Stannous Mesoporphyrin Research Articles

Role of heme oxygenase in the regulation of the renal hemodynamics in a model of sex-dependent programmed hypertension by maternal diabetes.

Intrauterine programming of cardiovascular and renal function occurs in diabetes because of the adverse maternal environment. Heme oxygenase 1 (HO-1) and -2 (HO-2) exert vasodilatory and antioxidant actions, particularly in conditions of elevated HO-1 expression or deficient nitric oxide levels. We evaluated whether the activity of the heme-HO system is differentially regulated by oxidative stress in the female offspring of diabetic mothers, contributing to the improved cardiovascular function in comparison with males. Diabetes was induced in pregnant rats by a single dose of streptozotocin (STZ, 50 mg/kg ip) in late gestation. Three-month-old male offspring from diabetic mothers (MODs) exhibited higher blood pressure (BP), higher renal vascular resistance (RVR), worse endothelium-dependent response to acetylcholine (ACH), and an increased constrictor response to phenylephrine (PHE) compared with those in age-matched female offspring of diabetic mothers (FODs), which were abolished by chronic tempol (1 mM) treatment. In anesthetized animals, stannous mesoporphyrin (SnMP; 40 µmol/kg iv) administration, to inhibit HO activity, increased RVR in FODs and reduced glomerular filtration rate (GFR) in MODs, without altering these parameters in control animals. When compared with MODs, FODs showed lower nitrotirosyne levels and higher HO-1 protein expression in renal homogenates. Indeed, chronic treatment with tempol in MODs prevented elevations in nitrotyrosine levels and the acute renal hemodynamics response to SnMP. Then, maternal diabetes results in sex-specific hypertension and renal alterations associated with oxidative stress mainly in adult male offspring, which are reduced in the female offspring by elevation in HO-1 expression and lower oxidative stress levels.

Trophoblast-induced spiral artery remodelling and uteroplacental haemodynamics in pregnant rats with increased blood pressure induced by heme oxygenase inhibition

IntroductionThe aim of the present study was to determine the contribution of the heme oxygenase (HO) system to the adaptation of the uteroplacental circulation to pregnancy in the rat, and its relationship with the maintenance of blood pressure during late gestation. MethodsThe HO inhibitor, stannous mesoporphyrin (SnMP), or vehicle were administered intraperitoneally to virgin and midpregnant rats. Mean arterial pressure (MAP) was measured before and after the treatment, in the conscious rats. Uterine and radial arteries blood flow velocities were obtained from pregnant rats at days 14 and 19 of gestation using high frequency ultrasonography. Trophoblast invasion and spiral arteries remodelling were analyzed in the mesometrial triangle of pregnant rats by immunohistochemistry. ResultsHO activity inhibition during late gestation induced a significantly increase in the MAP of pregnant rats (114 ± 1 mmHg vs 100 ± 2 mmHg, p < 0.05) but it did not affect this parameter in virgin rats (121 ± 2 mmHg vs 124 ± 3 mmHg). MAP elevation was associated with marked (p < 0.05) decreases in the systolic and diastolic flow velocities in uterine and radial arteries, as compared with pregnant control rats. Furthermore, spiral arteries of pregnant rats treated with SnMP showed lower (p < 0.001) proportion of lumen circumference covered by trophoblast (21 ± 3%) and a higher (p < 0.05) proportion of vascular smooth muscle (33 ± 5%) than control pregnant rats (59 ± 5% and 16 ± 5%, respectively) DiscussionThese data indicate that HO system play an important role in the adaptation of the uteroplacental circulation to pregnancy and in the blood pressure regulation during late gestation.

