SUPPLEMENTARY INFORMATION

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1 BASELINE ISCHAEMIA a b Phd2 +/- c d Collateral growth and maintenance SMC recruitment SMC proliferation Phd2 +/- NF- B off NF- B on NF- B on NF- B on Endothelial cell Smooth muscle cell Pro-arteriogenic macrophages Macrophage-derived soluble factors Supplementary figure 1: Scheme of collateral arterial growth in and Phd2 +/- mice. Collateral vessels are pre-existing conduits (shown in panel a) that undergo extensive remodeling in case of occlusion of the major arterial flow, in a process named arteriogenesis. Phd2 +/- mice display more abundant, larger, and thicker collateral arteries at baseline. Mechanistically, reduced levels of PHD2 in macrophages unleash the activation of canonical NF- B signals that induce a skew towards a pro-arteriogenic (M2-like) phenotype, which entails smooth muscle cell (SMC) recruitment and growth in response to their increased release of soluble factors (panel b). Femoral artery occlusion triggers the accumulation of pro-arteriogenic, M2- like macrophages in mice as well, fuelling collateral vessel maturation in a NF- B dependent manner (panel c). In Phd2 +/- mice, macrophage skewing and collateral remodeling after ischaemia are milder (panel d), likely because of their pre-adaptation at baseline. 1

2 b 8-OHdG 8-OHdG j 6 Phd2 Vessels/mm Baseline 12h k Baseline Proliferation 3d l Total vessel area +/ d 8 2 m Baseline 3d h 6 8-OHdG g Vessel density d 8-OHdG BrdU+ cells /mm2 8-OHdG+ area (%) i f Oxidative stress 14d n Endurance (min) e Phd2 Ligated c Lumen area (%) a +/- Viable area (%) Baseline BrdU BrdU Muscle viability (TTC) Treadmill running test at baseline Supplementary figure 2: Phd2 haplodeficiency prevents ischaemic damage. a-d, Staining for 8-hydroxy-2-deoxyguanosine (8-OHdG; red) and cell nuclei (DAPI, blue) in and crural muscles before and after ischaemia (12h). At baseline, 8-OHdG+ area is similar in both genotypes (a,b). After occlusion, (c), but not crural muscles present enhanced oxidative stress. Scale bars, 5 μm e, Quantification of the 8-OHdG+ area represented in a-d. f, g, Vessel density (f) and area (g) at baseline, 3 days and 14 days after femoral artery ligation in the soleus of and mice. h, Quantification of the TTC staining represented in i, j. i, j, Crural muscle viability by 2,3,5-tripheniltetrazolium chloride (TTC) staining is increased in mice (j) 72 hours after ischaemia. k, Quantification of BrdU+ cells in the crural muscle represented in l, m. l, m, Cell proliferation assessed by BrdU immunostaining indicates reduced muscle regeneration in mice (m) 72 hours post-ischaemia. n, Functional endurance (treadmill running test) in and mice at baseline. All bars and values show mean ± s.e.m. All experiments, n 5., P <.5 compared to., P <.5 towards baseline. 2

3 a Number of collaterals Secondary collaterals Phd2 +/- Baseline 12h 72h b Tertiary collaterals Number of collaterals Phd2 +/- 5 Baseline 12h 72h c d Phd2 +/- e Capillary density f Total capillary area g Small vessels in the thigh (diameter <2 µm) Capillaries/mm Phd2 +/- Lumen area (%) Phd2 +/- Number of vessels/ cross-section Phd2 +/- Supplementary figure 3: Phd2 haplodeficiency affects large but not small vessels. a, b, Phd2 +/- mice present increased number of secondary (a) and tertiary (b) collateral vessels, both at baseline and after ischaemia (12 and 72 hours post-ischaemia). c, d, Increased number of collaterals in (a) and Phd2 +/- (b) hindlimbs evaluated by X-ray radiography. e, f, Capillary density (e) and total capillary area (f) in the adductor of and Phd2 +/- mice at baseline. g, Number of small vessels (<2 µm in diameter) in the thigh of non-ligated and Phd2 +/- mice by micro-computed tomography. All bars and values show mean ± s.e.m. All experiments, n 6., P <.5 compared to., P <.5 towards baseline. 3

