Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice

<div><h3>Background</h3><p>Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types <em>in vivo</em>. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.</p> <h3>Methodology/Principal Findings</h3><p><em>Bcr</em> and <em>abr</em> null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia <em>bcr−/−</em> and <em>abr−/−</em> macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.</p> <h3>Conclusions</h3><p>Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.</p> </div

Hypoxia-induced pulmonary vascular remodeling in <i>bcr−/−</i> and <i>abr−/−</i> mice.

<p><b>A,</b> Hematoxylin and eosin staining on representative lung specimens from <i>bcr−/−, abr−/−</i> and <i>wt</i> mice under normoxia or hypoxia. Note that the walls of the pulmonary arteries of the <i>bcr−/−</i> and <i>abr−/−</i> mice are remarkably thicker than those of the <i>wt</i> mice after hypoxia. Magnification, 200×. <b>B,</b> Quantification of changes in the pulmonary artery walls. Percent wall thickness was determined on H&E stained sections as described in Methods on nine vessels of comparable size per mouse, with 6 mice per genotype per condition. *p<0.05 compared with the same genotype at normoxia. # p<0.05 compared with WT mice in the same exposure condition. Bars, mean ± SD. <b>C,</b> Immunostaining with α-SMA antibodies on pulmonary vessels from representative normoxia or hypoxia-treated mice. <b>D,</b> Quantification of areas for α-SMA-positive cells. Areas of α-SMA-positive cells were calculated using ImageJ software as described in the Materials and Methods.</p

Lack of Abr and Bcr function exacerbates hypoxia-induced leukocyte involvement in PH. A,

<p>Numbers of perivascular leukocytes surrounding terminal arterioles. Data are expressed as total leukocyte counts per high-power field (200× amplification). Bars, mean<u>+</u>SEM. n = 3/group. <b>B,</b> Peritoneal-elicited macrophages exposed to normoxia or hypoxia for 24 hours activated with 1 µM PMA. ROS production was measured by FACS 1 hr later. Controls, cells without PMA activation. Bars, mean<u>+</u>SEM. n  = 5 mice/group. *, p<0.05. MFI, mean fluorescent intensity. <b>C,</b> Vascular leakage. Evans blue was injected when mice had been exposed to normoxia or hypoxia for 3 weeks. Bars, mean<u>+</u>SEM. n = 3–4 mice/group. A and C<b>,</b> *p<0.05 comparison between animals of the same genotype at normoxia. #p<0.05 compared to WT mice exposed to the same condition.</p

Hypoxia-treated <i>bcr−/−</i> and <i>abr−/−</i> mice have higher RVSP and more severe right ventricular hypertrophy. A,

<p>RVSP from <i>bcr−/−, abr−/−</i> and <i>wt</i> mice with exposure to normoxia or hypoxia. <b>B,</b> Ratio of RV/LV+S calculated using the weight of RV, LV+S from the hearts of normoxic and hypoxic <i>bcr−/−, abr−/−</i> and <i>wt</i> mice. *p<0.05 compared to the values of mice with the same genotype at normoxia. # p<0.05 when compared to <i>wt</i> mice in the same exposure condition. Bars represent mean ±SD; n  = 6 mice per group.</p

Loss of Bcr or Abr promotes hypoxia-induced Rac1 activation <i>in vitro</i> and <i>in vivo</i>. A,

<p>Real-time RT-PCR analysis for quantification of <i>rac1</i>, <i>rac2</i> and <i>rac3</i> mRNA in PASMCs. <b>B,</b> Representative gel electrophoresis of RT-PCR products showing absence of <i>rac2</i> in PASMCs. Samples loaded are indicated above the lanes; spleen, positive control; RNA (−), no RNA, negative control. <b>C–F,</b> Assay for Rac1 activation. <b>C–D,</b> Analysis of activation of Rac1 <i>in vivo</i> in the lungs of <i>bcr−/−</i>, <i>abr−/−</i> and <i>wt</i> mice after normoxia or hypoxia exposure. <b>E–F,</b> Analysis of Rac1 activation in PASMC under normoxia or hypoxia. <b>C</b> and <b>E,</b> representative Western blots; <b>D</b> and <b>F,</b> quantification. Three independent samples of each genotype were tested and the entire experiment was repeated independently. To quantitate results, Western blots were scanned and the ratio of GTP-Rac/total Rac was determined (panels <b>D, F</b>). * p<0.05 when compared with the results of the same genotype under normoxia. # p<0.05 when compared with WT exposed to the same condition.</p

