Up-regulation of AGGF1 in response to acute MI and robust therapeutic effects of AGGF1 protein therapy on MI.

<p><b>(A)</b> Real-time reverse transcription (RT)-PCR analysis for <i>AGGF1</i> mRNA expression in mice with LAD ligation or sham operation (Sham) (<i>n</i> = 6/group). Pre, before surgery. <b>(B)</b> Western blot analysis for AGGF1 protein expression (<i>n</i> = 6 mice/group). <b>(C)</b> Immunostaining analysis for AGGF1 protein expression in cross-sections of hearts (<i>n</i> = 5). Scale bar = 50 μm. <b>(D)</b> AGGF1 (500 ng/ml) induces autophagy in HUVECs under a hypoxic condition for different time points (<i>n</i> = 3/group). <b>(E)</b> AGGF1-specific siRNA inhibited autophagy under normoxia or hypoxia. <b>(F)</b> Post-MI survival of mice with AGGF1 treatment (<i>n</i> = 20), mice with IgG treatment (<i>n</i> = 30), and sham mice (<i>n</i> = 14). <b>(G)</b> Representative M-mode echocardiograms. <b>(H)</b> Effects of AGGF1 protein therapy on myocardial function and contraction. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

AGGF1-induced angiogenesis is suppressed by autophagic flux inhibition.

<p><b>(A)</b> HUVEC proliferation induced by AGGF1 (500 ng/ml) was blocked by pretreatment with bafilomycin A1 (Baf) or chloroquine (CQ) for 2 h before treatment with IgG (control) or AGGF1 (<i>n</i> = 4/group). <b>(B)</b> Baf or CQ inhibited AGGF1-induced migration of HUVECs using scratch assays (<i>n</i> = 4/group). <b>(C)</b> Baf or CQ inhibited AGGF1-induced migration of HUVECs using Transwell migration assays (<i>n</i> = 4/group). <b>(D)</b> Baf or CQ inhibited AGGF1-induced endothelial tube formation (<i>n</i> = 4/group). Scale bar = 50 μm. <b>(E)</b> Baf or CQ inhibited AGGF1-induced sprout angiogenesis in an e<i>x vivo</i> aortic ring angiogenesis assay (<i>n</i> = 4/group). Scale bar = 125 μm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

<i>Atg5</i> KO blunted AGGF1-induced therapeutic angiogenesis, survival, and recovery of myocardial functions and contraction of MI mice.

<p><b>(A)</b> AGGF1-induced angiogenesis (CD31 immunostaining) in heart sections 28 d after MI in AAV9-GFP mice. <i>Atg5</i> KO blocks AGGF1-induced angiogenesis (CD31 immunostaining signal) in heart sections 28 d after treatment (<i>n</i> = 5/group, AAV9-CMV-Cre <i>Atg5</i> KO mice). Scale bar = 50 μm. (<b>B</b>) Four-week survival of MI mice under different treatments (Sham + AAV9-GFP + IgG, n = 14; MI + AAV9-GFP + IgG, n = 20; MI + AAV9-GFP + AGGF1, n = 20; Sham + AAV9-Cre + IgG, n = 15; MI + AAV9-Cre + IgG, n = 25; MI + AAV9-Cre + AGGF1, n = 25). (<b>C</b>) Effects of AGGF1 protein therapy under different treatments on the function of the left ventricle (Sham + AAV9-GFP + IgG, n = 13; MI + AAV9-GFP + IgG, n = 10; MI + AAV9-GFP + AGGF1, n = 14; Sham + AAV9-Cre + IgG, n = 13; MI + AAV9-Cre + IgG, n = 7; MI + AAV9-Cre + AGGF1, n = 8). (<b>D</b>) Effects of AGGF1 protein therapy under different treatments on cardiac fibrosis (Masson trichrome staining of cross-sections) and LV wall thickness 4 wk after treatment (<i>n</i> = 5/group). Scale bar = 1 mm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

Atg5 knockdown prevents activation of angiogenesis by AGGF1.

