Apo A1 Mimetic Rescues the Diabetic Phenotype of HO-2 Knockout Mice via an Increase in HO-1 Adiponectin and LKBI Signaling Pathway

Insulin resistance, with adipose tissue dysfunction, is one of the hallmarks of metabolic syndrome. We have reported a metabolic syndrome-like phenotype in heme oxygenase (HO)-2 knockout mice, which presented with concurrent HO-1 deficiency and were amenable to rescue by an EET analog. Apo A-I mimetic peptides, such as L-4F, have been shown to induce HO-1 expression and decrease oxidative stress and adiposity. In this study we aimed to characterize alleviatory effects of HO-1 induction (if any) on metabolic imbalance observed in HO-2 KO mice. In this regard, HO-2(−/−) mice were injected with 2 mg/kg/day L-4F, or vehicle, i.p., for 6 weeks. As before, compared to WT animals, the HO-2 null mice were obese, displayed insulin resistance, and had elevated blood pressure. These changes were accompanied by enhanced tissue (hepatic) oxidative stress along with attenuation of HO-1 expression and activity and reduced adiponectin, pAMPK, and LKB1 expression. Treatment with L-4F restored HO-1 expression and activity and increased adiponectin, LKB1, and pAMPK in the HO-2(−/−) mice. These alterations resulted in a decrease in blood pressure, insulin resistance, blood glucose, and adiposity. Taken together, our results show that a deficient HO-1 response, in a state with reduced HO-2 basal levels, is accompanied by disruption of metabolic homeostasis which is successfully restored by an HO-1 inducer

The Association of NOV/CCN3 With Obstructive Sleep Apnea (OSA): Preliminary Evidence of a Novel Biomarker in OSA

Obstructive sleep apnea (OSA) has a strong association with cardiovascular and metabolic abnormalities, although the mechanism driving this association is not well established. NOV/CCN3, a multifunctional extracellular matrix protein, may play a mechanistic and/or prognostic role in these associations. We hypothesized that patients with OSA, which primarily affects obese individuals, will have increased levels of NOV, and that NOV can serve as a biomarker in patients to predict OSA as well as metabolic and cardiac risk. Ten morbidly obese and 10 healthy lean subjects underwent overnight polysomnography (PSG) and clinical evaluation. Blood samples were analyzed for NOV levels, adiponectin and IL-6. OSA was found in nine obese subjects and three lean subjects. NOV levels were significantly higher in the OSA vs. no OSA group (2.1 +/- 0.9 vs. 1.3 +/- 0.8, p \u3c 0.03). NOV levels were significantly higher in the obese vs. lean group (2.2 +/- 0.3 vs. 1.4 +/- 0.2-fold change, p \u3c 0.03). Among lean subjects, NOV levels were significantly higher in the OSA vs. no OSA group (2.1 +/- 0.9 vs. 1.0 +/- 0.4, p \u3c 0.05). NOV and AHI were positively correlated (rho = 0.49, p = 0.033). IL-6 and adiponectin differences in obese vs. lean and OSA vs. no OSA were consistent with an inflammatory phenotype in obese subjects and OSA subjects. NOV is a novel biomarker of the presence and severity of OSA and a potential marker of future cardiovascular and metabolic disease in OSA patients

Development of NASH in Obese Mice is Confounded by adipose Tissue Increase in Inflammatory NOV and Oxidative Stress

Aim: Nonalcoholic steatohepatitis (NASH) is the consequence of insulin resistance, fatty acid accumulation, oxidative stress, and lipotoxicity. We hypothesize that an increase in the inflammatory adipokine NOV decreases antioxidant Heme Oxygenase 1 (HO-1) levels in adipose and hepatic tissue, resulting in the development of NASH in obese mice. Methods: Mice were fed a high fat diet (HFD) and obese animals were administered an HO-1 inducer with or without an inhibitor of HO activity to examine levels of adipose-derived NOV and possible links between increased synthesis of inflammatory adipokines and hepatic pathology. Results: NASH mice displayed decreased HO-1 levels and HO activity, increased levels of hepatic heme, NOV, MMP2, hepcidin, and increased NAS scores and hepatic fibrosis. Increased HO-1 levels are associated with a decrease in NOV, improved hepatic NAS score, ameliorated fibrosis, and increases in mitochondrial integrity and insulin receptor phosphorylation. Adipose tissue function is disrupted in obesity as evidenced by an increase in proinflammatory molecules such as NOV and a decrease in adiponectin. Importantly, increased HO-1 levels are associated with a decrease of NOV, increased adiponectin levels, and increased levels of thermogenic and mitochondrial signaling associated genes in adipose tissue. Conclusions: These results suggest that the metabolic abnormalities in NASH are driven by decreased levels of hepatic HO-1 that is associated with an increase in the adipose-derived proinflammatory adipokine NOV in our obese mouse model of NASH. Concurrently, induction of HO-1 provides protection against insulin resistance as seen by increased insulin receptor phosphorylation. Pharmacological increases in HO-1 associated with decreases in NOV may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NASH

CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 alpha Activation

We have previously shown that an Epoxyeicosatrienoic Acid (EET) -agonist has pleiotropic effects and reverses cardiomyopathy by decreasing inflammatory molecules and increasing antioxidant signaling. We hypothesized that administration of an EET agonist would increase Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha), which controls mitochondrial function and induction of HO-1 and negatively regulates the expression of the proinflammatory adipokines CCN3/NOV in cardiac and pericardial tissues. This pathway would be expected to further improve left ventricular (LV) systolic function as well as increase insulin receptor phosphorylation. Measurement of the effect of an EET agonist on oxygen consumption, fractional shortening, blood glucose levels, thermogenic and mitochondrial signaling proteins was performed. Control obese mice developed signs of metabolic syndrome including insulin resistance, hypertension, inflammation, LV dysfunction, and increased NOV expression in pericardial adipose tissue. EET agonist intervention decreased pericardial adipose tissue expression of NOV, while normalized FS, increased PGC-1alpha, HO-1 levels, insulin receptor phosphorylation and improved mitochondrial function, theses beneficial effect were reversed by deletion of PGC-1alpha. These studies demonstrate that an EET agonist increases insulin receptor phosphorylation, mitochondrial and thermogenic gene expression, decreased cardiac and pericardial tissue NOV levels, and ameliorates cardiomyopathy in an obese mouse model of the metabolic syndrome

The Role of Neutrophils in Corneal Wound Healing in HO-2 Null Mice

Our studies demonstrated that Heme oxygenase (HO), in particular, the constitutive HO-2, is critical for a self-resolving inflammatory and repair response in the cornea. Epithelial injury in HO-2 null mice leads to impaired wound closure and chronic inflammation in the cornea. This study was undertaken to examine the possible relationship between HO-2 and the recruitment of neutrophils following a corneal surface injury in wild type (WT) and HO-2 knockout (HO-2−/−) mice treated with Gr-1 monoclonal antibody to deplete peripheral neutrophils. Epithelial injury was performed by removing the entire corneal epithelium. Infiltration of inflammatory cell into the cornea in response to injury was higher in HO-2−/− than in WT. However, the rate of corneal wound closure following neutrophil depletion was markedly inhibited in both WT and HO-2−/− mice by 60% and 85%, respectively. Neutropenia induced HO-1 expression in WT but not in HO-2−/− mice. Moreover, endothelial cells lacking HO-2 expressed higher levels of the Midkine and VE-cadherin and displayed strong adhesion to neutrophils suggesting that perturbation in endothelial cell function caused by HO-2 depletion underlies the increased infiltration of neutrophils into the HO-2−/− cornea. Moreover, the fact that neutropenia worsened epithelial healing of the injured cornea in both WT and HO-2−/− mice suggest that cells other than neutrophils contribute to the exaggerated inflammation and impaired wound healing seen in the HO-2 null cornea

Development of NASH in Obese Mice is Confounded by Adipose Tissue Increase in Inflammatory NOV and Oxidative Stress

Aim. Nonalcoholic steatohepatitis (NASH) is the consequence of insulin resistance, fatty acid accumulation, oxidative stress, and lipotoxicity.We hypothesize that an increase in the inflammatory adipokine NOV decreases antioxidant Heme Oxygenase 1 (HO- 1) levels in adipose and hepatic tissue, resulting in the development of NASH in obese mice. Methods. Mice were fed a high fat diet (HFD) and obese animals were administered an HO-1 inducer with or without an inhibitor of HO activity to examine levels of adipose-derived NOV and possible links between increased synthesis of inflammatory adipokines and hepatic pathology. Results. NASH mice displayed decreased HO-1 levels and HO activity, increased levels of hepatic heme, NOV, MMP2, hepcidin, and increased NAS scores and hepatic fibrosis. IncreasedHO-1 levels are associated with a decrease in NOV, improved hepatic NAS score, ameliorated fibrosis, and increases in mitochondrial integrity and insulin receptor phosphorylation. Adipose tissue function is disrupted in obesity as evidenced by an increase in proinflammatory molecules such as NOV and a decrease in adiponectin. Importantly, increased HO-1 levels are associated with a decrease of NOV, increased adiponectin levels, and increased levels of thermogenic and mitochondrial signaling associated genes in adipose tissue. Conclusions.These results suggest that the metabolic abnormalities in NASH are driven by decreased levels of hepatic HO-1 that is associated with an increase in the adipose-derived proinflammatory adipokine NOV in our obese mouse model of NASH. Concurrently, induction of HO-1 provides protection against insulin resistance as seen by increased insulin receptor phosphorylation. Pharmacological increases in HO-1 associated with decreases in NOV may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NASH

