Real-time imaging of asthmatic epithelial cells identifies migratory deficiencies under type-2 conditions.

BACKGROUND: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear. OBJECTIVES: This study sought to develop and utilize primary multilayered polarized epithelial cell systems, derived from patients with asthma, to evaluate cell migration in response to wounding under type-2 and untreated conditions. METHODS: A novel wounding device for multilayered polarized cells, along with time-lapse live cell/real-time confocal imaging were evaluated under IL-13 and untreated conditions. The influence of inhibition of 15 lipoxygenase (15LO1), a type-2 enzyme, on the process was also addressed. Cell migration patterns were analyzed by high-dimensional frequency modulated Möbius for statistical comparisons. RESULTS: IL-13 stimulation negatively impacts wound healing by altering the total speed, directionality, and acceleration of individual cells. Inhibition 15LO1 partially improved the wound repair through improving total speed. CONCLUSIONS: Migration abnormalities contributed to markedly slower wound closure of IL-13 treated cells, which was modestly reversed by 15LO1 inhibition, suggesting its potential as an asthma therapeutic target. These novel methodologies offer new ways to dynamically study cell movements and identify contributing pathologic processes.

Characterization of Mice Ubiquitously Overexpressing Human 15-Lipoxygenase-1: Effect of Diabetes on Peripheral Neuropathy and Treatment with Menhaden Oil.

To rigorously explore the role of omega-3 polyunsaturated fatty acids (PUFA) in the treatment of diabetic peripheral neuropathy (DPN), we have created a transgenic mouse utilizing a Cre-lox promoter to control overexpression of human 15-lipoxygenase-1 (15-LOX-1). In this study, we sought to determine the effect of treating type 2 diabetic wild-type mice and transgenic mice ubiquitously overexpressing 15-LOX-1 with menhaden oil on endpoints related to DPN. Wild-type and transgenic mice on a C57Bl/6J background were divided into three groups. Two of each of these groups were used to create a high-fat diet/streptozotocin model for type 2 diabetes. The remaining mice were control groups. Four weeks later, one set of diabetic mice from each group was treated with menhaden oil for twelve weeks and then evaluated using DPN-related endpoints. Studies were also performed using dorsal root ganglion neurons isolated from wild-type and transgenic mice. Wild-type and transgenic diabetic mice developed DPN as determined by slowing of nerve conduction velocity, decreased sensory nerve fibers in the skin and cornea, and impairment of thermal and mechanical sensitivity of the hindpaw compared to their respective control mice. Although not significant, there was a trend for the severity of these DPN-related deficits to be less in the diabetic transgenic mice compared to the diabetic wild-type mice. Treating diabetic wild-type and transgenic mice with menhaden oil improved the DPN-related endpoints with a trend for greater improvement or protection by menhaden oil observed in the diabetic transgenic mice. Treating dorsal root ganglion neurons with docosahexanoic acid but not eicosapentaenoic acid significantly increased neurite outgrowth with greater efficacy observed with neurons isolated from transgenic mice. Targeting pathways that will increase the production of the anti-inflammatory metabolites of omega-3 PUFA may be an efficacious approach to developing an effective treatment for DPN.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

Cytochrome P450 1A1 enhances Arginase-1 expression, which reduces LPS-induced mouse peritonitis by targeting JAK1/STAT6.

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

Specific overexpression of 15-lipoxygenase in endothelial cells promotes cancer cell death in an in vivo Lewis lung carcinoma mouse model.

PURPOSE: Lipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. MATERIALS/METHODS: We used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. RESULTS: When LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. CONCLUSIONS: Taken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.

12(S)-hydroxyeicosatetraenoic acid impairs vascular endothelial permeability by altering adherens junction phosphorylation levels and affecting the binding and dissociation of its components in high glucose-induced vascular injury.

