Description: Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (〉200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3 , located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 ( Tnfaip3 ) gene, is an early-primary response gene controlled by nuclear factor-B (NF-B) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-B-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-B/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-B/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-B-induced lincRNA-Tnfaip3 to act as a coactivator of NF-B for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-B to modulate inflammatory gene transcription in mouse macrophages.

Description: A multitude of evidence suggests that iodinated contrast material causes nephrotoxicity; however, there have been no previous studies that use arterial spin labeling (ASL) blood flow functional magnetic resonance imaging (fMRI) to investigate the alterations in effective renal plasma flow between normointensive and hypertensive rats following injection of contrast media. We hypothesized that FAIR-SSFSE arterial spin labeling MRI may enable noninvasive and quantitative assessment of regional renal blood flow abnormalities and correlate with disease severity as assessed by histological methods. Renal blood flow (RBF) values of the cortex and medulla of rat kidneys were obtained from ASL images postprocessed at ADW4.3 workstation 0.3, 24, 48, and 72 h before and after injection of iodinated contrast media (6 ml/kg). The H&E method for morphometric measurements was used to confirm the MRI findings. The RBF values of the outer medulla were lower than those of the cortex and the inner medulla as reported previously. Iodinated contrast media treatment resulted in decreases in RBF in the outer medulla and cortex in spontaneously hypertensive rats (SHR), but only in the outer medulla in normotensive rats. The iodinated contrast agent significantly decreased the RBF value in the outer medulla and the cortex in SHR compared with normotensive rats after injection of the iodinated contrast media. Histological observations of kidney morphology were also consistent with ASL perfusion changes. These results demonstrate that the RBF value can reflect changes of renal perfusion in the cortex and medulla. ASL-MRI is a feasible and accurate method for evaluating nephrotoxic drugs-induced kidney damage.

Description: Reflux of acid and bile acids contributes to epithelial tissue injury in gastro-esophageal reflux disease. However, the influence of refluxed material on human esophageal stratified epithelial barrier function and tight junction (TJ) proteins has not been fully elucidated. Here, we investigated the influence of acid and bile acids on barrier function and TJ protein distribution using a newly developed air-liquid interface (ALI) in vitro culture model of stratified squamous epithelium based on primary human esophageal epithelial cells (HEECs). Under ALI conditions, HEECs formed distinct epithelial layers on Transwell inserts after 7 days of culture. The epithelial layers formed TJ, and the presence of claudin-1, claudin-4, and occludin were detected by immunofluorescent staining. The NP-40-insoluble fraction of these TJ proteins was significantly higher by day 7 of ALI culture. Exposure of HEECs to pH 2, and taurocholic acid (TCA) and glycocholic acid (GCA) at pH 3, but not pH 4, for 1 h decreased transepithelial electrical resistance (TEER) and increased paracellular permeability. Exposure of cell layers to GCA (pH 3) and TCA (pH 3) for 1 h also markedly reduced the insoluble fractions of claudin-1 and -4. We found that deoxycholic acid (pH 7.4 or 6, 1 h) and pepsin (pH 3, 24 h) significantly decreased TEER and increased permeability. Based on these findings, ALI-cultured HEECs represent a new in vitro model of human esophageal stratified epithelium and are suitable for studying esophageal epithelial barrier functions. Using this model, we demonstrated that acid, bile acids, and pepsin disrupt squamous epithelial barrier function partly by modulating TJ proteins. These results provide new insights into understanding the role of TJ proteins in esophagitis.

Description: The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-B pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-B target genes. Overexpression of NF-B subunits (p50 and p65) and NF-B target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-B pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-B inhibitor, BAY 11-7085 (20 mg·kg –1 ·day –1 ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-B-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-B pathway and impairs esophageal barrier function in mice and that targeting the NF-B pathway may strengthen esophageal barrier function against reflux.

Description: Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca 2+ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C +/– ) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C +/– hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C +/– hearts displayed decreased cross-bridge stiffness at half-maximal Ca 2+ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca 2+ activations (〈15% and 〈25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C +/– and wild-type myocardium. Intact ventricular myocytes from MyBP-C +/– hearts displayed abnormal sarcomere shortening but unchanged Ca 2+ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C +/– hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C +/– mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca 2+ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C +/– mice could increase their susceptibility to cardiac dysfunction and arrhythmia.

