Selective inhibition of heme oxygenase-2 activity by analogs of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole): Exploration of the effects of substituents at the N-1 position

Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many of the compounds were found to be potent and highly selective for the HO-2 isozyme (constitutive), and had substantially less inhibitory activity on the HO-1 isozyme (inducible). The compounds represent the first report of highly potent and selective inhibitors of HO-2 activity, and complement our suite of selective HO-1 inhibitors. The study has revealed many candidates based on the inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.     

Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury

We reported that carbon monoxide (CO) generated through heme oxygenase (HO) inhibits mitogen-induced proliferation of vascular smooth muscle cells (VSMCs). We report that balloon injury induces HO-1, the stress-inducible isozyme of HO, in VSMCs and inhibits neointimal formation through the action of endogenous CO. Northern blot analysis and immunohistochemistry revealed that HO-1 is markedly induced in the media as early as 1 day after injury, whereas only a little expression was detected in the intact carotid artery. The neointimal proliferative changes were augmented or inhibited by the HO inhibitors or inducer, respectively, and effects of these interventions were not altered by suppression of endogenous nitric oxide (NO), if any. To elucidate the mechanisms by which HO controls the proliferative changes, effects of alterations in the HO reaction were examined by determining angiotensin II-elicited VSMC proliferation in vitro: the HO inducer attenuated and its inhibitor restored the proliferative response to angiotensin II (1 nM and 100 nM). Hemoglobin, a reagent trapping both NO and CO, but not met-hemoglobin, which can capture NO but not CO, augmented the proliferative response. These data suggest that endogenous CO serves as a protective factor that limits the excessive VSMC proliferation associated with vascular diseases.     

Synthesis and evaluation of imidazole–dioxolane compounds as selective heme oxygenase inhibitors: Effect of substituents at the 4-position of the dioxolane ring

Several imidazole–dioxolane compounds were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds, which include a series of substituted thiophenol and substituted phenol derivatives of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[(phenylsulfanyl)methyl]-1,3-dioxolane hydrochloride (3), in addition to smaller functionalized derivatives, continue our structure–activity studies by exploration of the aminothiophenol region (‘northeastern region’) in our original target structure azalanstat (1). In vitro, most of the compounds in this series were found to be highly potent inhibitors of the stress-induced isozyme HO-1 and the constitutive isozyme HO-2, showing only moderate selectivity for HO-1. Nevertheless, a few of the compounds displayed higher selectivity toward HO-1. None of the compounds having a larger appendage in the northeastern region were inhibitors of CYP2E1, whereas a compound having a relatively small fluorine substituent in this region did inhibit CYP2E1; all of the compounds tested exhibited high inhibitory potency against CYP3A1/3A2.     

Heme oxygenase activity in fetal and adult sheep is not altered by acclimatization to high altitude hypoxia

Hypoxic stress has been reported to induce the expression of stress proteins such as heme oxygenase (HO), which catalyze the breakdown of heme to generate biliverdin, ferrous iron, and carbon monoxide. These degradation products play a role in the regulation of a variety of processes such as vascular tone, inflammation, and central nervous system function. In mammals, there are 2 catalytically functional HO isozymes, HO-1 (inducible) and HO-2 (constitutive). HO-1 expression is regulated by an array of nonphysiological and physiological stimuli including acute hypoxemia. As relatively little is known of the HO response to prolonged hypoxia in whole animals other than small laboratory rodents, the aim of this work was to examine the effect of long-term hypoxia on total HO activity in fetal and adult ovine tissue. Sheep were maintained at high altitude (3820 m), after which the following tissues were harvested from near-term fetal and non-pregnant ewes for in vitro measurement of HO activity: left ventricle, renal papilla, lung apex, pulmonary artery, carotid artery, mesenteric artery, placental cotyledon, spleen, and brain frontal cortex. There were no significant differences between HO activities in tissues from hypoxic fetal and adult sheep compared with their normoxic controls. Fetal heart HO activities were higher than those of adult tissue (p < 0.05), whereas adult spleen HO activity was significantly higher than that of fetal tissue (p < 0.05). In conclusion, these data indicate that long-term exposure to high altitude hypoxia does not have a persistent effect on HO activity in ovine tissues. Also, except for the spleen where there is a high expression of HO-1 under normal conditions, tissue HO activity is correlated with the expression of HO-2, the constitutive isozyme.     

Heme oxygenase and heme degradation

The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.     

Inhibitors of the heme oxygenase - carbon monoxide system: on the doorstep of the clinic?

The past decade has seen substantial developments in our understanding of the physiology, pathology, and pharmacology of heme oxygenases (HO), to the point that investigators in the field are beginning to contemplate therapies based on administration of HO agonists or HO inhibitors. A significant amount of our current knowledge is based on the judicious application of metalloporphyrin inhibitors of HO, despite their limitations of selectivity. Recently, imidazole-based compounds have been identified as potent and more selective HO inhibitors. This 'next generation' of HO inhibitors offers a number of desirable characteristics, including isozyme selectivity, negligible effects on HO protein expression, and physicochemical properties favourable for in vivo distribution. Some of the applications of HO inhibitors that have been suggested are treatment of hyperbilirubinemia, neurodegenerative disorders, certain types of cancer, and bacterial and fungal infections. In this review, we address various approaches to altering HO activity with a focus on the potential applications of second-generation inhibitors of HO.     

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3,5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.     

