Materials and reagents
Recombinant human BMP-2, α-tubulin antibody, protease inhibitor cocktail, and phosphatase inhibitor cocktail I and II were purchased from Sigma-Aldrich. PD98059 was obtained from EMD. Recombinant murine Wnt3a and human VEGF were bought from R&D Systems. FBS, PBS, MEM, optimized MEM, Medium 200, low serum growth supplement, gentamycin/amphotericin, polyvinylidene difluoride membranes, Lipofectamine 2000, Prolong Gold Antifade with DAPI, and Alexa Fluor 488– and 592–labeled antibodies were obtained from Invitrogen. Anti-VEGF and antisurvivin were obtained from Abcam. Anti-Dvl antibody was obtained from Santa Cruz Biotechnology, Inc. Anti–cyclin D1, Anti-pERK1/2 and –total ERK½, and GSK3-β were obtained from Cell Signaling Technology. Active β-C, Rac1, and RhoA antibodies, the TCF reporter assay system, and RhoA and Rac1 assay reagents were purchased from Millipore. Nucleofector II and reagents were purchased from Amaxa, Inc. Anti–c-myc antibody and tissue culture–treated chamber slides were obtained from BD. Cytodex 3 beads, HRP-conjugated rabbit and mouse secondary antibodies, and ECL and ECL Plus kits were ordered from GE Healthcare. Dual Luciferase and caspase 3/7 assay kits were obtained from Promega. Labtek II chambered coverslips and all siRNA duplexes were purchased from Thermo Fisher Scientific.
Primary hPAECs from large vessels (Invitrogen; and ScienCell) were grown in EC media supplemented with 2% FBS, 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin subcultured at a 1:4 ratio in 100-mm dishes (Corning) and used at passages 4–8. Cells were starved in Medium 200 with 0.1% FBS and gentamycin/amphotericin for 24 h before adding the agonist or the vehicle. Avoiding medium changes allows consistent determination of β-C and EC proliferation in response to the dose of BMP-2 used.
hPAECs were seeded at 25,000 cells per well on 24-well plates in growth medium and allowed to adhere overnight. Cells were washed three times with PBS and incubated in starvation media for 24 h followed by stimulation with agonists for 24 h. Cells were trypsinized and counted in a hemocytometer (Bright-Line; Hausser Scientific).
For caspase 3/7 assays, cells were seeded in a 96-well plate (4 wells per condition and 5,000 cells per well), allowed to attach overnight, and incubated for 24 h in serum-free media to induce apoptosis in the presence or absence of BMP-2 or Wnt3a. Cells were incubated for 1 h in 100 μl of Caspase 3/7 Luciferase Reagent Mix (Promega), and total luminescence was measured in a 20/20 luminometer (Turner Biosystems, Inc.).
Cytodex 3 cell culture beads were suspended in 1× PBS, autoclaved, and gently agitated in Petri dishes containing 10 ml of cell media followed by gentle agitation. Next, 1 ml hPAECs (105 cells/ml) was added and incubated overnight to allow attachment to the beads. Once cells had grown to confluence over the beads, 1 ml of cell-coated beads was added to a solution containing MEM and vitronectin, pH 7.6, and allowed to solidify. Cells were incubated for 30–60 min followed by the addition of 1.5 ml of cell media, with or without agonist, to the surface of each gel. After 48 h, cell migration was quantified by dividing each dish into four quadrants and selecting three beads in each for further analyses. Migration was quantified by the distance each cell traveled from the center of the bead. The mean distance traveled per quadrant was then used to calculate the mean distance migrated by cells in each plate. A microscope (DMRIRBA; Leica) with a headed channel switch Plan Apochromat 40× NA 0.85 objective was used.
