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Objective: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body’s response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient ( Hmox1 –/– ) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1 –/– mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O 2 ) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1 –/– mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1 –/– fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1 –/– fibroblasts in response to hypoxia. Conclusions: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1’s protection against ischemic injury independent of neovascularization.

Significance: Heme oxygenase-1 (HMOX1) plays a critical role in the protection of cells, and the inducible enzyme is implicated in a spectrum of human diseases. The increasing prevalence of cardiovascular and metabolic morbidities, for which current treatment approaches are not optimal, emphasizes the necessity to better understand key players such as HMOX1 that may be therapeutic targets. Recent Advances: HMOX1 is a dynamic protein that can undergo post-translational and structural modifications which modulate HMOX1 function. Moreover, trafficking from the endoplasmic reticulum to other cellular compartments, including the nucleus, highlights that HMOX1 may play roles other than the catabolism of heme. Critical Issues: The ability of HMOX1 to be induced by a variety of stressors, in an equally wide variety of tissues and cell types, represents an obstacle for the therapeutic exploitation of the enzyme. Any capacity to modulate HMOX1 in cardiovascular and metabolic diseases should be tempered with an appreciation that HMOX1 may have an impact on cancer. Moreover, the potential for heme catabolism end products, such as carbon monoxide, to amplify the HMOX1 stress response should be considered. Future Directions: A more complete understanding of HMOX1 modifications and the properties that they impart is necessary. Delineating these parameters will provide a clearer picture of the opportunities to modulate HMOX1 in human disease. Antioxid. Redox Signal. 20: 1723–1742.

Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.

Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high K(m). Apparent second-order rate constants, determined for concentrationsK(m), were 19.4, 23.8, 6.0, and 7.5 M(-1) min(-1) for glycine chloramine (Gly-Cl) and taurine, Gly-Cl and histamine, histamine chloramine and glycine, and taurine chloramine (Tau-Cl) and glycine, respectively. Thus with 10 mM amine concentrations, half-lives for chloramine exchange are on the order of a few minutes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity in cells was measured as an indicator of permeability of the chloramines. When endothelial or Jurkat cells were treated in Hanks' buffer, Gly-Cl inhibited GAPDH, whereas Tau-Cl, which does not penetrate the cells, did not. Adding glycine to Tau-Cl brought about inhibition, whereas taurine mitigated the effect of Gly-Cl. For cells in full medium, high chloramine concentrations were needed to inhibit GAPDH because of scavenging by methionine and other constituents. In methionine-free medium, chlorine exchange resulted in GAPDH inhibition by Tau-Cl, whereas Gly-Cl was less effective than in Hanks' buffer. Thus interchange between chloramines occurs readily and modulates their cellular effects.

Taurine is present in high concentrations in neutrophils, and when the cells are stimulated taurine can react with hypochlorous acid (HOCl) to form taurine-chloramine (Tau-Cl). This compound retains oxidant activity and can affect the neutrophil itself or surrounding tissue cells. We have investigated the effects of Tau-Cl on MAPK signaling in human umbilical vein endothelial cells (HUVEC). Tau-Cl caused no loss in intracellular glutathione or inactivation of the thiol-sensitive enzyme glyceraldehyde-3-phosphate dehydrogenase, indicating that it had not entered the cells. However, stimulation of HUVEC with Tau-Cl (20–100 μm) induced the rapid activation of ERK within 10 min. This activation was abolished by inhibition of MEK by U0126, indicating that it was not because of direct oxidation of ERK. No activation of p38 was detected. These results suggest that Tau-Cl reacts with a cell membrane target that results in intracellular ERK activation. Tau-Cl over the same concentration range and time scale stimulated epidermal growth factor (EGF) receptor tyrosine phosphorylation in A431 cells and HUVEC. The EGF receptor inhibitor PD158780 significantly attenuated Tau-Cl-induced phosphorylation of both the EGF receptor and ERK. This implicates the EGF receptor in the upstream activation of ERK. The Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine had no effect on Tau-Cl-induced EGF receptor or ERK activation. We propose that Tau-Cl acts on an oxidant-sensitive target on the cell surface, this being either the EGF receptor itself or another target that can interact with the EGF receptor, with consequential activation of ERK.

