Inhibition Of HO Activity Research Articles

Erythropoietin Attenuates Pulmonary Vascular Remodeling in Experimental Pulmonary Arterial Hypertension through Interplay between Endothelial Progenitor Cells and Heme Oxygenase.

Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease with a high mortality, characterized by typical angio-proliferative lesions. Erythropoietin (EPO) attenuates pulmonary vascular remodeling in PAH. We postulated that EPO acts through mobilization of endothelial progenitor cells (EPCs) and activation of the cytoprotective enzyme heme oxygenase-1 (HO-1). Rats with flow-associated PAH, resembling pediatric PAH, were treated with HO-1 inducer EPO in the presence or absence of the selective HO-activity inhibitor tin-mesoporphyrin (SnMP). HO activity, circulating EPCs and pulmonary vascular lesions were assessed after 3 weeks. In PAH rats, circulating EPCs were decreased and HO activity was increased compared to control. EPO treatment restored circulating EPCs and improved pulmonary vascular remodeling, as shown by a reduced wall thickness and occlusion rate of the intra-acinar vessels. Inhibition of HO activity with SnMP aggravated PAH. Moreover, SnMP treatment abrogated EPO-induced amelioration of pulmonary vascular remodeling, while surprisingly further increasing circulating EPCs as compared with EPO alone. In experimental PAH, EPO treatment restored the number of circulating EPCs to control level, improved pulmonary vascular remodeling, and showed important interplay with HO activity. Inhibition of increased HO activity in PAH rats exacerbated progression of pulmonary vascular remodeling, despite the presence of restored number of circulating EPCs. We suggest that both EPO-induced HO-1 and EPCs are promising targets to ameliorate the pulmonary vasculature in PAH.

Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet.

BackgroundOxidative stress underlies the etiopathogenesis of nonalcoholic fatty liver disease (NAFLD), obesity and cardiovascular disease (CVD). Heme Oxygenase-1 (HO-1) is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. Sirtuin1 (SIRT1) belongs to the family of NAD-dependent de-acyetylases and is modulated by cellular redox.HypothesisWe hypothesize that fructose-induced obesity creates an inflammatory and oxidative environment conducive to the development of NAFLD and metabolic syndrome. The aim of this study is to determine whether HO-1 acts through SIRT1 to form a functional module within hepatocytes to attenuate steatohepatitis, hepatic fibrosis and cardiovascular dysfunction.Methods and ResultsWe examined the effect of fructose, on hepatocyte lipid accumulation and fibrosis in murine hepatocytes and in mice fed a high fructose diet in the presence and absence of CoPP, an inducer of HO-1, and SnMP, an inhibitor of HO activity. Fructose increased oxidative stress markers and decreased HO-1 and SIRT1 levels in hepatocytes (p<0.05). Further fructose supplementation increased FAS, PPARα, pAMPK and triglycerides levels; CoPP negated this increase. Concurrent treatment with CoPP and SIRT1 siRNA in hepatocytes increased FAS, PPARα, pAMPK and triglycerides levels suggesting that HO-1 is upstream of SIRT1 and suppression of SIRT1 attenuates the beneficial effects of HO-1. A high fructose diet increased insulin resistance, blood pressure, markers of oxidative stress and lipogenesis along with fibrotic markers in mice (p<0.05). Increased levels of HO-1 increased SIRT1 levels and ameliorated fructose-mediated lipid accumulation and fibrosis in liver along with decreasing vascular dysfunction (p<0.05 vs. fructose). These beneficial effects of CoPP were reversed by SnMP.ConclusionTaken together, our study demonstrates, for the first time, that HO-1 induction attenuates fructose-induced hepatic lipid deposition, prevents the development of hepatic fibrosis and abates NAFLD-associated vascular dysfunction; effects that are mediated by activation of SIRT1 gene expression.

Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa.

