Human umbilical cord vein endothelial cells (HUVECs) and human bone marrow-derived endothelial cells (HBMECs) were isolated as previously described37
. Human aortic endothelial cells (HAECs) and human microvascular endothelial cells (HMVECs) were obtained from Invitrogen. Cells were cultured in endothelial cell growth medium (Medium 199, 20% (v/v) fetal bovine serum (FBS), 20 μg ml−1
endothelial cell growth supplement (Hallway), 1% (w/v) antibiotics (Hallway), and 20 units ml−1
Virus plasmids and virus generation
To construct constitutively active RAF (CA-Raf), the human RAF
open reading frame was amplified using HUVEC cDNA and cloned into pCCL.PGK.CAAX vector, which added the K-RAS
carboxy-terminal localization signal to the C terminus of RAF
. The E4ORF1
gene of adenovirus (serotype 5), mouse constitutively active Akt1
(myristoylated Akt: myrAkt
, a gift from L. E. Benjamin26
, human constitutive active K-RAS
), and polyoma middle-T antigen
, a gift from W. Sessa, Yale University School of Medicine, USA) were amplified by PCR and cloned into pCCL.PGK lentivirus vector17
-resistance(Neo) genes were also cloned into pCCL.PGK vector and used as a control for lentiviral infection of gene expression and HSPC expansion assay, respectively. Akt-resistant FoxO1 (FoxO1-TM
) cDNA was obtained from Addgene (Addgene plasmid 13508; ref. 38
) and cloned into pCCL.PGK vector by PCR. Lentiviral plasmids expressing shRNA against mTOR
were obtained from Addgene (Addgene plasmid 1855 (#1) and 1856 (#2); ref. 39
) and those against Jagged-1
, and scrambled-sequence control shRNA (pLKO-scramble
) were purchased from Open biosystems. Lentiviruses were generated as described previously17
and cells were transduced with lentivirus at multiplicity of infection (MOI) of 10, and maintained in endothelial cell growth medium. For shRNA experiments, cells were cultured in the presence of 500 ng ml−1
puromycin during a selection period of 4 days.
Tube formation assay
PECs were starved in M199 for 6 h and then 100,000 cells were cultured on 250 μl of Matrigel (BD bioscience) in assay medium (Medium 199, 2% (v/v) fetal calf serum (FCS), 20 ng ml−1 VEGF-A, 10 ng ml−1 FGF2 and heparin) for 16 h in 24-well plate. The degree of tube formation was quantified by measuring the length of tubes in three randomly chosen fields from each well using the angiogenic activity quantification programme (Kurabo).
Monoclonal Antibodies (mAbs) recognizing the following markers were used for flow cytometric analyses: c-Kit (2B8; APC-conjugated), Sca-1 (D7; PE–Cy-7-conjugated), CD34 (RAM34; FITC (fluorescein isothiocyanate)-conjugated), Flt-3 (A2F10.1; PE-conjugated), CD45.1(A20; PE-conjugated), CD45.2(104; FITC-conjugated), TER-119 (TER-119; APC-conjugated), Gr-1 (PE-conjugated), CD11b (PE-conjugated), CD41 (PE-conjugated), CD3 (PE-conjugated) and CD19 (PE-conjugated). All mAbs were purchased from BD Biosciences. For multi-lineage engrafted analysis non-labelled Gr-1, CD11b, B220 and CD3 antibodies were labelled with Pacific Blue and used together with CD45.1 (PE-conjugated), CD45.2 (FITC-conjugated), and TER-119 (APC-conjugated). Haematopoietic cells were analysed using a LSRII flow cytometer (BD Biosciences).
