Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. In this study, we tested the hypothesis that localized arterial infection at the time of balloon angioplasty with an adenovirus (ADV-tk) encoding the herpes simplex virus thymidine kinase gene (HSV-tk), followed by systemic ganciclovir administration, can inhibit VSMC proliferation and neointima formation in a well-characterized model of arterial injury and restenosis. MATERIALS AND METHODS: The left carotid arteries of 31 male Sprague-Dawley rats were subjected to balloon angioplasty and immediately infected with 2 x 10(9) pfu of either ADV-tk or a control adenovirus that does not encode a recombinant protein (ADV-delta E1). Twenty-four hours after injury, animals from each experimental group were randomized to receive a course of systemic ganciclovir (ADV-tk/+GC, ADV delta E1/+GC) or saline (ADV-tk/-GC, ADV-delta E1/-GC). VSMC DNA synthesis was measured by 5'-bromodeoxuridine (BrdU) incorporation 2-4 days after balloon injury. The extent of restenosis, expressed as the neointima to media (I/M) area ratio was determined by digital planimetry 20 days after balloon injury in each of the four treatment groups. Immunohistochemistry using a mAb to von Willebrand factor (vWF) was used to determine the effects of ADV-tk infection and ganciclovir treatment on re-endothelialization of the carotid arteries 20 days following balloon angioplasty. RESULTS: Forty-one percent of the medial VSMCs in the ADV-tk/-GC arteries were labeled with BrdU 4 days after balloon injury. In contrast, ADV-tk infected animals that were treated with systemic ganciclovir (ADV-tk/+GC) displayed a 40% reduction in BrdU-staining medial VSMCs (p < 0.03). I/M area ratios of the three control groups were 1.17 +/- 0.18 (ADV-tk/-GC, n = 5), 1.15 +/- 0.10 (ADV-delta E1/+GC, n = 6), and 0.91 +/- 0.08 (ADV-delta E1/-GC, n = 6). These differences were not statistically significant (p > 0.05). In contrast, the ADV-tk/+GC animals (n = 6) displayed an I/M area ratio of 0.49 +/- 0.13 which was significantly lower than that seen in each of the three control groups (p < 0.02). None of the treated animals showed evidence of significant organ toxicity at autopsy. A regenerated endothelium was observed in the ADV-tk/+GC animals 20 days after balloon injury. CONCLUSIONS: Localized arterial infection with ADV-tk at the time of balloon angioplasty followed by systemic ganciclovir therapy reduces VSMC proliferation and neointimal expansion in the rat carotid artery injury model. Moreover, combined treatment with ADV-tk and systemic ganciclovir does not result in systemic toxicity and appears to selectively eliminate proliferating VSMCs, while preserving the capacity of the injured arterial segments to re-endothelialize within 3 weeks of injury. Taken together, these results support the feasibility of using this gene therapy approach for the treatment of human vascular proliferative disorders.     

Dynamic regulation of vascular myosin light chain (MYL9) with injury and aging

Background

Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels.

Methodology/Principal Findings

We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI''s GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers.

Conclusions/Significance

The consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.     

Divergent Cardiopulmonary Actions of Heme Oxygenase Enzymatic Products in Chronic Hypoxia

Background

Hypoxia and pressure-overload induce heme oxygenase-1 (HO-1) in cardiomyocytes and vascular smooth muscle cells (VSMCs). HO-1^−/− mice exposed to chronic hypoxia develop pulmonary arterial hypertension (PAH) with exaggerated right ventricular (RV) injury consisting of dilation, fibrosis, and mural thrombi. Our objective was to indentify the HO-1 product(s) mediating RV protection from hypoxic injury in HO-1^−/− mice.

Methodology/Principal Findings

HO-1^−/− mice were exposed to seven weeks of hypoxia and treated with inhaled CO or biliverdin injections. CO reduced right ventricular systolic pressure (RVSP) and prevented hypoxic pulmonary arteriolar remodeling in both HO-1^−/− and control mice. Biliverdin had no significant effect on arteriolar remodeling or RVSP in either genotype. Despite this, biliverdin prevented RV failure in the hypoxic HO-1^−/− mice (0/14 manifested RV wall fibrosis or thrombus), while CO-treated HO-1^−/− mice developed RV insults similar to untreated controls. In vitro, CO inhibited hypoxic VSMC proliferation and migration but did not prevent cardiomyocyte death from anoxia-reoxygenation (A-R). In contrast, bilirubin limited A-R-induced cardiomyocyte death but did not inhibit VSMC proliferation and migration.