Despite substantial evidence showing the ammonium-altered redox homeostasis in plants, the involvement and molecular mechanism of heme-heme oxygenase 1 (heme-HO1), a novel antioxidant system, in the regulation of ammonium tolerance remain elusive. To fill in these gaps, the biological function of rice HO1 (OsSE5) was investigated. Results showed that NH4 Cl up-regulated rice OsSE5 expression. Oxidative stress and subsequent growth inhibition induced by excess NH4 Cl was partly mitigated by pretreatment with carbon monoxide (CO, a by-product of HO1 activity) or intensified by zinc protoporphyrin (ZnPP, a potent inhibitor of HO1 activity). Pretreatment with HO1 inducer hemin, not only up-regulated OsSE5 expression and HO activity, but also rescued the down-regulation of antioxidant transcripts, total and related isozymatic activities, thus significantly counteracting the excess NH4 Cl-triggered reactive oxygen species overproduction, lipid peroxidation and growth inhibition. OsSE5 RNAi transgenic rice plants revealed NH4 Cl-hypersensitive phenotype with impaired antioxidant defence, both of which could be rescued by CO but not hemin. Transgenic Arabidopsis plants over-expressing OsSE5 also exhibited enhanced tolerance to NH4 Cl, which might be attributed to the up-regulation of several antioxidant transcripts. Altogether, these results illustrated the involvement of heme-HO1 system in ammonium tolerance by enhancing antioxidant defence, which may improve plant tolerance to excess ammonium fertilizer.

Abstract 18792: Treatment With an EET Agonist in a Post Myocardial Infarction Model Increases Left Ventricular Ejection Fraction and Attenuates Cardiac Remodeling by Induction of HMOX1

Rationale: Myocardial Infarction (MI) leads to a progressive decline in cardiac function, an increase in patient morbidity and is an event expanding burden on the health care system. Failure of myocardial regeneration, insufficient blood flow and enhanced oxidative stress all contribute to this pathological outcome. Objective: Since epoxyeicosatrienoic acids (EETs) agonists increase angiogenesis and vasodilation in ischemic heart, we examined if cross talk between EET and heme oxygenase-1(HMOX1) could reduce post infarcted cardiac remodeling resulting in an improvement of cardiac function in a model of post-MI. Methods: C57B16 mice were divided into 4 groups: sham, mice with myocardial infarction (MI) via LAD ligation and mice with MI treated with an EET-agonist (NUDSA) with and without the HO activity inhibitor stannous mesoporphyrin (SnMP). NUDSA was administered 5 days after MI for 30 days (0.5 mg/kg). Myocardial echocardiography was performed to assess blood volume, velocity, LVend-diastolic and ejection fractions. Microvessel density, inflammation and pericardial fibrosis were compared among the groups. Results: Echocardiography showed that left ventricle dilatation, measured as end diastolic area (EDA), was reduced in the NUDSA treated group compared to the MI group (EDA: MI: 0.314 ±0.01 cm 2 ; MI+NUDSA: 0.193±0.02 cm 2 ; p&lt;0.01). Cardiac Index, decreased by MI, was restored by NUDSA. NUDSA treatment increased serum adiponectin levels and decreased IL-6 and MCP-1 levels (p&lt;0.05 vs. MI), reduced fibrosis in the peri-infarct myocardium and decreased apoptosis. Immunoblot analysis of cardiac tissue demonstrated increased expression of HMOX1, VEGF and FGF along with decreased expression of TXA2 and 3NT in mice treated with NUDSA as compared to the MI group (p&lt;0.02). All these beneficial effects were reversed by SnMP. Conclusion: This study demonstrates that cross talk between EET and HMOX1 ameliorates cardiac remodeling in the post infarcted myocardium and increases angiogenesis, suggesting EET as a potential therapeutic strategy in the preservation of cardiac function in this animal model of post MI.

Penicillinolide A: A New Anti-Inflammatory Metabolite from the Marine Fungus Penicillium sp. SF-5292

In the course of studies on bioactive metabolites from marine fungi, a new 10-membered lactone, named penicillinolide A (1) was isolated from the organic extract of Penicillium sp. SF-5292 as a potential anti-inflammatory compound. The structure of penicillinolide A (1) was mainly determined by analysis of NMR and MS data and Mosher’s method. Penicillinolide A (1) inhibited the production of NO and PGE2 due to inhibition of the expression of iNOS and COX-2. Penicillinolide A (1) also reduced TNF-α, IL-1β and IL-6 production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), a competitive inhibitor of HO activity, it was verified that the inhibitory effects of compound 1 on the production of pro-inflammatory mediators and NF-κB DNA binding activity were partially associated with HO-1 expression through Nrf2 nuclear translocation.

