Hypoxia alters ocular drug transporter expression and activity in rat and calf models: implications for drug delivery.

Chronic hypoxia, a key stimulus for neovascularization, has been implicated in the pathology of proliferative diabetic retinopathy, retinopathy of prematurity, and wet age related macular degeneration. The aim of the present study was to determine the effect of chronic hypoxia on drug transporter mRNA expression and activity in ocular barriers. Sprague-Dawley rats were exposed to hypobaric hypoxia (PB = 380 mmHg) for 6 weeks, and neonatal calves were maintained under hypobaric hypoxia (PB = 445 mmHg) for 2 weeks. Age matched controls for rats, and calves were maintained at ambient altitude and normoxia. The effect of hypoxia on transporter expression was analyzed by qRT-PCR analysis of transporter mRNA expression in hypoxic and control rat choroid-retina. The effect of hypoxia on the activity of PEPT, OCT, ATB(0+), and MCT transporters was evaluated using in vitro transport studies of model transporter substrates across calf cornea and sclera-choroid-RPE (SCRPE). Quantitative gene expression analysis of 84 transporters in rat choroid-retina showed that 29 transporter genes were up regulated or down regulated by ≥1.5-fold in hypoxia. Nine ATP binding cassette (ABC) families of efflux transporters including MRP3, MRP4, MRP5, MRP6, MRP7, Abca17, Abc2, Abc3, and RGD1562128 were up-regulated. For solute carrier family transporters, 11 transporters including SLC10a1, SLC16a3, SLC22a7, SLC22a8, SLC29a1, SLC29a2, SLC2a1, SLC3a2, SLC5a4, SLC7a11, and SLC7a4 were up regulated, while 4 transporters including SLC22a2, SLC22a9, SLC28a1, and SLC7a9 were down-regulated in hypoxia. Of the three aquaporin (Aqp) water channels, Aqp-9 was down-regulated, and Aqp-1 was up-regulated during hypoxia. Gene expression analysis showed down regulation of OCT-1, OCT-2, and ATB(0+) and up regulation of MCT-3 in hypoxic rat choroid-retina, without any effect on the expression of PEPT-1 and PEPT-2. Functional activity assays of PEPT, OCT, ATB(0+), and MCT transporters in calf ocular tissues showed that PEPT, OCT, and ATB(0+) functional activity was down-regulated, whereas MCT functional activity was up-regulated in hypoxic cornea and SCRPE. Gene expression analysis of these transporters in rat tissues was consistent with the functional transport assays except for PEPT transporters. Chronic hypoxia results in significant alterations in the mRNA expression and functional activity of solute transporters in ocular tissues.

Peripheral blood mononuclear cell gene expression in chronic obstructive pulmonary disease.

Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.

Polymorphisms in the SUFU gene are associated with organ injury protection and sepsis severity in patients with Enterobacteriacea bacteremia.

BACKGROUND: Organ injury including acute kidney injury (AKI) and acute lung Injury (ALI) are major contributors to mortality and morbidity in the setting of sepsis. Hedgehog pathway has been recognized as an important mediator in repair of organ injury. There are some clinical predictors associated with the development of organ injury in sepsis; however few host genetic risk factors have been identified and candidate genes for organ injury susceptibility and severity are largely unknown. METHODS: A prospective cohort study in a tertiary care hospital included 250 adult hospitalized patients with Enterobacteriacea bacteremia. We selected a panel of 69 tagging SNPs for genes in the Hedgehog signaling pathway using the TagSNP functionality of the SNPInfo web server and designed a panel on the GoldenGate Veracode genotyping assay (Illumina). We confirmed Illumina data using Taqman allelic discrimination assays. We assessed SNPs in combination with clinical variables for associations with outcomes and organ injury. RESULTS: Significant associations were identified using logistic regression models, controlling for age, race and gender. From the 69 tagging SNPs, 5 SNPs were associated with renal function and 2 with APACHEII score after false discovery rate correction. After multivariate analysis SNPs rs10786691 (p=0.03), rs12414407 (p=0.026), rs10748825 (p=0.01), and rs7078511 (p=0.006), all in the suppressor of fused homolog (SUFU) gene, correlated with renal function. Likewise, SUFU SNPs rs7907760 (p=0.009) and rs10748825 (p=0.029) were associated with APACHEII score. SNPs rs12414407 and rs1078825 are in linkage disequilibrium (LD) with rs2296590, a SNP in the 5'-UTR region that is within a predicted transcription factor bind site for CCAAT-enhancer-binding proteins. In multivariate analyses functional SNP rs2296590 was correlated with renal function (p=0.004) and APACHEII score (p=0.049). CONCLUSIONS: Host susceptibility factors play an important role in sepsis development and sepsis related organ injury. Polymorphisms in the SUFU gene (encoding for a negative regulator of the hedgehog signaling pathway) are associated with protection from Enterobacteriacea bacteremia related organ injury and sepsis severity.

Ozone enhances pulmonary innate immune response to a Toll-like receptor-2 agonist.

Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases Toll-like receptor-4 (TLR4)-mediated responses to subsequent stimulation with LPS. To explore the pulmonary innate immune response to ozone exposure further, we investigated the effects of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Whole-lung lavage (WLL) and lung tissue were harvested from C57BL/6 mice after exposure to ozone or filtered air, followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the WLL, the surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. We demonstrated an increased WLL cell influx, increased IL-6 and chemokine KC (Cxcl1), and decreased macrophage inflammatory protein (MIP)-1α and TNF-α in response to Pam3CYS as a result of ozone pre-exposure. We also observed the increased cell surface expression of TLR4, TLR2, and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in the expression of genes related to injury repair and the cell cycle as a result of ozone alone or in combination with Pam3CYS. Our results extend previous findings with ozone/LPS to other TLR ligands, and suggest that the ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level.