Abstract P338: Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin- a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO) is a stress response system, the induction of which reduces oxidative stress thereby abating patho-physiological conditions such as obesity and metabolic syndrome.8 week old male obese (ob) mice and their age- and sex-matched lean mice were used as controls. CoPP was administered intraperitoneally once a week (3 mg/kg) for 6 weeks to obese mice. CoPP plus stannous mesoporphyrin (SnMP) was administered intraperitoneally three times a week (20 mg/kg) for 6 weeks. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in tissues from lean and obese mice. Obese mice exhibited hyperglycemia along with increased levels of pro-inflammatory cytokines (MCP-1, IL-6, p&lt;0.05), oxidative stress and increased hepatic hepcidin levels (p&lt;0.05). Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p&lt;0.05). Further, our results showed attenuation of insulin receptor phosphorylation and attenuation of metabolic regulators including pAMPK, pAKT and pLKB1. Cobalt protoporphyrin (CoPP)-induced HO-1 up-regulation in obese mice and reversed these pathophysiological alterations (p&lt;0.05) while attenuating hepatic hepcidin levels and enhancing ferritin expression. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP) (p&lt;0.05). Taken together, our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin in conjunction with the rescue of cellular ferritin levels. Therefore, these findings may prove an effective strategy in treating the metabolic consequences of obesity including alteration of liver iron homeostasis.

Featured article: induction of heme oxygenase with hemin improves pericardial adipocyte morphology and function in obese Zucker rats by enhancing proteins of regeneration.

Oxidative stress and inflammation are implicated in tissue remodeling, hypertrophy, and organ malfunction. Since heme-oxygenase (HO) is a cytoprotective enzyme with effects against oxidative stress and inflammation, we investigated the effects of upregulating HO with hemin on adipocyte hypertrophy, proteins of repair/regeneration including beta-catenin, Oct3/4 and Pax2 as well as pro-fibrotic/remodeling proteins like osteopontin and transforming growth factor-beta (TGF-β) in pericardial adipose tissue from obese Zucker rats (ZRs). Treatment with hemin significantly reduced pericardial adipose tissue inflammation/oxidative stress, suppressed osteopontin and TGF-β, and attenuated pericardial adipocyte hypertrophy in obese ZRs. These were associated with enhanced expression of the stem/progenitor-cell marker cKit; the potentiation of several proteins of regeneration including beta-catenin, Oct3/4, Pax2; and improved pericardial adipocyte morphology. Interestingly, the amelioration of adipocyte hypertrophy in hemin-treated animals was accompanied by improved adipocyte function, evidenced by increased levels of pericardial adipose tissue adiponectin. Furthermore, hemin significantly reduced hypertriglyceridemia and hypercholesteromia in obese ZRs. The protective effects of hemin were accompanied by robust potentiation HO activity and the total antioxidant capacity, whereas the co-administration of hemin with the HO inhibitor, stannous mesoporphyrin abolished the effects of hemin. These data suggest that hemin improves pericardial adipocyte morphology and function by enhancing proteins of repair and regeneration, while concomitantly abating inflammatory/oxidative insults and suppressing extracellular-matrix/profibrotic and remodeling proteins. The reduction of hypertriglyceridemia, hypercholesteromia, pericardial adiposity, and pericardial adipocyte hypertrophy with corresponding improvement of adipocyte morphology/function in hemin-treated animals suggests that HO inducers may be explored for the design of novel remedies against cardiac complications arising from excessive adiposity.

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.

The heme oxygenase system rescues hepatic deterioration in the condition of obesity co-morbid with type-2 diabetes.

