Pathological neoangiogenesis depends on oxidative stress regulation by ATM

Journal name:
Nature Medicine
Volume:
18,
Pages:
1208–1216
Year published:
DOI:
doi:10.1038/nm.2846
Received
Accepted
Published online

Abstract

The ataxia telangiectasia mutated (ATM) kinase, a master regulator of the DNA damage response (DDR), acts as a barrier to cellular senescence and tumorigenesis. Aside from DDR signaling, ATM also functions in oxidative defense. Here we show that Atm in mice is activated specifically in immature vessels in response to the accumulation of reactive oxygen species (ROS). Global or endothelial-specific Atm deficiency in mice blocked pathological neoangiogenesis in the retina. This block resulted from increased amounts of ROS and excessive activation of the mitogen activated kinase p38α rather than from defects in the canonical DDR pathway. Atm deficiency also lowered tumor angiogenesis and enhanced the antiangiogenic action of vascular endothelial growth factor (Vegf) blockade. These data suggest that pathological neoangiogenesis requires ATM-mediated oxidative defense and that agents that promote excessive ROS generation may have beneficial effects in the treatment of neovascular disease.

At a glance

Figures

  1. Atm is activated specifically in newly formed pathological vessels in response to ROS accumulation.
    Figure 1: Atm is activated specifically in newly formed pathological vessels in response to ROS accumulation.

    (ae) Immunohistochemistry to detect pAtm (phosphorylated at Ser1987), isolectin (iB4) and DAPI on whole-mount retinal samples at P16 in the normoxic condition (a,b) or in ischemic retinopathy (ce). Closed arrowheads indicate pAtm in the nuclei of neovascular endothelial cells. Open arrowheads indicate pAtm-negative normal endothelial cells, including tip cells migrating into the central avascular area. The boxed area in a is shown enlarged in b, and the boxed areas in c are shown enlarged in d and e. (f) FACS plot and quantification (n = 4 mice in each group (normoxia and ischemic retinopathy)) of DCF-DA staining in CD31+ endothelial cells isolated from mice with ischemic retinopathy. (g,h) Dihydroethidium and acrolein staining of retinas of P16 mice with ischemic retinopathy. Closed arrowheads indicate abundant ROS accumulation in the neovascular tufts. Open arrowheads indicate normal endothelial cells. (i) Immunocytochemistry of HUVECs stained for pATM, VE-cadherin (VE-cad) and DAPI (left) and quantification of the percentage of pATM+ HUVECs (right; n = 6 independent experiments for each condition) under the indicated conditions. Arrowheads indicate pATM-positive cells. Cont, PBS control. (j) Immunohistochemistry of retinas of P16 mice with ischemic retinopathy systemically treated with vehicle or the antioxidant NAC between P11 and P15. All data are mean ± s.e.m. Scale bars, a,c, 500 μm; b,d,e,gj, 50 μm. *P < 0.05, **P < 0.01 by two-tailed Student's t test.

  2. Endothelial Atm is required for retinal pathological angiogenesis.
    Figure 2: Endothelial Atm is required for retinal pathological angiogenesis.

    (ac) Isolectin staining (a) and stereomicroscopy (b) of whole-mount retinas from Atm−/− and Atm+/+ mice with ischemic retinopathy at P16 and quantification of the neovascular tuft (NVT) and avascular areas (n = 8 mice per genotype) (c). In the insets of a, the red area indicates NVTs and the yellow area indicates the avascular area. Open arrowheads in b indicate retinal hemorrhage. (d) Short-term BrdU incorporation assay and TUNEL staining in the retinas of Atm−/− and Atm+/+ mice and quantification of the results (n = 4 mice per genotype). ECs, endothelial cells. (e) Isolectin staining of whole-mount retinal samples of Atm−/− and Atm+/+ mice at P8 and at P11 in the ischemic retinopathy model (left) and quantification of the vaso-obliterated area at P11 (right; n = 6 mice per group). (f) Isolectin staining of whole-mount retinas of AtmVEC-KO and control mice at P16 in the ischemic retinopathy model and quantification of the results (n = 6 mice per group). The control group in f and g were Cre-negative mice (Online Methods). (g) Isolectin staining of the retinas of AtmVEC-KO and control mice at P4. Open arrowheads indicate the area of vessel sparsity in AtmVEC-KO mice. (h) Immunocytochemistry of HUVECs to detect BrdU and TUNEL staining after treatment with the ATM inhibitor KU-55933 (KU) or DMSO and quantification of the results (n = 4 mice per group). Closed arrowheads indicate cell proliferation and open arrowheads indicate cell apoptosis. (i) Quantification of the percentage of viable HUVECs cultured in various concentrations of KU-55933 over the indicated time course (n = 4 mice per group). (j) Immunocytochemistry of primary lung endothelial cells from Atm−/− and Atm+/+ mice to detect VE-cadherin (VE-cad), BrdU and caspase 3 (Casp3) and quantification of the results (n = 4 mice per group). Closed arrowheads indicate proliferating endothelial cells, and open arrowheads indicate apoptotic endothelial cells. All data are means ± s.e.m. Scale bars, a,eg, 500 μm; d,h,j, 50 μm. *P < 0.05, **P < 0.01, NS, not significant by two-tailed Student's t test.

  3. Atm promotes endothelial proliferation by suppressing ROS accumulation.
    Figure 3: Atm promotes endothelial proliferation by suppressing ROS accumulation.

    (a) Immunocytochemistry of HUVECs treated with KU-55933 (KU) or DMSO to detect pH2AX and quantification of the results (n = 4 independent experiments for each condition). Arrowheads indicate pH2AX-positive cells. (b) Immunohistochemistry of whole-mount retinas of Atm−/− and Atm+/+ mice at P14 in the ischemic retinopathy model. Arrowheads indicate pH2AX expression in neovascular tufts. (c) Isolectin staining of p53−/− and p53+/+ whole-mount retinas at P16 in the ischemic retinopathy model and quantification of the results (n = 6 mice per group). (d) Quantification of DCF-DA and MitSOX staining in KU-55933–treated or DMSO-treated HUVECs and in CD31+ endothelial cells isolated from Atm−/− or Atm+/+ mice at P14 in the ischemic retinopathy model (n = 6 mice per group). (e) BrdU staining of HUVECs treated as indicated and quantification of the results (n = 4 independent experiments for each condition). (f,g) Quantification of the percentage of TUNEL-positive HUVECs after KU-55933 or DMSO treatment (f) and quantification of the percentage of viable HUVECs after the indicated treatments and over the indicated time course (g; n = 4 independent experiments per condition). (h) Isolectin staining of whole-mount retinas of Atm−/− and Atm+/+ mice treated systemically with NAC or PBS at P16 in the ischemic retinopathy model and quantification of the results (n = 6 mice per group). (i) Schematic diagram of the enzymatic activities of Sod, catalase and Gpx1 (top) and measurement of their activities in Atm−/− or Atm+/+ endothelial cells (bottom; n = 4 mice per group). GSH, glutathione; GSSG, glutathione disulfide. All data are mean ± s.e.m. Scale bars, c,h, 500 μm; a,b,e, 50 μm. *P < 0.05, **P < 0.01, NS, not significant by two-tailed Student's t test.

  4. p38[alpha] acts downstream of Atm in endothelial cells.
    Figure 4: p38α acts downstream of Atm in endothelial cells.

    (a) Western blotting of HUVECs with the indicated treatments to detect the indicated proteins. (b) Immunocytochemistry of HUVECs after the same treatments as in a and quantification of the number of p-p38–positive cells (n = 4 independent experiments per condition). Arrowheads indicate p-p38–positive HUVECs. KU, KU-55933; VE-cad, VE-cadherin. (c) Quantitative PCR analysis of the indicated CDK inhibitors in KU-55933–treated or DMSO-treated HUVECs (n = 4 independent experiments per condition). (d) Immunocytochemistry of HUVECs after the treatments with KU-55933 or DMSO and siRNAs as indicated to detect BrdU and TOTO3 and quantification of the results (n = 4 indepenedent experiments per condition). si, siRNA targeting the indicated proteins. (e) Isolectin staining of whole-mount retinas of Atm−/− and Atm+/+ mice treated with the p38 kinase inhibitor SB203580 (SB) or vehicle (DMSO) as indicated at P16 in the ischemic retinopathy model and quantification of the results (n = 6 mice per group). Mice were intraperitoneally treated with SB203580 or vehicle daily from P11 to P15. (f) Isolectin staining of whole-mount wild-type retinas treated with the p38 activator anisomycin (Aniso) or the vehicle control at P16 in the ischemic retinopathy model and quantification of the results (n = 6 mice per group). Mice were intraperitoneally treated with anisomycin or vehicle (DMSO) at P14. (g) Isolectin staining of whole-mount retinas of control mice or of mice with endothelial-specific deficiency of Atm (Cre+; Atmflox/flox) and/or p38α (Cre+; Mapk14flox/flox) at P16 in the ischemic retinopathy model and quantification of the results (n = 7 mice per group). The control group was Cre+; Atmflox/+; Mapk14flox/+ mice. All data are mean ± s.e.m. Scale bars, eg, 500 μm; b,d, 50 μm. *P < 0.05, **P < 0.01, NS, not significant by two-tailed Student's t test.