Abstract 49: Targeting Endothelial Cells with HO-1 Attenuated Vascular and Adipocyte Dysfunction in Mice Fed High Fat Diet

Introduction: Obesity or high fat diet (HF) are risk factors for the development of hypertension. We examined the hypothesis that targeting the vascular system with human heme oxygenase -1 (HO-1) may attenuate both vascular and adipocyte dysfunction in vivo and in vitro. Methods: Lentivirus (Lenti) construct expressing human HO-1 under the control of endothelium specific promoters VE-cadherin (VECAD-HO-1) and VECAD −GFP (control) were used to treat mice, using a bolus injection into the renal artery, and kept on a high fat diet for 26 weeks. Human HO-1 gene expression was sustained for 9 months. For in vitro studies, human EC were cultured with Lenti- VECAD-HO-1 and added to Lenti-VECAD-GFP or control. The conditioned media (CM) from ECs was harvested and 10% CM was added to adipogenic media to measure paracrine effect on adipogenesis in human Mesenchymal Stem Cells (MSCs) derived adipocytes. Signaling molecules were measured by western blot. Results: Lentiviral transduction with VECAD-HO-1 construct attenuated the increase in blood pressure (from 149.9 ± 2.4 to 118 ± 2.0 mmHg, p&lt;0.01) and prevented body weight gain by 39% (p&lt;0.05) in obese mice and increased plasma adiponectin (from 2.9 ± 0.2 to 6.5 ± 0.1 μg/ml, p&lt;0.05). CM derived from EC lenti HO-1 decreased adipogenesis compared to control (from 17.0 ± 0.2 to 10.2 ± 0.1, p&lt;0.05) and resulted in an increase of β-catenin and Wnt but a decrease in PPARγ (p&lt;0.05). These beneficial effects were reversed by treating the cell with SnMP, an inhibitor of HO activity (p&lt;0.01). EC-HO-1 increased angiopoietin-1 (ANG-1), vascular endothelial growth factor A (VEGF), and platelet derived growth factor (PDGF)-AA,−BB (p&lt;0.05). The addition of ANG-1 to adipogenic media resulted in the inhibition of adipogenesis (8.1 ± 0.1, p&lt;0.01). Conclusion: Targeting HO-1 to ECs resulted in the attenuation of blood pressure and the prevention of body weight gain resulting in increased ANG-1, PDGF, and VGEF levels and the reprogramming of MSCs derived adipocytes to produce healthy and smaller adipocytes with the release of adiponectin.

Abstract 55: PPAR-δ Binding to Heme Oxygenase 1 Promoter Prevents Angiotensin II Induced Vascular and Adipocyte Dysfunction in a Model of Renovascular Hypertension

Introduction: Renin-Angiotensin System (RAS) induces oxidative stress and contributes to various pathological conditions including insulin resistance and the metabolic syndrome. Recent studies have shown the role of PPARδ-agonist in attenuation of Angiotensin II induced oxidative stress.The aim of this study was to explore the effectiveness of a PPARδ-agonist in the prevention of Ang II-induced vascular and adipocyte dysfunction and the possible interaction between PPARδ and HO-1 system in a model of enhanced oxidative stress, the Goldblatt 2K1C model. Methods: We first established a direct stimulatory effect of the PPARδ-agonist (GW 501516) on the HO-1 gene by demonstrating increased luciferase activity in COS-7 cells transfected with a luciferase-HO-1 promoter construct. SD rats were divided in 4 groups: sham operated animals, 2K1C rats and 2K1C rats treated with GW 501516, in the absence or presence of the HO activity inhibitor, stannous mesoporphyrin (SnMP). Results: 2K1C animals had increased blood pressure, visceral adiposity, adipocyte hypertrophy, increased inflamatory cytokines, increased circulatory and adipose tisssue levels of renin and Ang II along with increased adipose tissue gp91 phox expression (p&lt;0.05) when compared to sham operated animals. Treatment with GW 501516 increased adipose tissue HO-1 and adiponectin levels (p&lt;0.01) along with enhancement of Wnt10b and β-catenin expression. HO-1 induction was accompanied by the decreased expression of Wnt 5b, Mest and C/EBPα levels and an increased number of small adipocytes (p&lt;0.05). These effects of GW501516 were reversed in 2K1C animals exposed to SnMP (p&lt;0.05). Conclusion: This novel study corroborates the existence of a PPARδ-HO-1 axis whose direct stimulation via the administration of an exogenous PPARδ-agonist induces HO-1 expression, abates RAS associated adipocyte and vascular dysfunction and induces adipocyte remodeling with smaller adipocytes while decreasing inflammation and increasing adiponectin secretion.

