Komal Sodhi

Epoxyeicosatrienoic Acid Agonist Rescues the Metabolic Syndrome Phenotype of HO-2-Null Mice

Heme oxygenase (HO) and cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) participate in vascular protection, and recent studies suggest these two systems are functionally linked. We examined the consequences of HO deficiency on P450-derived EETs with regard to body weight, adiposity, insulin resistance, blood pressure, and vascular function in HO-2-null mice. The HO-2-null mice were obese, displayed insulin resistance, and had high blood pressure. HO-2 deficiency was associated with decreases in cyp2c expression, EET levels, HO-1 expression, and HO activity and with an increase in superoxide production and an impairment in the relaxing response to acetylcholine. In addition, HO-2-null mice exhibited increases in serum levels of tumor necrosis factor (TNF)-α and macrophage chemoattractant protein (MCP)-1 and a decrease in serum adiponectin levels. Treatment of HO-2-null mice with a dual-activity EET agonist/soluble epoxide hydrolase inhibitor increased renal and vascular EET levels and HO-1 expression, lowered blood pressure, prevented body weight gain, increased insulin sensitivity, reduced subcutaneous and visceral fat, and decreased serum TNF-α and MCP-1, while increasing adiponectin and restoring the relaxing responses to acetylcholine. The decrease in cyp2c expression and EETs levels in HO-2-null mice underscores the importance of the HO system in the regulation of epoxygenase levels and suggests that protection against obesity-induced cardiovascular complications requires interplay between these two systems. A deficiency in one of these protective systems may contribute to the adverse manifestations associated with the clinical progression of the metabolic syndrome.

Adipocyte Heme Oxygenase-1 Induction Attenuates Metabolic Syndrome in Both Male and Female Obese Mice

Increases in visceral fat are associated with increased inflammation, dyslipidemia, insulin resistance, glucose intolerance, and vascular dysfunction. We examined the effect of the potent heme oxygenase (HO)-1 inducer, cobalt protoporphyrin (CoPP), on regulation of adiposity and glucose levels in both female and male obese mice. Both lean and obese mice were administered CoPP intraperitoneally (3 mg/kg once per week) for 6 weeks. Serum levels of adiponectin, tumor necrosis factor &agr; (TNFa), interleukin (IL)-1&bgr; and IL-6, and HO-1, PPAR&ggr;, pAKT, and pAMPK protein expression in adipocytes and vascular tissue were measured. While female obese mice continued to gain weight at a rate similar to controls, induction of HO-1 slowed the rate of weight gain in male obese mice. HO-1 induction led to lowered blood pressure levels in obese male and female mice similar to that of lean male and female mice. HO-1 induction also produced a significant decrease in the plasma levels of IL-6, TNF&agr;, IL-1&bgr;, and fasting glucose of obese females compared to untreated female obese mice. HO-1 induction increased the number and decreased the size of adipocytes of obese animals. HO-1 induction increased adiponectin, pAKT, pAMPK, and PPAR&ggr; levels in adipocyte of obese animals. Induction of HO-1 in adipocytes was associated with an increase in adiponectin and a reduction in inflammatory cytokines. These findings offer the possibility of treating not only hypertension, but also other detrimental metabolic consequences of obesity including insulin resistance and dyslipidemia in obese populations by induction of HO-1 in adipocytes.

Systematic Review of Metabolic Syndrome Biomarkers: A Panel for Early Detection, Management, and Risk Stratification in the West Virginian Population

Introduction: Metabolic syndrome represents a cluster of related metabolic abnormalities, including central obesity, hypertension, dyslipidemia, hyperglycemia, and insulin resistance, with central obesity and insulin resistance in particular recognized as causative factors. These metabolic derangements present significant risk factors for cardiovascular disease, which is commonly recognized as the primary clinical outcome, although other outcomes are possible. Metabolic syndrome is a progressive condition that encompasses a wide array of disorders with specific metabolic abnormalities presenting at different times. These abnormalities can be detected and monitored via serum biomarkers. This review will compile a list of promising biomarkers that are associated with metabolic syndrome and this panel can aid in early detection and management of metabolic syndrome in high risk populations, such as in West Virginia. Methods: A literature review was conducted using PubMed, Science Direct, and Google Scholar to search for markers related to metabolic syndrome. Biomarkers searched included adipokines (leptin, adiponectin), neuropeptides (ghrelin), pro-inflammatory cytokines (IL-6, TNF-α), anti-inflammatory cytokines (IL-10), markers of antioxidant status (OxLDL, PON-1, uric acid), and prothrombic factors (PAI-1). Results: According to the literature, the concentrations of pro-inflammatory cytokines (IL-6, TNF-α), markers of pro-oxidant status (OxLDL, uric acid), and prothrombic factors (PAI-1) were elevated in metabolic syndrome. Additionally, leptin concentrations were found to be elevated in metabolic syndrome as well, likely due to leptin resistance. In contrast, concentrations of anti-inflammatory cytokines (IL-10), ghrelin, adiponectin, and antioxidant factors (PON-1) were decreased in metabolic syndrome, and these decreases also correlated with specific disorders within the cluster. Conclusion: Based on the evidence presented within the literature, the aforementioned biomarkers correlate significantly with metabolic syndrome and could provide a minimally-invasive means for early detection and specific treatment of these disorders. Further research is encouraged to determine the efficacy of applying these biomarkers to diagnosis and treatment in a clinical setting.

