Carbon monoxide rescues heme oxygenase-1-deficient mice from arterial thrombosis in allogeneic aortic transplantation.

Heme oxygenase-1 (HO-1) catalyzes the conversion of heme into carbon monoxide (CO), iron, and biliverdin. In preliminary studies, we observed that the absence of HO-1 in aortic allograft recipients resulted in 100% mortality within 4 days due to arterial thrombosis. In contrast, recipients normally expressing HO-1 showed 100% graft patency and survival for more than 56 days. Abdominal aortic transplants were performed using Balb/cJ mice as donors and either HO-1(+/+) or HO-1(-/-) (C57BL/6xFVB) mice as recipients. Light and electron microscopy revealed extensive platelet-rich thrombi along the entire length of the graft in HO-1(-/-) recipients at 24 hours. Treatment of recipients with CORM-2, a CO-releasing molecule (10 mg/kg of body weight intravenously), 1 hour prior and 1, 3, and 6 days after transplantation, significantly improved survival (62% at >56 days, P < 0.001) compared with HO-1(-/-) recipients treated with inactive CORM-2 (median survival 1 day). Histological analyses revealed that CO treatment markedly reduced platelet aggregation within the graft. Adoptive transfer of wild-type platelets to HO-1(-/-) recipients also conferred protection and increased survival. Aortic transplants from either HO-1(-/-) or HO-1(+/+) C57BL/6 donors into HO-1(+/+) (Balb/cJ) mice did not develop arterial thrombosis, surviving more than 56 days. These studies demonstrate an important role for systemic HO-1/CO for protection against vascular arterial thrombosis in murine aortic allotransplantation.

Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions.

Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitrolinoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a -4.5 kb human HO-1 promoter construct, whereas a -4.0 kb construct with deletion of 500 bp from the 5' region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression, whereas the addition of a third mutation in the proximal E-box sequence completely abolished LNO2-induced HO-1 expression. Chromatin immunoprecipitation assays confirmed CREB (CRE-binding protein)-1 binding to the CRE (located at -4.0 kb) and E-box regions (located at -44 bp) of the human HO-1 promoter. A 3C (Chromosome Conformation Capture) assay of intact cells showed LNO2-induced interactions between the CRE- and E-box- containing regions. These observations indicate that regulation of human HO-1 expression by LNO2 requires synergy between CRE, AP-1 and E-box sequences and involves the participation of CREB-1.

Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid mediates protective effects through regulation of the ERK pathway.

In vivo and in vitro studies revealed that nitroalkenes serve as protective mediators in the lung by inducing the cytoprotective enzyme heme oxygenase-1 (HO-1). Nitrolinoleic acid (LNO2) increased HO-1 mRNA, protein, and activity in cultured pulmonary epithelial cells treated with 5 to 50 microM LNO2 and in lungs of rats injected intraperitoneally with 2.6 mg/kg LNO2 twice daily for 20 days. Western blotting revealed that HO-1 protein increased significantly within 4 h of in vitro LNO2 addition and was preceded by an increase in HO-1 mRNA, consistent with transcriptional regulation of HO-1 expression by LNO2. LNO2 also dephosphorylated and activated eukaryotic initiation factor 2alpha, a key translational regulatory protein, indicating that increased translation may also contribute to LNO2-induced increases in HO-1. Exposure of cells to LNO2 activated ERK and JNK, as evidenced by increased phosphorylation. Downstream targets of ERK and JNK, Elk-1 and c-Jun, respectively, were also phosphorylated in response to LNO2 exposure. However, inhibitor studies revealed that only the ERK pathway is necessary for the LNO2-mediated increase in HO-1 mRNA and protein. These data reveal that LNO2 induces pulmonary epithelial HO-1 expression and downstream adaptive responses to inflammation via both transcriptional and translational regulatory mechanisms.

JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells.

Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper-sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB(-)(/)(-) and junD(-)(/)(-) cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene.

Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid potently activates endothelial heme oxygenase 1 expression.

Nitroalkenes are a class of cell signaling mediators generated by NO and fatty acid-dependent redox reactions. Nitrated fatty acids such as 10- and 12-nitro-9,12-octadecadienoic acid (nitrolinoleic acid, LNO(2)) exhibit pluripotent antiinflammatory cell signaling properties. Heme oxygenase 1 (HO-1) is up-regulated as an adaptive response to inflammatory mediators and oxidative stress. LNO(2) (1-10 microM) induced HO-1 mRNA and protein up to 70- and 15-fold, respectively, in human aortic endothelial cells. This induction of HO-1 occurred within clinical LNO(2) concentration ranges, far exceeded responses to equimolar amounts of linoleic acid and oxidized linoleic acid, and rivaled that induced by hemin. Ex vivo incubation of rat aortic segments with 25 microM LNO(2) resulted in a 40-fold induction of HO-1 protein that localized to endothelial and smooth muscle cells. Actinomycin D inhibited LNO(2) induction of HO-1 in human aortic endothelial cells, and LNO(2) activated a 4.5-kb human HO-1 promoter construct, indicating transcriptional regulation of the HO-1 gene. The peroxisome proliferator-activated receptor gamma (PPARgamma) receptor antagonist GW9662 did not inhibit LNO(2)-mediated HO-1 induction, and a methyl ester derivative of LNO(2) with diminished PPARgamma binding capability also induced HO-1, affirming a PPARgamma-independent mechanism. The NO scavengers 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and oxymyoglobin partially reversed induction of HO-1 by LNO(2), revealing that LNO(2) regulates HO-1 expression by predominantly NO-independent mechanisms. In summary, the metabolic and inflammatory signaling actions of nitroalkenes can be transduced by robust HO-1 induction.

