Supplementary Information Pathological neoangiogenesis depends on oxidative stress regulation by ATM

Yuji Okuno1, Ayako Nakamura-Ishizu2, Kinya Otsu3,4, Toshio Suda2 and Yoshiaki

Kubota1

1Center for Integrated Medical Research, 2Department of Cell Differentiation, The

Sakaguchi Laboratory, School of Medicine, Keio University, 35 Shinanomachi,

Shinjuku-ku, Tokyo 160-8582, Japan. 3Department of Cardiovascular Medicine, Osaka

University, Graduate School of Medicine, Osaka 565-0871, Japan. 4Cardiovascular

Division, King’s College London, London SE5 9NU, UK

Address correspondence to: Yoshiaki Kubota, M.D., Ph.D.

Center for Integrated Medical Research, School of Medicine, Keio University, 35

Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

TEL: 81-3-5363-3475, FAX: 81-3-5363-3475

E-mail: ykubo33@a3.keio.jp

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 1. Atm is specifically activated in newly formed pathological

vessels. Separate images of each color channel for the images shown in Fig. 1 a‒e.

Closed arrowheads indicate pAtm in the nuclei of neovascular endothelial cells. Open

arrowheads indicate pAtm-negative endothelial cells in normal endothelial cells

including tip cells migrating into the central avascular area. Scale bars: 50 m.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 2. Endothelial ATM is deleted in Atm－/－ and Atm VEC-KO mice.

(a) Western blotting analysis for pAtm, Atm and Actin in VE-cadherin+ endothelial cells

isolated from the retina at P14 in ischemic retinopathy. (b) Immunohistochemistry of

the retina of VEcad-Cre+flox-CAT-EGFP+ mice at P14 in ischemic retinopathy. (c‒h)

Retinal immunohistochemistry of Atm+/+, Atm－/－ or Atm VEC-KO mice at P16 in ischemic

retinopathy. Arrowheads indicate pAtm in neovascular tufts. Arrows indicate slight

pAtm expression in neuroretinal cells. Scale bars: 50 m.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 3. Vascular phenotypes in Atm－/－ mice at various time

points during ischemic retinopathy and expression of angiogenic factors. (a, b)

Isolectin staining of whole-mount retinas at various time points in ischemic retinopathy

and quantification (n = 4). (c) Quantitative PCR analysis (n = 6) of retinal samples at

P14 in ischemic retinopathy. (d) Immunohistochemistry of whole-mount retinal samples

at P16 in ischemic retinopathy. Arrowheads indicate the expression of Vegfr2 detected

by crossing mice with the Vegfr2+/EGFP line. Scale bars: 500 m in a; 50 m in d; *P <

0.05; **P < 0.01.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 4. Decreased proliferation and increased apoptosis in

endothelial cells of Atm－/－ mice in ischemic retinopathy. Smears prepared with a

cytospin centrifuge of FACS-sorted VE-cadherin+ cells from mice treated

intraperitoneally with BrdU 2 h before sacrifice. The cytospin slides are stained with

BrdU or cleaved caspase3. Closed arrowheads indicate proliferating endothelial cells,

and open arrowheads do apoptotic endothelial cells. Scale bars: 10 m; **P < 0.01.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 5. No detectable abnormalities in perivascular cells of Atm－/－

mice. (a) Immunohistochemistry with desmin or ASMA on whole-mount retinal

samples at P16 in ischemic retinopathy. (b, c) Immunohistochemistry with F4/80 or

FACS analysis of CD11b and Gr1 for retinal tissues at P14 in ischemic retinopathy and

quantification (n = 4). (d) Isolectin stainings of whole-mount retinal samples at P16 in

ischemic retinopathy and quantification (n = 6). (e) Immunohistochemistry of

whole-mount retinal samples at P16 in ischemic retinopathy. Open arrowheads indicate

negativity for pAtm in Gfap+ astrocytes. (f‒i) Immunohistochemistry of whole-mount

retinal samples at P16 in ischemic retinopathy. Scale bars: 500 m in d, f, g; 50 m in a,

b, e, h, i.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 6. Vascular structures of Atm－/－ and AtmVEC-KO mice in early

developmental stages. (a) Immunohistochemistry of whole-mount retinal samples at

P4. Arrowheads indicate pATM very weakly expressed in endothelial stalk cells at the

same level in the neuroretinal cells (arrows). (b) Quantification at P4 (n = 5). (c‒h)