The prevalence of non-alcoholic fatty-liver disease (NAFLD) is increasing globally. NAFLD is a spectrum of related liver diseases that progressive from simple steatosis to serious complications like cirrhosis. The major pathophysiological driving of NAFLD includes elevated hepatic adiposity, increased hepatic triglycerides/cholesterol, excessive hepatic inflammation, and hepatocyte ballooning injury is a common histo-pathological denominator. Although heme-oxygenase (HO) is cytoprotective, its effects on hepatocyte ballooning injury have not been reported. We investigated the effects of upregulating HO with hemin or inhibiting it with stannous-mesoporphyrin (SnMP) on hepatocyte ballooning injury, hepatic adiposity and inflammation in Zucker-diabetic-fatty rats (ZDFs), an obese type-2-diabetic model. Hemin administration to ZDFs abated hepatic/plasma triglycerides and cholesterol, and suppressed several pro-inflammatory cytokines and chemokines including, TNF-α, IL-6, IL-1β, macrophage-inflammatory-protein-1α (MIP-1α) and macrophage-chemoattractant-protein-1 (MCP-1), with corresponding reduction of the pro-inflammatory M1-phenotype marker, ED1 and hepatic macrophage infiltration. Correspondingly, hemin concomitantly potentiated the protein expression of several markers of the anti-inflammatory macrophage-M2-phenotype including ED2, IL-10 and CD-206, alongside components of the HO-system including HO-1, HO-activity and cGMP, whereas the HO-inhibitor, SnMP abolished the effects. Furthermore, hemin attenuated liver histo-pathological lesions like hepatocyte ballooning injury and fibrosis, and reduced extracellular-matrix/profibrotic proteins implicated in liver injury such as osteopontin, TGF-β1, fibronectin and collagen-IV. We conclude that hemin restore hepatic morphology by abating hepatic adiposity, suppressing macrophage infiltration, inflammation and fibrosis. The selective enhancement of anti-inflammatory macrophage-M2-phenotype with parallel reduction of pro-inflammatory macrophage-M1-phenotype and related chemokines/cytokines like TNF-α, IL-6, IL-1β, MIP-1α and MCP-1 are among the multifaceted mechanisms by which hemin restore hepatic morphology.

Administration of Heme Arginate Ameliorates Murine Type 2 Diabetes Independently of Heme Oxygenase Activity

Amelioration of rodent type 2 diabetes by hemin has been linked to increased heme oxygenase (HO) activity, however alternative mechanisms have recently been proposed for its anti-diabetic effect. We sought to determine the anti-diabetic efficacy of heme arginate (HA), a clinically licensed preparation of heme, and whether its predominant mode of action is via increased HO activity. Intravenous administration of HA reduced hyperglycemia in diabetic (db/db) mice. Co-administration of the HO inhibitor stannous (IV) mesoporphyrin IX dichloride (SM) resulted unexpectedly in a further improvement in glycaemic control despite restoring HO activity to baseline levels. The anti-diabetic effects of HA±SM were associated with increased adiposity, increased serum adiponectin levels, reduced adipose tissue and islet inflammation and preservation of islet β-cell function. HO activity independent effects of HA on adipogenesis and β-cell inflammation were further confirmed in cell culture models using the 3T3-L1 pre-adipocyte and MIN6 β-cell lines, respectively. In conclusion, our work demonstrates that the heme component of HA ameliorates experimental type 2 diabetes by promoting metabolically favourable adipogenesis and preserving islet β-cell function, but this is not mediated via increased HO activity.

Abstract 395: Heme Oxygenase-1-mediated PPARd Improves Cardiac Fibrosis and Inflammation in SCID Mice via Induction of T Reg Cells

Introduction: Post-infarcted cardiac failure is the consequence of pathological remodeling after myocardial infarction (MI). A subtype of T lymphocytes, regulatory T (Treg) cells play an important role in improving cardiac function after MI via inhibition of inflammation and direct protection of cardiomyocytes. Heme oxygenase (HO)-1 induction improves heart function after ischemic damage by its anti-inflammatory, anti-oxidative and anti-fibrotic effects. To assess the role of Treg cells in post infarcted myocardium and on renal arteriolar vasoconstriction in immunosuppressed mice via the interplay of HO-1-PPAR δ axis. Methods: We examined the effect of HO-1 induction on post-ischemic heart failure induced by left anterior descending coronary artery ligation, in T-lymphocyte immunosuppressive (BALB SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with the HO-1 inducer cobalt protoporphyrin (CoPP) with and without the HO activity inhibitor stannous mesoporphyrin (SnMP). Thirty days after surgery all mice underwent cardiac and renal Doppler sonography. Cardiac fibrosis and necrosis were analyzed and densitometry analysis of HO-1, PPAR δ and thromboxane synthase (TxA2) in cardiac tissues was done. Results: Mice with MI had increased levels of cardiac fibrosis and necrosis (p&lt;0.05) compared to sham operated controls. Echocardiography showed that fractional area shortening (FAS) was increased in the CoPP−MI group compared to the MI group (p&lt;0.01). Renal pulsatiltiy index was increased in the MI group compared to the sham operated group (sham 0.72 ±0.08, MI 1.37±0.37, p&lt;0.05). CoPP improved renal vasoconstriction as compared to the MI group (p&lt;0.05). Increased expression of HO-1, PPAR-δ, pAMPK and pAKT and decreased expression of TxA2 was detected in cardiac tissues in CoPP-treated animals (p&lt;0.02) as compared to the MI group. All these beneficial effects were reversed by SnMP. Conclusion: Upregulation of HO-1 attenuates post myocardial fibrosis and inflammation and renal vasoconstriction in immunosuppressed mice via recruitment of Treg lymphocytes and PPAR delta activation.

