13 Tgfβ-Mediated Epithelial-Mesenchymal Transition is a Function of Cellular Redox Reprogramming Adam Franklin Prasanphanich1,2, and Melissa Lambeth Kemp1,2 1Georgia Institute of Technology, 2Emory University Fibrotic diseases of the lung, liver, kidney, eye, and heart arise through pathological disruption of epithelial cell layers. the process of epithelial-mesenchymal transition (EMT) is a driving force behind tissue fibrosis. during EMT, cells of epithelial origin acquire a mesenchymal phenotype, possessing aberrant regulation of the extracellular matrix that leads to fibrosis. the cytokine transforming growth factor beta (TGFβ) is a highly potent inducer of EMT, capable of mediating EMT in vitro on the order of days. Additionally, elevation of reactive oxygen species (ROS) is known to induce EMT. Here we report remodeling of cellular redox status as a necessary mechanism for induction of TGFβ-mediated EMT. A model for EMT consisted of A549 alveolar epithelial cells treated with 200pM TGFβ for 48 hours. EMT was monitored by observations a loss of E-cadherin and an increase in N-cadherin expression. in comparing the gene expression of the precursor epithelial cells with derived mesenchymal cells, a 50-fold upregulation (P<0.01) of the ROS-producing enzyme NADPH oxidase 4 (NOX4) mRNA was observed. Moreover, we observed an EMT-associated 4-fold downregulation (P<0.05) of multiple antioxidant enzymes, such as peroxiredoxin 2, thioredoxin reductase 1, and glutaredoxin. Thus, regulation of the intracellular oxidative environment during phenotypic switching occurs by controlling both ROS production and consumption. TGFβ treatment in the presence of 10 mM N-acetylcysteine (NAC) abrogated the ability of TGFβ to induce EMT as indicated by maintained E-cadherin and N-cadherin expression levels. Together, these findings suggest that TGFβ signaling is capable of reprograming the redox status of the cell, by blunting the antioxidant response and enhancing constitutive ROS production through changes in gene expression, in order for EMT to occur.

do i :10.1016/j.freeradbiomed.2011.10.035

14 Activation of Novel Cytoprotective Mechanisms in Nrf2-null Mice Subjected to Acute Exercise Stress Sellamuthu Subbanna Gounder1, Corey J Miller1, Kamal Sadhaasivam1, Vasanthi R Mutthusamy1, John R Hoidal1, and Rajasekaran N Soorappan1 1University of Utah Background: Skeletal muscle (SM) homoeostasis is maintained by verity of cytoprotective mechanisms. Age associated decline in antioxidant potential and accumulation of reactive oxygen/nitrogen species (ROS/RNS) are primary cause for multiple health problems, including muscular dystrophy/atrophy and sarcopenia. We hypothesize that Nrf2 (Nuclear erythroid 2 p45-related factor 2) independent cytoprotective mechanisms will be activated in Nrf2 null mice upon stress. Here, we investigated the loss-of-function mechanisms for Nrf2-dependent and independent cytoprotective pathways in skeletal muscle (SM). Methods: Wild type (WT) and Nrf2-/- mice (n=6-8/gp./experiment) were subjected to acute exercise stress (AES) and determined ROS generation, redox state and cytoprotective pathways using electron paramagnetic (EPR) spectroscopy, immunoblotting and qPCR analysis. Results: Under basal physiological conditions, disruption of Nrf2 showed significant down regulation of NQO1, catalase and G6PD in Nrf2-/- when compared to WT SM. Surprisingly, Nrf2-/- mice exhibited enhanced antioxidant (Gclm, G6pd and catalase; P<0.05) capacity identical to WT upon AES, suggesting activation

of Nrf2 independent protective mechanisms against oxidative stress. Next, we analyzed pro-survival pathways under basal and stressed states. Under basal condition, total and phospho Akt levels were diminished while p53, a repressor of Akt, was increased in Nrf2-/- when compared to WT. Interestingly, upon AES the Akt and p-Akt levels (>10 fold) were significantly (P<0.001) increased along with profound down regulation of p53 (P<0.01) in Nrf2-/- when compared to WT SM. Further, we found high levels of cytosolic p21 in AES compared to normal Nrf2-/- or WT mice, suggesting Akt mediated cytosolic shuttling of p21, which could prevent apoptotic events. Accordingly, we noticed a decrease of casp-3 and an increase of Bax proteins in AES when compared to normal Nrf2-/- mice SM. Conclusion: Our findings demonstrate the activation of Akt/p21 dependent cell survival and Nrf2-independent antioxidant mechanisms under Nrf2 ablation. Thus, Nrf2 is necessary, but not sufficient, to deregulate the antioxidant mechanisms in the skeletal muscle.

doi :10.1016/j.freeradbiomed.2011.10.036

15 The Role of Endothelial Nox4 in Angiogenesis Lili Chen1, Xiuyun Hou1, Richard Cohen1, and Xiaoyong Tong1 1Boston University Medical Center Vascular endothelial cells (ECs) play important roles in postnatal angiogenesis. Recent studies indicate Nox4 based NADPH oxidase might be one of the key players. This study tested the fundamental roles of endothelial Nox4 in angiogenesis by using the mouse lines to overexpress human Nox4 wild type (EWT) or its dominant negative form P437H (EDN) in endothelium driven by Tie-2 promoter. the blood flow recovery after hindlimb ischemia was enhanced in EWT but was impaired in EDN compared with littermate non-transgenic (NTg) mice. the clinical score also showed that the EDN had the severest hindlimb ischemia, while EWT had the least severity. in aortic ring growth assay, EWT had the most microvessel sprouting, while EDN had the fewest. in isolated heart ECs, EWT had more H2O2 production and EDN had less by amplex red assay. Both VEGF and TGFβ1 stimulated cell proliferation in EWT but failed to do so in EDN. the basal migration rate was increased in EWT compared with NTg, which was further increased by VEGF and TGFβ1 in both groups but not in EDN. the tube formation assay, an in vitro model of angiogenesis, showed that the basal tube formation was increased in EWT but decreased in EDN. Both VEGF and TGFβ1 increased more tube formation in NTg and EWT but not in EDN. 28 days after hindlimb ischemia, the gastrocnemius muscle of the ischemia limb was used for qPCR and western blot analysis. the critical angiogenesis regulating genes Nox2, VEGF-R2, eNOS, MCP1 were upregulated in EWT compared with NTg, while MCP1, Nox2 and TGFβ1 were downregulated in EDN compared with NTg. in human aortic ECs, overexpression Nox4 dramatically increased VEGF-R2, total eNOS, eNOS phosphorylation and TGFβ1 protein levels, while knockdown of Nox4 dramatically decreased TGFβ1 level and slightly decreased eNOS phosphorylation. the mouse lines with overexpression of Nox4 P437H specifically in smooth muscle showed no difference in hindlimb ischemia blood flow recovery compared with their litter-mate control. in summary, the endothelial Nox4 is essential for ischemia-initiated angiogenesis, its regulation on VEGF-R2, Nox2, eNOS and TGFβ1 might be the potential mechanisms.

doi :10.1016/j.freeradbiomed.2011.10.037

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