4 Desmin Collateral vessel area Bismuth+ vessel area (%) Remote Infarct myocardium j Collateral vessel density 6 4 e 8 4 Remote Infarct myocardium k h Large vessels in the heart (diameter >2 µm) 2 Capillaries/mm2 i 3 g d Capillary density (heart) l Small vessels in the heart (diameter <2 µm) Number of vessels/ cross-section f Myocardial infarction Number of vessels/ cross-section Desmin c Infarcted area (% of LV) b Bismuth+ vessels/mm2 a Supplementary figure 4: Phd2 haplodeficiency reduces myocardial damage after ischaemia. a, b, Cross-sections through the heart (desmin staining) in (a) and (b) mice 24 hours after coronary artery occlusion. Scale bars, 1 μm. c, The quantification of the infarcted area (% of left ventricular area [LV]) shows reduced infarct size in mice (b) compared to mice (a). d,e, Representative micrographs of infarcted areas (Sirius red) in (d) and (e) hearts upon gelatin-bismuth-based angiographies (black spots). Scale bars, 5 μm f, g, Collateral vessel area (f) and density (g) are increased in hearts (e) compared to (d) in both remote healthy myocardium and infarct site. h-j, Quantification of micro-computed tomographies at baseline (h) showing increased number of large vessels (>2 μm in diameter) in hearts (j) versus (i) hearts. k, Capillary density in and hearts at baseline. l, Number of small vessels (<2 µm in diameter) in and hearts at baseline by micro-computed tomography. All bars and values show mean ± s.e.m. All experiments, n 4., P <.5 compared to. 4

5 a Control b c Phd2 +/- d Migrated cells/mm 2 Macrophage sustained EC migration Control Phd2 +/- e Absorbance (a.u.) Macrophage sustained EC growth Phd2 +/- f Migrated cells/mm shsdf1 shpdgfb Macrophage sustained SMC migration Phd2 +/- $ + + $ g Absorbance (a.u.) shsdf1 shpdgfb Macrophage sustained SMC growth Phd2 +/- + + $ Supplementary figure 5: Sdf1 and Pdgfb silencing in Phd2 +/- macrophages reduces SMC migration and growth in vitro. a-d, Migration of primary endothelial cells (ECs) towards control medium (a), (b) and Phd2 +/- (c) macrophages. Quantification of transmigrating ECs is represented in d. e, EC growth in response to macrophage-derived soluble factors was comparable in both genotypes. f, g, and Phd2 +/- macrophages were transduced with lentiviral vectors carrying a shrna against Sdf1 or Pdgfb. f, Knockdown of Sdf1 or Pdgfb alone abrogates SMC migration induced by Phd2 +/- macrophages, whereas combined silencing is more effective. g, Knockdown of both Sdf1 and Pdgfb abrogates the enhanced growth of SMCs in response to Phd2 +/- macrophage-derived soluble factors, although inhibition of either factor alone is also greatly effective. A scramble shrna was used as control in f, g. All bars and values show mean ± s.e.m. All experiments, n 4., P <.5 compared to., P <.5 towards scramble. $, P <.5 towards the baseline and either treatment alone in panel f and versus shpdgfb in panel g. 5

6 a Treadmill running test at baseline (LysCre + ) b Treadmill running test at baseline Endurance (min) / lox/ lox/lox Endurance (min) HE Supplementary figure 6: Phd2 haplodeficiency in myeloid cells does not modify functional endurance at baseline. a, Functional endurance (treadmill running test) is similar in (Phd2 LysCre;/ ) or myeloidcell specifi c Phd2-haplodefi cient (Phd2 LysCre;lox/ ) or Phd2-null mice (Phd2 LysCre;lox/lox ) at baseline. b, The running capacity at baseline is comparable in recipient mice transplanted with either ( ) or Phd2 +/- (HE ) bone marrow. All bars and values show mean ± s.e.m. All experiments, n

7 Fold change relative to wt/wt Expression of PHDs in Phd2-haplodefi cient and Phd2-null macrophages (LysCre + ) Phd1 lox/ Phd2 lox/lox Phd3 Supplementary figure 7: PHD expression in Phd2-haplodeficient and Phd2-null macrophages. RNA levels of Phd1, Phd2, and Phd3 in macrophages from Phd2 LysCre;/, Phd2 LysCre;lox/, and Phd2 LysCre;lox/lox (labeled as /, lox/, and lox/lox respectively) mice. As expected, Phd2 levels were signifi cantly decreased in Phd2 LysCre;lox/ and Phd2 LysCre;lox/lox macrophages. Phd1 and Phd3 transcript levels were higher in Phd2-haplodefi cient and Phd2-null macrophages. All bars and values show mean ± s.e.m. n 4., P <.5 compared to / macrophages. 7