Description: MicroRNAs (miRNAs) comprise of a novel class of endogenous small noncoding RNAs that frequently downregulate the expression of target genes. Recent reports suggest that miRNA-200b prevents epithelial-to-mesenchymal transition (EMT) in cancer cells by targeting the E-box binding transcription factors Zinc finger E-box-binding homeobox 1 (ZEB1) and Zinc finger E-box-binding homeobox 2 (ZEB2). About 35% of active fibroblasts are derived from EMT which is central to the development of progressive renal fibrosis. Hence, this study was designed to assess the effect of miRNA-200b on transforming growth factor-β (TGF-β1)-induced fibrotic responses in renal tubular cells. Morphologically, human kidney-2 cells transfected with miRNA-200b retained their epithelial cell characteristics when exposed to TGF-β1. miRNA-200b significantly increased E-cadherin ( P 〈 0.001) and reduced fibronectin mRNA and protein expression (both P 〈 0.01) independent of phospho-Smad2/3 and phospho-p38 and p42/44 signaling. Increased E-cadherin expression was associated with decreased expression of ZEB1 and ZEB2 and repression of fibronectin was mediated through direct targeting of the fibronectin mRNA, demonstrated using pMIR luciferase reporter assay and site-directed mutagenesis. These results suggest that miRNA-200b suppresses TGF-β1-induced EMT via inhibition of ZEB1 and ZEB2 and the extracellular matrix protein fibronectin by directing targeting of its 3'UTR mRNA, independent of pathways directly involved in TGF-β1 signaling.

Description: Intervening in angiotensin (Ang)-II type 2 receptor (AT2) signaling may have therapeutic potential for bronchopulmonary dysplasia (BPD) by attenuating lung inflammation and preventing arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH). We first investigated the role of AT2 inhibition with PD123319 (0.5 and 2 mg·kg –1 ·day –1 ) on the beneficial effect of AT2 agonist LP2–3 (5 μg/kg twice a day) on RVH in newborn rats with hyperoxia-induced BPD. Next we determined the cardiopulmonary effects of PD123319 (0.1 mg·kg –1 ·day –1 ) in two models: early treatment during continuous exposure to hyperoxia for 10 days and late treatment starting on day 6 in rat pups exposed postnatally to hyperoxia for 9 days, followed by a 9-day recovery period in room air. Parameters investigated included lung and heart histopathology, fibrin deposition, vascular leakage, and differential mRNA expression. Ten days of coadministration of LP2–3 and PD123319 abolished the beneficial effects of LP2–3 on RVH in experimental BPD. In the early treatment model PD123319 attenuated cardiopulmonary injury by reducing alveolar septal thickness, pulmonary influx of inflammatory cells, including macrophages and neutrophils, medial wall thickness of small arterioles, and extravascular collagen III deposition, and by preventing RVH. In the late treatment model PD123319 diminished PAH and RVH, demonstrating that PAH is reversible in the neonatal period. At high concentrations PD123319 blocks the beneficial effects of the AT2-agonist LP2–3 on RVH. At low concentrations PD123319 attenuates cardiopulmonary injury by reducing pulmonary inflammation and fibrosis and preventing PAH-induced RVH but does not affect alveolar and vascular development in newborn rats with experimental BPD.

