Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

<div><p>Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 <i>versus</i> Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.</p></div

Additional file 1: Figure S1. of Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo

A representative section from the lumbar part of the spinal cord. The regions under the blue-lined rectangular boxes show the areas where CD4+ T cells and SMI32-positive axons were quantified and analyzed. (PPTX 6495 kb

mTORC2 inactivation inhibits focal adhesion kinase activity.

<p>HUVECs were transfected with siRict¹ or siAkt1, then stimulated with 20 ng/mL VEGF for 10 minutes as indicated. <b>A</b>) A representative Western blot of EC phospho-focal adhesion kinase (P-FAK), total FAK, total Akt1, total rictor and actin. <b>B)</b> Quantitation of P-FAK (n = 4 independent experiments, *<i>P</i><0.05 by ANOVA). <b>C)</b> A representative Western blot of EC phospho-eNOS, and phospho-Src, illustrates that mTORC2 disruption, but not Akt1 inactivation, blocks VEGF-stimulated Src activation (n = 3 independent experiments). Knockdown of either rictor or Akt1 similarly blunts eNOS phosphorylation. <b>D)</b> Quantitation of P-Src (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). <b>E)</b> The effect of sustained mTORC1 or mTORC1/2 inhibition on EC FAK activation. HUVECs were treated with PP242 or rapamycin and stimulated with VEGF overnight. A representative Western blot of EC P-FAK, and P-S6K (n = 3 independent experiments). <b>F)</b> Quantitation of P-FAK (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p

Sustained mTORC1, but not mTORC2, inhibition activates Akt.

<p>HUVECs were treated by rapamycin, two different small interfering RNAs against rictor (siRict¹ and siRict²) or raptor (siRapt), or non-silencing siRNA (siNS), then stimulated with 20 ng/mL VEGF as indicated. Akt phosphorylation and S6K phosphorylation were evaluated as described in Materials and Methods. <b>A</b>) A representative Western blot of the effect of Rapamycin pretreatment for 1 hour on VEGF-stimulated Akt and S6 kinase phosphorylation in HUVECs. <b>B)</b> Quantitation of phospho-S6K. <b>C)</b> Quantitation of phospho-Akt (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA). The effect of rapamycin treatment of HUVEC for 24 hours. <b>D)</b> A representative Western blot of HUVEC phospho-Akt, total-Akt1 and tubulin over a range of concentration of rapamycin exposure. <b>E)</b> Quantitation of phospho-Akt (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA). The effect of mTORC1 <i>versus</i> mTORC2 disruption on Akt signaling in EC. <b>F)</b> A representative Western blot of HUVEC rictor, raptor, phospho-Forkhead box protein O1/3 (P-FOXO1/3), phospho-Akt, total Akt1, phospho-S6K, total S6K, and actin after treatment with siRapt, siRict or siNS. <b>G)</b> Quantitation of phospho-Akt. <b>H)</b> Quantitation of phospho-S6K (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p

mTORC1/2 dual inhibition blocks VEGF-mediated angiogenesis.

<p>HUVEC-coated Cytodex beads were embedded in fibrin gels as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135245#pone.0135245.g002" target="_blank">Fig 2</a>, then the EC were stimulated with 50 ng/ml VEGF, and were treated with PP242 or carrier as indicated. <b>A)</b> Representative images of EC sprouts after 18 hours incubation. <b>B)</b> Quantitation of the number of sprouts per bead. <b>C)</b> Quantitation of the length of the sprouts (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA, scale bar = 95 um). <b>D)</b> Collagen gel onplants containing VEGF (100 ng/onplant) and PP242 or carrier, were placed on chicken embryo CAM as described in Methods. Quantitation of neovascularization after 64 hours of exposure to VEGF supplemented with 1, 5, 10 or 50 uM PP242 (n > 48 onplants or 16 chicken embryos per group, <i>*P</i><0.05 by ANOVA).</p

mTORC2 inactivation inhibits focal adhesion formation.

<p>HUVECs plated on gelatin matrix were transfected with small interfering RNA against rictor (siRict¹) or non-silencing siRNA (siNS). <b>A)</b> Representative fluorescence image of EC immuno-stained for the formation of vinculin-rich focal adhesions (green) and DNA (blue). <b>B)</b> Quantitation of the number of focal adhesions per cell, of 100 ECs pooled from three independent experiments.</p

mTORC1 inhibition primes endothelial cells to resist apoptosis and to respond to tumour-derived pro-angiogenic cues.

<p>HUVECs were treated with Rapamycin (5 nM), PP242 (1 uM), or Ku 0063794 (50 nM) for 24 hours. <b>A)</b> The EC were challenged with tumor necrosis factor-α (TNFα) + cycloheximide (CHX) for 4 hours in normal growth conditions. Active cleaved caspase-3 was detected by DEVD-FMK-FITC and analyzed by flow cytometry as described in Methods. Quantitation of active caspase-3 in mock-, rapamycin-, PP242-, and Ku 0063794-treated ECs (n = 4 independent experiments, *<i>P</i><0.05 by ANOVA). <b>B)</b> Evaluation of EC angiogenic sprouting to tumor-derived growth factors <i>in vitro</i>. HUVEC were pretreated with Rapamycin (5 nM), PP242 (1 uM), or Ku 0063794 (50 nM) for 24 hours, then mounted on Cytodex beads (green) and were embedded with renal cell carcinoma cell-coated beads (red) in 3D fibrin gels as described in Methods, then co-cultured without additional growth factor supplementation or inhibitors. Representative images of EC sprouts after 18 hours incubation. <b>C)</b> Quantitation of the number of sprouts per bead (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p

mTORC2 inactivation reduces EC migration, matrix adhesion, and actin polymerization.

<p>HUVECs were transfected with siRNA targeting Akt1 (siAkt1), rictor (siRict¹) or raptor (siRapt). <b>A)</b> The ECs were seeded on gelatin-coated electrodes at equal density to reach confluence. The recovery of electric impedance was measured following delivery of a high electric current through an electrode to create a defect in the EC monolayer as described in Methods. Data are represented as the relative rate of migration per hour <i>versus</i> the control (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). HUVECs were transfected with siRict¹, or siAkt1, or treated with PP242. <b>B)</b> The EC were seeded equally on gelatin-coated electrodes, then adhesion was evaluated using electrical impedance measurements. Data are represented as the relative rate of adhesion <i>versus</i> the controls (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). <b>C)</b> The EC were serum-starved overnight, and stimulated with VEGF for 10 minutes or not (upper left). Fluorescence images of phalloidin-stained filamentous (F)-actin (green) and DNA (blue) are representative of 3 independent experiments illustrating a marked decrease in VEGF-stimulated F-actin among mTORC2-inactivated EC. <b>D)</b> HUVECs were transfected with siAkt1, or siRict¹, or control siNS. The EC were stimulated with VEGF, then globular (G)-actin and F-actin were separated as described in Methods. A representative Western blot illustrates the relative abundance of the F-actin and G-actin in ECs. <b>E)</b> Quantitation of the F-/G-actin ratio (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA).</p