Crosstalk between EET and HO-1 downregulates Bach1 and adipogenic marker expression in mesenchymal stem cell derived adipocytes

Epoxygenase activity and synthesis of epoxyeicosatrienoic acids (EETs) have emerged as important modulators of obesity and diabetes. We examined the effect of the EET-agonist 12-(3-hexylureido)dodec-8(2) enoic acid on mesenchymal stem cell (MSC) derived adipocytes proliferation and differentiation. MSCs expressed substantial levels of EETs and inhibition of soluble epoxide hydrolase (sEH) increased the level of EETs and decreased adipogenesis. EET agonist treatment increased HO-1 expression by inhibiting a negative regulator of HO-1 expression, Bach-1. EET treatment also increased βcatenin and pACC levels while decreasing PPARγ C/EBPα and fatty acid synthase levels. These changes were manifested by a decrease in the number of large inflammatory adipocytes, TNFα, IFNγ and IL-1α, but an increase in small adipocytes and in adiponectin levels. In summary, EET agonist treatment inhibits adipogenesis and decreases the levels of inflammatory cytokines suggesting the potential action of EETs as intracellular lipid signaling modulators of adipogenesis and adiponectin.

Increased heme-oxygenase 1 expression in mesenchymal stem cell-derived adipocytes decreases differentiation and lipid accumulation via upregulation of the canonical Wnt signaling cascade

IntroductionHeme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes.MethodsTo verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining.ResultsDuring adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP.ConclusionsThis is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.

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 &bgr;-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, TNF&agr;, 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.

CYP4A2-Induced Hypertension Is 20-Hydroxyeicosatetraenoic Acid– and Angiotensin II–Dependent

We have shown previously that increased vascular endothelial expression of CYP4A2 leads to 20-hydroxyeicosatetraenoic (20-HETE)-dependent hypertension. The renin-angiotensin system is a key regulator of blood pressure. In this study, we examined possible interactions between 20-HETE and the renin-angiotensin system. In normotensive (110±3 mm Hg) Sprague-Dawley rats transduced with a lentivirus expressing the CYP4A2 cDNA under the control of an endothelial-specific promoter (VECAD-4A2), systolic blood pressure increased rapidly, reaching 139±1, 145±3, and 150±2 mm Hg at 3, 5, and 10 days after transduction; blood pressure remained elevated, thereafter, with maximum levels of 163±3 mm Hg. Treatment with lisinopril, losartan, or the 20-HETE antagonist 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid decreased blood pressure to control values, but blood pressure returned to its high levels after cessation of treatment. Endothelial-specific overexpression of CYP4A2 resulted in increased expression of vascular angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor and increased levels of plasma and tissue angiotensin II; all were attenuated by treatment with HET0016, an inhibitor of 20-HETE synthesis, or with 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid. In cultured endothelial cells, 20-HETE specifically and potently induced ACE expression without altering the expression of ACE2, angiotensinogen, or angiotensin II receptors. This is the first study to demonstrate that 20-HETE, a key constrictor eicosanoid in the microcirculation, induces ACE and angiotensin II type 1 receptor expression and increases angiotensin II levels, suggesting that the mechanisms by which 20-HETE promotes hypertension include activation of the renin-angiotensin system that is likely initiated at the level of ACE induction.

CYP2J2 Targeting to Endothelial Cells Attenuates Adiposity and Vascular Dysfunction in Mice Fed a High-Fat Diet by Reprogramming Adipocyte Phenotype