Haematopoietic cell isolation and co-culture on PECs
After culture for 10 days, whole bone marrow cells from C57BL/6J mice were isolated and Lin− cells were enriched by mouse Lineage Cell Depletion Kit (Miltenyi Biotec). Primary endothelial cells were cultured on gelatin-coated 6-well plates, and 7,500 of the isolated Lin− cells were plated onto PECs in 2 ml of X-vivo 20 media (Lonza) supplemented with 25 ng ml−1 of mouse Kit Ligand (Pepro Tech). Freshly prepared 500 μl of serum- and growth factor-free X-vivo 20 media with Kit-Ligand was added every other day. At the end of co-culture period, floating cells and attached cells were collected and endothelial cells were removed by human CD31 Microbead Kit (Miltenyi Biotec). Lin− cells and Lin+ cells were separated using mouse Lineage Cell Depletion Kit. Total cell number was determined using an Advia 120 (Bayer) automated haematological analyser or haemocytometer (BD Biosciences). Colony assays were performed by placing 1,000 cultured haematopoietic cells into methylcellulose-based media with cytokines (Methocult GF M3434; Stem Cell Technologies) and analysed at day 7 for total colony number. Cell cycle analysis was performed by staining the Lin− cells with 7-aminoactinomycin D (BD Biosciences), c-Kit (APC-conjugated) and Sca-1 (PE-Cy-7-conjugated), and measured by FACS. Data were analysed with ModFit LT software (Verity Software House).
Endothelial cell selective expression of myrAkt1 in vivo
The double transgenic mouse model that expresses myrAkt1
in endothelial cells under tetracycline control has been previously described26
. Briefly, the D4 line (VEcad–tTA
) and K8 line (Tet–myrAkt1
) were used in these experiments. To suppress myrAkt1
expression in embryos, neonates and adults, 1.5 mg ml−1
tetracycline with 5% (w/v) sucrose was given in the drinking water. To induce myrAkt1
expression, tetracycline was removed from the water and then analyses were done 3 weeks after tetracycline removal.
Spleen colony forming units (CFU-S) assay
Lethally irradiated (9 Gy) FVB mice were reconstituted with 500,000 whole bone marrow cells from wild type (n = 7) or myrAkt1 transgenic mice (n = 8). Ten days post-transplant, peripheral blood was analysed by retro-orbital blood collection and differential blood counts were obtained using an automated Advia 120 (Bayer). Mice were then killed and spleens were removed and put in Bouin's Fixative (Sigma Aldrich) over night at 4 °C. Spleens were thoroughly washed with 1 × PBS and splenic colonies were counted using a Zeiss dissecting microscope (Carl Zeiss).
Competitive reconstitution assay
In and , lethally irradiated (9.5 Gy) C57BL/6-CD45.1 congenic mice were reconstituted with 10,000, 3,000 or 1,000 Lin− cells (marked CD45.2) expanded on different PECs, in competition with 500,000 () or 200,000 () whole bone marrow cells from C57BL/6-CD45.1 mice. Reconstitution of donor-derived cells (CD45.2) was monitored by staining peripheral blood cells with mAbs against CD45.2 (FITC-conjugated), CD45.1 (PE-conjugated), TER-119 (APC-conjugated) and haematopoietic lineage positive markers (Gr-1, CD11b, B220, and CD3; conjugated with Pacific blue). TER-119 negative cells were gated and analysed.
In , lethally irradiated (9 Gy) FVB mice were reconstituted with 5 × 105 whole bone marrow cells harvested from wild-type or myrAkt1 mice in competition with 3 × 105 whole bone marrow cells from FVB-GFP mice. Reconstitution of donor-derived cells (CD45+GFP−) was monitored by staining peripheral blood cells with mAbs against CD45, TER-119, and haematopoietic lineage positive markers (Gr-1, CD11b, B220, and CD4). TER-119− and CD45+ cells were gated and analysed. For the limiting dilution CRU assay (), lethally irradiated (9 Gy) FVB female mice were reconstituted with the indicated number of whole bone marrow cells harvested from wild-type or myrAkt1 mice. Donor-derived cells (from male) were determined by detecting Y-chromosome-specific gene expression (frequency SRY) in peripheral blood cells with quantitative PCR.
PECs survival assay
The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based AlamarBlue reagent (Invitrogen) was used to assess cell survival. PECs were plated at a density of 1.5 × 104 cells per well on gelatin-coated 96-well plates and incubated in growth medium until the cells reached confluency. Cells were washed and cultured with growth medium as a control or with serum- and growth factor-free X-vivo 20 medium for the indicated days. Medium was changed to Medium 199 containing AlamarBlue reagent and the fluorescence (Ex530/Em590) was measured after incubation for 3 h.
