A total of 47,232 small RNA clone sequences distributed over 65 tissues, including the kidney glomerulus, were obtained from a recent survey . Two compendia with microarray data from mouse tissues, including lung [17, 18], were downloaded from the NCBI Gene Expression Omnibus repository. To ensure consistent mapping between datasets, clone/probe sequences were re-annotated against miRBase release 10.1  using a proprietary Matlab (Mathworks Inc. Natick, MA, USA) script. For each mature miRNA, a P-value for over-representation in the glomerulus library compared to the other tissues was calculated using Fisher's exact test. Likewise, P-values for differential expression in the lung compared to remaining adult tissues were determined using the Student's t-test. The t-test provides a useful metric of differential tissue expression, although the formal requirements for the underlying distribution of the data may not be completely met . Genomic localization of miRNAs was evaluated using data derived from the UCSC browser (July 2007 assembly) .
Isolation of CD31+ microvascular fragments and TaqMan qRT-PCR
Microvascular fragments were isolated from mouse tissues and embryonic stem cell cultures using mechanical and enzymatic digestion followed by incubation with magnetic Dynabeads coated with anti-CD31 (anti-platelet endothelial cell adhesion molecule (PECAM)). The procedure was performed essentially as described previously . All mice were adult (8 to 12 weeks old) males, either wild-type C57BL/6 or Pdgfb
backcrossed for seven generations onto a C57BL/6 background . RNA from vascular fragments and remaining tissue was prepared using miRNeasy Mini spin columns (Qiagen, Hilden, Germany). Samples were quantified with a NanoDrop spectrophotometer (Thermo Scientific Corporation, Waltham, MA, USA) and cDNA was synthesized using equal amounts of RNA in each reaction (High-Capacity Reverse Transcription Kit or MicroRNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA). Expression levels were determined using pre-designed TaqMan assays (Applied Biosystems) on a 7900 HT real-time PCR system, according to the manufacturer's instructions. Relative levels were calculated using the 2-Ct method. Fli1 mRNA levels were determined using SYBR Green quantitative qPCR (95°C, 55°C, 72°C, 40 cycles) using the following primers: 5'-TATCAGATCCTGGGGCCAAC-3' and 5'-CTCATCAGGGTCCGTCATTT-3'.
Differentiation of embryonic stem cells into vascular sprouts
The murine embryonic stem cell line R1  was routinely cultured on growth arrested mouse embryonic fibroblasts in stem cell medium composed of DMEM-Glutamax (Invitrogen, Carlsbad, CA, USA) supplemented with 25 mM HEPES pH 7.4, 1.2 mM sodium pyruvate, 19 mM monothioglycerol (Sigma-Aldrich, St. Louis, MO, USA), 15% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA), and 1,000 U/ml leukemia inhibitory factor (Chemicon International/Millipore, Billerica, MA, USA). EBs were generated by aggregation of stem cells in hanging drops in the absence of leukemia inhibitory factor, as described previously . Briefly, EBs were collected after 4 days and seeded into 12-well dishes onto a layer of 0.9 ml solidified collagen type I solution composed of Ham's F12 medium (Promocell, Heidelberg, Germany), 6.26 mM NaOH, 20 mM HEPES, 0.117% NaHCO3, 1% Glutamax-I (Gibco) and 1.5 mg/ml collagen I (PureCol, Advanced BioMatrix, San Diego, CA, USA). Immediately thereafter, a second layer of 0.9 ml collagen solution was added on top and allowed to polymerize. After 3 hours, 0.9 ml of stem cell medium supplemented with vascular endothelial growth factor A (VEGFA; PeproTech, Rocky Hill, NJ, USA), at a final concentration of 30 ng/ml, was added to induce angiogenic sprouting. The medium was replaced every second day. EBs were excised from the gels at day 14 and immediately processed for isolation of CD31+ vascular fragments, as described above. NG2+ cells were isolated with the same protocol using a rabbit anti-rat NG2 antibody (Chemicon; AB5320), after depletion of CD31+ cells from the cultures.
In situ hybridization and immunohistochemistry
In situ hybridization was performed using a 3' DIG-labeled miRCURY LNA probe to mouse miR-145 and miR-126 (Exiqon, Vedbaek, Denmark) as previously described . For dual detection of miR-145 and the pericyte marker NG2, the immunostaining was performed after development of the in situ signal. Slides were washed in phosphate-buffered saline, blocked with 3% donkey serum and 1% bovine serum albumin in phosphate-buffered saline, then incubated with rabbit anti-rat NG2 antibody (Chemicon; diluted 1/50) overnight at 4°C, washed in phosphate-buffered saline, then detected with Alexa488-conjugated donkey anti-rabbit IgG (Invitrogen; diluted 1/200).
Vascular aortic endothelial cell culture, scratch wound and proliferation assays
Mouse vascular aortic endothelial cells (VAECs; Dominion Pharmakine, Derio-Bizkaia, Spain) were cultured in RPMI 1640 media (Sigma) supplemented with 10% fetal calf serum (Gibco), 1 μg/ml dexamethasone, 10 U/ml heparin, 50 U/ml penicillin/streptomycin and 75 μg/ml EC growth factor supplement (Sigma). For scratch wound migration assays, cells were transfected by electroporation (Nucleofector system, Basic Endothelial Cell Kit, Amaxa Inc/Lonza group ltd, Basel, Switzerland) using 0.5 μg of synthetic mature miR-145 double-stranded RNA (dsRNA; Pre-miR-145; Applied Biosystems) or negative control dsRNA (Stealth siRNA negative control; Applied Biosystems), seeded onto 6-well plates and cultured for 48 hours. Scratch wounds were generated in the cell monolayer using a pipet tip and each wound was photographed at 0 and 24 hours. Wound widths were evaluated blindly at both time-points and the average amount of closure was determined for each replicate transfection. VAEC proliferation was measured by quantification of 5'-bromo-2'-deoxyuridine (BrdU) incorporation. Cells were pulsed for 4 hours with 20 μM BrdU and DNA synthesis was determined using a colorimetric ELISA (Calbiochem/Merck, Darmstadt, Germany) according to the manufacturer's instructions. Absorbance was measured at dual wavelengths of 450 to 540 nm.