Obesity is a global epidemic and a common risk factor for endothelial dysfunction and the subsequent development of diabetes mellitus and vascular diseases such as hypertension. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)–derived metabolites of arachidonic acid that contribute to vascular protection by stimulating vasodilation and inhibiting inflammation. Heme oxygenase-1 is a stress response protein that plays an important cytoprotective role against oxidative insult in diabetes mellitus and cardiovascular disease. We recently demonstrated interplay between EETs and heme oxygenase-1 in the attenuation of adipogenesis. We examined whether adipocyte dysfunction in mice fed a high-fat diet could be prevented by endothelial-specific targeting of the human CYP epoxygenase, CYP2J2. Tie2-CYP2J2 transgenic mice, fed a high-fat diet, had a reduction in body weight gain, blood glucose, insulin levels, and inflammatory markers. Tie2-CYP2J2 gene targeting restored HF-mediated decreases in vascular heme oxygenase-1, Cyp2C44, soluble epoxide hydrolase, phosphorylated endothelial nitric oxide synthase, phosphorylated protein kinase B, and phosphorylated adenosine monophosphate protein kinase protein expression, thus improving vascular function. These changes translated into decreased inflammation and oxidative stress within adipose tissue and decreased peroxisome proliferator–activated receptor-&ggr;, CCAAT/enhancer binding protein alpha, mesoderm-specific transcript, and adipocyte 2 expression and increased uncoupling protein 1 and uncoupling protein 2 expression, reflecting the effect of vascular EET overproduction on adipogenesis. The current study documents a direct link between endothelial-specific EET production and adipogenesis, further implicating the EET-heme oxygenase-1 crosstalk as an important cytoprotective mechanism in the amelioration of vascular and adipocyte dysfunction resulting from diet-induced obesity.

EET agonist prevents adiposity and vascular dysfunction in rats fed a high fat diet via a decrease in Bach 1 and an increase in HO-1 levels

Recent reports have shown interplay between EETs (epoxides) and the heme oxygenase (HO) system in attenuating adipogenesis in cell culture models; prompting an examination of the effectiveness of EET agonist on obesity and associated cardio-metabolic dysfunction. Patho-physiological effects of an EET agonist (NUDSA) were contrasted in the absence and in the presence of stannous mesoporphyrin (an HO inhibitor) in SD rats fed a high fat (58%, HF) for 16 weeks. Animals on HF diet exhibited enhanced oxidative stress, increased levels of inflammatory cytokines and decreased levels of adiponectin along with reduced vascular and adipose tissue levels of EETs, HO-1; as compared to control rats (11% dietary fat). Treatment with NUDSA not only reversed serum adiponectin and vascular and adipose tissue levels of EETs and HO-1, but also, decreased blood pressure, subcutaneous and visceral fat content and serum TNFα and IL-6 levels in rats on HF diet. Aortic endothelial function, peNOS expression and adipose tissue markers of energy homeostasis i.e. pAMPK, Sirt1 and FAS, impaired in rats fed a HF diet, were restored in animals treated with this EET agonist. That NUDSA enhanced HO-1 expression, was accompanied by increase in p-GSK-3β and pAKT levels along with attenuation of adipose tissue levels of Bach 1--the transcriptional suppresser of HO-1 expression. Prevention of these beneficial effects of NUDSA, in animals on HF diet and concurrently exposed to NUDSA and SnMP, supports the role of EET-HO interaction in mediating such effects. Taken together, our findings suggest that the EETs stimulate HO-1 expression via suppression of Bach 1 and interplay of these two systems affords vascular and metabolic protection in diet induced obesity.

Endothelial-specific CYP4A2 overexpression leads to renal injury and hypertension via increased production of 20-HETE

We have previously reported that adenoviral-mediated delivery of cytochrome P-450 (CYP) 4A2, which catalyzes the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), results in endothelial dysfunction and hypertension in Sprague-Dawley (SD) rats (Wang JS, Singh H, Zhang F, Ishizuka T, Deng H, Kemp R, Wolin MS, Hintze TH, Abraham NG, Nasjletti A, Laniado-Schwartzman M. Circ Res 98: 962-969, 2006). In this study, we targeted the vascular endothelium by using a lentivirus construct expressing CYP4A2 under the control of the endothelium-specific promoter VE-cadherin (VECAD-4A2) and examined the effect of long-term CYP4A2 overexpression on blood pressure and kidney function in SD rats. A bolus injection of VECAD-4A2 increased blood pressure (P < 0.001) by 26, 36, and 30 mmHg 10, 20, and 30 days postinjection, respectively. Arteries from VECAD-4A2-transduced rats produced increased levels of 20-HETE (P < 0.01), expressed lower levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) (P < 0.05), generated higher levels of superoxide anion, and displayed decreased relaxing responsiveness to acetylcholine (P < 0.05). Proteinuria increased by twofold in VECAD-4A2-transduced rats compared with controls. Treatment of VECAD-4A2-transduced rats with HET0016, an inhibitor of 20-HETE biosynthesis, not only attenuated the increase in blood pressure (P < 0.05) but also improved vascular function (acetylcholine-induced relaxations) and reduced plasma creatinine and proteinuria. HET0016 treatment decreased oxidative stress and increased the phosphorylated state of key proteins that regulate endothelial function, including eNOS, AKT, and AMPK. Collectively, these findings demonstrate that augmentation of vascular endothelial 20-HETE levels results in hypertension, endothelial dysfunction, and renal injury, which is offset by HET0016 through a reduction in vascular 20-HETE coupled with a lessening of oxidative stress and the amplification of pAKT, pAMPK, and p-eNOS levels leading to normalization of endothelial responses.