<p><b>(A)</b> HUVEC proliferation induced by AGGF1 (500 ng/ml) was blocked by <i>Atg5</i>-specific siRNA (<i>n</i> = 4/group). <b>(B)</b> <i>Atg5</i>-specific siRNA inhibited AGGF1-induced migration of HUVECs using scratch assays (<i>n</i> = 4/group). <b>(C)</b> <i>Atg5</i>-specific siRNA inhibited AGGF1-induced migration of HUVECs using Transwell migration assays (<i>n</i> = 4/group). <b>(D)</b> <i>Atg5</i>-specific siRNA inhibited AGGF1-induced endothelial tube formation (<i>n</i> = 4/group). Scale bar = 50 μm. <b>(E)</b> <i>Atg5</i>-specific siRNA inhibited AGGF1-induced sprout angiogenesis in an e<i>x vivo</i> aortic ring angiogenesis assay (<i>n</i> = 4/group). Scale bar = 125 μm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

Schematic diagram showing the molecular signaling pathway for AGGF1-induced autophagy and angiogenesis.

<p>Schematic diagram showing the molecular signaling pathway for AGGF1-induced autophagy and angiogenesis.</p

AGGF1 is involved with autophagy activity.

<p><b>(A)</b> Western blot analysis showing that AGGF1 at different concentrations induces autophagy (increased LC3-II/β-actin and LC3-II/I; decreased p62 expression) in HUVECs under a normoxic condition for 4 h (<i>n</i> = 3/group). <b>(B)</b> AGGF1 (500 ng/ml) induces autophagy in HUVECs (representative images of LC3-GFP punctuate) (<i>n</i> = 6/group). Scale bar = 10 μm. <b>(C)</b> Representative electronic microscopic images of HUVECs treated with AGGF1 or control IgG showing detection of AGGF1-induced autophagy. Scale bar = 1 μm. <b>(D)</b> Western blot analyses for autophagy markers in cardiac tissues from <i>AGGF1</i>^<i>+/-</i> mice and wild-type <i>AGGF1</i>^<i>+/+</i> mice (<i>n</i> = 6/group). <b>(E)</b> Western blot analysis for autophagy markers in cardiac ECs isolated from <i>AGGF1</i>^<i>+/-</i> mice and wild-type <i>AGGF1</i>^<i>+/+</i> mice (<i>n</i> = 6/group). Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

Autophagy is required for AGGF1-induced therapeutic angiogenesis, survival, and recovery of myocardial functions and contraction of MI mice.

<p><b>(A)</b> Bafilomycin A1 (Baf) blocked AGGF1-induced angiogenesis (CD31 immunostaining) in heart sections 28 d after treatment (<i>n</i> = 5/group). Scale bar = 50 μm. <b>(B)</b> Four-week survival of MI mice under different treatments (MI + Veh + IgG, n = 36; MI + Veh + AGGF1, n = 30; MI + Baf + IgG, n = 44; MI + Baf + AGGF1, n = 40). <b>(C)</b> Effects of AGGF1 protein therapy with and without bafilomycin A1 pretreatment on the function of the left ventricle (MI + Veh + IgG, n = 18; MI + Veh + AGGF1, n = 21; MI + Baf + IgG, n = 8; MI + Baf + AGGF1, n = 8). <b>(D)</b> Effects of AGGF1 protein therapy with and without bafilomycin A1 pretreatment on the cardiac fibrosis (Masson trichrome staining of cross-sections) and LV wall thickness 4 wk after treatment (<i>n</i> = 5/group). Scale bar = 1 mm. <b>(E)</b> CD31 immunostaining in autophagy-deficient <i>Becn1</i>^<i>+/-</i> mice after LAD ligation (<i>n</i> = 5/group). Scale bar = 50 μm. <b>(F)</b> AGGF1 protein therapy showed no effect on 4-wk survival after MI of <i>Becn1</i>^<i>+/-</i> mice (Sham + IgG, n = 15; Sham + AGGF1, n = 14; MI + IgG, n = 44; MI + AGGF1, n = 43). <b>(G)</b> Echocardiographic parameters at 4 wk after MI. Effects of AGGF1 protein therapy on myocardial functions and contraction in <i>Becn1</i>^<i>+/-</i> mice (Sham + IgG, n = 13; Sham + AGGF1, n = 12; MI + IgG, n = 16; MI + AGGF1, n = 17). <b>(H)</b> Effects of AGGF1 protein therapy on the cardiac fibrosis 4 wk after MI in <i>Becn1</i>^<i>+/-</i> mice (<i>n</i> as in A). Scale bar = 1 mm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

AGGF1 induces autophagy and angiogenesis and suppresses apoptosis in mice after MI.