Conclusions/Significance

CO and bilirubin have distinct protective actions in the heart and pulmonary vasculature during chronic hypoxia. Moreover, reducing pulmonary vascular resistance may not prevent RV injury in hypoxia-induced PAH; supporting RV adaptation to hypoxia and preventing RV failure must be a therapeutic goal.     

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-(S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 microM AA or 100 microM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.     

Heme oxygenase-1 protects against vascular constriction and proliferation

Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.     

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.     

Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background

Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated.

Methodology/Principal Findings

We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression.

Conclusions/Significance

This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain.     

Carbon monoxide promotes hypoxic pulmonary vascular remodeling

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.     

Inhibition of heme oxygenase augments tubular sodium reabsorption

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 μmol/kg ip), a photostable HO inhibitor, was given to control rats and N(G)-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg?1·day?1), 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.     

Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.     

Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30-50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.     

YC-1, a benzyl indazole derivative, stimulates vascular cGMP and inhibits neointima formation

The pathobiologic process of arterial stenosis following balloon angioplasty continues to be an enigmatic problem in clinical settings. This research project investigates the ability of YC-1, a benzyl indazole derivative that sensitizes sGC/cGMP, to stimulate endogenous cGMP and attenuate balloon injury-induced neointima (NI) formation in the rat carotid artery. Northern and Western blot analyses revealed enhanced acute expression of iNOS and inducible heme oxygenase (HO-1) mRNA and protein in the injured artery. The contralateral uninjured artery also demonstrated acute HO-1 mRNA and protein induction without detectable iNOS expression. Perivascular application of YC-1 immediately following injury significantly stimulated acute vessel wall cGMP compared to untreated controls. YC-1 treated sections demonstrated significant reduction in NI area (-74%), NI area/medial wall area (-72%), and NI thickness (-76%) 2 weeks post-injury. These results directly implicate YC-1 as a potent new therapeutic agent capable of reducing post-angioplasty stenosis through endogenous CO- and/or NO-mediated, cGMP-dependent processes.     

Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO exists as constitutive (HO-2, HO-3) and inducible isoforms (HO-1), the latter which responds to regulation by multiple stress-stimuli. HO-1 confers protection in vitro and in vivo against oxidative cellular stress. Although the redox active compounds that are generated from HO activity (i.e. iron, biliverdin-IXalpha, and bilirubin-IXa) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for CO. Though not reactive, CO regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. The latter effects of CO depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic 3':5'-guanosine monophosphate. CO potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. Recent progress indicates that CO exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p38 mitogen activated protein kinase (MAPK)-signaling pathway. By virtue of these effects, CO confers protection in oxidative lung injury models, and likely plays a role in HO-1 mediated tissue protection.     

Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression

Arterial remodeling in response to pathological insult is a complex process that depends in part on the balance between vascular cell apoptosis and proliferation. Studies in experimental models suggest that HO-1 mediates neointimal formation while limiting lumen stenosing, indicating a differential effect on vascular endothelial (EC) and smooth muscle cells (SMC). We investigated the effect of HO-1 expression on cell cycle progression in EC and SMC. The addition of SnMP (10 microM), an inhibitor of HO activity, to EC or SMC for 24h, resulted in significant abnormalities in DNA distribution and cell cycle progression compared to cells treated with the HO-1 inducers, heme (10 microM) or SnCl(2) (10 microM). SnMP increased G(1) phase and decreased S and G(2)/M phases in EC while heme or SnCl(2) decreased G(1) phase, but increased S and G(2)/M phases (p<0.05). Opposite effects were obtained in SMC. SnMP decreased G(1) phase and increased S and G(2)/M phases while heme or SnCl(2) increased G(1) phase but decreased S and G(2)/M phases (p<0.05). Our data demonstrate that HO-1 regulates the cell cycle in a cell-specific manner; it increases EC but decreases SMC cycle progression. The mechanisms underlying the HO-1 cell-specific effect on cell cycle progression within the vascular wall are yet to be explored. Nevertheless, these findings suggest that cell-specific targeting of HO-1 expression may provide a novel therapeutic strategy for the treatment of cardiovascular diseases.     

Hypothermic preconditioning of endothelial cells attenuates cold-induced injury by a ferritin-dependent process

Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0°C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0°C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0°C but did not increase BDI or oxidative stress further. HPC at 25°C for 15–72 h attenuated these changes by an amount achievable by pretreating cells with 10–20 μM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37°C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0°C but ferritin was protective. HPC increased iron maximally after 2 h at 25°C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0°C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.     