Description: This study aimed to test the hypothesis that the brain of Protopterus annectens expressed l -gulono--lactone oxidase ( gulo /Gulo), the enzyme catalyzing the last step of ascorbate biosynthesis, and could maintain high concentrations of ascorbate during estivation. We cloned and sequenced gulo from the kidney of P. annectens and performed quantitative PCR to determine its mRNA expression in kidney and brain. Gulo activity was assayed and its protein abundance was determined by Western blot using custom-made anti-Gulo antibody. Effects of estivation on concentrations of ascorbate and dehydroascorbate in the kidney and brain were also determined. Both brain and kidney, but not liver, of P. annectens expressed gulo /Gulo. Desiccation induced P. annectens to estivate, and 6 mo of estivation led to drastic decreases in gulo /Gulo expression and ascorbate concentration in the kidney. However, high concentrations of ascorbate and ascorbate + dehydroascorbate were maintained in the brain during estivation, probably resulting from in situ ascorbate synthesis. Control fish were placed in freshwater, where they were fully active in a favorable environment unlike estivation on land. The ability to synthesize ascorbate to ameliorate oxidative stress directly in the brain might contribute to the ability of P. annectens to undergo prolonged estivation on land.—Ching, B., Ong, J. L. Y., Chng, Y. R., Chen, X. L., Wong, W. P., Chew, S. F., Ip, Y. K. l -gulono--lactone oxidase expression and vitamin C synthesis in the brain and kidney of the African lungfish, Protopterus annectens.

Description: Vascular endothelial cell injury is considered to be the major factor inducing vascular complications in metabolic diseases and plays an important role in other organ damage. With diabetic and hyperlipidemic rats and cultured VSMCs, the present study was aimed at investigating whether the early damage of VSMCs during metabolic diseases plays a critical role in vascular dysfunction and the underlying mechanisms and would be a promising treatment target. With diabetic and hyperlipidemic rats and cultured VSMCs, the changes and relationships of vascular relaxation and contractile function to the vital organ damage and the underlying mechanisms were investigated; meanwhile, the protective and preventive effects of lowering blood lipid and glucose and inhibition of diabetes and hyperlipidemia-induced vascular hyperreactivity were observed. Diabetic and hyperlipidemic rats presented hyperreactivity in vascular contractile response in the early stages. Hyperglycemia and hyperlipidemia directly affected the contractile function of VSMCs. Early application of fasudil, a specific antagonist of Rho kinase, significantly alleviated diabetes and hyperlipidemia-induced organ damage by inhibiting vascular hyperreactivity. Diabetes and hyperlipidemia-induced inflammatory response could upregulate the expression of connexins and Rho kinase by selective downregulation of the expression of miR-10a, miR-139b, miR-206, and miR-222. These findings suggest that hyperglucose and lipid may directly impair VSMCs and induce vascular hyperreactivity in the early stages. Metabolic inflammation-induced changes in the miRNA-connexin/Rho kinase regulatory pathway are the main mechanism for vascular hyperreactivity and organ damage. Measures inhibiting vascular hyperreactivity are promising for the prevention of organ damage induced by metabolic diseases.

Description: Lipid metabolic disturbances are related to many diseases, such as obesity, diabetes, and certain cancers. Notably, lipid metabolic disturbances have been reported to be a risk factor for colorectal cancer. Nuclear receptors act as ligand-dependent transcription regulators and play key roles in the regulation of body lipid metabolism and the development of many cancers. Retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor and can regulate several lipid metabolism genes in certain cancers. Herein, we demonstrate that the conditioned medium from adipocytes has a proproliferative and promigratory effect on colorectal cancer cells and enhances angiogenesis in chicken embryonic chorioallantoic membranes. In addition, the conditioned medium leads to a decrease in the expression of RORα and its target genes. Meanwhile, RORα and its target gene expressions are lower in human colorectal tumor tissue compared with control colorectal tissue. Activation of RORα inhibits the effect of conditioned medium on the proliferation and migration of colorectal cancer cells as well as the angiogenesis in chicken embryonic allantoic membranes. In colorectal cancer cells, the putative ligand of RORα, cholesterol sulfate (CS), prevents cell cycle progression at the G 1 /S boundary and concurrently modulates the expression of cell cycle-regulatory genes in colorectal cancer cell. CS inhibits angiogenesis in chicken embryonic chorioallantoic membranes and concurrently decreases the mRNA expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α as well as the secretion of VEGF. In addition, lipogenic gene expression is higher in human colorectal tumor tissue compared with control colorectal tissue. CS inhibits the expression of lipogenic genes in colorectal cancer cells. These results suggest that RORα could represent a direct link between local lipid metabolism of colorectal tissue and colorectal cancer. Therefore, the reduction of the expression of RORα could represent a potential warning sign of colorectal cancer.