An analysis of the DOCA-salt model of hypertension in HO-1-/- mice and the Gunn rat

Heme oxygenase-1 (HO-1) is induced in the vasculature in the DOCA-salt model of hypertension in rats. Whereas the HO system and its products may exert vasodilator effects, recent studies have suggested that the HO system may predispose to hypertension. The present study examined the effects of selected components of the HO system, specifically, the HO-1 isozyme and the product bilirubin in the DOCA-salt model of systemic hypertension; the experimental approach employed mutant rodent models, namely, the HO-1(-/-) mouse and the hyperbilirubinemic Gunn rat. DOCA-salt induced HO-1 protein in the aorta in HO-1(+/+) mice and provoked a significant rise in systolic arterial pressure in HO-1(-/-) mice but not in HO-1(+/+) mice; this effect could not be ascribed to impaired urinary sodium excretion or impaired glomerular filtration rate in the DOCA-salt-treated HO-1(-/-) mice. The administration of DOCA salt to uninephrectomized rats significantly increased systolic arterial pressure in wild-type rats, an effect that was attenuated in the mutant Gunn rat; this reduction in systemic hypertension in the DOCA-salt-treated Gunn rat was not due to a greater induction of HO-1 in the vasculature or to a more avid urinary sodium excretion. DOCA-salt impaired endothelium-dependent and endothelium-independent vasorelaxation in wild-type rats but not in Gunn rats; prior exposure to bilirubin repaired the defect in endothelium-dependent vasorelaxation in aortic rings in DOCA-salt-treated rats. DOCA salt stimulated vascular production of superoxide anion in wild-type but not in Gunn rats. We suggest that HO-1 and the product bilirubin may exert a countervailing effect in the DOCA-salt model of systemic hypertension.     

Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.

The purpose of this study was to examine the expression of hemeoxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO-1 and HO-2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO-2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO-2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO-2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO-2. HO-1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO-1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose-dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.     

HO-1 induction attenuates renal damage and oxidative stress induced by K2Cr2O7

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme; its inducible isozyme HO-1 protects against some types of acute tissue injury. The expression and functional role of HO-1 in rats with renal injury induced by potassium dichromate (K(2)Cr(2)O(7)) was investigated in this work. Rats were studied 24 h after a single injection of K(2)Cr(2)O(7). To address the possible protective effect of HO-1 in this experimental model, this enzyme was induced by an injection of stannous chloride (SnCl(2)) 12 h before K(2)Cr(2)O(7) administration. The functional role of HO-1 in K(2)Cr(2)O(7) + SnCl(2)-treated animals was tested by inhibiting HO activity with an injection of zinc (II) protoporphyrin IX (ZnPP) 18 h before K(2)Cr(2)O(7). In K(2)Cr(2)O(7)-treated rats: (i) renal HO-1 content, measured by Western blot, increased 2.6-fold; and, (ii) renal nitrotyrosine and protein carbonyl content, markers of oxidative stress, increased 3.5- and 1.36-fold, respectively. Renal damage and oxidative stress were ameliorated and HO-1 content was increased in the K(2)Cr(2)O(7) + SnCl(2) group. The attenuation of renal injury and oxidative stress was lost by the inhibition of HO activity in K(2)Cr(2)O(7) + SnCl(2) + ZnPP-treated animals. Our data suggest that HO-1 overexpression induced by SnCl(2) is responsible for the attenuation of renal damage and oxidative stress induced by K(2)Cr(2)O(7).     

Heme oxygenase-carbon monoxide (HO-CO) system in rat uterus: effect of sexual steroids and prostaglandins

Pregnancy maintenance is a very complex phenomenon, and the mechanisms that allow the survival of the fetus within the maternal uterus are still poorly understood. Our objectives were to analyze heme oxygenase (HO) activity and expression in the pregnant rat and to study its association with steroid hormones and prostaglandins. Uterine tissues were obtained from non-pregnant and from time-mated rats at days 5, 13, 18-22 of pregnancy and postpartum. HO activity was significantly higher at days 5 and 20 while HO-1 protein levels measured by Western blot, were significantly elevated from days 19 to 22. In ovariectomized rats, estrogen and progesterone in estrogenized animals increased HO activity and expression. Cyclooxygenase inhibitors augmented HO activity and HO-1 expression. Pre-incubation with prostaglandin F2alpha (PGF2alpha) diminished the enzymatic activity in ovariectomized rat uterus. Tin protoporphyrin IX, an HO inhibitor, significantly decreased uterine cGMP accumulation. Bilirubin decreased uterine thiobarbituric acid substances levels (an index of lipid peroxidation). These results demonstrate a uterine gestational pattern of HO activity and expression in the rat. In addition, these results suggest that uterine HO activity could regulate uterine quiescence in pregnancy via cGMP and it may contribute to the defense against oxidative stress.     

Heme oxygenase expression in selected regions of term human placenta

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Heme oxygenase inhibition by 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes: effect of halogen substitution in the phenyl ring

A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, imidazole-dioxolanes, and imidazole-alcohols substituted with halogens in the phenyl ring were synthesized and evaluated as novel inhibitors of heme oxygenase which are structurally distinct from metalloporphyrins. The entire library of compounds was found to be highly active, with the bromine- and iodine-substituted derivatives being the most potent. The imidazole-dioxolanes were all selective for the HO-1 isozyme (inducible) and exhibited substantially lower activity toward the HO-2 isozyme (constitutive). The corresponding imidazole-ketones and imidazole-alcohols showed selectivity toward HO-1 to a lesser degree than the similarly substituted imidazole-dioxolanes.