Abstract 396: Enhanced VEGF and ETS-1 Recruitment by T Reg- Heme Oxygenase- 1 Module Increases Blood Flow in Post-infarction Myocardium in SCID Mice

Introduction: Post-infarction cardiomyopathy has been ascribed to chronic inflammation, increased oxidative stress and insufficient angiogenesis. T lymphocyte mediated immune response plays an important role in post-infarct cardiac remodeling. T regulatory lymphocytes (Treg) are a subpopulation of T helper lymphocytes with a regulatory and immunosuppressive role. Heme oxygenase (HO)-1 induction improves heart function after ischemic damage by its anti-oxidative effect. To assess the role of the HO-1 mediated increased levels of T-reg lymphocytes in immunosuppressed mice in a model of post-infarcted heart failure. Methods: We compared the effect of HO-1 induction on post-infarction myocardium induced by left anterior coronary artery ligation in T-lymphocyte immunosuppressed mice (BALB SCID) . Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with the HO-1 inducer cobalt protoporphyrin (CoPP) with and without the HO activity inhibitor stannous mesoporphyrin (SnMP). Thirty days after surgery all mice underwent echocardiography, immunohistochemistry and an assessment of T-lymphocyte subpopulations (Treg cells) via fluorescence activated cell sorting (FACS). Results: Mice with MI had increased levels of inflammatory cytokines (p&lt;0.05) and lower capillary density (SCID: 1.2±0.7% vs control: 4.3±0.9%; p&lt;0.01). Left ventricle end diastolic area (EDA) was reduced in CoPP treated compared to the MI group (EDA: MI: 0.22 ±0.02 cm; MI+CoPP: 0.17±0.03 cm; p&lt;0.01). In CoPP treated SCID mice the T-reg subpopulation measured by FACS analysis (20±3% vs 5±3%; p&lt;0.01) and immunohistochemistry (p&lt;0.02) was significantly increased as compared to the MI group. Increased expression of the angiogenic markers VEGF, FGF and ETS-1 (p&lt;0.02) was found in CoPP treated animals as compared to MI animals. These beneficial effects were reversed by SnMP. Conclusion: Upregulation of HO-1 increases T regulatory cells, reverses dysfunctional remodeling and enhances angiogenesis and peri-infarct survival in the post-infarction myocardium in immunosuppressed mice. Targeted enhancement of T-reg cells via HO-1 induction suggests adjuvant avenues for reducing morbidity and mortality in patients with post-MI heart failure.

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.

Abstract 397: HMOX1 Ameliorates Fatty Liver and Metabolic Syndrome by Reduction of Hepatic Heme and PGC1a

