Dominant Role for Regulatory T Cells in Protecting Females Against Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. OBJECTIVE: To evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH. METHODS AND RESULTS: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. CONCLUSIONS: In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

The contribution of carbon monoxide to vascular tonus.

OBJECTIVE: The aim of this descriptive study was to examine the contribution of CO in the maintenance of vascular tonus in different organs and different vessel segments; the underlying mechanism of CO-induced vasodilation was investigated. METHODS: Sixty Wistar albino rats, aged 6-8 months, were used in this study. Response to CO by isolated arteries from the thoracic and abdominal aorta and mesenteric, renal, gastrocnemius, and gracilis muscles as well as heart, lung, and brain vascular beds was endogenously and exogenously studied using organ baths or myograph. In addition, HO-2 protein expression was assessed using Western blot analysis in isolated vessel segments. RESULTS: Although CO was shown to contribute to the regulation of vascular tonus in all feed arteries except those of the gracilis vascular bed, no effect was observed in the resistance arteries, with the sole exception of the pial artery. No relationship between HO-2 protein level and CO contribution to endogenous vascular tonus was observed. CONCLUSIONS: While the vasodilator effect of CO in vessels smaller than 600 µm in diameter was found to be mediated via potassium channels, in vessels larger than 600 µm in diameter, the effect was through both the potassium channels and the cGMP pathway.

Heme oxygenase levels and metaflammation in benign prostatic hyperplasia patients.

PURPOSE: To investigate the relationship between intra-prostatic levels of heme oxygenase (HO), metaflammation in benign prostatic hyperplasia (BPH) tissue in patients with MetS and moderate-severe lower urinary tract symptoms (LUTS). METHODS: Between January 2012 and June 2013, 132 consecutive patients, who underwent transurethral resection of the prostate for moderate-severe LUTS, secondary to clinical BPH, were enrolled. Prostate samples were investigated for the presence of an inflammatory infiltrate, according to the Irani score, and for HO-1 and HO-2 levels measurements. Patients were evaluated for the presence of metabolic syndrome (MetS) defined by the International Diabetes Federation. RESULTS: We observed that subjects with MetS exhibited greater Irani score (3.0 vs. 2.0; p < 0.05), Irani grade (2.0 vs. 1.0; p < 0.05) and lower value of HO-1 (4.55 vs. 6.01; p < 0.05) and HO-2 (0.81 vs. 2.66; p < 0.05). HO-1 (3.91 vs. 5.67; p < 0.05) and HO-2 (1.06 vs. 1.37; p < 0.05) were significantly reduced in patients with high intra-prostatic inflammation (Irani score ≥4). At the multivariate logistic regression analysis, HO-1 reduction (OR 0.588; p < 0.01), waist circumference (OR 1.09; p < 0.01), triglycerides (OR 1.013; p < 0.05) and HDL (OR 0.750; p < 0.05) were independent predictors of high intra-prostatic inflammation. We also found that HO-1 reduction (OR 0.598; p < 0.01) and the presence of MetS (OR 34.846; p < 0.01) were associated with Irani score ≥4. CONCLUSION: MetS-induced inflammation may play a key role in BPH. In detail, prostate metaflammation is inversely related to intra-prostatic HO-1 levels, serum HDL and positively with triglycerides.

A novel and sensitive assay for heme oxygenase activity.

Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.

Alteration of hepatic structure and oxidative stress induced by intravenous nanoceria.

