sj-docx-1-jet-10.1177_15266028241232915 – Supplemental material for Long-Term Outcomes of Endovascular Repair Within and Outside the Instructions for Use in Korean Patients With Abdominal Aortic Aneurysm

Supplemental material, sj-docx-1-jet-10.1177_15266028241232915 for Long-Term Outcomes of Endovascular Repair Within and Outside the Instructions for Use in Korean Patients With Abdominal Aortic Aneurysm by Joonpyo Lee, Pyung Chun Oh, Albert Youngwoo Jang, Chul-Min Ahn, Donghoon Choi, Young-Guk Ko and Woong Chol Kang in Journal of Endovascular Therapy</p

Therapeutic application of rPOA in rat MI model with VEGF expression.

<p>(A) VEGF expression in the rat myocardium 1 week after the injection of PEI/pVEGF or rPOA/pVEGF complexes, as visualized by anti-VEGF antibody labeling (200×). Red arrows indicated brown staining of VEGF expression. (B) Quantitative analysis of VEGF immunoreactivity per mm² tissue. Average values were obtained from five random high magnification fields in the infarct border zone from each animal. Data represent mean ± SD. *P < 0.05.</p

Histological analysis of gene therapy-treated MI hearts.

<p>(A) Representative picture of myocardial sections stained with 2,3,5-triphenyltetrazolium chloride (TTC). Pale yellow indicates infarct region. (B) Ratio of infarcted to non-infarcted left ventricular myocardium (%) from LAD-ligated rats injected with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.0005, **P < 0.00001 vs. saline. (C) Representative myocardium sections stained with Masson’s trichrome (lower panel, 200×). Scale bar, 200 μm. (D) Percent of myocardial collagen fibrosis expressed as the ratio of fibrotic area to left ventricle area in LAD-ligated rats injected with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.005, **P < 0.00001. (E) Myocardial sections labeled with an antibody against CD31 to detect neovascularization 1 week after injection (400×). Scale bar, 100 μm. (F) Quantitative analysis of CD31-positive cells in ischemic myocardia treated with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.05, **P < 0.005.</p

<i>In vitro</i> transfection efficiency and cytotoxicity of rPOA as a gene carrier.

<p>(A) Optimization of pCMV-Luc transfection into H9c2 cardiomyocytes using three different weight ratios (1:1, 1:2, and 1:4) of rPOA and PEI25K (1:1) as a positive control. Transfection efficiencies were measured by luciferase assay. Data are expressed as the mean ± SD of three experiments. *P < 0.001 vs. PEI. (B) Green fluorescence protein (GFP) expression in rPOA-transfected H9c2 cardiomyocytes. pEGFP-C1 plasmids were mixed with indicated carrier and ratio. A 465–495 nm excitation filter was used to detect GFP expression. (C) GFP-expressing cells were analyzed by flow cytometry to determine the percentage of GFP-expressing cells. (D) Cytotoxicity of rPOA vs. PEI in H9c2 cells. Polymer/pDNA complexes were transfected into H9c2 cells, and cell viability was measured 24 h later by MTT assay. Data are expressed as the mean ± SD. *P < 0.005 vs. control and PEI transfections.</p

<i>In vivo</i> transfection efficiency and cytotoxicity of rPOA as a gene carrier.

<p>(A) Representative cardiac tissue sections with H&E staining and GFP fluorescence analysis 3 days after injection of pEGFP-C1, PEI/GFP, or rPOA/GFP into rat hearts (400× magnification). Scale bar, 100 μm. (B) Quantitative analysis of GFP expression across different groups. *P < 0.005, **P < 0.001 vs. control. (C) H&E staining of saline-, PEI-, or rPOA-injected rat myocardial tissue. (D) Quantitative analysis of nuclear stained cells as a measure of left ventricular cellular infiltration post injection with saline (n = 4), PEI (n = 5), or rPOA (n = 6). **P < 0.001 between groups.</p

Insulin Protects Cardiac Myocytes from Doxorubicin Toxicity by Sp1-Mediated Transactivation of Survivin

<div><p>Insulin inhibits ischemia/reperfusion-induced myocardial apoptosis through the PI3K/Akt/mTOR pathway. Survivin is a key regulator of anti-apoptosis against doxorubicin-induced cardiotoxicity. Insulin increases survivin expression in cardiac myocytes to mediate cytoprotection. However, the mechanism by which survivin mediates the protective effect of insulin against doxorubicin-associated injury remains to be determined. In this study, we demonstrated that pretreatment of H9c2 cardiac myocytes with insulin resulted in a significant decrease in doxorubicin-induced apoptotic cell death by reducing cytochrome c release and caspase-3 activation. Doxorubicin-induced reduction of survivin mRNA and protein levels was also significantly perturbed by insulin pretreatment. Reducing survivin expression with survivin siRNA abrogated insulin-mediated inhibition of caspase-3 activation, suggesting that insulin signals to survivin inhibited caspase-3 activation. Interestingly, pretreatment of H9c2 cells with insulin or MG132, a proteasome inhibitor, inhibited doxorubicin-induced degradation of the transcription factor Sp1. ChIP assay showed that pretreatment with insulin inhibited doxorubicin-stimulated Sp1 dissociation from the <i>survivin</i> promoter. Finally using pharmacological inhibitors of the PI3K pathway, we showed that insulin-mediated activation of the PI3K/Akt/mTORC1 pathway prevented doxorubicin-induced proteasome-mediated degradation of Sp1. Taken together, insulin pretreatment confers a protective effect against doxorubicin-induced cardiotoxicity by promoting Sp1-mediated transactivation of survivin to inhibit apoptosis. Our study is the first to define a role for survivin in cellular protection by insulin against doxorubicin-associated injury and show that Sp1 is a critical factor in the transcriptional regulation of survivin.</p></div

Effect of insulin on the doxorubicin-induced changes in Sp1 and p53.