Reciprocal Effects of Oxidative Stress on Heme Oxygenase Expression and Activity Contributes to Reno-Vascular Abnormalities in EC-SOD Knockout Mice

Heme oxygenase (HO) system is one of the key regulators of cellular redox homeostasis which responds to oxidative stress (ROS) via HO-1 induction. However, recent reports have suggested an inhibitory effect of ROS on HO activity. In light of these conflicting reports, this study was designed to evaluate effects of chronic oxidative stress on HO system and its role in contributing towards patho-physiological abnormalities observed in extracellular superoxide dismutase (EC-SOD, SOD3) KO animals. Experiments were performed in WT and EC-SOD(−/−) mice treated with and without HO inducer, cobalt protoporphyrin (CoPP). EC-SOD(−/−) mice exhibited oxidative stress, renal histopathological abnormalities, elevated blood pressure, impaired endothelial function, reduced p-eNOS, p-AKT and increased HO-1 expression; although, HO activity was significantly (P \u3c 0.05) attenuated along with attenuation of serum adiponectin and vascular epoxide levels (P \u3c 0.05). CoPP, in EC-SOD(−/−) mice, enhanced HO activity (P \u3c 0.05) and reversed aforementioned pathophysiological abnormalities along with restoration of vascular EET, p-eNOS, p-AKT and serum adiponectin levels in these animals. Taken together our results implicate a causative role of insufficient activation of heme-HO-adiponectin system in pathophysiological abnormalities observed in animal models of chronic oxidative stress such as EC-SOD(−/−) mice

Antioxidants Condition Pleiotropic Vascular Responses to Exogenous H2O2: Role of Modulation of Vascular TP Receptors and the Heme Oxygenase System

Aims: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants. Results: Exogenous H(2)O(2) (0.1-10.0 μM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p\u3c0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation. Innovations: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 μM), thromboxane (TP) synthase (CGS13080-10 μM), and TP receptor antagonist (SQ29548-1 μM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats. Conclusions: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2)

Ablation of Soluble Epoxide Hydrolase Reprogram White Fat to Beige-Like Fat Through an Increase in Mitochondrial Integrity, HO-1-Adiponectin in vitro and in vivo

We have shown that epoxyeicosatrienoic acids (EETs), specifically 11,12- and 14,15-EETs, reduce adipogenesis in human mesenchymal stem cells and mouse preadipocytes (3T-3L1). In this study, we explore the effects of soluble epoxide hydrolase (sEH) deletion on various aspects of adipocyte-function, including programing for white vs. beige-like fat, and mitochondrial and thermogenic gene-expressions. We further hypothesize that EETs and heme-oxygenase 1 (HO-1) form a synergistic, functional module whose effects on adipocyte and vascular function is greater than the effects of sEH deletion alone. In in vitro studies, we examined the effect of sEH inhibitors on MSC-derived adipocytes. MSC-derived adipocytes exposed to AUDA, an inhibitor of sEH, exhibit an increased number of small and healthy adipocytes, an effect reproduced by siRNA for sEH. in vivo studies indicate that sEH deletion results in a significant decrease in adipocyte size, inflammatory adipokines NOV, TNFalpha, while increasing adiponectin (p \u3c 0.05). These findings are associated with a decrease in body weight (p \u3c 0.05), and visceral fat (p \u3c 0.05). Importantly, sEH deletion was associated with a significant increase in Mfn1, COX 1, UCP1 and adiponectin (p \u3c 0.03). sEH deletion was manifested by a significant increase in EETs isomers 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET and an increased EETs/DHETEs ratio. Notably, activation of HO-1 gene expression further increased the levels of EETs, suggesting that the antioxidant HO-1 system protects EETs from degradation by ROS. These results are novel in that sEH deletion, while increasing EET levels, resulted in reprograming of white fat to express mitochondrial and thermogenic genes, a phenotype characteristic of beige-fat. Thus, EETs agonist(s) and sEH inhibitors may have therapeutic potential in the treatment of metabolic syndrome and obesity