Effects of intratracheal tumor necrosis factor-alpha plasmid vector on lipopolysaccharide lethality and lung injury in mice.

Bacterial lipopolysaccharide (LPS) causes acute lung injury (ALI) and contributes to inflammation in the acute respiratory distress syndrome (ARDS) and sepsis, making mechanisms of resistance to LPS critically important in clinical settings. The authors postulated that intratracheal administration of a plasmid (pcDNA3. 0-rTNFalpha) encoding rat tumor necrosis factor-alpha (TNF-alpha) would increase resistance of mice to LPS-induced ALI or mortality. They investigated the time course and dose-response for development of LPS-induced ALI in C57/BL6 mice and sought possible protective effects of 100 microg pcDNA3.0-rTNFalpha intratracheally 1, 2, or 3 weeks before LPS challenge. Lung myeloperoxidase (MPO) activity and alveolar lavage fluid (BALF) cell counts increased significantly 48 hours after intraperitoneal (IP) LPS challenges. After pcDNA3.0-rTNFalpha pretreatment, mice challenged with LPS had lower lung/body weight ratios than mice treated with pcDNA3.0; however, other indices of lung injury did not differ. Survival of mice challenged with lethal IP LPS 2 weeks after intratracheal pcDNA3.0-rTNFalpha vector improved significantly, compared to mice pretreated with the control vector, pcDNA3.0. However, pcDNA3.0-pretreated mice tolerated LPS challenge less well than saline-pretreated controls. LPS causes neutrophilic lung injury and mortality, but pcDNA3.0-TNFalpha does not prevent ALI due to LPS. Intratracheal pcDNA3.0-rTNFalpha pretreatment significantly improves survival of mice after LPS challenge, compared to those pretreated with pcDNA3.0.

p38mapk and MEK1/2 inhibition contribute to cellular oxidant injury after hypoxia.

Lung epithelial cells produce increased reactive oxygen species (ROS) after hypoxia exposure, and they are more susceptible after hypoxia to injury by agents that generate superoxide [O2-; e.g., 2,3-dimethoxy-1,4-naphthoquinone (DMNQ)]. Cellular GSH and MnSOD both decrease in hypoxic lung epithelial cells, altering the redox state. Because ROS participate in signaling pathways involved in cell death or survival, we tested the hypothesis that mitogen-activated protein kinases (MAPK) were involved in a protective response against cellular injury during reoxygenation. Human lung epithelial A549 cells were incubated in hypoxia (<1% O2 for 24 h) and then reoxygenated by return to air. p38mapk and MKK3 phosphorylation both decreased after hypoxia. During reoxygenation, cells were incubated with DMNQ (0-50 microM), a redox cycling quinone that produces O2-. Hypoxia preexposure significantly increased epithelial cell lysis resulting from DMNQ. Addition of the p38mapk inhibitors SB-202190 or SB-203580 markedly increased cytotoxicity, as did the mitogen/extracellular signal-regulated kinase (MEK) 1/2 inhibitor PD-98059 (all 10 microM), suggesting a protective effect of downstream molecules activated by the kinases. Transfection of A549 cells with a dominant active MKK3 plasmid (MKK3[Glu]) partially inhibited cytolysis resulting from DMNQ, whereas the inactive MKK3 plasmid (MKK3[Ala]) had less evident protective effects. Stress-related signaling pathways in epithelial cells are modulated by hypoxia and confer protection from reoxygenation, since hypoxia and chemical inhibition of p38mapk and MEK1/2 similarly increase cytolysis resulting from O2-.

Reactive species mediated injury of human lung epithelial cells after hypoxia-reoxygenation.

This study tested the hypothesis that hypoxia exposure predisposed lung epithelial cells to reactive oxygen species-(ROS) mediated cellular injury. Human lung carcinoma cells (ATCC line H441) having epithelial characteristics (including lamellar bodies, surfactant protein [SP]-A, and SP-B) were cultured in air (air/5% CO(2)) or hypoxia (< 1% O(2)/5% CO(2)) for 0 to 24 hours before imposition of oxidant stress. Cellular manganese superoxide dismutase (MnSOD) activity (units/mg protein) decreased significantly after 24 hours of hypoxia. In normoxic culture after hypoxia, the cells produced increased ROS, detected as dichlorofluorescein (DCF) fluorescence and H(2)O(2) accumulation in medium. Antioxidants N-acetylcysteine (N-Ac) and ebselen inhibited increased DCF fluorescence after hypoxia. To test their ability to tolerate oxidant stress, some cells were incubated with antimycin A (100 microM) and trifluorocarbonylcyanide phenylhydrazone (10 microM) (anti A + FCCP), a mitochondrial complex III inhibitor and respiratory chain uncoupler, which together increase mitochondrial superoxide (O(2)(-)) and H(2)O(2) production. Lung epithelial cells preexposed to hypoxia released more lactate dehydrogenase (LDH) than normoxic controls in response to increased O(2)(-) production. Increased LDH release from hypoxia-preexposed cells treated with anti A + FCCP was inhibited by 1 mM N-Ac. Rotenone and myxothiazole increased DCF oxidation more in hypoxic than in normoxic cells, suggesting that mitochondrial electron transport complex I may have been altered by hypoxia preexposure.