<p>(A, D) H9c2 cardiac myocytes were left untreated or treated with the indicated concentration of doxorubicin (<i>Doxo</i>) for 12 h, and (B, C, E) serum-deprived cells were left untreated or pretreated with insulin (200 nM) for 1 h prior to treatment with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting with anti-Sp1, anti-phospho-p53 (Ser¹⁵), anti-p53 and anti-GAPDH antibodies. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (A, **<i>p</i> < 0.01, n = 3; B, ***<i>p</i> < 0.001, n = 5). (D, E) Sp1 mRNA amount was determined by RT-PCR (28 cycles). Note that the blots in <i>panel C</i> represent one of three independent experiments.</p

Effect of doxorubicin and/or insulin on the transcriptional activity of Sp1 and p53.

<p>(A) Multiple sequence alignment (ClustalW2) of the proximal promoter regions of the rat, mouse and human <i>survivin</i> genes. Black arrow indicates the transcriptional start site and +1 points out the translation start codon. Canonical p53, Sp1, Sp1-like sites of the mouse and human <i>survivin</i> genes are boxed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref038" target="_blank">38</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref040" target="_blank">40</a>]. Two gray bars indicate the primers for ChIP analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref031" target="_blank">31</a>]; a red bar corresponds to human CHR sequences; two blue bars highlight human CDE regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref050" target="_blank">50</a>]. (B) Serum-deprived cells were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. Cross-linked cell lysates were subjected to ChIP analysis with anti-Sp1 or anti p53-antibody. RT-PCR (36 cycles) was performed with ChIP primers as listed in Materials and Methods (n = 5). (C–E) One day after transfection with Sp1 siRNA (20 nM), cells were left untreated or pretreated with insulin (200 nM) for 1 h and treated with doxorubicin (1 μM) for 24 h. Whole cell lysates were immunoblotted with anti-Sp1, anti-survivin and anti-caspase-3 (active form) antibodies (C, D), and total RNA was analyzed by RT-PCR (28 cycles) with specific primers to <i>survivin</i> gene (E). Note that these results represent one of three independent experiments.</p

Inhibitory effect of insulin on the doxorubicin-induced Sp1 degradation via PI3K/Akt/mTORC1 pathway in H9c2 cardiac myocytes.

<p>(A) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. (B–D) Cells were pretreated with 2 μM PI3K inhibitor LY294002 (<i>LY</i>) (B), 1 μM mTORC1 inhibitor rapamycin (<i>Rapa</i>) (C), or 5 μM p70S6K inhibitor PF4708671 (<i>PF</i>). (D) for 1 h, followed by treatment with insulin (200 nM) for 1 h and then with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting for protein levels or phosphorylation status of Akt, mTORC1 and p70S6K and for protein levels of Sp1 using antibodies listed in Materials and Methods. Note that these blots represent one of three independent experiments. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (*<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3).</p

Protective effect of insulin on the doxorubicin-induced cell death in H9c2 cardiac myocytes.

<p>(A) H9c2 cardiac myocytes were left untreated or treated with 1 μM doxorubicin (<i>Doxo</i>) for 24 h, 48 h, and 72 h. (B) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with the indicated concentration of insulin (<i>Ins</i>) for 1 h prior to treatment with 1 μM doxorubicin (<i>Doxo</i>) for 24 h. Cell viability was assessed by the MTT assay (**<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3 performed in triplicates). (C–F) Serum-deprived cells were left untreated or pretreated with insulin (200 nM) for 1 h prior to treatment with doxorubicin (1 μM) for 24 h. (C) Represented images of the TUNEL assay (100×). Treated cells were incubated with TUNEL reaction mixture, followed by staining with DAPI. (D) The TUNEL-stained cells were counted under fluorescence microscopy and presented as a bar graph (***<i>p</i> < 0.001, n = 3 performed in triplicates). (E) Caspase-3 activity was determined by immunoblot analysis of active form of caspase-3. (F) Mitochondrial (<i>Mito</i>) and cytosolic (<i>Cyto</i>) fractions were separated by SDS-PAGE gel and analyzed by immunoblotting with anti-cytochrome C (<i>Cyt C</i>) antibodies. GAPDH or β-actin bands show that equal amounts of sample were loaded and VDAC1 is used as a loading control for mitochondrial fraction. Note that blots represent one of three independent experiments. Values are mean ±S.D.</p