediators, histamine and bradykinin. Cellular data shows that high heme or low HO-1 induces mast cell degranulation and the release of histamine and bradykinin. Together, these data suggest that heme-dependent mast cell degranulation and the release of pain mediators may be contributing CWP in HIV. Future studies will focus on determining strategies to stabilize mast cell membrane in HIV and reduce hypersensitivity. Grant Number: RO1DA049657 Grant Name: Chronic Widespread Pain in HIV: Novel Mechanisms and Therapeutics.

Endothelin A receptor antagonist attenuated renal iron accumulation in iron overload heme oxygenase-1 knockout mice.

Progressive iron accumulation and renal impairment are prominent in both patients and mouse models of sickle cell disease (SCD). Endothelin A receptor (ETA) antagonism prevents this iron accumulation phenotype and reduces renal iron deposition in the proximal tubules of SCD mice. To better understand the mechanisms of iron metabolism in the kidney and the role of the ETA receptor in iron chelation and transport, we studied renal iron handling in a nonsickle cell iron overload model, heme oxygenase-1 (Hmox-1-/-) knockout mice. We found that Hmox-1-/- mice had elevated plasma endothelin-1 (ET-1), cortical ET-1 mRNA expression, and renal iron content compared with Hmox-1+/+ controls. The ETA receptor antagonist, ambrisentan, attenuated renal iron deposition, without any changes to anemia status in Hmox-1-/- mice. This was accompanied by reduced urinary iron excretion. Finally, ambrisentan had an important iron recycling effect by increasing the expression of the cellular iron exporter, ferroportin-1 (FPN-1), and circulating total iron levels in Hmox-1-/- mice. These findings suggest that the ET-1/ETA signaling pathway contributes to renal iron trafficking in a murine model of iron overload.

Effects of Heme Oxygenase-1 on c-Kit-Positive Cardiac Cells

Heme oxygenase-1 (HO-1) is one of the most powerful cytoprotective proteins known. The goal of this study was to explore the effects of HO-1 in c-kit-positive cardiac cells (CPCs). LinNEG/c-kitPOS CPCs were isolated and expanded from wild-type (WT), HO-1 transgenic (TG), or HO-1 knockout (KO) mouse hearts. Compared with WT CPCs, cell proliferation was significantly increased in HO-1TG CPCs and decreased in HO-1KO CPCs. HO-1TG CPCs also exhibited a marked increase in new DNA synthesis during the S-phase of cell division, not only under normoxia (21% O2) but after severe hypoxia (1% O2 for 16 h). These properties of HO-1TG CPCs were associated with nuclear translocation (and thus activation) of Nrf2, a key transcription factor that regulates antioxidant genes, and increased protein expression of Ec-SOD, the only extracellular antioxidant enzyme. These data demonstrate that HO-1 upregulates Ec-SOD in CPCs and suggest that this occurs via activation of Nrf2, which thus is potentially involved in the crosstalk between two antioxidants, HO-1 in cytoplasm and Ec-SOD in extracellular matrix. Overexpression of HO-1 in CPCs may improve the survival and reparative ability of CPCs after transplantation and thus may have potential clinical application to increase efficacy of cell therapy.