Microfluidic migration chamber
Migration of HUVECs in response to a stable gradient of VEGFA-165 (PeproTech; 0 to 50 ng/ml over a distance of 400 μm) or BJ-hTERT (human foreskin fibroblast) cells in response to platelet-derived growth factor (PDGF)-BB (0-20 ng/ml) was examined using a microfluidic chemotaxis chamber, essentially as previously described . HUVECs were transferred to 3-cm culture dishes coated with type A gelatin from porcine skin (Sigma) and were allowed to attach to the dish in EGM-2MV medium (Lonza) with serum and supplement growth factors. After 2 hours the medium was aspirated and the cells were transfected with 0.5 μg of Pre-miR negative control, Pre-miR-145, Anti-miR negative control or Anti-miR-145 (Applied Biosystems) using siPORT NeoFX (Ambion, Austin, TX, USA) in serum and growth factor free EBM-2 medium (Lonza) containing 0.2% bovine serum albumin. After 24 hours the gradient experiment was initiated. BJ-hTERT cells were cultured in minimal essential medium (MEM, Invitrogen) containing 10% fetal calf serum (Gibco), 1 mM sodium pyruvate (Gibco) and non-essential amino acids (Gibco). Cells were transfected using electroporation (0.5 μg, Nucleofector system, Amaxa) and were allowed to rest between 24 and 48 h before being seeded onto gelatin A-coated culture dishes and serum starved overnight, before onset of gradient. VEGFA-165 or the PDGF-BB gradients were generated in serum-free cell medium. Cell migration was tracked during 3 hours (HUVECs) or 4 hours (BJ-hTERT cells) using a Cell Observer System (Carl Zeiss AB, Stockholm, Sweden) fitted with a Zeiss Axiovert 200 microscope, an AxioCam MRm camera, a motorized X/Y stage, and an XL incubator with equipment for temperature and CO2 control (Zeiss). Cells were kept in a humidified atmosphere of 5% CO2 in air at 37°C during all experiments. AxioVision software (Zeiss) was used for time-lapse imaging and cell tracking.
Luciferase reporter assays
Oligonucleotides (65 bp) harboring wild-type or mutated miR-145 binding sites from the mouse Fli1 3' UTR (Additional data file 1) were annealed and ligated into the HindIII and SpeI sites of the pMIR-REPORT CMV-firefly luciferase reporter vector (Applied Biosystems). All constructs were verified by sequencing. HEK293 cells were seeded onto 24-well plates at a density of 50,000 cells/well and cultured overnight in DMEM (10% fetal calf serum) without antibiotics. Cells were transfected with 60 ng of pMIR-REPORT, 8 ng of pRL-SV40 renilla luciferase control vector and 10 pmol of Pre-miR negative control or Pre-miR-145 using Lipofectamine 2000 (Invitrogen) and luciferase activity was assayed after 48 hours using the Dual-Luciferase Reporter System (Promega, Madison, WI, USA).
Long mouse and human Fli1 3' UTR fragments were amplified by PCR using the following primers (numbers indicate the position starting from the stop codon): 5'-AACTAACACCAGTTGGCCTTC-3' and 5'-CGTCAGGAGTGTCTGAGTTTG-3' (1-704); 5'-GCTTCTTCTAGCTGAAGCCCATC-3' and 5'-GTCAAATTATTTTACAACATGG-3' (3-1,391). Amplimers were cloned in psiCHECK-2 (Promega) to generate Renilla luciferase-3' UTR reporter constructs. Basal expression of firefly luciferase from the same plasmid served as an internal control. HEK293T cells seeded in 96-well plates were cotransfected with plasmid (50 ng per well) and synthetic miRNA (0.25 to 2.5 pmol per well; Biomers, Ulm, Germany) using Lipofectamine 2000. Luciferase activity was assayed 24 hours after transfection as described . Nucleotides 2 and 4 in the seed region of three predicted miR-145 sites within the mouse 3' UTR fragment were mutated using Multisite-Quickchange (Stratagene/Agilent, Santa Clara, CA, USA). Results represent Renilla/firefly luciferase ratios from four independent experiments performed in triplicates. Statistical significance was evaluated using Student's t-test.
Western blot analysis
VAECs were electroporated with either Pre-miR-145 or Pre-miR negative control as described above. Nuclear extracts were prepared using the CelLytic NuCLEAR kit (Sigma) at 72 hours post-transfection. Western blotting was performed using a Fli1 antibody (Sc-356; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 2 mg/ml and ECL reagents (Amersham Biosciences/GE Healthcare Bio-Sciences, Uppsala, Sweden). As a loading control, the membrane was stripped and reprobed using a lamin A/C antibody (Sc-7293, Santa Cruz Biotechnology) at a dilution of 1/1,500. Densitometric analysis was performed using ImageJ software.