AIMS/INTRODUCTION: Diabetes is an important risk factor for atherosclerotic disease. The initiating factor of atherosclerosis is local endothelial cell injury. The arachidonic acid metabolite, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), might be involved in this process. In recent years, some studies have discussed the effect of 12(S)-HETE on vascular endothelial cell function. In the present study, we investigated the effect of 12(S)-HETE on vascular endothelial cell function in high-glucose conditions and the mechanisms involved. MATERIALS AND METHODS: Human umbilical vein endothelial cells were cultured in conventional M199 medium and high-glucose M199 medium. Human umbilical vein endothelial cells were stimulated with 12(S)-HETE and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (a 12/15-lipoxygenases inhibitor). A type 1 diabetes mellitus model was established in C57BL/6 or 12/15-lipoxygenases knockout mice with streptozotocin. Aortic tissue was harvested for subsequent testing. The transmembrane transport of dextran and human acute monocytic leukaemia cell line (THP-1) cells was measured. The adherens junction protein, IkBα, nuclear factor kappa Bp65 (P65), intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression and phosphorylation, and the binding/dissociation of endothelial cell components were observed. RESULTS: Transendothelial migration of dextran and THP-1 cells was significantly increased by stimulation of human umbilical vein endothelial cell monolayers with high glucose and 12(S)-HETE (P < 0.05). High glucose and 12(S)-HETE altered the vascular endothelial cadherin and ß-catenin phosphorylation level, and promoted the dissociation of ß-catenin and vascular endothelial cadherin. Expression levels of P-Ikbα, P-P65, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 were elevated in high glucose and 12(S)-HETE treated cells and diabetic mice compared with controls (P < 0.05). CONCLUSIONS: The lipoxygenases metabolite, 12(S)-HETE, can impair vascular endothelial permeability by altering adherens junction phosphorylation levels, and affecting the binding and dissociation of its components in high-glucose conditions.

Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice.

15-lipoxygenase is involved in the generation of specialized pro-resolving lipid mediators that play essential roles in resolution and inflammatory responses. Here, we investigated anti-inflammatory role of Alox15 in skin homeostasis. We demonstrated that knockout (KO) of Alox15 led to hair loss and disrupted the structural integrity of the dorsal skin. Alox15 KO resulted in loss of hair follicle stem cells and abnormal transition of dermal adipocytes into fibroblasts. Alox15 deficiency increased infiltration of proinflammatory macrophages and upregulated proinflammatory and necroptotic signaling in dermal adipose tissue in the dorsal skin. Lipidomic analysis revealed severe loss of resolvin D2 in the dorsal skin of Alox15 KO mice compared to wild type controls. Treatment with resolvin D2 reduced skin inflammation in Alox15 KO mice. Collectively, these results indicate that Alox15-mediated production of resolvin D2 is required to maintain skin integrity by suppressing dermal inflammation.

12/15-Lipoxygenase Inhibition Reverses Cognitive Impairment, Brain Amyloidosis, and Tau Pathology by Stimulating Autophagy in Aged Triple Transgenic Mice.

BACKGROUND: The 12/15-lipoxygenase (12/15-LO) enzyme is upregulated in the brains of patients with Alzheimer's disease (AD), and its expression levels influence the onset of the AD-like phenotype in mouse models. However, whether targeting this pathway after the neuropathology and behavioral impairments have been established remains to be investigated. METHODS: Triple transgenic (3xTg) mice received either PD146176-a selective and specific pharmacological inhibitor of 12/15-LO-or placebo starting at 12 months of age for 12 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. RESULTS: At the end of the study, mice in the control group showed a worsening of memory and learning abilities, whereas mice receiving PD146176 were undistinguishable from wild-type mice. The same group also had significantly lower amyloid beta levels and deposition, less tau neuropathology, increased synaptic integrity, and autophagy activation. Ex vivo and in vitro genetic and pharmacological studies found that the mechanism involved in these effects was the activation of neuronal autophagy. CONCLUSIONS: Our findings provide new insights into the disease-modifying action of 12/15-LO pharmacological inhibition and establish it as a viable therapeutic approach for patients with AD.

The role of 15-lipoxygenase-1 expression and its potential role in the pathogenesis of endometrial hyperplasia and endometrial adenocarcinomas.