8 a NF-κB activity in ECs after TNF-α stimulation b Pdgfb and Sdf1 expression after NF-κB inhibition Phd2 +/- Vehicle Vehicle NF-κB inhibitor NF-κB inhibitor Luminiscence (a.u) Control TNF-α Fold change relative to vehicle Phd2 +/- Phd2 +/- Pdgfb Sdf1 Supplementary figure 8: NF-κB is selectively activated in Phd2 +/- macrophages. a, Histogram showing comparable NF-κB activity in and Phd2 +/- ECs at baseline; stimulation with TNF-α (2 ng/ml, 8 hours) unleashes NF-κB activity to the same level in both genotypes. b, Pharmacological inhibition of NF-κB activity by the exposure of macrophages to 6-amino-4-(4- phenoxyphenylethylamino)quinazoline reduces the expression of Pdgfb, and Sdf1 in Phd2 +/- macrophages compared to vehicle exposed cells. All bars and values show mean ± s.e.m. All experiments, n 4., P <.5 towards vehicle treated macrophages., P <.5 compared to control or vehicle treated cells. 8

9 SUPPLEMENTARY TABLES SUPPLEMENTARY TABLE 1: GENE EXPRESSION IN AND PHD2 +/- ECS. Phd2 +/- GENE BASELINE LIGATED BASELINE LIGATED Egln1 / Phd2 1 ± ±.17.5 ±.3.5 ±.9 Tek / Tie2 1 ± ± ±.13.9 ±.4 Ang1 1 ±.25 1 ± ± ±.75 Ang2 1 ± ± ± ±.16 Cxcl1 1 ± ± ± ±.8 Cxcl2 n.d. n.d. n.d. n.d. Cxcr4 1 ±.11.6 ±.11.9 ±.17.5 ±.8 Cx3cr1 1 ±.13.6 ±.13.9 ±.13.6 ±.11 Hgf 1 ± ± ± ±.25 Pdgfb 1 ±.6.9 ±.8.9 ±.15.9 ±.19 Plgf 1 ± ± ± ±.56 Rantes n.d. n.d. n.d. n.d. Ccl17 n.d. n.d. n.d. n.d. Ccl22 n.d. n.d. n.d. n.d. Kdr / Flk1 1 ±.14.4 ±.9.86 ±.9.4 ±.4 Flt1 1 ±.26.7 ±.17.9 ±.47.7 ±.24 sflt1 1 ±.1.7 ±.3 1. ±.1.7 ±.12 Nrp1 1 ±.4.4 ±.8.9 ±.38.5 ±.5 Cdh5 1 ±.6.8 ± ±.17.8 ±.17 Vegfa 1 ±.5 1 ±.3.8 ± ±.2 Mmp2 1 ± ± ± ±.36 Mmp9 1 ±.48.6 ± ±.68.5 ±.11 Cxcl12 / Sdf1 1 ± ±.26.8 ±.12 1, ±

10 Tgfb 1 ±.4.8 ±.5.9 ±.9 1. ±.38 Il1b n.d. n.d. n.d. n.d. Il6 1 ± ± ± ±.31 Nos3 1 ±.17.8 ±.11.8 ±.16.8 ±.14 Ccl2 1 ± ±.13.9 ± ±.13 Tnfa 1 ±.21.8 ± ±.18.7 ±.15 Cxcl1 1 ± ± ± ±.27 Il12a n.d. n.d. n.d. n.d. Ifnb n.d. n.d. n.d. n.d. Cox2 1 ± ± ± ±.2 Jagged1 1 ±.9.9 ± ±.1 1. ±.15 Icam1 1 ±.11.8 ±.13.8 ±.9.6 ±.8 The data represent the expression analysis of ECs, freshly sorted from and Phd2 +/- adductor muscles at baseline and 72 hours after femoral artery occlusion. Data are normalized towards the expression levels of ECs at baseline; n.d., not determined. All numbers show mean ± s.e.m.; n = 4-8. P <.5 towards. P <.5 towards baseline. 1