German constitutional development in the 19th century

German constitutional development in the 19th century The purpose of my thesis is to analyse the development of the constitutional arrangements and the constitutional institutions of German states and describe the constitutional foundations of the German unification in the 19th century. The thesis is composed of seven chapters. The first chapter consists of two subchapters and describes the constitutional character of the Holy Roman Empire of German Nation at the end of the 18th century and the constitutional nature of its territories. The second chapter is subdivided into five subchapters analysing the development that followed the outbreak of the French revolution, the consequences of the Final Recess of the Imperial Deputation, the downfall of the Holy Roman Empire of German Nation, the Confederation of the Rhine and the constitutionally significant reforms in Prussia at the beginning of the 19th century. Chapter three consists of two large parts. Part one analyses the constitution and institutions of the German Confederation and the way it influenced the constitutions of its member states. One subpart is also concerned with the repressive laws the German Confederation enacted in order to supress the German national and liberal movement. Second part of chapter three is concerned with some liberal..

Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia-5

<p><b>Copyright information:</b></p><p>Taken from "Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia"</p><p>http://www.molecular-cancer.com/content/6/1/67</p><p>Molecular Cancer 2007;6():67-67.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2169263.</p><p></p>diated MEFs and further treated with 5, 50 or 100 μM AG490 () or 20 nM nilotinib (), respectively. In () and (), in addition to nilotinib, treatment with 5, 50 or 75 μM AG490 was initiated as indicated by an arrow. As a control for the effect of AG490, single treatment with DMSO only () or 20 nM nilotinib () was analyzed, respectively. Fresh drug was added (depending on proliferation of the leukemia cells) every second or third day with the change of medium. Viability was assessed by propidium iodide uptake using a FACScan. Each point represents the average of triplicate values ± SEM

Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia-6

<p><b>Copyright information:</b></p><p>Taken from "Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia"</p><p>http://www.molecular-cancer.com/content/6/1/67</p><p>Molecular Cancer 2007;6():67-67.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2169263.</p><p></p>sue culture plates in the presence of E14.5 irradiated MEFs and cultured for 3 days. All cultures were simultaneously treated with the indicated concentrations of nilotinib or imatinib. Viability is defined as the percentage of viable cells/of the total number of cells. Each point represents mean of triplicate values ± standard error of the mean

Evaluation of selumetinib and trametinib Mek1/2 inhibitors as mono-treatment on BCP-ALL cell viability in the presence and absence of stroma.

<p>The indicated ALLs were treated with different μM concentrations of selumetinib or trametinib for 72 hours in the presence (A) or absence (B) of irradiated OP9 stromal support. Note: “0” values were determined on one sample set but are shown twice, for selumetinib and trametinib, for clarity. (C) 72-hour treatment of primary LAX56 with selumetinib as indicated. Viability (left panels) and cell counts (right panels) were determined by Trypan Blue exclusion. Error bars, SD of values measured in triplicate wells. One of two experiments for US7 and TXL2 with similar results. *p<0.05 **p<0.01, one-way ANOVA.</p

Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia-0

<p><b>Copyright information:</b></p><p>Taken from "Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia"</p><p>http://www.molecular-cancer.com/content/6/1/67</p><p>Molecular Cancer 2007;6():67-67.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2169263.</p><p></p>sue culture plates in the presence of E14.5 irradiated MEFs and cultured for 3 days. All cultures were simultaneously treated with the indicated concentrations of nilotinib or imatinib. Viability is defined as the percentage of viable cells/of the total number of cells. Each point represents mean of triplicate values ± standard error of the mean