HO-1 Induction Improves The Type-1 Cardiorenal Syndrome in Mice With Impaired Angiotensin II–Induced Lymphocyte Activation

Type-1 cardiorenal syndrome, characterized by acute kidney dysfunction secondary to cardiac failure and renal arteriolar vasoconstriction, is mediated by the renin–angiotensin–aldosterone axis and sympathetic nervous system activation. Previous reports indicate that angiotensin II modulates immune function and causes recruitment and activation of T-lymphocytes. The goal of this study was to evaluate the effects of postischemic heart failure on renal morphology and circulation and the beneficial effects of heme oxygenase-1 (HO-1) induction in T-lymphocyte–suppressed severe combined immune deficiency (SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with an HO-1 inducer, cobalt protoporphyrin, and with or without stannous mesoporphyrin, an inhibitor of HO activity. Heart and kidney function were studied 30 days after surgery. Fractional area change was reduced 30 days after surgery in both the C57 and SCID MI–groups as compared with their respective controls ( P &lt;0.01). Renal Pulsatility Index and renal injury were increased in C57 and SCID MI–groups compared with the sham group. HO-1 induction improved renal vasoconstriction as well as ameliorated renal injury in both the SCID and C57 MI–groups ( P &lt;0.01). However, improvement was more evident in SCID mice. In addition, our results showed that plasma creatinine, angiotensin II, and renin were significantly increased in the C57 and SCID MI–groups as compared with their respective controls. HO-1 induction decreased these parameters in both MI groups. Stannous mesoporphyrin reversed the beneficial effect of cobalt protoporphyrin in both mouse strains. The study demonstrates that T-lymphocyte suppression facilitated the HO-1–dependent improvement in the attenuation of type-1 cardiorenal syndrome.

PPARδ binding to heme oxygenase 1 promoter prevents angiotensin II-induced adipocyte dysfunction in Goldblatt hypertensive rats.

Objective:Renin–angiotensin system (RAS) regulates adipogenic response with adipocyte hypertrophy by increasing oxidative stress. Recent studies have shown the role of peroxisome proliferator-activated receptor-δ (PPARδ) agonist in attenuation of angiotensin II-induced oxidative stress. The aim of this study was to explore a potential mechanistic link between PPARδ and the cytoprotective enzyme heme oxygenase-1 (HO-1) and to elucidate the contribution of HO-1 to the adipocyte regulatory effects of PPARδ agonism in an animal model of enhanced RAS, the Goldblatt 2 kidney 1 clip (2K1C) model.Method:We first established a direct stimulatory effect of the PPARδ agonist (GW 501516) on the HO-1 gene by demonstrating increased luciferase activity in COS-7 cells transfected with a luciferase-HO-1 promoter construct. Sprague-Dawley rats were divided into four groups: sham-operated animals, 2K1C rats and 2K1C rats treated with GW 501516, in the absence or presence of the HO activity inhibitor, stannous mesoporphyrin (SnMP).Results:2K1C animals had increased visceral adiposity, adipocyte hypertrophy, increased inflammatory cytokines, increased circulatory and adipose tisssue levels of renin and Ang II along with increased adipose tissue gp91 phox expression (P<0.05) when compared with sham-operated animals. Treatment with GW 501516 increased adipose tissue HO-1 and adiponectin levels (P<0.01) along with enhancement of Wnt10b and β-catenin expression. HO-1 induction was accompanied by the decreased expression of Wnt5b, mesoderm specific transcript (mest) and C/EBPα levels and an increased number of small adipocytes (P<0.05). These effects of GW501516 were reversed in 2K1C animals exposed to SnMP (P<0.05).Conclusion:Taken together, our study demonstrates, for the first time, that increased levels of Ang II contribute towards adipose tissue dysregulation, which is abated by PPARδ-mediated upregulation of the heme-HO system. These findings highlight the pivotal role and symbiotic relationship of HO-1, adiponectin and PPARδ in the regulation of metabolic homeostasis in adipose tissues.

Heme Oxygenase-1 and Breast Cancer Resistance Protein Protect Against Hemeinduced Toxicity

Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating oxidative and inflammatory injury. Protective mechanisms against free heme are therefore pivotal for cellular survival. We postulated that overexpression of Heme Oxygenase-1 (HO-1) and Breast Cancer Resistance Protein (BCRP) would protect against heme-induced cytotoxicity. HO-1 is a heme-degrading enzyme generating carbon monoxide, iron, and biliverdin/bilirubin, while BCRP is a heme efflux transporter. Human embryonic kidney cells were transduced using a baculovirus system as a novel strategy to efficiently overexpress HO-1 and BCRP. Exposing cells to heme resulted in a dose-dependent increase in reactive oxygen species formation, DNA damage and cell death. Heme-induced cell death was significantly attenuated when cells overexpressed HO-1, BCRP, or both. The protective effects of HO-1 overexpression were most pronounced, while co-treatment with the HO-activity inhibitor tin mesoporphyrin reversed these protective effects. Also cells treated with the anti-oxidants N-acetylcysteine or HO-effector molecule bilirubin showed protection against heme insults, which may explain the increased protection by HO-1 compared to BCRP. In conclusion, both HO-1 and BCRP protect against heme-induced toxicity and may thus form novel therapeutic targets for heme-mediated pathologies.

UP-REGULATING HO-1 IMPROVES POST-INFARCTION HEART FUNCTION OF SPONTANEOUS HYPERTENSIVE RATS VIA ANTI-INFLAMMATION, ANTI-OXIDATION, LOWERING BLOOD GLUCOSE AND IMPROVING ENDOTHELIAL FUNCTION

ObjectivesOur previous results showed that hame oxygenase 1 (HO-1) is involved in the regulation of endothelial function by the modulation of NOS isoforms and control of oxidative stress. HO-1, in...

Abstract 61: Adipose Tissue Specific HO-1 Induction Regulates the Crosstalk between Wnt and β-catenin Pathways and Leads to Restoration of Vascular Endothelial Function in Mice Fed a High Fat Diet

The principal goal of the present study is to examine the contributions of adipocyte specific over expression of the cytoprotective gene heme oxygenase-1 (HO-1) in modulating vascular function in an animal model of diet induced obesity. A lentiviral construct of the human HO-1 was synthesized under the control of an aP2-activated promoter so as to ensure HO-1 targeting to murine adipocytes. Lentiviruses (50 μl, 2x109 TU/ml in saline) were injected into the C57BL/6 mice by a single intracardiac injection. Mice (6-7 wks old) were divided into 4 groups: Control, high fat diet (HF) mice receiving the control Lenti-aP2-GFP (HF-GFP) and high fat diet mice receiving the lenti-aP2-HO-1 (HF-HO-1) with and without treatment with SnMP. Transduction of lenti-aP2-HO-1 increased human HO-1 expression in adipose tissues without affecting endogenous mice HO-1 (p&lt;0.01). In mice fed a HF diet, lenti-aP2-HO-1 transduction attenuated the increases in body weight (from 52.0±1.0 g to 35.6±2.1g; p&lt;0.01), blood glucose (from 255±3.5 g to 140±2.9mg/dl), blood pressure (from 135±2.8mmHg to 101±2.2mmHg; p&lt;0.01) and inflammatory cytokines as well as the content of both visceral (from 5.5±0.15g to 2.9±0.22g; p&lt;0.05) and subcutaneous fat (p&lt;0.05). Transduction of lenti-aP2-HO-1 increased the numbers of adipocytes of small cell size (p&lt;0.05), insulin sensitivity (p&lt;0.05), adiponectin levels (from 32.4±1.9μg/ml to 19.9±2.1μg/ml; p&lt;0.05) as well as vascular relaxation to acetylcholine ( p&lt;0.05) compared to HF mice administered the lenti-aP2- GFP. Adipocytes of mice fed a HF diet expressed high levels of PPARγ, aP2, C/EBP and Wnt5b proteins and displayed marked increases in Peg1/Mest (p&lt;0.03). Lenti-aP2-HO-1 transduction lowered the elevated levels of these proteins and increased Shh, Wnt10b and β-catenin (p&lt;0.05). Inhibition of HO activity by administration of tin mesoporphyrin (SnMP) to HF-fed mice transduced with the lenti-aP2-HO-1 reversed the decrease in Peg 1/Mest, TNFα and MCP-1 levels. This novel study demonstrate that adipocyte-specific overexpression of HO-1 attenuates HF-mediated adiposity and vascular dysfunction, increases insulin sensitivity and improves adipocyte function by increasing adiponectin, Shh and WNT10b and decreasing inflammatory cytokines.

Abstract 649: Heme-oxygenase-1 Induction Improves Type-1 Cardiorenal Syndrome Only In Mice With Impaired AngII-induced Lymphocyte Activation (SCID Mice)

Rational: The absence of lymphocyte activity protects SCID mice from AngII-induced hypertension facilitating blood pressure-induced sodium excretion, possibly via the stimulation of eNOS- and COX-2-dependent pathways. Type-1 cardiorenal syndrome (CRS-1), characterized by acute kidney dysfunction secondary to deterioration in cardiac function, is caused by renal arteriolar vasoconstriction, mediated by the activation of renin-angiotensin and sympathetic nervous systems (RAAS, SNS). Heme Oxygenase-1 (HO-1) induction improves renal function, but not renal vasoconstriction, in AngII-induced hypertension, and causes the desensitization of vascular smooth muscle to phenylephrine. Objectives: We evaluated whether AngII resistant SCID mice develop CRS-1 as occurs in control mice, and the differential effects of HO1 induction on renal hemodynamics in CRS-1. Methods: Post ischemic heart failure was induced in C57 and SCID mice by left anterior coronary artery ligation. Mice were divided in 4 groups: sham, myocardial infarction (MI), MI treated with cobalt protoporphyrin (CoPP), an inducer of HO-1, in the presence and absence of HO activity inhibitor, stannous mesoporphyrin (SnMP). All mice underwent echocardiography (ejection fraction, EF) and renal echoDoppler (arterial pulsatility index, K-PI) examination 30 days after surgery. Results: EF was significantly reduced both in control and SCID mice after MI (C57: sham 0.60±0.07, MI 0.45±0.04, p&lt;0.05; SCID: sham 0.60±0.06, MI: 0.46±0.1, p&lt;0.01). K-PI was significantly increased in MI groups compared to sham groups (C57: sham 0.98±0.05, MI 1.12±0.11, p&lt;0.05; SCID: sham 0.72±0.08, MI 1.37±0.37, p&lt;0.05). HO1 induction improved renal vasoconstriction only in SCID mice (SCID K-PI: MI+CoPP 0.9±0.19 p&lt;0.5çC57 K-PI: MI+CoPP 1.05±0.15, n.s.). In SCID mice SnMP reversed the effect of CoPP on renal vasoconstriction. Conclusion: CRS-1 is similar in SCID and control mice and is associated with increased renal arterial resistance. HO-1 induction improves renal vasoconstriction only in SCID (AngII resistant) mice with CRS-1, suggesting that increased HO-1 activity cannot protect the kidney from AngII-induced lymphocyte activation, but only from SNS-induced vasoconstriction.

Heme Oxygenase Gene Targeting to Adipocytes Attenuates Adiposity and Vascular Dysfunction in Mice Fed a High-Fat Diet

We examined the hypothesis that adipocyte dysfunction in mice fed a high-fat (HF) diet can be prevented by lentiviral-mediated and adipocyte specific-targeting delivery of the human heme oxygenase-1 (aP2-HO-1). A bolus intracardial injection of aP2-HO-1 resulted in expression of human HO-1 for up to 9.5 months. Transduction of aP2-HO-1 increased human HO-1 expression in fat tissues without affecting murine HO-1. In mice fed a HF diet, aP2-HO-1 transduction attenuated the increases in body weight, blood glucose, blood pressure, and inflammatory cytokines, as well as the content of both visceral and subcutaneous fat. Transduction of aP2-HO-1 increased the numbers of adipocytes of small cell size (P<0.05), insulin sensitivity (P<0.05), adiponectin levels, as well as vascular relaxation to acetylcholine compared with HF mice administered the aP2-green fluorescent protein. Adipocytes of mice fed a HF diet expressed high levels of peroxisome proliferator activator receptor, aP2, C/EBP, and Wnt5b proteins and displayed marked increases in Peg1/Mesoderm specific transcript (P<0.03). Transduction of aP2-HO-1 lowered the elevated levels of these proteins and increased Sonic hedgehog, Wnt10b, and β-catenin (P<0.05). Inhibition of HO activity by administration of tin mesoporphyrin to HF-fed mice transduced with the aP2-HO-1 reversed the decrease in Peg1/Mesoderm-specific transcript, TNFα, and MCP-1 levels. Collectively, this novel study demonstrates that adipocyte-specific overexpression of HO-1 attenuates HF-mediated adiposity and vascular dysfunction; increases insulin sensitivity; and improves adipocyte function by increasing adiponectin, Shh, and WNT10b, and by decreasing inflammatory cytokines. These effects are reversed by the HO activity inhibitor, stannous mesoporphyrin.

Enhanced Expression of Heme Oxygenase‐1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 μm) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Salvianolic Acid B Exerts Vasoprotective Effects through the Modulation of Heme Oxygenase-1 and Arginase Activities

Salvia miltiorrhiza (Danshen), a traditional Chinese herbal medicine, is commonly used for the prevention and treatment of cardiovascular disorders including atherosclerosis. However, the mechanisms responsible for the vasoprotective effects of Danshen remain largely unknown. Salvianolic acid B (Sal B) represents one of the most bioactive compounds that can be extracted from the water-soluble fraction of Danshen. We investigated the effects of Danshen and Sal B on the inflammatory response in murine macrophages. Danshen and Sal B both induced the expression of heme oxygenase-1 (HO-1) and inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Inhibition of HO activity using Sn-protoporphyrin-IX (SnPP) abolished the inhibitory effect of Sal B on NO production and iNOS expression. Sal B increased macrophage arginase activity in a dose-dependent manner and diminished LPS-inducible tumor necrosis factor-α production. These effects were also reversed by SnPP. These data suggest that HO-1 expression plays an intermediary role in the anti-inflammatory effects of Sal B. In contrast to the observations in macrophages, Sal B dose-dependently inhibited arginase activity in murine liver, kidney, and vascular tissue. Furthermore, Sal B increased NO production in isolated mouse aortas through the inhibition of arginase activity and reduction of reactive oxygen species production. We conclude that Sal B improves vascular function by inhibiting inflammatory responses and promoting endothelium-dependent vasodilation. Taken together, we suggest that Sal B may represent a potent candidate therapeutic for the treatment of cardiovascular diseases associated with endothelial dysfunction.

ApoA1

Insulin resistance is a risk factor in the development of type 2 diabetes and is a major cause of atherosclerosis. Reduction in heme oxygenase (HO-1) has been shown to exacerbate vascular dysfunction and insulin resistance in obese mice and involves a decrease in adiponectin levels. Adiponectin is released from mesenchymal stem cell (MSC)-derived adipocytes, its levels are decreased in type 2 diabetes. We hypothesized that the apoA1 mimetic peptide, L-4F, will target the expression of the HO-1-adiponectin axis and reverse adipocyte dysfunction both in vivo and in vitro. The administration of L-4F [2 mg/Kg/daily (i.p.) for 4-week to 8-week-old obese (ob) mice restored adipocyte function, increased adiponectin release (p < 0.05) and decreased the levels of IL-1 and IL-6 (p < 0.05)]. These perturbations were associated with an increase in insulin sensitivity (p < 0.01 vs. untreated ob mice) and decreased glucose levels (309 + 42 vs. 201 + 8 mg/d after L-4F treatment). Treatment of both mesenchymal stem cell (MSC)-derived adipocytes with L-4F (50 μg/ml) increased adiponectin (p < 0.05), decreased IL-1 and IL-6 (p < 0.05) levels and increased MSC-derived adipocyte cell numbers by 50% in S phase (p < 0.05). MSC-derived adipocytes treated with L-4F increased WNT10b and decreased Peg 1/Mest. Inhibition of HO activity reversed the decrease in the adipogenic response gene, Peg 1/Mest. An increase of HO-1 expression by L-4F increased insulin-receptor phosphorylation. These findings support the hypothesis that L-4F increases early adipocyte markers, HO-1-adiponectin, WNT10b and decreases Peg1/Mest, negative regulators of adipocyte differentiation.