Beyond the traditional use of ceria as an abrasive, the scope of nanoceria applications now extends into fuel cell manufacturing, diesel fuel additives, and for therapeutic intervention as a putative antioxidant. However, the biological effects of nanoceria exposure have yet to be fully defined, which gave us the impetus to examine its systemic biodistribution and biological responses. An extensively characterized nanoceria (5 nm) dispersion was vascularly infused into rats, which were terminated 1 h, 20 h or 30 days later. Light and electron microscopic tissue characterization was conducted and hepatic oxidative stress parameters determined. We observed acute ceria nanoparticle sequestration by Kupffer cells with subsequent bioretention in parenchymal cells as well. The internalized ceria nanoparticles appeared as spherical agglomerates of varying dimension without specific organelle penetration. In hepatocytes, the agglomerated nanoceria frequently localized to the plasma membrane facing bile canaliculi. Hepatic stellate cells also sequestered nanoceria. Within the sinusoids, sustained nanoceria bioretention was associated with granuloma formations comprised of Kupffer cells and intermingling CD3⁺ T cells. A statistically significant elevation of serum aspartate aminotransferase (AST) level was seen at 1 and 20 h, but subsided by 30 days after ceria administration. Further, elevated apoptosis was observed on day 30. These findings, together with increased hepatic protein carbonyl levels on day 30, indicate ceria-induced hepatic injury and oxidative stress, respectively. Such observations suggest a single vascular infusion of nanoceria can lead to persistent hepatic retention of particles with possible implications for occupational and therapeutic exposures.

[Heme oxygenase-2 neurons brain and spinal cord of human].

Immune localization of heme oxygenase-2 in neurons of some nuclei of the spinal cord and brain stem in 6 men 18-44 years old who died from causes unrelated to injury of central nervous system was studied. Neurons with positive reaction are determined for all studied regions of the brain where their contents in various nuclei ranging from 0.5 to 16% of the total number of cells detected by methylene blue. In all the sensory nuclei there is a high proportion of small neurons with a high or moderate density of reaction produce deposits. Large cells of motor nuclei often exhibit negative or low intensive enzyme reaction.

[The distribution of heme oxygenase-2 in the brainstem nuclei of rats].

Immunocytochemical method was used to determine the distribution of neurons expressing heme oxygenase-2 (HO-2-positive neurons) in the nuclei of various parts of the brainstem of 16 male Wistar rats. The sizes of neurons and the optical density of the product of histochemical reaction in their cytoplasm were determined in the nuclei studied. HO-2-positive neurons, differing in shape, size and numbers, were identified in the nuclei of the medulla oblongata, pons and midbrain. HO-2-positive cells were found 3-5 times more frequently in the sensory nuclei as compared to the motor ones. At the same time, relatively large number of nuclei was detected, which contained either no or a few HO-2-positive neurons.

Heme oxygenase regulates renal arterial resistance and sodium excretion in cirrhotic rats.

BACKGROUND & AIMS: Heme oxygenase (HO) catabolizes heme into biliverdin, carbon monoxide (CO), and free iron. CO generated in endothelial and smooth muscle layers of blood vessels modulates vascular tone by inducing relaxation of vascular smooth muscle cells. The aim of this study was to verify the role played by HO in regulating renal arterial resistance and Na(+) excretion in cirrhosis. METHODS: Twenty control rats and 20 rats with CCl(4)(-) induced cirrhosis, 10 of which were chronically treated with the HO inducer cobalt-protoporphyrin (CoPP), were studied. Pressurized renal interlobar arteries were challenged with increasing doses of phenylephrine (PE) and acetylcholine (ACh). Dose-response curves were evaluated under basal conditions and after inhibition of HO with chromium-mesoporphyrin (CrMP). HO-1 (inducible form) and HO-2 (constitutive form) expression was measured in the main and interlobar renal arteries. Serum and urinary levels of Na(+) and creatinine were also evaluated. RESULTS: In renal interlobar arteries from cirrhotic rats, the response to PE was increased, while that to ACh was blunted. After HO inhibition, the responsiveness to these vasoactive substances was comparable in the two groups. In cirrhotic rats, HO-1 expression was impaired in the main and the interlobar renal arteries. Chronic HO induction normalized the response to the vasoconstrictor, but not to the vasodilator. Cirrhotic rats treated with CoPP showed higher urinary Na(+) concentration and fractional Na(+) excretion, compared to both untreated cirrhotic and control rats. CONCLUSIONS: In cirrhotic rats, an impaired HO-1 expression promotes vasoconstriction of renal interlobar arteries. Chronic HO induction normalizes the sensitivity to PE and promotes Na(+) excretion.