Paradoxical enhancement of oxidative cell injury by overexpression of heme oxygenase-1 in an anchorage-dependent cell ECV304

There has been increasing evidence suggesting the potent anti-inflammatory roles of heme oxygenase-1 (HO-1) in protecting renal tubular epithelial cells, vascular endothelial cells, and circulating monocytes. Based on these findings, novel therapeutic interventions have been proposed to control the expression of endothelial HO-1 levels to ameliorate various vascular diseases. We evaluated the effect of HO-1 gene transfer into an anchorage-dependent cell, ECV304. Effect of HO-1 production on the cell injury induced by hydrogen peroxide was evaluated after hemin stimulation and after HO-1 gene transfection. Morphological changes and the induction of various anti-apoptotic proteins were examined at the same time. Levels of HO-1 expression were variable in different clones of HO-1-transfected ECV304 cells. Among these, the clones with moderate levels of HO-1 expression were significantly more resistant to oxidative stress. In contrast, those with the highest levels of HO-1 exhibited paradoxically enhanced susceptibility to oxidative injury. Interestingly, the cell survival after oxidative stress was in parallel with the levels of Bcl-2 expression and of fibronectin receptor, alpha5 integrin. It is suggested from these results, that excessive HO-1 not only leads to enhanced cell injury, but also prolongs the repair process of the injured endothelial tissue. However, HO-1 reduces the oxidative cell injury and protects the endothelial cells, if its expression is appropriately controlled. Copyright 2004 Wiley-Liss, Inc.     

Heme oxygenase-1 induction depletes heme and attenuates pulmonary artery relaxation and guanylate cyclase activation by nitric oxide

This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-1 (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24-h organ culture with 0.1 mM cobalt chloride (CoCl2) or 30 microM Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by approximately two- to fourfold and lowered heme levels by 40-45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 microM chromium mesoporphyrin during the 24-h organ culture (but not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the beta(1)-subunit of sGC. However, the increase in activity seen in the presence of 1 microM spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-1 had decreased levels of superoxide detected by the chemiluminescence of 5 microM lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO.     

转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用

本实验构建含人血红素加氧酶-1（hHO-1)基因的逆转录病毒载体XM-6/hHO-1,将其导入离体培养的大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC),观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用,结果表明：（1）hHO-1基因可在靶细胞中明显表达,转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍;（2）转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,表现为细胞存活率增加和乳酸脱氢酶（LDH）漏出减少,上述保护作用可被HO特异性抑制剂锌原卟啉IX（Zinc-proto-porphyrinIX,ZnPP-IX)所阻断,研究结果提示,外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。     

Multiple Factors from Bradykinin-Challenged Astrocytes Contribute to the Neuronal Apoptosis: Involvement of Astroglial ROS, MMP-9, and HO-1/CO System

Bradykinin (BK) has been shown to induce the expression of several inflammatory mediators, including reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), in brain astrocytes. These mediators may contribute to neuronal dysfunction and death in various neurological disorders. However, the effects of multiple inflammatory mediators released from BK-challenged astrocytes on neuronal cells remain unclear. Here, we found that multiple factors were released from brain astrocytes (RBA-1) exposed to BK in the conditioned culture media (BK-CM), including ROS, MMP-9, and heme oxygenase-1 (HO-1)/carbon monoxide (CO), leading to neuronal cell (SK-N-SH) death. Exposure of SK-N-SH cells to BK-CM or H₂O₂ reduced cell viability and induced cell apoptosis which were attenuated by N-acetyl cysteine, indicating a role of ROS in these responses. The effect of BK-CM on cell viability and cell apoptosis was also reversed by immunoprecipitation of BK-CM with anti-MMP-9 antibody (MMP-9-IP-CM) or MMP2/9 inhibitor, suggesting the involvement of MMP-9 in BK-CM-mediated responses. Astroglial HO-1/CO in BK-CM induced cell apoptosis and reduced cell viability which was reversed by hemoglobin. Consistently, the involvement of CO in these cellular responses was revealed by incubation with a CO donor CO-RM2 which was reversed by hemoglobin. The role of HO-1 in BK-CM-induced responses was confirmed by overexpression of HO-1 in SK-N-SH infected with Adv-HO-1. BK-CM-induced cell apoptosis was due to the activation of caspase-3 and cleavage of PARP. Together, we demonstrate that BK-induced several neurotoxic factors, including ROS, MMP-9, and CO released from astrocytes, may induce neuronal death through a caspase-3-dependent apoptotic pathway.