11 SUPPLEMENTARY TABLE 2: COLLATERALIZATION IN MICE HAPLODEFICIENT FOR PHD2 IN HAEMATOPOIETIC CELLS AND/OR ECS. Donor Phd2 Tie2Cre;/ Phd2 Tie2Cre;lox/ Phd2 Tie2Cre;/ Phd2 Tie2Cre;lox/ Recipient Phd2 Tie2Cre;/ Phd2 Tie2Cre;/ Phd2 Tie2Cre;lox/ Phd2 Tie2Cre;lox/ 2 nd generation collaterals 3 rd generation collaterals 5.9 ± ± ± ± ± ± ± ± 1. Reciprocal bone marrow (BM) transplantation in lethally irradiated mice reveals that enhanced arteriogenesis of Phd2-haplodeficient mice is specifically caused by loss of one Phd2 allele in BM derived haematopoietic cells (green column) but not in ECs (pink column) compared to controls (yellow column). Combined deletion of one Phd2 allele in both the haematopoietic and EC lineage (light blue column) does not modify the biological effect elicited on collateral arteries by Phd2-haplodeficient haematopoietic cells alone. All numbers show mean ± s.e.m; n = 5-8. P <.5 towards Phd2 Tie2Cre;/ Phd2 Tie2Cre;/. P <.5 towards Phd2 Tie2Cre;/ Phd2 Tie2Cre;lox/. 11

12 SUPPLEMENTARY TABLE 3: PHD2 HAPLODEFICIENCY IN ECS OR SMCS DOES NOT CONFER COLLATERAL PRECONDITIONING. Phd2 Cre;/ Phd2 Cre;lox/ Promoter of Cre Secondary Tertiary Secondary Tertiary recombinase collaterals collaterals collaterals collaterals VE-Cadherin 9. ± ± ± ± 2. PDGFRB 8.2 ± ± ± ± 1.6 The data represent the number of secondary (light grey column) and tertiary (light blue column) collateral branches in mice haplodeficient for Phd2 in ECs or SMCs at baseline. Mice carrying one floxed Phd2 allele were intercrossed with deleters expressing the Cre recombinase under an EC specific promoter, that is, VE-Cadherin, or a SMC specific promoter, that is, PDGFRB. All numbers show mean ± s.e.m; n =

13 SUPPLEMENTARY TABLE 4: LIST OF PRIMERS USED FOR QPCR. GENE (ACCESSION) Nos2 (NM_1927.3) PROBE FORWARD REVERSE ACT-ATA-ACT- CCA-TCA-AAA- AGC-CCG-GGA- TGA-AGC-CGC- GGA-GTG-GCT- CTT-CAT-CAA-TC TGC-TCA-TGA-G CCC-AG Nos3 ACT-ATA-ACT- CCA-TCA- AGC-CCG-GGA- TGA-AGC-CGC- (NM_8713.4) AAAGGA-GTG- GCT-CCC-AG CTTCAT-CAA-TC TGCTCA-TGA-G Icam1 (NM_1493.2) CCT-CAT-GCA- AGG-AGG-ACC- TCA-GCC-T GTC-CGT-GCA- GGT-GAA-CTG- TTC CTT-TCA-GCC- ACT-GAG-TCT- CCA-A Pdgfb (NM_1157.3) CCC-ATC-TTC- AAG-AAG-GCC- ACA-GTG-ACC-T CGG-TCC-AGG- TGA-GAA-AGA- TTG CGT-CTT-GGC- TCG-CTG-CTC Egln1 / Phd2 (NM_5327.2) ACG-AAA-GCC- ATG-GTT-GCT- TGT-TAC-CCA GCT-GGG-CAA- CTA-CAG-GAT- AAA-C CAT-AGC-CTG- TTC-GTT-GCC-T Flt4 (NM_829.3) CGG-CGA-GCC- CCA-CTT-GTC- CA GGT-TCC-TGA- TGG-GCA-AAG-G TCA-GTG-GGC- TCA-GCC-ATA- GG sflt1 (NM_1228.3) TTT-GCC-GCA- GTG-CTCACC- TCT-AAC-G GAA-GAC-ATC- CTTCGG-AAG- CAC-GAA TTG-GAG-ATC- CGAGAG-AAA- ATG-G Cxcl12 / Sdf1 CGG-TAA-ACC- CCG-CGC-TCT- GCG-ATG-TGG- 13