[Expression of heme oxygenase-1 and inducible nitric oxide synthase in the lungs of hyperoxia-exposed preterm rats].

OBJECTIVE: To study the expression and the role of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) in preterm rats with hyperoxia-induced lung injuries. METHODS: Sixty-four three-day-old preterm Sprague-Dawley rats were randomly assigned to a hyperoxia group (90% oxygen exposure) and a control group (room air exposure), with 32 rats in each group. After 3 days or 7 days of exposure, the lung activity of HO-1 and nitric oxide (NO) contents in bronchoalveolar lavage fluid (BALF), pulmonary histopathologic changes, and the cellular distribution and expression of HO-1 and iNOS in the lungs were measured. RESULTS: After 3 days and 7 days of exposure, the hyperoxia group showed acute lung injuries characterized by the presence of hyperaemia, red cell extravasation and inflammatory infiltration. The NO contents in BALF and the iNOS expression in the lungs increased significantly in the hyperoxia group compared with those in the control group 3 and 7 days after exposure. The expression of HO-1 in macrophages in the lungs increased significantly in the hyperoxia group compared with that in the control group 3 and 7 days after exposure. The NO contents in BALF and the iNOS and HO-1 expression in the lungs increased significantly 7 days after hyperoxia exposure compared with 3 days after hyperoxia exposure. CONCLUSIONS: iNOS and HO-1 levels in the lungs increase in preterm rats with hyperoxia-induced lung injuries, suggesting that iNOS and HO-1 may play roles in hyperoxia-induced pulmonary injuries.

Expression of heme oxygenase-1 in type II pneumocytes protects against heatstroke-induced lung damage.

Heatstroke (HS) is an acute clinical disease characterized by abnormal hyperthermia and multi-organ dysfunction. Heme oxygenase (HO)-1, also called heat shock protein (HSP)32, is induced by hyperthermia and also plays protective roles in many lung disease models. Based on this phenomenon, we investigated the protective role of endogenous HO-1 in heat-induced lung damage in rats. Male Sprague-Dawley (SD) rats were separated into three groups: (a) normothermic sham, (b) HS, and (c) SnPP (inhibitor of HO-1) pretreatment rats. In the HS group, rats were killed at various time points (1, 3, 6, and 12 h after heat exposure) in order to analyze messenger ribonucleic acid (mRNA) and protein levels. Lung sections were examined for tissue damage and localization of HO-1 using immunofluorescence double labeling. We found that HS induced lung pathology (congested and thickened lung septa). The level of HO-1 mRNA was increased at 1 h, and the protein level peaked at 6 h after heat exposure. Pretreatment with SnPP (tin-protoporphyrin IX, 30 mg/kg, intraperitoneal injection for 1 h before heat exposure) aggravated the lung damage. Furthermore, we demonstrated HO-1 expression in lung type II pneumocytes. Our results suggest that endogenous HO-1 is protective against HS-induced lung damage. Induction of HO-1 may be a potential therapeutic strategy for treating heat-related diseases.

N-acetylcysteine attenuates glycerol-induced acute kidney injury by regulating MAPKs and Bcl-2 family proteins.

BACKGROUND: Rhabdomyolysis-induced acute kidney injury (AKI) accounts for about 10 to 40% of all cases of AKI. It is known that N-acetylcysteine (NAC) is effective in various experimental renal injury models; however, little information is available about the rat model of glycerol-induced rhabdomyolysis. In this study, we hypothesize that NAC plays a renoprotective role via the anti-apoptotic pathway. METHODS: Male Sprague-Dawley rats were divided into four groups: (i) saline control group, (ii) NAC-treated group (N-acetylcysteine) (150 mg/kg), (iii) glycerol-treated group (50%, 8 ml/kg, IM) and (iv) NAC plus glycerol-treated group. Rats were sacrificed at 24 h after glycerol injection, and the blood and renal tissues were harvested. RESULTS: Glycerol administration caused severe renal dysfunction, which included marked renal oxidative stress, significantly increased blood urea nitrogen (BUN) and serum creatinine levels. Histopathological findings, such as cast formation and tubular necrosis, confirmed renal impairment. We noted a marked activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p-38, in the glycerol-treated group. We also observed high expression of Bax and Bad but only weak expression of Bcl-2 and Bcl-xL in the glycerol-treated group. However, NAC pretreatment significantly improved renal function and decreased the activation of ERK, JNK, Bax and Bad, whereas it increased Bcl-2 and Bcl-xL. CONCLUSION: These results demonstrate that NAC protects against renal dysfunction, morphological damage and biochemical changes via the anti-apoptotic pathway in the glycerol-induced rhabdomyolysis model in rats.