Crosstalk between central pathways of nitric oxide and carbon monoxide in the hypertensive action of cyclosporine

Although the intermediary role of central neurons in the hypertensive and sympathoexcitatory actions of cyclosporine (CSA) has been recognized in previous studies including our own, the underlying mechanism remains obscure. In this study, we tested the hypothesis that central pathways of nitric oxide (NO) and carbon monoxide (CO) modulate the blood pressure (BP) response elicited by CSA in conscious rats. Hemodynamic effects of CSA were evaluated in absence and presence of maneuvers that inhibit or facilitate biosynthesizing enzymes of NO (NOS) or CO (heme oxygenase, HO). CSA (20mg/kg i.v.) produced abrupt increases in BP that peaked in 5min and maintained for at least 45min. The hypertensive effect of CSA disappeared in rats pretreated intracisternally (i.c.) with Nω-nitro-l-arginine methyl ester (L-NAME, nonselective NOS inhibitor), N5-(1-iminoethyl)-l-ornithine (L-NIO, selective eNOS inhibitor), Nω-propyl-l-arginine (NPLA, selective nNOS inhibitor), or 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, guanylate cyclase inhibitor), suggesting the importance of central eNOS/nNOS/GC cascade in CSA-induced hypertension. L-NAME also abolished the hypotension caused by the sympatholytic drug moxonidine, indicating a tonic sympathoinhibitory action for NO. The inhibition of HO activity by zinc protoporphyrin IX (ZnPP) abrogated the hypertensive action of CSA. The abolition by L-NAME or ZnPP of CSA hypertension was compromised upon simultaneous i.c. exposure to hemin (HO substrate) and l-arginine (NOS substrate), respectively. Together, the interruption of the mutually facilitated NOS/NO and HO/CO pathways and coupled GC/cGMP in central neuronal pools accounts, at least partly, for the hypertensive and perhaps sympathoexcitatory actions of CSA.

Heme Oxygenase 1-Induced Resistance to Imatinib In Chronic Myelogenous Leukemia Cells

Abstract 4410Heme oxygenase 1 (HO-1) is the rate-limiting enzyme in heme degradation leading to the generation of free iron, biliverdin and carbon monoxide (CO). In this study, K562 cells were incubated for 24 h with imatinib (1 μM) alone or in combination with: 1) cobalt protoporphyrin (CoPP, 10 μM), a potent inducer of HO-1; or 2) tin mesoporphyrin (SnMP, 10 μM), an inhibitor of HO activity. CoPP was able to overcome the inhibitory effect of imatinib while SnMPP restored the cytotoxicity. Interestingly, neither bilirubin nor CO (using the CO-releasing agent CORM-3) were able to protect K562 cells from imatinib-induced toxicity. The protective effect of CoPP was mitigated by addition of a protease inhibitor (Ed64), which was able to stop HO-1 nuclear translocation. We also analyzed 96 kinase genes and found that addition of CoPP in combination with imatinib leads to at least seven kinases being significantly increased while the expression of these genes was reduced in cells treated with imatinib alone or in the presence of SnMP. All of them are able to activate mitogenic signals. We also found that 1 μM imatinib was able to increase the formation of cellular reactive oxygen species and this effect was inhibited by CoPP and restored in presence of SnMP. In conclusion, the protective effect of HO-1 on imatinib-induced cytotoxicity does not involve the action of its catabolites, but rather a translocation of HO-1 to the nucleus after proteolytic cleavage. Migration to the nucleus activates several kinases able to induce mitogenic signals thus enabling HO-1 to reduce oxidative stress induced by imatinib. Disclosures:Di Raimondo:celgene: Honoraria.

Heme oxygenase-1 induction stimulates host defense mechanism against infections (110.3)

Abstract Innate immune response is pivotal for host defense against pathogenic infections. It is, however, often ineffective to provide protection against infections and fails to efficiently respond to the existing conditions. Induction of host response or direct targeting of the invading pathogens then becomes desirable to reduce the disease progression. Activation of HO-1 by a natural substrate hemin effectively enhanced the ability of human macrophage to resist infections by several pathogens, including HIV-1, dengue virus, West Nile virus, poxvirus, and Leishmania donovani. HO-1 expression in a prostate cancer cell line susceptible to infection by XMRV resulted in substantial reduction in virus production by infected cells. Reduction of intracellular pathogens in HO-1-activated cells was attenuated by pretreatment of cells with SnPP IX, a specific inhibitor of HO-1 activity, indicating a key role of this endogenous enzyme in the host defense mechanism. Considering effective treatment of these infections is a continuous daunting challenge due to the emergence of drug resistant mutants, unlocking of HO-1-dependent regulatory genes may provide novel biomarkers and therapeutic targets for treating multiple infections and disease conditions.