Cyp2c44 Gene Disruption Exacerbated Pulmonary Hypertension and Heart Failure in Female but Not Male Mice

Epoxyeicosatrienoicacids (EETs), synthesized from arachidonic acid by epoxygenases of the CYP2C and CYP2J gene subfamilies, contribute to hypoxic pulmonary vasoconstriction (HPV) in mice. Despite their roles in HPV, it is controversial whether EETs mediate or ameliorate pulmonary hypertension (PH). A recent study showed that deficiency of Cyp2j did not protect male and female mice from hypoxia-induced PH. Since CYP2C44 is a functionally important epoxygenase, we hypothesized that knockout of the Cyp2c44 gene would protect both sexes of mice from hypoxia-induced PH. We tested this hypothesis in wild-type (WT) and Cyp2c44 knockout (Cyp2c44 (-/-)) mice exposed to normoxia (room air) and hypoxia (10% O2) for 5 weeks. Exposure of WT and Cyp2c44 (-/-) mice to hypoxia resulted in pulmonary vascular remodeling, increased pulmonary artery resistance, and decreased cardiac function in both sexes. However, in female Cyp2c44 (-/-) mice, compared with WT mice, (1) pulmonary artery resistance and right ventricular hypertrophy were greater, (2) cardiac index was lower, (3) left ventricular and arterial stiffness were higher, and (4) plasma aldosterone levels were higher, but (5) there was no difference in levels of EET in lungs and heart. Paradoxically and unexpectedly, we found that Cyp2c44 disruption exacerbated hypoxia-induced PH in female but not male mice. We attribute exacerbated PH in female Cyp2c44 (-/-) mice to elevated aldosterone and as-yet-unknown systemic factors. Therefore, we suggest a role for the human CYP2C genes in protecting women from severe PH and that this could be one of the underlying causes for a better 5-year survival rate in women than in men

Thromboxane A2 is a key regulator of pathogenesis during Trypanosoma cruzi infection

Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A2 (TXA2). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA2 is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA2 accounts for up to 90% of the circulating levels of TXA2 in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA2 receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA2 synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA2 in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA2 may result in novel therapeutic targets for a disease with limited treatment options

Lipid Mediator Informatics and Proteomics in Inflammation-Resolution

Lipid mediator informatics is an emerging area denoted to the identification of bioactive lipid mediators (LMs) and their biosynthetic profiles and pathways. LM informatics and proteomics applied to inflammation, systems tissues research provides a powerful means of uncovering key biomarkers for novel processes in health and disease. By incorporating them with system biology analysis, we review here our initial steps toward elucidating relationships among a range of bimolecular classes and provide an appreciation of their roles and activities in the pathophysiology of disease. LM informatics employing liquid chromatography-ultraviolet-tandem mass spectrometry (LC-UV-MS/MS), gas chromatography-mass spectrometry (GC-MS), computer-based automated systems equipped with databases and novel searching algorithms, and enzyme-linked immunosorbent assay (ELISA) to evaluate and profile temporal and spatial production of mediators combined with proteomics at defined points during experimental inflammation and its resolution enable us to identify novel mediators in resolution. The automated system including databases and searching algorithms is crucial for prompt and accurate analysis of these lipid mediators biosynthesized from precursor polyunsaturated fatty acids such as eicosanoids, resolvins, and neuroprotectins, which play key roles in human physiology and many prevalent diseases, especially those related to inflammation. This review presents detailed protocols used in our lab for LM informatics and proteomics using LC-UV-MS/MS, GC-MS, ELISA, novel databases and searching algorithms, and 2-dimensional gel electrophoresis and LC-nanospray-MS/MS peptide mapping

Lipid Mediator Informatics and Proteomics in Inflammation Resolution

Lipid mediator informatics is an emerging area denoted to the identification of bioactive lipid mediators (LMs) and their biosynthetic profiles and pathways. LM informatics and proteomics applied to inflammation, systems tissues research provides a powerful means of uncovering key biomarkers for novel processes in health and disease. By incorporating them with system biology analysis, we review here our initial steps toward elucidating relationships among a range of bimolecular classes and provide an appreciation of their roles and activities in the pathophysiology of disease. LM informatics employing liquid chromatography-ultraviolet-tandem mass spectrometry (LC-UV-MS/MS), gas chromatography-mass spectrometry (GC-MS), computer-based automated systems equipped with databases and novel searching algorithms, and enzyme-linked immunosorbent assay (ELISA) to evaluate and profile temporal and spatial production of mediators combined with proteomics at defined points during experimental inflammation and its resolution enable us to identify novel mediators in resolution. The automated system including databases and searching algorithms is crucial for prompt and accurate analysis of these lipid mediators biosynthesized from precursor polyunsaturated fatty acids such as eicosanoids, resolvins, and neuroprotectins, which play key roles in human physiology and many prevalent diseases, especially those related to inflammation. This review presents detailed protocols used in our lab for LM informatics and proteomics using LC-UV-MS/MS, GC-MS, ELISA, novel databases and searching algorithms, and 2-dimensional gel electrophoresis and LCnanospray-MS/MS peptide mapping

