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Blood, Vol. 114, Issue 2, 469-477, July 9, 2009
Endothelial deletion of hypoxia-inducible factor–2 (HIF-2) alters vascular function and tumor angiogenesis
Blood Skuli et al.
114: 469
Supplemental materials for: Skuli et al
Antibodies
Antibodies were purchased from commercial sources and used according to manufacturers’ instructions. Specifically: CD31 (Clone MEC13.3, BD Pharmingen), Actin, F4/80, and Ki67 (Abcam), smooth muscle actin (clone 1A4, Sigma), S100 (Lab Vision), HIF-1α (Transduction Labs) and HIF-2α (Novus). Alexa 488 conjugated anti-mouse, rabbit, and goat IgG were purchased from Invitrogen. Generation of HIF-2α, VE-Cadherin Cre mice
Endothelial specific deletion of HIF-2α was generated by breeding Hif-2αfl∕fl mice with transgenic mice expressing Cre recombinase under the VE-cadherin promoter to obtain Hif-2αfl∕+, Cre control mice (designated “C”), as described in Fig. S1A. These mice were bred to Hif-2α+∕Δ mice harboring global deletion of one Hif-2α allele to obtain experimental Hif-2αfl∕Δ, Cre (KO) mice. All animal experiments were conducted in accordance with National Institutes of Health guidelines for use and care of live animals and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). Hif-2α wild type, conditional floxed (fl), and deleted (Δ) genomic alleles can be distinguished using a multiplex PCR (1 and Fig. S1B). Genotyping for VE-cadherin Cre and ROSA26 mice has been described previously (2). Preparation of EC populations
Lungs from control or KO mice were macerated in sterile PBS, filtered through a 75 µM mesh and digested with collagenase at 37°C for 1 hr as described (3). Following hemolysis with ACK buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.4) for 5 min at room temperature, cells were pelleted, resuspended in EGM media (Lonza) and plated on fribonectin-coated 35 mm2 plates (R&DSystems). Confluent cultures were trypsinized and incubated with CD31 antibody-conjugated Dynal beads (Invitrogen). Selected cells were replated, immortalized by infection with the pMigR1 vector encoding green fluorescence protein-conjugated polyomavirus middle T antigen. Immortalized cells were subjected to two additional rounds of CD31 selection. Purity of the endothelial cells was determined by their ability to take up Dil-Ac-LDL (10 µg/ml) (Biomedial Technology Inc), or flow cytometry following phycoerythrin-CD31 (BD Pharmingen) staining (FACS Scan, Becton Dickson). Endothelial cells with HIF-2α acute deletion (Cont3/KO3)
Lungs from Hif-2α2L∕1L, Ubc-CreERT2 mice were isolated and processed as described above to obtain endothelial cells. The Ubc-Cre-ERT2 transgene (4) expresses the Cre recombinase under the control of the human Ubiquitin C promoter. Cre-ERT2 recombinase was activated in cells by adding Tamoxifen (1µM; MP Biomedicals) in culture media. DNA extracted from cells shows that recombination is highly efficient. Matrigel and Fibronectin adhesion assay
KO and related control ECs were seeded into 24-well plates coated with Matrigel (10 µg/ml, BD Biosciences) or Fibronectin (1 µg/ml, Upstate) and incubated for 1h at 37°C under normoxic or hypoxic conditions. Cells were then carefully washed 3 times with PBS, fixed and counted. The number of adherent cells was counted in 6 representative HPFs (high power fields). The data are representative of three independent experiments. Vascular permeability assessment
For chronic permeability studies, control and KO mice were anesthetized with Avertin and tail veins injected with Evans blue dye (Sigma) at 50 mg/kg (0.1 ml of 0.5% solution/10 g). After 6 hours, mice were perfused with 30 ml PBS from the left ventricle to remove the dye in the circulation, and organs removed. Tissues were minced, dried (55°C, 24 hr), weighed and extracted with formamide (1 ml) at 56°C for 48 hours. Extracted dye was measured by absorbance at 620nm and calculated against a standard curve of known concentrations. Data were normalized to the tissue weight (N=6). For acute permeability studies, mice were injected with Evans blue dye as described above. Recombinant murine VEGF (50 ng in 50 µl volume) (RDI) or PBS vehicle control (50 µl) was then injected intradermally into the mouse back skin. After 30 minutes, mice were sacrificed and back skin removed, photographed and weighed. Leaked dye was quantified as described previously (N=8 for each age). Lung plasma protein leakage was assessed by H&E staining of lung sections. Echocardiography and determination of heart parameters
