Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes

Increased heme oxygenase (HO)-1 activity attenuates endothelial cell apoptosis and decreases superoxide anion (O2-) formation in experimental diabetes by unknown mechanisms. We examined the effect of HO-1 protein and HO activity on extracellular SOD (EC-SOD), catalase, O2-, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) levels and vascular responses to ACh in control and diabetic rats. Vascular EC-SOD and plasma catalase activities were significantly reduced in diabetic compared with nondiabetic rats (P < 0.05). Upregulation of HO-1 expression by intermittent administration of cobalt protoporphyrin, an inducer of HO-1 protein and activity, resulted in a robust increase in EC-SOD but no significant change in Cu-Zn-SOD. Administration of tin mesoporphyrin, an inhibitor of HO-1 activity, decreased EC-SOD protein. Increased HO-1 activity in diabetic rats was associated with a decrease in iNOS but increases in eNOS and plasma catalase activity. On the other hand, aortic ring segments from diabetic rats exhibited a significant reduction in vascular relaxation to ACh, which was reversed with cobalt protoporphyrin treatment. These data demonstrate that an increase in HO-1 protein and activity, i.e., CO and bilirubin production, in diabetic rats brings about a robust increase in EC-SOD, catalase, and eNOS with a concomitant increase in endothelial relaxation and a decrease in O2-. These observations in experimental diabetes suggest that the vascular cytoprotective mechanism of HO-1 against oxidative stress requires an increase in EC-SOD and catalase.     

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 PGF₂ 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 PGF₂, 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.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Beneficial effect of heme oxygenase-1 expression on myocardial ischemia-reperfusion involves an increase in adiponectin in mildly diabetic rats

Transient reduction in coronary perfusion pressure in the isolated mouse heart increases microvascular resistance (paradoxical vasoconstriction) by an endothelium-mediated mechanism. To assess the presence and extent of paradoxical vasoconstriction in hearts from normal and diabetic rats and to determine whether increased heme oxygenase (HO)-1 expression and HO activity, using cobalt protoporphyrin (CoPP), attenuates coronary microvascular response, male Wistar rats were rendered diabetic with nicotinamide/streptozotocin for 2 wk and either CoPP or vehicle was administered by intraperitoneal injection weekly for 3 wk (0.5 mg/100 g body wt). The isolated beating nonworking heart was submitted to transient low perfusion pressure (20 mmHg), and coronary resistance (CR) was measured. During low perfusion pressure, CR increased and was associated with increased lactate release. In diabetic rats, CR was higher, HO-1 expression and endothelial nitric oxide synthase were downregulated, and inducible nitric oxide synthase and O(2)(-) were upregulated. After 3 wk of CoPP treatment, HO activity was significantly increased in the heart. Upregulation of HO-1 expression and HO activity by CoPP resulted in the abolition of paradoxical vasoconstriction and a reduction in oxidative ischemic damage. In addition, there was a marked increase in serum adiponectin. Elevated HO-1 expression was associated with increased expression of cardiac endothelial nitric oxide synthase, B-cell leukemia/lymphoma extra long, and phospho activator protein kinase levels and decreased levels of inducible nitric oxide synthase and malondialdehyde. These results suggest a critical role for HO-1 in microvascular tone control and myocardial protection during ischemia in both normal and mildly diabetic rats through the modulation of constitutive and inducible nitric oxide synthase expression and activity, and an increase in serum adiponectin.     

Long-Lasting Expression of HO-1 Delays Progression of Type I Diabetes in NOD Mice

Heme oxygenase-1 (HO-1) is crucial in regulating oxidative injury. The present study was designed to assess whether HO-1 upregulation by cobalt protoporphyrin IX (CoPP) moderates or prevents the diabetic state in non-obese diabetic (NOD) mice, an animal model for Type 1 diabetes (T1D). HO-1 expression and HO activity were upregulated in the pancreas by the intermittent administration of CoPP. This was associated with decreases in blood glucose and pancreatic O2-, but increased pAKT and BcL-XL and cell survival. A considerable number of beta cells were preserved in the islets of CoPP-treated NOD mice, while none were found in untreated diabetic mice. The number of CD11c+ dendritic cells was decreased in the pancreas of CoPP-treated NOD mice (p     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors.