PURPOSE OF INVESTIGATION: To investigate the presence of 15-lipoxygenase-1 (15-LOX-1) expression and its potential role in the pathogenesis of endometrial hyperplasia and endometrial adenocarcinomas. MATERIALS AND METHODS: The authors investigated the presence of 15-LOX-1 expression in samples from patients diagnosed with normal endometrium (n = 12), endometrial hyperplasia (n = 12), and endometrial cancer (n = 12). The immunohistochemical stainings were scored by three independent pathologists. A Western blot of 15- LOX-1 determined the presence of protein expression in normal endometrium. A Kolmogorov-Smirnov test was used to evaluate the data's distribution pattern. For pairwise comparisons of the combined scores between groups, the Mann-Whitney U test was used. RESULTS: Based on the combined scores for 15-LOX-1 expression, strong immunochemistry staining was observed in samples diagnosed with normal endometrium. There was a significant difference in 15-LOX-1 expression between normal endometrium and endometrial adenocarcinoma (p = 0.03). Comparing tissues from normal endometrium and endometrial hyperplasia, there was a decline in the expression from normal endometrium to endometrial hyperplasia. However, the difference was not statistically significant. CONCLUSION: The present results show that a decrease of 15-LOX-1 expression in the endometrial tumorigenesis process, starting from normal endometrium to hyperplasia and endometrial cancer, might be a trigger. Further studies are required to determine its potential use as a marker in a larger randomized multicenter study.

A role for 12/15-lipoxygenase-derived proresolving mediators in postoperative ileus: protectin DX-regulated neutrophil extravasation.

Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1(+)/Ly6C(+) infiltrating monocytes rather than Ly6G(+) neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G(+)/Ly6C(+) neutrophil extravasation after protectin DX treatment, whereas Ly6G(-)/Ly6C(+) monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.

15-Lipoxygenases regulate the production of chemokines in human lung macrophages.

BACKGROUND AND PURPOSE: 15-Lipoxygenase (15-LOX) activity is associated with inflammation and immune regulation. The objectives of the present study were to investigate the expression of 15-LOX-1 and 15-LOX-2 and evaluate the enzymes' roles in the polarization of human lung macrophages (LMs) in response to LPS and Th2 cytokines (IL-4/-13). EXPERIMENTAL APPROACH: LMs were isolated from patients undergoing surgery for carcinoma. The cells were cultured with a 15-LOX inhibitor (PD146176 or ML351), a COX inhibitor (indomethacin), a 5-LOX inhibitor (MK886) or vehicle and then stimulated with LPS (10 ng · mL(-1)), IL-4 (10 ng · mL(-1)) or IL-13 (50 ng · mL(-1)) for 24 h. Levels of ALOX15 (15-LOX-1) and ALOX15B (15-LOX-2) transcripts were determined by real-time quantitative PCR. Immunoassays were used to measure levels of LPS-induced cytokines (TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL10) and Th2 cytokine-induced chemokines (CCL13, CCL18 and CCL22) in the culture supernatant. KEY RESULTS: Stimulation of LMs with LPS was associated with increased expression of ALOX15B, whereas stimulation with IL-4/IL-13 induced the expression of ALOX15. PD146176 and ML351 (10 µM) reduced the release of the chemokines induced by LPS and Th2 cytokines. The effects of these 15-LOX inhibitors were maintained in the presence of indomethacin and MK886. Furthermore, indomethacin revealed the inhibitory effect of PD146176 on TNF-α release. CONCLUSIONS AND IMPLICATIONS: Inhibition of the 15-LOX pathways is involved in the down-regulation of the in vitro production of chemokines in LMs. Our results suggest that the 15-LOX pathways have a role in the pathogenesis of inflammatory lung disorders and may thus constitute a potential drug target.

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival.

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.

12/15-lipoxygenase during the regulation of inflammation, immunity, and self-tolerance.

12/15-Lipoxygenase (12/15-LO) catalyzes the oxidation of free and esterified fatty acids thereby generating a whole spectrum of bioactive lipid mediators. This enzyme is involved in the regulation of various homeostatic processes as well as in the pathogenesis of multiple diseases. During the innate and adaptive immune response, 12/15-LO and its products exert both pro- and anti-inflammatory effects. Likewise, this enzyme has been implicated in the pathogenesis of autoimmune disease as well as in the maintenance of self-tolerance. This review will summarize our current knowledge about the role of 12/15-LO and will try to examine the two faces of this enzyme within the context of inflammation and immunity.

Transcriptional regulation of ß-secretase-1 by 12/15-lipoxygenase results in enhanced amyloidogenesis and cognitive impairments.

OBJECTIVE: 12/15-Lipoxygenase (12/15-LO) is an enzyme widely distributed in the central nervous system, and it has been involved in the neurobiology of Alzheimer disease (AD). However, the mechanism involved remains elusive. METHODS: We investigated the molecular mechanism by which 12/15-LO regulates amyloid ß (Aß)/Aß precursor protein (APP) metabolism in vivo and in vitro by genetic and pharmacologic approaches. RESULTS: Here we show that overexpression of 12/15-LO leads to increased levels of ß-secretase-1 (BACE1) mRNA and protein, a significant elevation in Aß levels and deposition, and a worsening of memory deficits in AD transgenic mice. In vitro and in vivo studies demonstrate that 12/15-LO regulates BACE1 mRNA expression levels via the activation of the transcription factor Sp1. Thus, 12/15-LO-overexpressing mice had elevated levels of Sp1 and BACE1, whereas 12/15-LO-deficient mice had reduced levels of both. Preventing Sp1 activation by pharmacologic inhibition or dominant-negative mutant blocks the 12/15-LO-dependent elevation of Aß and BACE1 levels. INTERPRETATION: Our findings demonstrate a novel pathway by which 12/15-LO increases the amyloidogenic processing of APP through a Sp1-mediated transcriptional control of BACE1 levels that could have implications for AD pathogenesis and therapy.

Adipose tissue-specific deletion of 12/15-lipoxygenase protects mice from the consequences of a high-fat diet.

Type 2 diabetes is associated with obesity, insulin resistance, and inflammation in adipose tissue. 12/15-Lipoxygenase (12/15-LO) generates proinflammatory lipid mediators, which induce inflammation in adipose tissue. Therefore we investigated the role of 12/15-LO activity in mouse white adipose tissue in promoting obesity-induced local and systemic inflammatory consequences. We generated a mouse model for fat-specific deletion of 12/15-LO, aP2-Cre; 12/15-LO(loxP/loxP), which we call ad-12/15-LO mice, and placed wild-type controls and ad-12/15-LO mice on a high-fat diet for 16 weeks and examined obesity-induced inflammation and insulin resistance. High-fat diet-fed ad-12/15-LO exhibited improved fasting glucose levels and glucose metabolism, and epididymal adipose tissue from these mice exhibited reduced inflammation and macrophage infiltration compared to wild-type mice. Furthermore, fat-specific deletion of 12/15-LO led to decreased peripheral pancreatic islet inflammation with enlarged pancreatic islets when mice were fed the high-fat diet compared to wild-type mice. These results suggest an interesting crosstalk between 12/15-LO expression in adipose tissue and inflammation in pancreatic islets. Therefore, deletion of 12/15-LO in adipose tissue can offer local and systemic protection from obesity-induced consequences, and blocking 12/15-LO activity in adipose tissue may be a novel therapeutic target in the treatment of type 2 diabetes.

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling.

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

Lack of association between aspirin-triggered 15-hydroxyeicosatetraenoic acid release and mast cell/eosinophil activation in nasal polyps from aspirin-sensitive patients.