14 (NM_ ) AGT-CAG-CCT- GAG-CTA-CCG GCAT-CAG-T CTC-TCGAAG-A Tgfb (NM_9368.3) GCG-GAC-TAC-T TGC-TAA-AGA- GGT-CAC-CCG- CGT-GCT GAG-CCC-GAA- GCG-TTG-TTG- CGG-TCC-AC For the following genes with sequence ID (enclosed between brackets), commercially available primers were ordered from Applied Biosystems ( Actb (Mm67939_s1), Ang1 (Mm456498_m1), Ang2 (Mm545822_m1), Arg1 (Mm475991_m1), Cnn1 / Calponin-1 (Mm48732_m1), Ccl17 (Mm516136_m1), Ccl22 (Mm436439_m1), Cox2 (Mm478374_m1), Cxcl1 (Mm433859_m1), Cxcl1 (Mm _m1), Cxcl2 (Mm43645_m1), Cx3cr1 (Mm26211_s1), Cxcr4 (Mm _m1), Fizz (Mm44519_m1), Flt1 (Mm121866_m1), Hgf (Mm _m1), Ifnb (Mm439552_s1), Il1a (Mm434169_m1), Il1b (Mm _m1), Il6 (Mm121733_m1), Ccl2 (Mm441242_m1), Mmp2 (Mm43956_m1), Mmp9 (Mm442991_m1), Nfkb1 / p5 (Mm476361_m1), Rela / p65 (Mm51346_m1), Nrp1 (Mm125321_m1), Myh1 / NMHC-B (Mm85131_m1), Egln2 / Phd1 (Mm51967_m1), Egln3 / Phd3 (Mm4722_m1), Plgf (Mm435613_m1), Rantes (Mm132428_m1), Tagln / Sm22 (Mm44166_m1), Smtn / Smoothelin (Mm449973_m1), Tek / Tie2 (Mm443243_m1), Tnfa (Mm443258_m1), Vegfa (Mm43734_m1), Ym1 (Mm657889_mH), Acta2 / SMA (Mm _m1), Jagged1 (Mm49692_m1), Kdr / Flk1 (Mm _m1), Cdh5 / VE-cadherin (Mm486938_M1). 14

15 SUPPLEMENTARY NOTES SUPPLEMENTARY NOTE 1: To measure the silencing of Sdf1 and Pdgfb, we performed qpcr on and Phd2 +/- pmø at 4 days after transduction with a lentiviral vector carrying a shrna against Sdf1 or Pdgfb. Compared to their scramble control, the knockdown for Sdf1 was 77 ± 5.% and 71 ± 2.6%, and for Pdgfb 81 ± 3.2% and 87 ± 5.9% in and Phd2 +/- pmø, respectively (n = 4-6; P <.1). SUPPLEMENTARY NOTE 2: Collateral arterial growth was quantified 5 weeks after BM transplantation, when haematopoietic reconstitution by GFP + blood cells was, on average, 82 ± 8% and differential white blood counts were comparable in all the groups (not shown). SUPPLEMENTARY NOTE 3: Five weeks after injection of BM cells from Phd2 Rosa26CreERT;lox/ mice into irradiated recipients (HE Rosa26CreERT ), transplanted mice were treated with vehicle or tamoxifen (1 mg/mouse) for 5 days. Administration of tamoxifen induced about 5% reduction of Phd2 levels in macrophages (copies of Phd2 / 1 5 copies -actin: 62.7 ± 6.8 in vehicle versus 34 ± 6.9 in tamoxifen treated mice; n = 4; P =.1). SUPPLEMENTARY NOTE 4: To address whether the induction of Phd3 rescued the activation of NF- B pathway by loss of Phd2, we silenced Phd3 in Phd2 LysCre;/, Phd2 LysCre;lox/ and Phd2 LysCre;lox/lox macrophages carrying the NF- B-responsive luciferase reporter. The knockdown of Phd3 was 63 ±.3%, 6 ±.4%, and 37 ±.1% in Phd2 LysCre;/, Phd2 LysCre;lox/, and Phd2 LysCre;lox/lox macrophages, respectively, compared to their scramble controls (n = 4; P <.1). SUPPLEMENTARY NOTE 5: To dissect the axis of NF- B activation in Phd2 +/- macrophages, we silenced the main components of the canonical pathway, that is, p65 (Rela) and p5 (Nfkb1) in macrophages carrying the NF- B-responsive luciferase reporter. The efficiency of knockdown for Rela was 52.7% ± 1.4% and 5.2% ±.4% in and Phd2 +/- macrophages compared to their scramble control. Respectively, the 15

16 efficiency of knockdown for Nfkb1 was 49.6% ±.9% and 59.1% ± 1.2% in and Phd2 +/- macrophages compared to their scramble control (n = 4, P <.1). SUPPLEMENTARY NOTE 6: Noteworthy, Phd2 haplodeficiency did not affect collaterogenesis and ischaemic necrosis in a Swiss/129 mixed background (not shown and 11 ). This is consistent with the observation that strain-related genetic differences profoundly influence the collateral vasculature

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