Human umbilical vein endothelial cells (HUVECs) were exposed to low (5?mmol/l) and high (25?mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10?μmol/l), HO antagonist (SnPPIX, 10?μmol/l), and NO synthase blocker (l-NAME, 200?μmol/l) with or without NO donor (arginine, 1?mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF).

In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

A significant role for the heme oxygenase-1 gene in endothelial cell cycle progression

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression, using a retroviral vector, on cell cycle progression in the presence and absence of pyrrolidine dithiocarbamate (PDTC). The addition of PDTC (25 and 50 microM) to human microvessel endothelial cells over 24 h resulted in significant (P < 0.05) abnormalities in DNA distribution and cell cycle progression compared to cells overexpressing the HO-1 gene. The addition of PDTC resulted in a significantly decreased G(1) phase and an increased G(2)/M phase in the control cells, but not in cells transduced with the human HO-1 gene (P < 0.05). Further, PDTC had a potent effect on DNA distribution abnormalities in exponentially grown cells compared to subconfluent cells. Upregulation of HO activity in endothelial cells, as a result of overexpressing human HO-1, prevented PDTC-mediated abnormalities in DNA distribution. Inhibition of HO activity by tin-mesoporphyrin (SnMP) (30 microM) resulted in enhancement of PDTC-mediated abnormalities in cell cycle progression. Bilirubin or iron did not mediate DNA distribution. We conclude that an increase in endothelial cell HO-1 activity with subsequent generation of carbon monoxide, elicited by gene transfer, reversed the PDTC-mediated abnormalities in cell cycle progression and is thus a potential therapeutic means for attenuating the effects of oxidative stress-causing agents.     

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-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells

Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 microM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased (P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% (P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants (P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content (r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.     

Erythropoietin induces heme oxygenase-1 expression and attenuates oxidative stress

Recent studies have established that erythropoietin (EPO) is a pleiotropic cytokine. In this study we investigated whether pleiotropic effects of EPO may involve regulation of heme oxygenase (HO)-1, an anti-oxidative stress protein. A stimulatory effect of EPO on HO-1 expression was demonstrated in cultured renal endothelial cells, in which EPO decreased intracellular oxidative stress and provided cytoprotection against H(2)O(2). These beneficial effects were partially reversed by a HO-1 inhibitor. We then evaluated whether EPO induces HO-1 and ameliorates renal injury in vivo. Administration of EPO to Dahl salt-sensitive (DS) rats with low salt diet, a model of chronic tubulointerstitial injury, reduced proteinuria, and renal injury including peritubular capillaries rarefaction as compared to vehicle-treated DS rats. This renoprotection was associated with up-regulation of HO-1 in the kidney. In conclusion, EPO-induced HO-1 expression is likely to provide cytoprotection against oxidative stress.     

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.     

Diminished heme oxygenase potentiates cell death: pyrrolidinedithiocarbamate mediates oxidative stress

Pyrrolidinedithiocarbamate (PDTC) is a metal-chelating compound that exerts both pro-oxidant and antioxidant effects and is widely used as an antitumor and anti-inflammatory agent. Heme oxygenase-1 (HO-1) is a redox-sensitive-inducible protein that provides efficient cytoprotection against oxidative stress. Because it has been reported that several angiogenic stimulating factors upregulating HO-1 in endothelial cells cause a significant increase in angiogenesis, we investigated the effect of PDTC on cell proliferation and angiogenesis and the effect of overexpression and underexpression of HO-1. The evaluation of PDTC (20 or 50 micro M) in endothelial cells resulted in significant increase in HO-1 mRNA and protein (P < 0.001), but a decrease in cell proliferation. Pretreatment of endothelial cells with SnCl(2) (10 micro M), an inducer of HO-1 attenuated the PDTC-mediated decrease in cell proliferation (P < 0.05). In contrast, pretreatment with SnMP, an inhibitor of HO activity, magnified the inhibiting effect of PDTC on cell proliferation. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated the PDTC-induced decrease in cell proliferation. Underexpression of HO-1, by delivery of the human HO-1 in antisense orientation, enhanced the PDTC-mediated decrease in cell proliferation. The decrease, by PDTC, in proliferation of cells underexpressing HO-1 is related to an increase in O(-)(2) production. Collectively, these results demonstrate that upregulation of HO-1 was able to attenuate the PDTC-mediated cell proliferation, but was unable to reverse the high concentration of PDTC-induced decrease in angiogenesis.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.