Introduction: Nonalcoholic fatty liver (NAFLD) occurs in a setting of high fat diets, insulin resistance, obesity and dyslipidemia. Individuals with NAFLD have an increased risk of developing metabolic syndrome. Heme oxygenase-1 (HMOX1), a major cytoprotective enzyme, attenuates oxidative stress and obesity and increases insulin sensitivity. The antioxidant effect of HMOX1 is due to an increase in ferritin, and bilirubin and a decrease in heme, a pro-oxidant. The aim of this study was to examine the role of increased hepatic HMOX activity in decreasing steatosis, adiposity and vascular dysfunction and to determine the mechanism underlying these metabolic changes in obese mice. Methods: Obese mice were administered cobalt protoporphyrin (CoPP) and HMOX activity inhibitor stannous mesoporphyrin (SnMP) for 6 weeks. Blood pressure, body weight and blood glucose levels were measured in all the groups. Glycogen content, hepatic fibrosis, heme levels, fatty acid synthase (FAS) and lipid droplet size in liver were also assessed. Results: CoPP administration increased hepatic HMOX1 protein levels and HMOX activity, decreased blood pressure, body weight, blood glucose levels, hepatic heme content (p&lt;0.05) as compared to obese mice. Our results also showed that HMOX1 induction causes a significant decrease in lipid steatosis ( lipid droplet size and FAS levels; p&lt;0.01) as compared to obese mice. Densitometry analysis showed increased expression of PPARα and Glut 1 along with decreased expression of PGC1α in hepatic tissue. These beneficial effects were reversed by administration of SnMP. Conclusion: This novel study demonstrates the role of hepatic HMOX1 in attenuating the fatty liver and metabolic homeostasis by decreasing PGC1α and heme content and enhancing glycogen levels. Pharmacological agents that increase HMOX1 levels or gene targeting of HMOX1 offer a promising therapeutic target for NAFLD and suggest the existence of a significant link between the heme-HMOX system and the extent and severity of heme-dependent fatty liver.

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.

Hemin therapy suppresses inflammation and retroperitoneal adipocyte hypertrophy to improve glucose metabolism in obese rats co‐morbid with insulin‐resistant type‐2 diabetes

Visceral adiposity and impaired glucose metabolism are common patho-physiological features in patients co-morbid with obesity and type-2 diabetes. We investigated the effects of the heme-oxygenase (HO) inducer hemin and the HO blocker stannous-mesoporphyrin (SnMP) on glucose metabolism, adipocyte hypertrophy and pro-inflammatory cytokines/mediators in Zucker diabetic fatty (ZDF) rats, a model characterized by obesity and type-2 diabetes. Histological, morphological/morphometrical, Western immunoblotting, enzyme immunoassay, ELISA and spectrophotometric analysis were used. Treatment with hemin enhanced HO-1, HO activity and cGMP, but suppressed retroperitoneal adiposity and abated the elevated levels of macrophage-chemoattractant protein-1 (MCP-1), ICAM-1, tumour necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), IL-1β, NF-κB, c-Jun-NH2-terminal-kinase (JNK) and activating-protein (AP-1), with parallel reduction of adipocyte hypertrophy. Correspondingly, important proteins of lipid metabolism and insulin-signalling such as lipoprotein lipase (LPL), insulin-receptor substrate-1 (IRS-1), GLUT4, PKB/Akt, adiponectin, the insulin-sensitizing and anti-inflammatory protein and adenosine-monophosphate-activated protein kinase (AMPK) were significantly enhanced in hemin-treated ZDF rats. Elevated retroperitoneal adiposity and the high levels of MCP-1, ICAM-1, TNF-α, IL-6, IL-1β, NF-κB, JNK and AP-1 in untreated ZDF are patho-physiological factors that exacerbate inflammatory insults, aggravate adipocyte hypertrophy, with corresponding reduction of adiponectin and deregulation of insulin-signalling and lipid metabolism. Therefore, the suppression of MCP-1, ICAM-1, TNF-α, IL-6, IL-1β, NF-κB, JNK, AP-1 and adipocyte hypertrophy, with the associated enhancement of LPL, adiponectin, AMPK, IRS-1, GLUT4, PKB/Akt and cGMP in hemin-treated ZDF are among the multifaceted mechanisms by which the HO system combats inflammation to potentiate insulin signalling and improve glucose and lipid metabolism. Thus, HO inducers may be explored in the search of novel remedies against the co-morbidities of obesity, dysfunctional lipid metabolism and impaired glucose metabolism.

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.

Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-β (transforming growth factor β) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

The Risk of Heart Failure and Cardiometabolic Complications in Obesity May Be Masked by an Apparent Healthy Status of Normal Blood Glucose

Although many obese individuals are normoglycemic and asymptomatic of cardiometabolic complications, this apparent healthy state may be a misnomer. Since heart failure is a major cause of mortality in obesity, we investigated the effects of heme-oxygenase (HO) on heart failure and cardiometabolic complications in obese normoglycemic Zucker-fatty rats (ZFs). Treatment with the HO-inducer, hemin, reduced markers of heart failure, such as osteopontin and osteoprotegerin, abated left-ventricular (LV) hypertrophy/fibrosis, extracellular matrix/profibrotic proteins including collagen IV, fibronectin, TGF-β1, and reduced cardiac lesions. Furthermore, hemin suppressed inflammation by abating macrophage chemoattractant protein-1, macrophage-inflammatory protein-1 alpha, TNF-α, IL-6, and IL-1β but enhanced adiponectin, atrial-natriuretic peptide (ANP), HO activity, insulin sensitivity, and glucose metabolism. Correspondingly, hemin improved several hemodynamic/echocardiographic parameters including LV-diastolic wall thickness, LV-systolic wall thickness, mean-arterial pressure, arterial-systolic pressure, arterial-diastolic pressure, LV-developed pressure, +dP/dt, and cardiac output. Contrarily, the HO-inhibitor, stannous mesoporphyrin nullified the hemin effect, exacerbating inflammatory/oxidative insults and aggravated insulin resistance (HOMA-index). We conclude that perturbations in insulin signaling and cardiac function may be forerunners to overt hyperglycemia and heart failure in obesity. Importantly, hemin improves cardiac function by suppressing markers of heart failure, LV hypertrophy, cardiac lesions, extracellular matrix/profibrotic proteins, and inflammatory/oxidative mediators, while concomitantly enhancing the HO-adiponectin-ANP axis.

Abstract 309: Role of Hemeoxygenase in the Reno-Protective Effects of Soluble Epoxide Hydrolase Inhibition In Diabetic Spontaneously Hypertensive Rats

We previously showed that inhibition of soluble epoxide hydrolase (sEH) increased epoxyeicosatrienoic acids (EETs) levels and reduced renal injury in diabetic mice and these changes were associated with induction of hemeoxygenase-1 (HO-1). The present study determines whether the inhibition of HO negates the reno-protective effect of sEH inhibition in diabetic spontaneously hypertensive rats as a model of diabetic nephropathy in which hypertension coexists with diabetes. After six weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor, trans -4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (AUCB), treated with the HO inhibitor, stannous mesoporphyrin (SnMP), and treated with both inhibitors for four more weeks; non diabetic SHR served as a control group. Although inhibition of sEH increased renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR, it did not significantly alter blood pressure (plasma EETs/DHETEs ratio was 0.5± 0.1 in AUCB-treated vs. 0.1± 0.01 in untreated diabetic SHR, P&lt;0.05). Treatment of diabetic SHR with AUCB reduced the elevation in urinary albumin and nephrin excretion (albuminuria was 6.5± 0.5 in AUCB-treated diabetic SHR vs. 9± 1.7 mg/day in untreated diabetic SHR and nephrinuria was 70±11 in AUCB-treated diabetic SHR vs. 111± 9 μg/day in untreated diabetic SHR, P&lt;0.05) whereas co-administration of SnMP with AUCB prevented these changes (albuminuria was 10.6± 0.6 mg/day and nephrinuria was 91±11 μg/day). Immunohistochemical analysis revealed elevations in renal fibrosis and apoptosis as evidenced by increased renal TGF-β, fibronectin and annexin V expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with AUCB prevented its ability to reduce renal fibrosis and apoptosis in diabetic SHR. In addition, SnMP treatment also prevented AUCB-induced decreases in renal macrophage infiltration and renal TGF-β, NFκB and MCP-1 levels in diabetic SHR. These data suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

Abstract 636: Angiotensin II-Mediated Endothelial Cell Dysfunction Contributes to Adipocyte Stem Cell Dysfunction and Adiposity