Carbon monoxide: a putative neural messenger.

Carbon monoxide, an activator of guanylyl cyclase, is formed by the action of the enzyme heme oxygenase. By in situ hybridization in brain slices, discrete neuronal localization of messenger RNA for the constitutive form of heme oxygenase throughout the brain has been demonstrated. This localization is essentially the same as that for soluble guanylyl cyclase messenger RNA. In primary cultures of olfactory neurons, zinc protoporphyrin-9, a potent selective inhibitor of heme oxygenase, depletes endogenous guanosine 3',5'-monophosphate (cGMP). Thus, carbon monoxide, like nitric oxide, may be a physiologic regulator of cGMP. These findings, together with the neuronal localizations of heme oxygenase, suggest that carbon monoxide may function as a neurotransmitter.

Protective effect of heme oxygenase-1 on lung injury induced by erythrocyte instillation in rats.

BACKGROUND: Intratracheal instillation of blood induces self-repaired acute lung injury. However, the mechanism of repair has been unclear. Heme-oxygenase (HO)-1, which catalyzes heme breakdown, acts as an inducible defense against oxidative stress and plays an important role in inflammation. The objective of this study was to test the role of HO-1 in lung injury caused by intratracheal instillation of red cells. METHODS: Forty healthy, male Sprague-Dawley rats were randomly divided into five groups: normal group, saline group, erythrocyte group, erythrocyte+zinc-protoporphyrin (ZnPP, HO-1 inhibitor) group and saline+ZnPP group. At 2 days after intratracheal instillation of red cells, lung tissues and lavage samples were isolated for biochemical determinations and histological measurements. RESULTS: Histological analysis revealed that administration of ZnPP worsened the acute lung injury induced by instilled erythrocytes. HO-1 was over-expressed in the erythrocyte group and in the erythrocyte + ZnPP group. Compared with the erythrocyte + ZnPP group, the levels of total protein, lactate dehydrogenase and tumor necrosis factor-alpha in the lavage were lower (P < 0.01), while the level of interleukin-10 was higher in the erythrocyte group (P < 0.01). CONCLUSION: HO-1 protects against erythrocyte-induced inflammatory injury in lung.

Protective effects of hemin pretreatment combined with ulinastatin on septic shock in rats.