  5. Loss of Atm decreases tumor angiogenesis and enhances the antiangiogenic activity of Vegf blockade.
    Figure 5: Loss of Atm decreases tumor angiogenesis and enhances the antiangiogenic activity of Vegf blockade.

    (a) Quantification of DCF-DA staining in CD31+ endothelial cells isolated from B16 tumors or from normal subcutaneous tissue (n = 5 mice per group). (b) Immunohistochemistry to detect pAtm, CD31 and DAPI on in tissue sections from B16 tumors or normal subcutaneous tissue. Arrowheads indicate pAtm-positive endothelial cells in tumors, and asterisks indicate pAtm-positive tumor cells. (c) Macroscopic appearance of B16 tumors implanted in Atm−/− or control (Cre) mice. (d) Quantification of tumor weight 10 d after transplantation (n = 6 mice per group). (e) Immunohistochemistry to detect BrdU, collagen IV (Col IV) and DAPI in tumors 10 d after transplantation and quantification of the results (n = 6 mice per group). The boxes in the images in the top row indicate areas of high magnification shown in the images in the bottom row. Arrowheads indicate BrdU-positive endothelial cells. (f) Immunohistochemistry to detect FITC, CD31, ASMA and DAPI in tumors of mice injected intracardially with FITC-dextran (left) and quantification of the results (right; n = 5 mice per group). Open arrowheads indicate immature vessels (lacking blood flow or ASMA-positive pericytes) and closed arrowheads indicate mature vessels (with blood flow or ASMA-positive pericytes). (g) Quantification of tumor weight and vessel density 10 d after transplantation in mice with endothelial-specific deficiency of Atm and/or p38α (n = 4–6 in each genotype). (h) Quantification of tumor volume in tumors of control or AtmVEC-KO mice treated with DMSO or the Vegfr2 inhibitor SU1498 as indicated (n = 5 mice per group). The values on the x axis indicate days after transplantation. (i) Quantification of tumor vessel density in the same tumors as in h at 10 d after transplantation. In e, g and i, vessel density is shown as the qualitative intensity of collagen IV staining. All data are mean ± s.e.m. Scale bars, 50 μm. *P < 0.05, **P < 0.01 by two-tailed Student's t test.

  6. Atm deficiency does not affect the maintenance of healthy vessels in adult mice.
    Figure 6: Atm deficiency does not affect the maintenance of healthy vessels in adult mice.

    (a) Whole-mount immunohistochemistry of CD31 in the indicated tissues of Atm−/− and Atm+/+ mice at P30. (b) Proposed model for the selective activation of ATM in immature vessels and for the proangiogenic function of ATM through decreasing oxidative stress and p38α activation. Scale bar, 500 μm.

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Affiliations

  1. Center for Integrated Medical Research, School of Medicine, Keio University, Tokyo, Japan.

    • Yuji Okuno &
    • Yoshiaki Kubota
  2. Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.

    • Ayako Nakamura-Ishizu &
    • Toshio Suda
  3. Department of Cardiovascular Medicine, Osaka University, Graduate School of Medicine, Osaka, Japan.

    • Kinya Otsu
  4. Cardiovascular Division, King's College London, London, UK.

    • Kinya Otsu

Contributions

Y.O. and A.N.-I. performed experiments and analyzed data. T.S. interpreted results and assisted in manuscript preparation. K.O. provided experimental materials. Y.K. designed experiments, interpreted results and wrote the paper.

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The authors declare no competing financial interests.

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