Protocols for murine transthoracic echocardiography have been previously described (5). Briefly, mice were anesthetized using an integrated isoflurane-based system that maintains a dose of 1.0–2.0% isoflurane. Animals were kept warm on a heated platform, and the temperature and surface ECG were continuously monitored and recorded. Transthoracic echocardiography was performed using a Vevo 770 Visual Sonic machine (Toronto, Canada) equipped with a linear 30-MHz probe (RMV 707B). Two-dimensional images were recorded in the parasternal long- and short-axis views to guide M-mode records obtained at the mid-ventricular level in both views. Digital images were obtained at a frame rate 180 images/sec. The diastolic thicknesses of interventricular septum (IVS) and left ventricular posterior wall (LVPW), and LV diastolic dimensions (LVIDd) were measured and the average value from both views reported. The diameter of the pulmonary artery was measured during systole at the parasternal short-axis view of the great vessels. The thickness and diameter of the right ventricle (RV) were measured during diastole by rotating the transducer 30° counterclockwise from the right parasternal short axis view. Measurement of pulmonary arterial (PA) pressure
Following anesthetization with Avertin, trachea was cannulated, and mice ventilated using a MiniVent Type 845 (Harvard Apparatus). The chest cavity was opened to expose the heart and a Micro-Tip Catheter Transducer SPR-1000 (Millar Instruments) was inserted into the right ventricle (RV) and subsequently threaded into the pulmonary artery (PA). Systolic PA pressure was then measured, recorded on a PowerLab 4/30 instrument (ADInstruments), and analysed using Chart 5 Pro software (ADInstruments). The transducer was calibrated prior to obtaining measurements for each mouse and the quality of the pressure wave was monitored. Pressure measurements associated with heart rates outside the range of 300–500 bpm were excluded from analysis. For each mouse, 2–4 measurements were analysed, each corresponding to the average of 10–20 individual data points. Mice were then sacrificed and hearts were weighed to establish the heart-to-body weight ratios (N=12 for each age). Tumor volume measurements
Tumor dimensions were measured with a caliper on days indicated and volume calculated using the formula: v=π/6 × A × B2, where A is the larger diameter of the tumor and B is the smaller diameter of the tumor. Immunohistochemistry and immunofluorescence
Vessel density was calculated by counting CD31 positive vessels in 5 representative fields at 20× magnification (N=6 for each genotype). Tumor cell proliferation in xenografts was determined by immunohistochemistry for Ki67. For quantification, positively stained cells in 5 consecutive fields at 40× magnification were counted from the edge towards the center of each section (N=6). Hypoxia and apoptosis levels in tumor sections were quantified using color thresholding techniques in Metamorph software (Molecular Devices) and values expressed as a percentage of the entire image. Knockdown of HIF-1α and HIF-2α using siRNAs
Control siRNA (Qiagen) and siRNAs targeting HIF-1α and HIF-2α (respectively purchased from Qiagen and Dharmacon) were used according to manufacturer’s instructions. Briefly, MS1 cells (60% conlfluency) were transfected with siRNAs at a final concentration of 20 nM by using DharmaFECT transfection reagent. 24 hrs post-transfection, cells were cultured at 21% or 0.5% O2 for 16 hrs prior to harvesting protein and mRNA. Primer sequences for QRT-PCR
β-actin
Fwd, 5′-TTTGCAGCTCCTTCGTTGC-3′, Rev, 5′-CATCCATGGCGAACTGGTG-3′
PGK
Fwd, 5′-GGAAGCGGGTCGTGATGA-3′; Rev, 5′-GCCTTGATCCTTTGGTTGTTTG-3′
Ang-1
Fwd, 5′-GTATGTTGCAAATGCGCTCTCA-3′, Rev, 5′-AGGCATCGAACCACCAACC-3′
Ang-2
Fwd, 5′-ACTGACTGATGTGGAAGCCCA-3′, Rev, 5′-AGCTCGAGTCTTGTCGTCTGG-3′
Dll4
Fwd, 5′-AGCTGGGTGTCTGAGTAGGC-3′; Rev, 5′-AGAAGGTGCCACTTCGGTTA-3′
VEGF
Fwd, 5′-TACCTCCACCATGCCAAGTG-3′, Rev, 5′-ACAGGACGGCTTGAAGATGTA-3′
ET-1
Fwd, 5′-TTCCCGTGATCTTCTCTCTGCT-3′; Rev, 5′-TCTGCTTGGCAGAAATTCCA-3′
ET-B
Fwd, 5′-TCAACACGGTGGTGTCCTGC-3′; Rev, 5′-ACTGAATAGCCACCAATCTT-3′
FN1
Fwd, 5′-GCGACTCTGACTGGCCTTAC-3′; Rev, 5′-CCGTGTAAGGGTCAAAGCAT-3′ REFERENCES 1. Gruber, M., et al. (2007) Acute postnatal ablation of Hif-2alpha results in anemia Proc Natl Acad Sci U S A 104: 2301–2306.