BACKGROUND: Urinary trypsin inhibitor inhibits the enhanced production of pro-inflammatory molecules. Hemeoxygenase-1 induction protects against ischemia/reperfusion injury, oxidative stress, inflammation, transplant rejection, apoptosis, and other conditions. However, it is unknown if a combined hemin and ulinastatin pretreatment could result in protective effects for septic shock. In this study, we investigated the role of hemin pretreatment combined with ulinastatin on septic shock in rats. METHODS: Eighty healthy, male Sprague-Dawley rats were randomly divided into four groups: group S, group H, group U and group HU. Groups S and U received 1 ml normal saline intraperitoneally, while groups H and HU both received 1 ml (100 mg /kg) hemin. Twenty-four hours later, 0.5 ml (10 mg/kg) E. coli lipopolysaccharide was injected intravenously to replicate the experimental model of septic shock. After an initial 25% decrease in the mean arterial pressure, corresponding to time point 0, groups HU and U received 0.5 ml 10 000 U/kg ulinastatin intravenously, and the others received 0.5 ml normal saline. RESULTS: The number of deaths in groups H and U was lower than that in the group S (P < 0.05), and was higher than that in group HU (all P < 0.05) respectively. The mean arterial pressure (MAP) in the group S was significantly greater than that in group H (P < 0.05), and was lower than that in group HU and group U (P < 0.05). The plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and blood urea nitrogen (BUN), the malondial-dehyde (MDA) of liver, kidney and lung, and the lung Evans blue (EB) contents in groups H and U, were greater than that in group HU (all P < 0.05), and were lower than that in group S (all P < 0.05). In contrast, the plasma levels of CO in groups H and HU were higher than that in groups S and U (all P < 0.05), and SOD of liver, kidney and lung in groups H and U were higher than that in group S, and were lower than that in group HU (all P < 0.05). The levels of TNF-alpha, IL-6, IL-8 and beta-glucuronidase (GCD) activity of plasma in groups U and HU were lower than those in groups H and S, all having a P < 0.05, while there were no significant differences between group H and group S, or between group HU and group U (all P > 0.05). The HO-1 mRNA and HO-1 protein levels from hepatic, renal, and pulmonary tissue in groups S and U were lower than those in groups H and HU (all P < 0.05), but there were no significant differences between groups S and U, or between groups H and HU (all P > 0.05). The HO-2 mRNA and HO-2 protein were not significantly different among the four groups (all P > 0.05). CONCLUSIONS: Combined pretreatment with hemin and ulinastatin in septic shock rats results in an improved response by the upregulation of HO-1 protein followed by increasing CO with resistance to increased oxidative stress, restraining the release of inflammatory mediators, and inhibiting beta-GCD activity.

Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2.

Heme oxygenase (HO) activity leads to accumulation of the antioxidant bilirubin, and degradation of the prooxidant heme. Moderate overexpression of the inducible form, HO-1, is associated with protection against oxidative injury. However, the role of HO-2 in oxidative stress has not been explored. We evaluated survival, indices of oxidative injury, and lung and HO expression in HO-2 null mutant mice exposed to > 95% O2 compared with wild-type controls. Similar basal levels of major lung antioxidants were observed, except that the knockouts had a twofold increase in total glutathione content. Despite increased HO-1 expression from HO-1 induction, knockout animals were sensitized to hyperoxia-induced oxidative injury and mortality, and also had significantly increased markers of oxidative injury before hyperoxic exposure. Furthermore, during hyperoxia, lung hemoproteins and iron content were significantly increased without increased ferritin, suggesting accumulation of available redox-active iron. These results demonstrate that the absence of HO-2 is associated with induction of HO-1 and increased oxygen toxicity in vivo, apparently due to accumulation of lung iron. These results suggest that HO-2 functions to augment the turnover of lung iron during oxidative stress, and that this function does not appear to be compensated for by induction of HO-1 in the knockouts.

Aging is associated with an increased susceptibility to ischaemic necrosis due to microvascular perfusion failure but not a reduction in ischaemic tolerance.

In the present study in a murine model of chronic ischaemia, we analysed: (i) whether aging was associated with an increased susceptibility to ischaemic necrosis, and (ii) whether this was based on microvascular dysfunction or reduced ischaemic tolerance. An ischaemic pedicled skin flap was created in the ear of homozygous hairless mice. The animals were assigned to three age groups, including adolescent (2+/-1 months), adult (10+/-2 months) and senescent (19+/-3 months). Microvascular perfusion of the ischaemic flap was assessed over 5 days by intravital microscopy, evaluating FCD (functional capillary density), capillary dilation response and the area of tissue necrosis. Expression of the stress-protein HO (haem oxygenase)-1 was determined by immunohistochemistry and Western blotting. Induction of chronic ischaemia stimulated a significant expression of HO-1 without a significant difference between the three age groups. This was associated with capillary dilation, which, however, was more pronounced in adolescent (10.5+/-2.8 microm compared with 3.95+/-0.79 microm at baseline) and adult (12.1+/-3.1 microm compared with 3.36+/-0.45 microm at baseline) animals compared with senescent animals (8.5+/-1.7 microm compared with 3.28+/-0.69 microm at baseline; P value not significant). In senescent animals, flap creation further resulted in complete cessation of capillary flow in the distal area of the flap (FCD, 0+/-0 cm/cm(2)), whereas adult (11.9+/-13.5 cm/cm(2)) and, in particular, adolescent animals (58.4+/-33.6 cm/cm(2); P<0.05) were capable of maintaining residual capillary perfusion. The age-associated microcirculatory dysfunction resulted in a significantly increased flap necrosis of 49+/-8% (P<0.05) and 42+/-8% (P<0.05) in senescent and adult animals respectively, compared with 31+/-6% in adolescent mice. Of interest, functional inhibition of HO-1 by SnPP-IX (tin protoporphyrin-IX) in adolescent mice abrogated capillary dilation, decreased functional capillary density and aggravated tissue necrosis comparably with that observed in senescent mice. Thus aging is associated with an increased susceptibility to tissue necrosis, which is due to a loss of vascular reactivity to endogenous HO-1 expression, rather than a reduction in ischaemic tolerance.

HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism.

This study investigated whether inducible heme oxygenase-1[corrected] (HO-1) [corrected] is targeted to mitochondria and its putative effects on oxidative metabolism in rat liver. Western blot and immune-electron microscopy in whole purified and fractionated organelles showed basal expression of HO-1 protein in both microsomes and mitochondria (inner membrane), accompanied by a parallel HO activity. Inducers of HO-1 increased HO-1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase, supporting that both enzymes are in the same compartmentalization. Induction of mitochondrial HO-1 was associated with a decrease of mitochondrial heme content and selective reduction of protein expression of cytochrome oxidase (COX) subunit I, which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability; these changes resulted in decreased COX spectrum and activity. Mitochondrial HO-1 induction was also associated with down-regulation of mitochondrial-targeted NO synthase expression and activity, resulting in a reduction of NO-dependent mitochondrial oxidant yield; inhibition of HO-1 activity reverted these effects. In conclusion, we demonstrated for the first time localization of HO-1 protein in mitochondria. It is surmised that mitochondrial HO-1 has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis, in which substantially increased mitochondrial NO and oxidant production have been implicated.

Toxicogenomics of drug-induced hemolytic anemia by analyzing gene expression profiles in the spleen.

Hemolytic anemia is a serious adverse effect of therapeutic drugs that is caused by increased destruction of drug-damaged erythrocytes by macrophages in the spleen and liver. We previously applied a toxicogenomic approach to the toxicity by analyzing microarray data of the liver of rats dosed with two hemolytic agents: phenylhydrazine and phenacetin. In the present study, we analyzed gene expression profiles in the spleen, the primary organ for destruction of damaged erythrocytes, of the same models in order to identify splenic gene expression alterations that could be used to predict the hematotoxicity. Microarray analyses revealed hundreds of genes commonly deregulated under all severe hemolytic conditions, which included genes related to splenic events characteristic of the hematotoxicity, such as proteolysis and iron metabolism. Eleven upregulated genes were selected as biomarker candidates, and their expression changes were validated by quantitative real-time PCR. The transcript levels of most of these genes showed strong correlation with the results of classical toxicological assays (e.g., histopathology and hematology). Furthermore, hierarchical clustering analysis suggested that altered expression patterns of the 11 genes sensitively reflected the erythrocyte damage even under a condition that caused no decrease in erythrocyte counts. Among the selected genes, heme oxygenase 1 was one of the most promising biomarker candidates, the upregulation of which on the protein level was confirmed by immunohistochemistry. These results indicate that altered splenic expression of a subset of genes may allow detection of drug-induced hemolytic anemia, with better sensitivity than that of erythrocyte counts in the blood.

Carbon monoxide from heme oxygenase-2 Is a tonic regulator against NO-dependent vasodilatation in the adult rat cerebral microcirculation.

Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium-expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.

Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes.

Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF(2alpha) and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF(2alpha), P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 +/- 0.8 cells/ml blood in control rats vs. 48 +/- 4.8 cells/ml blood in diabetic rats (P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 +/- 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 +/- 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 +/- 2.3 (P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 +/- 2.3 to 29 +/- 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes.