PHD2 regulates arteriogenic macrophages through TIE2 signalling

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.


Methods
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Tie2 silencing in mature hematopoietic cells: To silence Tie2 expression specifically in mature hematopoietic cells, HSPC isolated as described above were transduced with LVs carrying an artificial microRNA either against Tie2 (amiR(Tie2)) or a control microRNA targeting Luciferase (amiR(Luc)), as described above. Transduced cells were then superinfected with a second LV carrying the reverse tetracycline transactivator (rtTA-m2-miR-126T) (Mazzieri et al, 2011). Sixteen hours after transduction, 10 6 cells were infused into the tail vein of lethally irradiated, 6-week-old, female Balb/C mice (radiation dose: 7.5 Gy). Twenty-five days after irradiation, doxycyline (Sigma) or vehicle was administered three times a week at a dose of 25 mg per kg of body weight.
Ligation was performed 6 weeks after irradiation.

TEM depletion:
To deplete TEMs, we infused 6-week-old, lethally irradiated female Balb/C mice with 10 6 HSPCs co-transduced with two LVs, Tie2:tk (to deplete TEMs) and PGK:GFP (to label all BM-derived cells in transplanted mice), using LV doses equivalent to 10 8 LV Transducing Units/ml for each vector, as described above (De Palma et al, 2005). Depletion of TEMs was achieved by oral gavage of ganciclovir (GCV, 50 mg/kg daily or vehicle as control) 4 weeks after transplantation for 10 days before and 6 days after femoral artery ligation.
Administration of sTIE2: The BM of WT or Phd2 +/mice was transplanted into lethally irradiated WT mice. After 5 weeks, mice were injected both systemically (5x10 11 vp in the tail vein) and locally (5x10 9 vp directly in two sites of the adductor) with an AAV9 encoding the mouse extracellular domain of TIE2 (AAV-sTIE2) (Holopainen et al, 2012). AAV9 encoding for serum albumin (AAV-Alb) was used as control. One week after injection of the viral vector, mice were subjected to femoral artery ligation. Blood and adductor samples were harvested at baseline and 72h post-ligation and used to analyze bone marrow reconstitution by FACS or sort F4/80 + GFP + macrophages, respectively.
Overexpression of ANG1 or ANG2: WT mice were injected locally (5x10 9 vp directly in two sites of the adductor) with an AAV9 encoding ANG1 or ANG2 (Anisimov et al, 2013). AAV9:Albumin was used as negative control. 14 days after injection, mice were sacrificed, and adductors were harvested for FACS sorting of adductor macrophages or for RNA extraction from whole muscle.
Vector copy number analysis: Transduced HSPCs were cultured and collected after 9 days, whereas blood from the transplanted mice was collected 4 weeks after HSPC transplantation to measure the number of integrated LV copies/cell genome (vector copy number, VCN) by qRT-PCR, as previously described (De Palma et al, 2005 ELISA quantification of sTIE2: sTIE2 was quantified in plasma using the Quantikine mouse TIE2 ELISA kit (R&D systems) according to the manufacturer's instructions. Plasma was prepared from blood samples that were collected at the time of sacrifice by retro-orbital bleeding.
Necrotic areas in the crural muscle were identified by staining the sections with haematoxylin and eosin (H&E). Necrotic cells display a more glassy homogeneous appearance in the cytoplasm with increased eosinophilia, whereas the nuclear changes are reflected by karyolysis, pyknosis and karyorrhexis. Necrotic area was defined as the percentage of area that includes these necrotic myocytes, inflammatory cells and interstitial cells, compared to the total soleus area. After Macrophage preparation: To harvest peritoneal macrophages (pMØ), the peritoneal cavity was washed with 5 ml of RPMI 10% FBS. The pooled cells were then seeded in RPMI 10% FBS in 24well plates (5x10 5 cells/well). After 6 hours of incubation at 37°C in a moist atmosphere of 5% CO2 and 95% air, non-adhering cells on each plate were removed by rinsing with phosphate-buffered saline (PBS). Adherent macrophages were cultured in RPMI 10% FBS for 16 hours. When higher amounts of cells were needed, macrophages were derived from bone marrow precursors (bone marrow derived macrophages, BMDM) as described before (Meerpohl et al, 1976). Briefly, bone marrow cells (2x10 6 cells/ml) were cultured in a volume of 5 ml in a 10 cm Petri dish (non tissue culture treated, bacterial grade) for 7 days in DMEM supplemented with 20% FBS and 30% L929 conditioned medium as a source of M-CSF. The cells obtained in those cultures are uniformly macrophages.

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The siRNA sequences used are the following: after inhibition of NF-kB pathway were measured by qRT-PCR on pMØ exposed for 12 hours to 500 nM 6-amino-4-(4-phenoxyphenylethylamino)quinazoline.