The discovery of metabolite-phenotype associations may highlight candidate biomarkers and metabolic pathways altered in disease states. We sought to identify novel metabolites associated with obesity and one of its major complications, nonalcoholic fatty liver disease (NAFLD), using a liquid chromatography-tandem mass spectrometry method. In 997 individuals in Framingham Heart Study Generation 3 (FHS Gen 3), we identified an association between anandamide (AEA) and BMI. Further examination revealed that AEA was associated with radiographic hepatic steatosis. In a histologically defined NAFLD cohort, AEA was associated with NAFLD severity, the presence of nonalcoholic steatohepatitis, and fibrosis. These data highlight AEA as a marker linking cardiometabolic disease and NAFLD severity.

Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning.

Virtually all organisms seek to maximize fitness by matching fuel availability with energy expenditure. In vertebrates, glucose homeostasis is central to this process, with glucose levels finely tuned to match changing energy requirements. To discover new pathways regulating glucose levels in vivo, we performed a large-scale chemical screen in live zebrafish and identified the small molecule alexidine as a potent glucose-lowering agent. We found that alexidine inhibits the PTEN-like mitochondrial phosphatase PTPMT1 and that other pharmacological and genetic means of inactivating PTPMT1 also decrease glucose levels in zebrafish. Mutation of ptpmt1 eliminates the effect of alexidine, further confirming it as the glucose-lowering target of alexidine. We then identified succinate dehydrogenase (SDH) as a substrate of PTPMT1. Inactivation of PTPMT1 causes hyperphosphorylation and activation of SDH, providing a possible mechanism by which PTPMT1 coordinates glucose homeostasis. Therefore, PTPMT1 appears to be an important regulator of SDH phosphorylation status and glucose concentration.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

Spectrin α2 (αII-spectrin) is a scaffolding protein encoded by the Spna2 gene and constitutively expressed in most tissues. Exon trapping of Spna2 in C57BL/6 mice allowed targeted disruption of αII-spectrin. Heterozygous animals displayed no phenotype by 2 years of age. Homozygous deletion of Spna2 was embryonic lethal at embryonic day 12.5 to 16.5 with retarded intrauterine growth, and craniofacial, neural tube and cardiac anomalies. The loss of αII-spectrin did not alter the levels of αI- or βI-spectrin, or the transcriptional levels of any β-spectrin or any ankyrin, but secondarily reduced by about 80% the steady state protein levels of βII- and βIII-spectrin. Residual βII- and βIII-spectrin and ankyrins B and G were concentrated at the apical membrane of bronchial and renal epithelial cells, without impacting cell morphology. Neuroepithelial cells in the developing brain were more concentrated and more proliferative in the ventricular zone than normal; axon formation was also impaired. Embryonic fibroblasts cultured on fibronectin from E14.5 (Spna2(-/-)) animals displayed impaired growth and spreading, a spiky morphology, and sparse lamellipodia without cortical actin. These data indicate that the spectrin-ankyrin scaffold is crucial in vertebrates for cell spreading, tissue patterning and organ development, particularly in the developing brain and heart, but is not required for cell viability.

BACKGROUND: Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs). However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies. METHODOLOGY/PRINCIPAL FINDINGS: Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry (WNT16, ST14, Pcsk1, Jumonji, Morca2a, TRPC5, and others) were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins (WNT16, ADAM15 and NOGO-A/B) was demonstrated in an ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs. CONCLUSIONS/SIGNIFICANCE: The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin-independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of alphavbeta3 integrin receptor and resulted in decreased alphavbeta3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT.

Nitric oxide (NO) participates in a diverse array of biological functions in mammalian organ systems. Depending on the biochemical environment, the production of NO may result in cytoprotection or cytotoxicity. The paradoxical actions of NO arise from the complexities generated by the redox milieu, NO concentration/bioavailability, and tissue/cell context, which ultimately result in the wide range of regulatory roles observed. Additionally, in physiological versus pathological states, NO often displays diametrically opposing affects in several organ systems. Here, we will discuss the roles of NO during reproduction, organ system development, in particular, the cardiovascular system, and its potential implications in diabetes-induced fetal defects.

OBJECTIVES: The study of isolated microvascular endothelial cells from mice has long been impeded due to the many difficulties encountered in isolating and culturing these cells. We focused on developing a method to isolate microvascular endothelial cells from the skin fragments of newborn mice. We also aimed at establishing optimal culture conditions to sustain the growth of these cells. METHODS AND RESULTS: Isolation of murine dermal microvascular endothelial cells (mDMEC) from P3 newborn mice was based first on enzymatic separation of the skin epidermal layer from the dermis using dispase and then on disaggregating dermal cellular elements using collagenase. The cells obtained from the dermis were subjected to a continuous density gradient centrifugation. Cells situated between densities 1.033 and 1.047 were then cultured on collagen IV-coated culture flasks using optimized growth culture conditions. Cells were characterized by endothelial appearance and by the presence and genetic expression of endothelial markers like CD31, NOS3, VEGFR-2 and Tie-2. Uptake of acetylated low-density lipoprotein (Ac-LDL) was used as a functional assay. CONCLUSIONS: The methodology described herein for isolation and culture of murine microvascular endothelium offers a distinctive advantage for those using mouse models to study endothelial cell biology.

The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-gamma and IFN-alpha treatments revealed dramatic changes in binding locations between the two treatments. The IFN-alpha induction revealed nonconserved STAT1 occupancy at IFN-gamma-induced sites, as well as novel sites of STAT1 binding not evident in IFN-gamma-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states.