Reverse transcriptase PCR (RT–PCR)
Virus-transduced PECs were grown for 16 h in serum- and growth factor-free X-vivo 20 medium, and total RNA was extracted using RNeasy Mini Kit (Invitrogen). cDNA was produced with SuperScript™ III First-Strand Synthesis Kit (Invitrogen) by using random-sequence hexamer primers. Real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) in 7500 Fast Real-Time PCR System (Applied Biosystems). Amplification of 36B4 (for in vitro experiment) and 18S rRNA (for in vivo experiment in ) was used for sample normalization. Primer sequences are as follow: 36B4, 5′- CGACCTGGAAGTCCAACTAC -3′, 5′- ATCTGCTGCATCTGCTTG -3′; Ang1, 5′- AGAGCTACCACCAACAACAGTGTC -3′, 5′- GCTTGATATACATCTGCACAGTCTC -3′; Ang2, 5′- AGGCTGCAAGTGCTGGAGAAC -3′, 5′- CCGTCTGGTTCTGTACTGCATTCTG -3′; BMP4, 5′- AGGAAGAGCAGATCCACAGCAC -3′, 5′- GCAGAGTTTTCACTGGTCCCTGG -3′; DKK1, 5′- ATGCGTCACGCTATGTGCTG -3′, 5′- AGAACCTTCTTGTCCTTTGGTGTG -3′; DLL1, 5′- TTGCTGTGTCAGGTCTGGAG -3′, 5′- TTCTGTTGCGAGGTCATCAG -3′; DLL4, 5′- CCTCTCCAACTGCCCTTCAATTTC -3′, 5′- ATGAGTGCATCTGGTGGCAAGG -3′, FGF2, 5′- AGCAGAAGAGAGAGGAGTTGTGTC -3′, 5′- ACCAACTGGTGTATTTCCTTGACCG -3′; IGFBP2, 5′- CGAGGGCACTTGTGAGAAGC -3′, 5′- ATGTTCATGGTGCTGTCCACG -3′; IGFBP3, 5′- CTCTGCGTCAACGCTAGTGC -3′, 5′- GTGGAACTTGGGATCAGACACC -3′, Jagged-1, 5′- TGACCAGAATGGCAACAAAA -3′, 5′- GTTGGGTCCTGAATACCCCT -3′; Murine Akt1; 5′- GGCTGCTCAAGAAGGACCC -3′, 5′- CCACACACTCCATGCTGTCAT -3′; transgene myrAkt1, 5′- GCCATGGGGAGCAGCAAGAGCAAG -3′, 5′- CCTCAGGCGTTTCCACATGGAAG -3′; Murine SRY, 5′- GGGGAGTGTTGGCATAGGTA -3′, 5′- AGCTGACATCACTGGTGAGC -3′; Murine 18S rRNA; 5′- AAGTCCCTGCCCTTTGTACACA -3′, 5′- GCCTCACTAAACCATCCAATCG -3′.
Microarray gene expression profile
The Affymetrix Human Genome U133 Plus 2.0 array was used to analyze gene expression. In brief, confluent virus-transduced PECs were grown for 16 h in serum- and growth factor-free X-vivo 20 medium and total RNA was extracted using RNeasy Mini Kit (Invitrogen). The probe arrays were scanned with the Genechip System confocal scanner, data was processed with Affymetrix Microarray Suite 4.0 (Affymetrix) and analysis was performed with Genespring GX (Agilent). The microarray data is deposited at http://www.ncbi.nlm.nih.gov/geo
repository (record number: GSE24093).
Preparation of conditioned medium
Confluent PECs were cultured in X-vivo 20 medium for 3 days and conditioned media were collected. The conditioned media were condensed 8-fold with Amicon Ultra-15 (MILLIPORE). In co-culture experiments, one-fourth volume of each conditioned medium per total culture medium was added.
Cells were cultured in X-vivo20 medium for 16 h, lysed with RIPA (radio-immunoprecipitation assay) buffer (1 × TBS (tris-buffered saline), 1% (v/v) Nonidet P-40, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS (sodium dodecyl sulfate), 1 mM sodium orthovanadate, 10 mM NaF and protease inhibitor cocktail), and equal amounts of proteins were subjected to SDS–PAGE (SDS–polyacrylamide gel electrophoresis) with 4–12% (v/v) Bis-Tris gel (Invitrogen). Proteins were transferred onto nitrocellulose membrane and subjected to standard immunoblotting with antibodies against phosphorylated Akt (S473), Akt, phosphorylated p42/44 ERK, ERK or mTOR (Cell Signalling).
All results are presented as the mean and s.d. of independent experiments. Statistical analyses were performed using Student's t-test.