BACKGROUND: The mechanism of aspirin sensitivity in patients with asthma and rhinosinusitis has been attributed to arachidonic acid metabolism abnormalities. OBJECTIVE: We aimed to test whether aspirin-triggered generation of 15-hydroxyeicosatetraenoic acid (15-HETE) in nasal polyp dispersed cells (NPDCs) from aspirin-sensitive patients is associated with activation of inflammatory cells. METHODS: Polyps were obtained from 11 aspirin-sensitive and 19 aspirin-tolerant patients with chronic rhinosinusitis. NPDCs were stimulated by aspirin or calcium ionophore. Levels of 15-HETE, leukotriene (LT) C4, eosinophil cationic protein (ECP), and tryptase were measured in NPDC supernatant. RESULTS: NPDCs from aspirin-sensitive patients contained more eosinophils (14% vs 9%, P < .05) and released 2.4-fold more ECP (P < .01) at baseline. Stimulation with aspirin (200 microM) resulted in a significant increase in 15-HETE generation only in tissue from aspirin-sensitive patients (mean increase, 82%) but did not induce any increase in the release of LTC4, ECP, or tryptase. Preincubation with calcium ionophore resulted in significantly enhanced generation of 15-HETE, ECP, tryptase, and LTC4 in patients from both groups. Incubation of NPDCs with misoprostol inhibited aspirin-induced 15-HETE generation in aspirin-sensitive patients and calcium ionophore-induced 15-HETE, ECP, and tryptase release in both aspirin-sensitive and aspirin-tolerant patients. CONCLUSION: Our study demonstrated that aspirin-induced 15-HETE generation in nasal polyps from aspirin-sensitive patients is not associated with activation of mast cells and eosinophils. Misoprostol has a potent inhibitory effect on the activation of cells derived from the site of nasal mucosal inflammation, regardless of sensitivity to aspirin.

Disruption of the 12/15-lipoxygenase gene (Alox15) protects hyperlipidemic mice from nonalcoholic fatty liver disease.

UNLABELLED: We have shown that Alox15, the gene encoding for 12/15-lipoxygenase (12/15-LO), is markedly up-regulated in livers from apolipoprotein E-deficient (ApoE(-/-)) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE(-/-) mice with a targeted disruption of the Alox15 gene to assess the role of 12/15-LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE(-/-) mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15-LO double-knockout (ApoE(-/-)/12/15-LO(-/-)) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-18, and IL-6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high-fat diet-induced insulin resistance, up-regulated insulin receptor substrate-2, and exerted opposite effects on hepatic c-Jun amino-terminal kinase and adenosine monophosphate-activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin-resistant adipokines MCP-1, TNFα, and resistin while increasing the expression of glucose transporter-4. Interestingly, compared with ApoE(-/-) mice, which exhibited increased hepatic caspase-3 staining, ApoE(-/-)/12/15-LO(-/-) mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE(-/-) mice were more vulnerable to TNFα-induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. CONCLUSION: Collectively, our data suggest a potentially relevant mechanism linking 12/15-LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin.

White tea extract induces apoptosis in non-small cell lung cancer cells: the role of peroxisome proliferator-activated receptor-{gamma} and 15-lipoxygenases.

Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non-small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-gamma with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-gamma activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-gamma activation and upregulation of PPAR-gamma mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non-small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-gamma and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer.

Resolution, the grail for healthy ocular inflammation.

Acute inflammation is a frequent, essential and beneficial response to maintain normal tissue function. PMN are the primary effector cells of acute inflammatory responses and their timely resolution by macrophages from an injured, stressed or infected tissues are required for the successful execution of this routine tissue response. Dysregulation of this fundamental program is a major factor in the global disease burden and contributes to many ocular diseases. Counter-regulatory signals are critical to the controlled activation of innate and adaptive immune responses in the eye and recent studies have identified two circuits in the cornea, uvea and/or retina, namely 15-lipoxygenase and heme-oxygenase, which control inflammation, promote resolution of PMN and afford neuroprotection. The role of these counter-regulator and pro-resolution circuits may provide insight into ocular inflammatory diseases and opportunities to restore stressed ocular tissue to a pre-inflammatory state, namely homeostasis, rather than limiting therapeutic options to palliative inhibition of pro-inflammatory circuits.