2. Alva, J. A., et al. (2006) VE-Cadherin-Cre-recombinase transgenic mouse: a tool for lineage analysis and gene deletion in endothelial cells Dev Dyn 235: 759–767.
3. Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J. & Lechler, R. I. (2000) Isolation of endothelial cells from murine tissue J Immunol Methods 244: 205–15.
4. Ruzankina Y., Pinzon-Guzman, C., Asare, A., et. al. (2007) Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1: 113–26.
5. Trivedi CM, Luo Y, Yin Z, et al. Hdac2 regulates the cardiac hypertrophic response by modulating Gsk3 beta activity. Nat Med. 2007;13:324–331.
Files in this Data Supplement:
- Figure S1. Generation and characterization of the Hif-2α conditional allele (JPG, 185 KB)
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(A) Breeding scheme used to obtain endothelial-specific Hif-2α KO (Hif-2αfl∕Δ, VE-cadherin-Cre) and Control (Hif-2αfl∕+, VE-cadherin-Cre) mice. fl = floxed allele; Δ = 1− deleted allele; + = wild type allele. (B) Diagram of the Hif-2α alleles used. Black arrowheads represent loxP sequences. PCR primers (blue arrows) distinguish Hif-2α WT (400 bp, primers 1, 2), Hif-2α fl (434 bp, primers 1, 2) and Hif-2α Δ (300 bp, primers 1, 3) alleles. Primers 1 and 3 fail to amplify products from WT or Hif-2α fl alleles. Red bar indicates position of Southern blot probe. (C) Hif-2αfl∕Δ, Cre (KO) embryos dissected at E11.5 with or without yolk sac. No apparent defects were detected for KO embryos and yolk sacs. (D) Analysis for whole blood by complete blood count indicating no significant differences between control and KO mice. WBC, white blood cells; NE, neutrophils; LY, lymphocytes; MO, monocytes; RBC, red blood cells; HB, hemoglobin; HCT, hematocrit; MCHC; mean corpuscular hemoglobin concentration. N=9.
- Figure S2. Increased vessel permeability in KO mice (JPG, 118 KB)
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Photographs of Evan’s blue dye leakage 30 minutes following intradermal injection of either PBS or VEGF into the shaved dorsal skin of 3 month-old (A) or 6 month-old (B) control and KO mice. N=8 for each age.
- Figure S3. Decreased metastasis in KO mice (JPG, 484 KB)
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(A) H&E stain of normal lungs and lungs with metastasis from control mice. B16F10 tumor metastasis (black arrows) is shown with 2 different magnifications (50× upper panel and 200× lower panel). (B) Staining for S100, a melanoma cell marker, in normal lungs and lungs with metastasis from control mice. B16F10 tumor metastasis (black arrows) is shown with 2 different magnifications (50× upper panel and 200× lower panel). (C) Percentage of control and KO mice with B16F1 (poorly metastatic) / B16F10 (highly metastatic) metastasis. N=12 for B16F1 cells and N=9 for B16F10 cells. ** P<0.01.
- Figure S4. Dll4, VEGF, Ang2, Flk-1, and Flt-1 gene expression is regulated by HIF-α subunits under hypoxia in murine endothelial MS1 cells (JPG, 181 KB)
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(A) Western blot analysis of HIF-1α and HIF-2α protein expression in MS1 cells transfected with siRNAs and subjected to 21% (N), or 0.5% (H) O2 for 16 hrs. (B) Hypoxic induction of Dll4, VEGF, Ang2, Flk-1, and Flt-1 gene expression in MS1 cells transfected with specific siRNAs targeting HIF-1α and/or HIF-2α. Specific siRNAs compared to control scrambled siRNA * P<0.05.
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