Role of HO-1 in B Lymphocyte Proliferation and Development in Mice

Background: B lymphocytes are essential for an efficient immune response against a variety of pathogens. Not only a large fraction of hematologic malignancies is of B cell origin, but the immunity reconstruction is also depended on its normal growth. Then, it's very important to pay more attention on tightly regulating the development and activation of B cells. Heme oxygenase-1 (HO-1) is thought initially to be a key enzyme that provides potent cyto-protection, cell proliferation, malignancy development and drug resistance. In our primary studies, we have already reported its roles in various of hematological malignancies. However, As we noticed thatObjective: Herein, there is something different with before we observed. The count of B lymphocyte in HO-1 deficient mice decreased significantly. It never be reported yet. Therefore, we aimed to explore the regulation of HO-1 to B lymphocyte development in HO-1 knockout mice model.Method: Originally, HO-1 deficient C57BL6 mice model was constructed by CRISPR/Cas9 assay for examining the effect of HO-1 on bone marrow-derived cells. After immune-phenotyping by flow cytometry and colony formation assay, we accidently noticed that its quantity of CD19+ cells in peripheral blood was less than HO-1 wild type mice, while the other cells kept relative balance. Then, to further explore which stage was the direct target for HO-1, the absolute quantity of B lymphocyte in each stage from bone marrow cells and mature B lymphocyte from spleen were detected by flow cytometry. Our staining strategy to distinguish between pro-B (B220+IgM−CD43+) and pre-B (B220+IgM−CD43−) lymphocytes revealed the presence of cells expressing intermediate CD43 levels in bone marrow from HO-1-deficient mice. B220+ IgM+ immature B cells, and B220+ IgM+ IgD+ mature recirculating B cells. CD21brightCD23+ for marginal zone B cells, CD21+CD23brightCD93− for follicular B cells and CD21+CD23brightCD93+ for transitional B cells. In additional, to get an insight into the mechanisms underlying the early B lymphocyte development, we detected cell proliferation, cell cycle and cell death in pro-B subsets in the absence of HO-1. And PCR Array was used to explore the pathway involved in B cells development. Real-time PCR and Western blot were used to identify it.Results: It revealed that absence of HO-1 resulted in a block in B cell development at the pro-B to pre-B cell transition. The absolute count of pro-B cells in HO-1 deficient mice was 71.2% compare with control(p<0.05). Fewer B cells in spleen and peripheral blood were observed comparing with control group. In particular, the analysis of immature B cells, marginal zone B cells, follocular B cells and transtional B cells revealed that the absolute numbers of all B cell subtypes were significantly lower in HO-1-deficient mice. Proliferation rate of pro-B cells were decreased significantly, while cell cycle and death were not sensibly in the absence of HO-1. In addition, we also observed intracellular IgHμexpression to test whether HO-1 is required for V(D)J recombination. The results still revealed that the effect of HO-1 on B lymphocyte development depends on promoting cells proliferation. Then, PCR array was preliminary used and showed that MAPK signaling pathway was possibly involved in regulation of early B lymphocyte development induced by HO-1 deficiency. And BAFF expression was possible to relate with HO-1 knockout. Lenti-virus with HO-1 was injected into HO-1 deficient mice through tail vein. We found that absolute quantity of B cells increased significantly in vivo.Conclusion: In conclusion, all results indicate HO-1 is a crucial role in early B lymphocyte proliferation and development.Disclosures: No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.

Exacerbation of Neonatal Hemolysis and Impaired Renal Iron Handling in Heme Oxygenase 1-Deficient Mice.

In most mammals, neonatal intravascular hemolysis is a benign and moderate disorder that usually does not lead to anemia. During the neonatal period, kidneys play a key role in detoxification and recirculation of iron species released from red blood cells (RBC) and filtered out by glomeruli to the primary urine. Activity of heme oxygenase 1 (HO1), a heme-degrading enzyme localized in epithelial cells of proximal tubules, seems to be of critical importance for both processes. We show that, in HO1 knockout mouse newborns, hemolysis was prolonged despite a transient state and exacerbated, which led to temporal deterioration of RBC status. In neonates lacking HO1, functioning of renal molecular machinery responsible for iron reabsorption from the primary urine (megalin/cubilin complex) and its transfer to the blood (ferroportin) was either shifted in time or impaired, respectively. Those abnormalities resulted in iron loss from the body (excreted in urine) and in iron retention in the renal epithelium. We postulate that, as a consequence of these abnormalities, a tight systemic iron balance of HO1 knockout neonates may be temporarily affected.

Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.

Club Cell Heme Oxygenase-1 Deletion: Effects in Hyperoxia-Exposed Adult Mice.

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro. The influence of club cell HO-1 on lung development and lung injury responses is poorly understood. The present studies characterized the effects of hyperoxia on club cell-specific HO-1 knockout (KO) mice. These mice were generated by crossing Hmox1 flox mice with transgenic mice expressing cre recombinase under control of the club cell-specific Scgb1a1 promoter. Baseline analyses of lung architecture and function performed in age-matched adult wild-type and KO mice indicated an increased alveolar size and airway resistance in HO-1 KO mice. In subsequent experiments, adult wild-type and HO-1 KO mice were either continuously exposed to >95% hyperoxia or room air for 72 h or exposed to >95 hyperoxia for 48 h followed by recovery in room air for 48 h. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg). Analyses indicated an independent effect of hyperoxia but not genotype on right lung/body weight ratios in both wild-type and HO-1 KO mice. The magnitude of increases in right lung/body weight ratios was similar in mice of both genotypes. In the recovery model, an independent effect of hyperoxia but not genotype was also detected. In contrast to the continuous exposure model, right lung/body weight ratio mice were significantly elevated in HO-1 KO but not wild-type mice. Though club cell HO-1 does not alter hyperoxic sensitivity in adult mice, it significantly influences lung development and resolution of lung injury following acute hyperoxic exposure.

