The purpose of the current study was to evaluate the effect of 12/15- lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2^Akita mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.

The ascending prevalence of obesity in recent decades is commonly associated with soaring morbidity and mortality rates, resulting in increased health-care costs and decreased quality of life. A systemic state of stress characterized by low-grade inflammation and pathological formation of reactive oxygen species (ROS) usually manifests in obesity. The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) is the master regulator of the redox homeostasis and plays a critical role in the resolution of inflammation. Here, we show that the natural isothiocyanate and potent NRF2 activator sulforaphane reverses diet-induced obesity through a predominantly, but not exclusively, NRF2-dependent mechanism that requires a functional leptin receptor signaling and hyperleptinemia. Sulforaphane does not reduce the body weight or food intake of lean mice but induces an anorectic response when coadministered with exogenous leptin. Leptin-deficient Lep^ob/ob mice and leptin receptor mutant Lepr^db/db mice display resistance to the weight-reducing effect of sulforaphane, supporting the conclusion that the antiobesity effect of sulforaphane requires functional leptin receptor signaling. Furthermore, our results suggest the skeletal muscle as the most notable site of action of sulforaphane whose peripheral NRF2 action signals to alleviate leptin resistance. Transcriptional profiling of six major metabolically relevant tissues highlights that sulforaphane suppresses fatty acid synthesis while promoting ribosome biogenesis, reducing ROS accumulation, and resolving inflammation, therefore representing a unique transcriptional program that leads to protection from obesity. Our findings argue for clinical evaluation of sulforaphane for weight loss and obesity-associated metabolic disorders.

Type 2 diabetes (T2DM) is a multi-factorial disease with a complex pathogenic mechanism; however a complete understanding of precise biochemical alterations accompanying the onset and progression of T2DM is lacking. Using a combination of untargeted and targeted metabolomic profiling approach we were able to delineate significantly altered metabolites in the diabetic (T2DM) group. Our results indicate significant perturbations in amino acid metabolism, TCA cycle and glycerol-phospholipid metabolism possibly impacting the overall glucose homeostasis in T2DM. A systems approach offers promise towards identification of clinically relevant markers of T2DM and novel molecular targets to foster drug discovery for effective therapeutic development for diabetes.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by an exacerbated fibrotic response. Although molecular and cellular determinants involved in the onset and progression of this devastating disease are largely unknown, an aberrant remodeling of the pulmonary vasculature appears to have implications in IPF pathogenesis. Here, we demonstrated for the first time that an increase of reactive oxygen species (ROS) generation induced by sera from IPF patients drives both collagen type I deposition and proliferation of primary human pulmonary artery smooth muscle cells (HPASMCs). IPF sera‐induced cellular effects were significantly blunted in cells exposed to the NADPH oxidase inhibitor diphenyleneiodonium (DPI) proving the causative role of ROS and suggesting their potential cellular source. Contrary to IPF naive patients, sera from Pirfenidone‐treated IPF patients failed to significantly induce both ROS generation and collagen synthesis in HPASMCs, mechanistically implicating antioxidant properties as the basis for the in vivo effect of this drug.