<p><b>(A)</b> Western blot analysis for LC3-II in cardiac tissues from mice with LAD ligation (<i>n</i> = 6/group). <b>(B)</b> CD-31 immunostaining. Densities of CD31-positive vessels are quantified and graphed (<i>n</i> = 5/group). HPF, high-power field. Scale bar = 50 μm and 25 μm. <b>(C)</b> TUNEL staining of cross-sections of mouse hearts. The number of TUNEL-positive myocytes in the infarct area is quantified and graphed (<i>n</i> = 5/group). Caspase-3 activity was measured in myocardial tissues (<i>n</i> = 5/group). Scale bar = 50 um. <b>(D)</b> Western blot analysis for cleaved PARP, Bax, and Bcl-2 from myocardial protein lysates (<i>n</i> = 3/group) and real-time RT-PCR for the ratio of <i>Bax/Bcl-2</i> mRNA levels (<i>n</i> = 3/group). Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

Molecular signaling pathway for AGGF-induced autophagy.

<p><b>(A)</b> Western blot analysis for phosphorylation of JNK with protein extracts from HUVECs treated with different concentrations of AGGF1 for 4 h. <b>(B)</b> Activation of JNK in HUVECs treated with AGGF1 protein (500 ng/ ml) for different time points. <b>(C)</b> JNK inhibitors II and III inhibited AGGF1-induced autophagy (LC3-II levels) and activation of JNK. <b>(D)</b> Co-immunoprecipitation to assess the assembly of Beclin1-Vps34-Atg14L complex. Immunoprecipitation, beclin 1 antibody, or control IgG; Western blot, individual antibodies for Vsp34, Atg14L, or beclin 1. <b>(E)</b> Vps34 lipid kinase activity with ELISA assays (<i>n</i> = 3/group). <b>(F)</b> Autophagic activity assay for membrane-associated PI(3)K (GFP-2xFYVE dots). HUVECs were transfected with GFP-2xFYVE for 24 h and then treated with JNK inhibitor II or control DMSO, followed by treatment with AGGF1 or IgG (<i>n</i> = 6/group). Scale bar = 10 μm. <b>(G)</b> Autophagic activity assay for degradation of long-lived, damaged proteins by ³H-leucine release in HUVECs (<i>n</i> = 6/group). (<b>H</b>) <i>JNK1</i> siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of JNK1. (<b>I</b>) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by <i>JNK1</i> siRNA. (<b>J</b>) <i>Atg14L</i> siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of Atg14L. (<b>K</b>) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by <i>Atg14L</i> siRNA. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

Reduced autophagy, decreased survival, larger infarct areas, compromised cardiac function and contraction, and impaired angiogenesis in <i>AGGF1</i>^<i>+/-</i> knockout mice after MI.

<p><b>(A)</b> Four-week survival of <i>AGGF1</i>^<i>+/-</i> mice (<i>n</i> = 40) and <i>AGGF1</i>^<i>+/+</i> mice (<i>n</i> = 14) under MI as compared to <i>AGGF1</i>^<i>+/-</i> mice (<i>n</i> = 16) and <i>AGGF1</i>^<i>+/+</i> mice (<i>n</i> = 8) with sham operation. <b>(B)</b> Cardiac function, contraction, and structure were impaired in <i>AGGF1</i>^<i>+/-</i> mice compared to wild-type mice with or without LAD ligation. <b>(C)</b> Masson trichrome staining of cross-sections of hearts to measure fibrotic sizes of LV and LV wall thickness (<i>n</i> = 5/group). Scale bar = 1 mm. <b>(D)</b> CD31 immunostaining of myocardial sections 28 d after ligation (<i>n</i> = 5/group). Scale bar = 50 μm. <b>(E)</b> Western blot analysis for autophagy markers in <i>AGGF1</i>^<i>+/-</i> mice (<i>n</i> = 6) and <i>AGGF1</i>^<i>+/+</i> mice (<i>n</i> = 6) after MI or sham operation. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p