Hepatocellular heme oxygenase 1 deficiency does not affect inflammatory hepcidin regulation in mice.

Hepcidin is an iron regulatory peptide hormone that is secreted from hepatocytes and inhibits iron efflux from tissues to plasma. Under inflammatory conditions, hepcidin is transcriptionally induced by IL-6/STAT3 signaling and promotes hypoferremia, an innate immune response to infection. If this pathway remains unresolved, chronic overexpression of hepcidin contributes to the anemia of inflammation, a common medical condition. Previous work showed that carbon monoxide (CO) releasing drugs (CORMs) can attenuate inflammatory induction of hepcidin. Because CO is physiologically generated during heme degradation by heme oxygenase 1 (HO-1), an IL-6-inducible enzyme with anti-inflammatory properties, we hypothesized that hepatocellular HO-1 may operate as a physiological feedback regulator of hepcidin that resolves inflammatory signaling. To address this, we generated and analyzed hepatocyte-specific HO-1 knockout (Hmox1Alb-Cre) mice. We show that these animals mount appropriate hepcidin-mediated hypoferremic response to LPS-induced inflammation, with kinetics similar to those of control Hmox1fl/fl mice. Likewise, primary hepatocytes from Hmox1Alb-Cre and Hmox1fl/fl mice exhibit similar degree and kinetics of hepcidin induction following IL-6 treatment. We conclude that hepatocellular HO-1 has no physiological function on hepcidin regulation by the inflammatory pathway.

11 - Lung epithelial specific heme oxygenase-1 deletion does not worsen hyperoxic lung injury in adult mice

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2(Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro. To evaluate the contribution of HO-1 toward the protective effects of TXNRD1 inhibitors in vivo, a club cell specific HO-1 knockout (KO) mouse is needed. The present studies characterized the effects of hyperoxia on club cell specific HO-1 KO mice that were generated by crossing HO-1 floxed mice with transgenic mice expressing cre recombinase under control of a club cell specific promoter. Twelve-week old wildtype (WT) and KO mice were continuously exposed to >95% hyperoxia (HYP) or room air (RA) for 72h. Following exposure, mice were sacrificed and weighed. The left lung was inflation fixed and the right lung was collected and weighed. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg) and qualitatively assessed in HE features that appeared to be more prominent in KO/HYP mice. In conclusion, lung epithelial cell HO-1 deletion does not alter hyperoxic lung injury in adult mice. Future studies will utilize this unique transgenic model to evaluate the contribution HO-1 toward the protective effects of TXNRD1 inhibitors in vivo.

Reprint of: Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1−/−) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1.The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1−/− mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1−/− mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPβ (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPβ expression and activity in HO-1−/− myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1−/− granulocytes were functionally competent as determined by oxidative burst capacity.In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPβ protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

Sex-Dependent Effects of HO-1 Deletion from Adipocytes in Mice

Induction of heme oxygenase-1 (HO-1) has been demonstrated to decrease body weight and improve insulin sensitivity in several models of obesity in rodents. To further study the role of HO-1 in adipose tissue, we created an adipose-specific HO-1 knockout mouse model. Male and female mice were fed either a control or a high-fat diet for 30 weeks. Body weights were measured weekly and body composition, fasting blood glucose and insulin levels were determined every six weeks. Adipocyte-specific knockout of HO-1 had no significant effect on body weight in mice fed a high-fat diet but increased body weight in female mice fed a normal-fat diet. Although body weights were not different in females fed a high fat diet, loss of HO-1 in adipocytes resulted in significant alterations in body composition. Adipose-specific HO-1 knockout resulted in increased fasting hyperglycemia and insulinemia in female but not male mice on both diets. Adipose-specific knockout of HO-1 resulted in a significant loss of HO activity and a decrease in the protein levels of adiponectin in adipose tissue. These results demonstrate that loss of HO-1 in adipocytes has greater effects on body fat and fasting hyperglycemia in a sex-dependent fashion and that expression of HO-1 in adipose tissue may have a greater protective role in females as compared to males.

Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1−/−) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1.The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1−/− mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1−/− mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPβ (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPβ expression and activity in HO-1−/− myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1−/− granulocytes were functionally competent as determined by oxidative burst capacity.In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPβ protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

Taurine Chloramine Stimulates Efferocytosis Through Upregulation of Nrf2-Mediated Heme Oxygenase-1 Expression in Murine Macrophages: Possible Involvement of Carbon Monoxide

To examine the pro-resolving effects of taurine chloramine (TauCl). TauCl injected into the peritoneum of mice enhanced the resolution of zymosan A-induced peritonitis. Furthermore, when the macrophages obtained from peritoneal exudates were treated with TauCl, their efferocytic ability was elevated. In the murine macrophage-like RAW264.7 cells exposed to TauCl, the proportion of macrophages engulfing the apoptotic neutrophils was also increased. In these macrophages treated with TauCl, expression of heme oxygenase-1 (HO-1) was elevated along with increased nuclear translocation of the nuclear factor E2-related factor 2 (Nrf2). TauCl binds directly to Kelch-like ECH association protein 1 (Keap1), which appears to retard the Keap1-driven degradation of Nrf2. This results in stabilization and enhanced nuclear translocation of Nrf2 and upregulation of HO-1 expression. TauCl, when treated to peritoneal macrophages isolated from either Nrf2 or HO-1 wild-type mice, stimulated efferocytosis (phagocytic engulfment of apoptotic neutrophils by macrophages), but not in the macrophages from Nrf2 or HO-1 knockout mice. Furthermore, transcriptional expression of some scavenger receptors recognizing the phosphatidylserines exposed on the surface of apoptotic cells was increased in RAW264.7 cells treated with TauCl. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 gene in RAW264.7 cells abolished the TauCl-induced efferocytosis, whereas both overexpression of HO-1 and treatment with carbon monoxide (CO), the product of HO, potentiated the efferocytic activity of macrophages. This work provides the first evidence that TauCl stimulates efferocytosis by macrophages. The results of this study suggest the therapeutic potential of TauCl in the management of inflammatory disorders. TauCl can facilitate resolution of inflammation by increasing the efferocytic activity of macrophages through Nrf2-mediated HO-1 upregulation and subsequent production of CO.

In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation but whether such control occurs in vivo is not known. To enable such analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome Conformation Capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases.

Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection

Ginkgo biloba extract, EGb 761, a popular and standardized natural extract, contains 24% ginkgo-flavonol glycosides and 6% terpene lactones. EGb 761 is used worldwide to treat many ailments, and although a number of studies have shown its neuroprotective properties, the mechanisms of action have not been elucidated fully. We hypothesize that EGb 761 and some of its bioactive components [Bilobalide (BB), Ginkgolide A (GA), Ginkgolide B (GB), and Terpene Free Material (TFM)] could provide neuroprotection in ischemic conditions through heme oxygenase 1 (HO1). Mice were subjected to permanent distal middle cerebral artery occlusion ( pMCAO) and survived for 7 days. HO1 knockout (HO1 −/−) mice showed significantly higher ( P<0.05) infarct volume and Neurologic Deficit Scores (NDS) as compared to their wildtype (WT) counterparts. In another cohort, WT mice subjected to pMCAO and treated at 4 h of pMCAO with 100 mg/kg EGb 761, 6 mg/kg BB, GA, GB, or 10 mg/kg TFM showed significantly lower ( P<0.05) infarct volumes (BB; 29.0±3.9%, GA; 31.3±4.0%, GB; 32.0±3.8%, TFM; 32.5±3.5%, and EGb 761; 27.4±4.5%) than those in the vehicle-treated mice (46.0±3.7%). Similarly, NDS were lower in BB; 7.1±1.8, GA; 7.4±2.1, GB; 7.9±1.8, TFM; 7.7±1.7, and EGb 761; 6.8±2.0 groups as compared with the vehicle-treated group (13.8±1.5). Interestingly, the protective effect of EGb 761 was essentially lost when HO1 knockout mice were treated with EGb 761. In another cohort, HO1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) protein levels in the brain cortices appeared to be higher in EGb 761 and BB but not in GA, GB and TFM treated groups. Together, these results suggest that HO1 plays, at least in part, an important role in the neuroprotective mechanism of EGb 761 and in delayed ischemia. Targeting this pathway could lead to neuroprotective agents against ischemic stroke.

Human Heme Oxygenase-1 Deficiency Presenting With Hemolysis, Nephritis, and Asplenia

Heme oxygenase-1 (HO-1) is a stress-induced enzyme that catalyses the oxidation of heme to biliverdin. The primary deficiency of this enzyme has been shown in HO-1 knockout mice, and is characterized by intrauterine death and chronic inflammation. The first case of human HO-1 deficiency was reported in 1999. Human HO-1 deficiency has been observed to involve the endothelial cells more severely, resulting in hemolysis and disseminated intravascular coagulation. We report another case of human HO-1 deficiency in a young girl with congenital asplenia, who presented with severe hemolysis, inflammation, nephritis, which was refractory to therapy with corticosteroids, cyclophosphamide, and rituximab.