Immunohistochemistry for while-mount retinas with isolectin (c‒f) or frozen sections

with CD31 (g, h) at P30. P, primary plexus; ID inner deeper plexus; OD, outer deeper

plexus; CH, choroidal plexus. (i) Quantification of capillary density in whole-mounts

samples at P30 (n = 4). Scale bars: 500 m in right panels of a and c, d; 50 m in e‒h;

10 m in left panels of a; *P < 0.05.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 7. Atm inhibition does not affect cell proliferation in

non-endothelial cell lines. (a) BrdU staining in B16 melanoma cells (B16), Lewis lung

carcinoma cells (LLC) and human dermal fibroblasts (HDF). (b) Quantification (n = 4).

Scale bar: 50 m.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 8. NAC rescues increased apoptosis and decreased

proliferation in Atm － / － lung endothelial cells. (a ‒ c) Quantification in

immunocytochemistry for primary lung endothelial cells in the presence or absence of

NAC (n = 4). *P < 0.05; **P < 0.01.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 9. Adenoviral delivery of Catalase partially abrogates the

vascular phenotypes in Atm－/－ mice in ischemic retinopathy. (a‒d) Isolectin stainings

of whole-mount retinal samples at P16 in ischemic retinopathy. Mice were

intra-ocularly treated with Adeno- gal or Adeno-Catalase at P12. (e, f) Quantification of

the amount of neovascular tufts or the avascular area (n = 5). (g, h) Quantification of

the amount of neovascular tufts or the avascular area at P16 in ischemic retinopathy (n

= 5). Mice were intra-ocularly treated with PBS or a Gpx inhibitor, Mercaptosuccinate

at P12. Scale bar: 500 m; *P < 0.05.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 10. Analysis of the downstream cascade of ATM in

KU55933-treated HUVECs. (a) Western blotting analysis for key molecules in DDR

pathway in HUVECs treated with or without 10 M of KU55933. (b) Representive

images for DNA single-cell electrophoresis in HUVECs. For a positive control, cells

were irradiated at a dose of 10 Gy. (c) Quantification of relative ratio of tail DNA (n =

4). (d) Western blotting analysis for molecules known as the downstream of the ATM

signaling. Scale bar: 50 m.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 11. Treatment with caspase inhibitors partially abrogates

the vascular phenotypes in AtmVEC-KO mice in ischemic retinopathy. (a) Quantitative

PCR analysis of pro-apoptotic genes (n = 4). (b) Isolectin stainings of whole-mount

retinal samples at P16 in ischemic retinopathy. (c, d) Quantification of the amount of

neovascular tufts or the avascular area (n = 5). Scale bar: 500 m. *P < 0.05.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 12. Excessive activation of p38 in Atm－/－ mice is abrogated

by NAC treatment. (a) Western blotting analysis in HUVECs treated with H2O2 for 30

min. (b, c) Immunohistochemistry of whole-mount retinas at P14 in ischemic

retinopathy and quantification (n = 4). Arrowheads indicate phosphorylation of p38 in

the neovascular tufts. Scale bar: 50 m. **P < 0.01.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 13. Endothelial-specific p38 knockout does not affect the

phenotypes in ischemic retinopathy. Isolectin staining of whole-mount retinas and

quantification (n = 5) at P16 in ischemic retinopathy. Scale bar: 500 m.

Nature Medicine doi:10.1038/nm.2846

Supplementary Figure 14. Loss of p38 abrogates the decreased vascularity and

proliferation in tumors transplanted into AtmVEC-KO mice. Section

immunohistochemistry and quantification (n = 4) in the tumors 10 d after

transplantation. Scale bars: 50 m; *P < 0.05.

Nature Medicine doi:10.1038/nm.2846