Lipid Mediator Informatics and Proteomics in Inflammation-Resolution

Lipid mediator informatics is an emerging area denoted to the identification of bioactive lipid mediators (LMs) and their biosynthetic profiles and pathways. LM informatics and proteomics applied to inflammation, systems tissues research provides a powerful means of uncovering key biomarkers for novel processes in health and disease. By incorporating them with system biology analysis, we review here our initial steps toward elucidating relationships among a range of bimolecular classes and provide an appreciation of their roles and activities in the pathophysiology of disease. LM informatics employing liquid chromatography-ultraviolet-tandem mass spectrometry (LC-UV-MS/MS), gas chromatography-mass spectrometry (GC-MS), computer-based automated systems equipped with databases and novel searching algorithms, and enzyme-linked immunosorbent assay (ELISA) to evaluate and profile temporal and spatial production of mediators combined with proteomics at defined points during experimental inflammation and its resolution enable us to identify novel mediators in resolution. The automated system including databases and searching algorithms is crucial for prompt and accurate analysis of these lipid mediators biosynthesized from precursor polyunsaturated fatty acids such as eicosanoids, resolvins, and neuroprotectins, which play key roles in human physiology and many prevalent diseases, especially those related to inflammation. This review presents detailed protocols used in our lab for LM informatics and proteomics using LC-UV-MS/MS, GC-MS, ELISA, novel databases and searching algorithms, and 2-dimensional gel electrophoresis and LC-nanospray-MS/MS peptide mapping

Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators

Atherosclerosis is now recognized as an inflammatory disease involving the vascular wall. Recent results indicate that acute inflammation does not simply passively resolve as previously assumed but is actively terminated by a homeostatic process that is governed by specific lipid-derived mediators initiated by lipoxygenases. Experiments with animals and humans support a proinflammatory role for the 5-lipoxygenase system. In contrast, results from animal experiments show a range of responses with the 12/15-lipoxygenase pathways in atherosclerosis. To date, the only two clinical epidemiology human studies both support an antiatherogenic role for 12/15-lipoxygenase downstream actions. We tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation by analyzing apolipoprotein E-deficient mice with 1) global leukocyte 12/15-lipoxygenase deficiency, 2) normal enzyme expression, or 3) macrophage-specific 12/15-lipoxygenase overexpression. Results from these indicate that 12/15-lipoxygenase expression protects mice against atherosclerosis via its role in the local biosynthesis of lipid mediators, including lipoxin A4, resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.—Merched, A. J., Ko, K., Gotlinger, K. H., Serhan, C. N. Chan, L. Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators

Elevated 20-HETE Impairs Coronary Collateral Growth in Metabolic Syndrome Via Endothelial Dysfunction

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of metabolic syndrome (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished, and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists ((collateral-dependent zone)CZ/(normal zone)NZ flow ratio was 0.76±0.07 in JCR+20-SOLA, 0.84±0.05 in JCR+20-HEDGE vs. 0.11±0.02 in JCR vs. 0.84±0.03 in normal rats). In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, eNOS Ser1179 phosphorylation, eNOS-dependent NO.- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS

20-HETE-Induced Mitochondrial Superoxide Production and Inflammatory Phenotype in Vascular Smooth Muscle Is Prevented by Glucose-6-Phosphate Dehydrogenase Inhibition

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD

Heme Oxygenase (HO-1) Rescue of Adipocyte Dysfunction in HO-2 Deficient Mice via Recruitment of Epoxyeicosatrienoic Acids (EETs) and Adiponectin

Background/Aims: HO-1 and EETs are functionally linked and their interactions influence body weight, insulin sensitivity, and serum levels of inflammatory cytokines in metabolic syndrome phenotype of HO-2 null mice. The HO-2 isozyme is essential for regulating physiological levels of ROS. Recent studies have suggested a potential role of EET in modifying adipocyte differentiation through up-regulation of HO-1-adiponectin-AkT signaling in human mesenchymal stem cells (MSCs). Our aim was to examine the consequences of HO deficiency on MSC-derived adipogenesis in vitro using MSC derived from HO-2 null and WT mice in vivo. Methods: Four-month-old HO-2 null (HO-2-/-) and B6/129SF2/J (WT) mice were divided into three groups (four mice/group): WT, HO-2-/-, and HO-2-/- +CoPP. Adipogenesis was performed on purified MSC-derived adipocytes cultured in adipogenic differentiation media and an EET-agonist was added every 3 days. Results: HO-2 depletion of MSC adipocytes resulted in increased adipogenesis (p\u3c0.01) and increased levels of inflammatory cytokines including (TNF)-alpha (p\u3c0.05), (MCP)-1 (p\u3c0.05), and (IL-1)-beta (p\u3c0.05). These results were accompanied by decreases in HO-1 (p\u3c0.05) and subsequently EET and HO activity (p\u3c0.05). Up-regulation of HO-1 resulted in decreased MSC-derived adipocyte differentiation, decreased production of TNF-alpha and MCP-1 and increased levels of adiponectin (p\u3c0.05). Cyp2J5 (p\u3c0.05), HO-1 (p\u3c0.05), and adiponectin mRNA levels (p\u3c0.05) were also decreased in visceral adipose tissue isolated from HO-2 null compared to WT mice. EET agonist stimulation of MSC adipocytes derived from HO-2 null mice yielded similar results. Conclusion: Increased levels of EET and HO-1 are essential for protection against the adverse effects of adipocyte hypertrophy and the ensuing metabolic syndrome. These results offer a portal into therapeutic approaches for the prevention of the metabolic syndrome