Human Heme Oxygenase-1 Deficiency Presenting with Hemolysis, Bleeding, Nephritis, Asplenia and Inflammation.

Abstract 3013Poster Board II-989 Introduction:Heme oxygenase-1 (HO-1) is a stress induced enzyme that catalyses the oxidation of heme to biliverdin and bilirubin. HO-1 is not constitutively produced and has been shown to be an endogenous factor against inflammation. Deficiency of HO-1 in HO-1 knockout mice as well as in one human case has been found to have a pro-inflammatory phenotype. To the best of our knowledge, previously there is only one published case of human HO-1 deficiency in literature. We report a patient who presented with massive hemolysis, bleeding, nephritis and inflammation and was proved to have HO-1 deficiency. Case report:A 15-year-old previously well girl presented with high-grade fever for 2 weeks and cervical and axillary lymphadenopathy. She had an insignificant family history and had no growth or developmental retardation. Initial evaluation was unremarkable except for thrombocytosis noted from first week of fever and asplenia that was noted on CT scan. There was no past history of any major infection or thrombocytosis. She developed intravascular hemolysis from the second week of illness followed by nephritis as evidenced by hematuria, proteinuria (2950 mg over 24 hr), hyaline cast with hypertension. C3 levels were normal. IgA levels were increased to 502 mg/dl. Her blood was agglutinating at room temperature and serum could be separated with great difficulty. Workup for hemolysis showed evidence of cold antibody mediated immune hemolysis. Her direct combs test was positive. LDH was 9262 IU/l. Urine was positive for hemoglobinuria. Her serum ferritin was 4219 ng/ml, ESR was 75mm/1st hr, platelets were increased to 1200,000/ul and interleukin-6 levels were increased to 139 pg/ml. Blood cultures were negative repetitively. However etiology including infections, lymphoreticular malignancies was ruled out. She was started on treatment with methylprednisolone (20 mg/kg x 3 days) followed by oral prednisolone at 2mg/kg/day. A month later, she developed vasculitic skin rash. In renal biopsy, glomeruli showed normal cellularity with mild focal mesangeal prominence, normal basement membrane and open capillary loops compatible with IgA nephropathy or minimal change disease. Immunoflourescence was non-contributory due to absence of any glomerulus. Bone marrow was normal and skin biopsy was non-contributory. Hemolysis persisted despite giving steroids. She developed bleeding diathesis in the form of uncontrolled epistaxis, diffuse alveolar hemorrhage and skin bleeds. Evaluation for coagulopathy showed high platelet count and normal PT (14.9 sec), INR 1.24 and APTT (32 sec). Von Willebrand antigen was raised to 308 %; Fibrinogen was 2.4 g/l and FDP level was increased to 8.0 mg/l by screening method. Epistaxis was controlled with repeated transfusion of fresh plasma. In view of hemolysis and nephritis non-responsive to steroids, she received a pulse of cyclophosphamide (500mg/m2) followed by two doses of Rituximab (375 mg/m2 each). Her condition deteriorated further over the next few weeks. She developed intracranial hemorrhage in right parietal lobe and required Factor VIIa for control of bleeding. She died with fungal sepsis (Trichosporon asahii) after prolonged hospitalization within 5 months from onset of the illness. Atypical features noticed in this case were near normal serum bilirubin despite severe intravascular hemolysis and presence of asplenia. In view of a similar case with hemolysis, asplenia and nephritis reported from Japan as the first case of human HO-1 deficiency, our patient's DNA was sent to the same lab for HO-1 gene mutation. Mutation analysis revealed homozygous mutations in exon 2 (R44X) on chromosome 22q12 which would result in absence of functional protein HO. Renal biopsy for HO-1 immunostaining showed little if any detectable HO-1 in renal tubules, despite the presence of massive hemolysis. Conclusion:HO-1 has been found to have cytoprotective effect against stress mediated organ damage. Inhibition of HO-1 has been shown to increase the vulnerability of an individual to stressful stimuli like infections. Lack of this enzyme might explain the varied clinical features and exaggerated response to stress noted in our patient. However it is not clear whether the entire spectrum of manifestations noted in our patient can be explained solely by HO-1 deficiency. Further cases are required to characterize the clinical and pathological manifestations of HO-1 deficiency. Disclosures:Off Label Use: Use of activated factor VII for control of intracranial hemorrhage.