Rationale: Crosstalk between perivascular adipose tissue and endothelial cells has been proposed to modulate vascular homeostasis. However, paracrine regulatory effects of the stressed endothelium on adipose tissue are relatively unknown. Objective: We examined the hypothesis that redox mechanisms in endothelial cells can regulate adipocyte function, in a paracrine fashion, via suppression of the Wnt10b/β-catenin-dependent pathways. METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with AngII (1-50 μM) in the absence and in the presence of the heme oxygenase (HO-1) inducer_cobalt protoporphyrin (CoPP)_or to HO-1 activity inhibitor_stannous mesoporphyrin (SnMP). Adipogenesis was then induced in mesenchymal stem cells, in the presence (10%) or absence of conditioned media (CM) from HUVECs. Results: MSCs exposed to CM from AngII-treated HUVEC displayed enhanced adipogenesis (0.026±0.001 vs. 0.031±0.001, n=6, p&lt;0.0405). This effect was reversed in MSCs supplemented with CM from HUVECs treated with AngII and CoPP (0.021±0.001, n=6, p&lt;0.0024). This beneficial effect of CoPP in MSCs exposed to CM is reversed by SnMP (0.030±0.001, n=6, p&lt;0.0462) and corroborates the ability of HO-1 in attenuating AngII-dependent adipocyte dysfunction. Complementary immunoblot analysis showed an attenuative effect of AngII on Wnt10b and β-catenin expression (p&lt;0.05). Additionally, AngII enhanced the expression of adipogenic regulators including PPARY, CEBPα, and aP2, which was decreased by CoPP and increased by SnMP. Conclusion: Our results show that EC dysfunction, as a result of treatment with AngII, contributes to increased adipogenesis in MSCs by the release of negative regulators of adipogenesis. We also show that up-regulation of antioxidant HO-1 ameliorates these effects of AngII. This novel observation suggests that paracrine crosstalk between HUVECs and adipocytes increases endothelial stress to affect adipocyte structure and function. These phenomena might contribute to the metabolic imbalance frequently encountered in conditions associated with chronic endothelial dysfunction and adiposity.

Abstract 648: Heme-oxygenase-1 Gene Expression Ameliorates Cardiac Dysfunction Following Myocardial Infarction In C57 Mice By Recruitment Of Angiogenic Factors And Angiogenesis In C57 Mice

Rational: Angiogenesis is essential in order to increase blood circulation in infarcted tissue of MI (Myocardial infarction). Increased Heme-Oxygenase (HO)-1 gene expression increases angiogenic proteins, e.g. VEGF, bFGF, EGF, angiopoietin and adiponectin. Objective: To investigate whether increased levels of HO-1, after the occurrence of a MI, improves angiogenesis and capillary formation in ischemic myocardium, thereby improving cardiac function. METHODS: Experimental MI was induced by LAD (Left anterior descending artery) ligation. C57BL6 mice were divided into 4 groups: Sham; MI; 5 days after MI treated with the HO-1 inducer, cobalt protoporphyrinIX (CoPP); and, CoPP in the presence of the HO activity inhibitor, Stannous Mesoporphyrin (SnMP). HO-1 downstream signaling proteins were determined including VEGF, CD31 and adiponectin. Echocardiography was performed weekly for 4 weeks after surgery. Results: 5 days after MI, CoPP treatment significantly increased VEGF (p&lt;0.05 vs.MI), CD31 (p&lt;0.05 vs.MI), and adiponectin levels (p&lt;0.05 vs.MI). These findings were associated with a significant increase in capillary formation and blood flow in CoPP-treated animals (p&lt;0.05 vs.MI). Echocardiography showed that left ventricle dilatation, measured as end diastolic area (EDA), was significantly reduced in CoPP- treated animals compared to MI groups (EDA: MI: 0.216±0.02cm2; MI+CoPP: 0.172±0.03 cm2; (-13%) p&lt;0.01). This was associated with a significant decrease in apoptosis and fibrosis (P&lt;0.05). These beneficial effects were reversed by SnMP administration. Conclusion: HO-1 improved cardiac function and enhanced angiogenesis via the recruitment of pro-angiogenic factors.

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.