The first mature cells to arise in the developing mammalian embryo belong to the erythroid lineage. This highlights the immediacy of the need for red blood cells during embryogenesis and for survival. Linked with this pressure is the necessity of the embryo to obtain and transport iron, synthesize hemoglobin, and then dispose of the potentially toxic heme via the stress-induced protein heme oxygenase-1 (HO-1, encoded byHmox1in the mouse). Null mutation ofHmox1results in significant embryonic mortality as well as anemia and defective iron recycling. Here, we discuss the interrelated nature of this critical enzyme with iron trafficking, erythroid cell function, and embryonic survival.

Objectives— Induction of heme oxygenase-1 (HO-1) protects against atherosclerotic disease in part by promoting reendothelialization. As endothelial progenitor cells (EPCs) contribute to reendothelialization, we examined the role of HO-1 on bone marrow and circulating EPCs. Methods and Results— In a rabbit model of aortic balloon injury, pharmacological induction of HO-1 enhanced reendothelialization at sites with and without adjacent blood vessels, the latter indicative of a contribution by EPCs. Coinciding with maximal HO-1 induction in the injured vessel, plasma concentrations of bilirubin and the numbers of circulating progenitor cells were elevated. Both processes were abolished by cotreatment of the animals with an inhibitor of HO-1. Inducers of HO-1 promoted bone marrow cells to form progenitor cell colonies, and Flk1 + /Sca-1 + -cells to adhere to the luminal surface of the injured vessel. In noninjured mice, HO-1 inducers also increased bone marrow and circulating EPCs, and the ability of these cells to differentiate and form colonies. Compared to wild-type mice, bone marrow cells from HO-1 −/− mice generated fewer endothelial colony-forming cells, and HO-1 inducers failed to promote CFU-Hill colony formation. Conclusions— These findings suggest that HO-1 contributes to vascular repair by increasing circulating EPCs derived from the bone marrow.

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.

The chemopreventive properties of the isothiocyanates have been attributed to their ability to inhibit phase I enzymes that activate procarcinogens, induce phase II protective enzymes and trigger apoptosis in transformed cells. In this study we provide evidence for a new mechanism of chemoprevention, wherein sublethal doses of phenethyl isothiocyanate (PEITC) sensitize cells to Fas-mediated apoptosis. The phenomenon was observed in the Fas-resistant T24 bladder carcinoma cell line and in Jurkat T cells overexpressing the anti-apoptotic protein Bcl-2. Caspase-3-like activity was increased up to 20-fold of that observed with either PEITC or anti-Fas antibody alone. While PEITC activated ERK, JNK and p38, inhibitors of these MAP kinases did not block apoptosis. PEITC transiently depleted cellular glutathione, providing a putative mechanism for sensitizing the cells to apoptosis. However, lowering glutathione with buthionine sulfoximine did not mimic the effect of PEITC. Instead, we propose that PEITC promotes apoptosis by directly modifying intracellular thiol proteins. The ability of PEITC to sensitize cells to receptor-mediated apoptosis provides an additional mechanism to explain its chemopreventive properties.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 microM HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 microM and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 microM HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H2O2 and the lack of JNK activation contrasts with the activation frequently seen with H2O2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N-terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N-terminus of several oligopeptides but it was unable to cleave peptide bonds on the N-terminal side of a proline residue.

Males (n= 13) Females (n= 7) Noradrenaline 0.73 ± 0.25 M 0.23 ± 0.40 M Phenylephrine 3.36 ± 0.29 M 0.36 ± 0.22 M* U46619 11.29 ± 0.12 nM 2.32 ± 0.41 nM* Serotonin 4.08 ± 0.27 M 0.16 M ± 0.10 M* Endothelin 9.36 ± 1.05 nM 7.65 ± 1.49 nM Conclusion: The internal mammary arteries of women are more sensitive to constriction by thromboxane, phenylephrine and serotonin, comparedwithmen.Ongoing studies are evaluating the molecular mechanisms responsible for these differences. doi:10.1016/j.hlc.2011.05.035