Cells were plated in four-chamber polystyrene glass slides (15,000 cells per chamber). For stimulation experiments, cells were starved for 24 h and stimulated with BMP as specified in the figure legends. Next, cells were fixed for 10 min in 4% paraformaldehyde followed by three washes with PBS. For experiments of native Dvl distribution in hPAECs, cells were permeabilized with ice-cold 1× PBS containing 0.1% Triton X-100 and donkey serum for 20 min followed by overnight incubation with goat anti-Dvl (Santa Cruz Biotechnology, Inc.) at RT, washed in 1× PBS, and incubated with Alexa Fluor 555 donkey anti–goat antibody (Invitrogen) for 1 h at RT followed by Alexa Fluor 488–labeled phalloidin for 20 min to stain actin filaments. For experiments of cells nucleofected with the various GFP-tagged Dvl constructs, actin staining was performed by incubating Alexa Fluor 555–phalloidin (Invitrogen) for 20 min after permeabilization with 0.1% Triton X-100 in PBS. Before mounting, slides were treated with Gold Antifade solution containing DAPI and stored at 4°C until analysis.
Confocal analysis was performed on a confocal laser-scanning microscope (SP2 AOBS; Leica) using a headed channel switch Plan Apochromat 63× NA 1.32–0.60 oil objective to locate areas of interest in the slides, and image acquisition was performed using the confocal build 1347 software (version 2.5; Leica) installed in the Leica confocal PC (Windows XP operating system). Images were processed and saved in JPEG format using Photoshop Creative Suite 2 (Adobe Systems).
To quantify distribution of GFP in PAECs transfected with the various Dvl constructs, GFP signal intensity in the cellular processes and the perinuclear area was measured in six randomly selected sectors using the ImageJ software (National Institutes of Health). A mean of the values was calculated, and a ratio of peripheral to central GFP intensity was plotted and used for statistical analyses. A total of four cells was used for each condition. The results were analyzed using Prism software (GraphPad Software, Inc.).
hPAECs were washed three times with ice-cold 1× PBS, and lysates were prepared by adding boiling lysis buffer (10 mM Tris HCl, 1% SDS, and 0.2 mM PMSF) containing protease and phosphatase inhibitors, scraping into a 1.5-ml microcentrifuge tube, and boiling for 10 min before centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and stored at −80°C. The protein concentration was determined by the Lowry assay (Bio-Rad Laboratories). Equal amounts of protein were loaded onto each lane of a 4–12% Bis-Tris gel and subjected to electrophoresis under reducing conditions. After blotting, polyvinylidene difluoride membranes were blocked for 1 h (5% milk powder in 0.1% TBS/Tween) and incubated with primary antibodies overnight at 4°C. The binding of secondary HRP antibodies was visualized by ECL or ECL Plus. Normalizing for total cell protein was performed by reprobing the membrane with a mouse monoclonal antibody against α-tubulin (Sigma-Aldrich).
Dvl plasmids and transfection methods
Plasmids encoding the WT, ΔDEP, ΔDIX, and DEP+ forms of Dvl were linked to a GFP tag and cloned in pCS vectors as described previously (Axelrod et al., 1998
). An adenovirus containing a dominant-negative form of ERK1/2 was purchased from Seven Hills Bioreagents. Mutant constructs of Smad1 and Smad3 containing three serine to alanine substitutions in their C-terminal sequence and cloned in pCMV5 vectors were supplied by J. Massague (Memorial Sloan-Kettering Institute, New York, NY). Transfection of plasmids was performed using a Nucleofector II (program T-23) with the Basic Endothelial Cell Nucleofection kit (Amaxa, Inc.). All experiments were performed 24 h after nucleofection.
To achieve gene knockdown, siRNA duplexes specific for β-C (On-Target Plus; Invitrogen; GenBank/EMBL/DDBJ accession no. NM_001012329 and NM_020248), ActRIIa (On-Target Plus; Invitrogen; GenBank/EMBL/DDBJ accession no. NM_001616), and BMPRII (On-Target Plus; Invitrogen; GenBank/EMBL/DDBJ accession no. NM_001204) were transfected into PAECs using nucleofection as described in the previous section. Knockdown efficiency was evaluated 48 h after nucleofection by measuring protein levels in cell lysates using a Western immunoblot.
For measurements of β-C–mediated changes in gene expression, we used a TOPflash/FOPflash TCF/LEF reporter assay. The TOPflash construct contains a promoter with eight TCF/LEF1-binding domains found in the promoters of β-C target genes linked to a luciferase gene, whereas the FOPflash construct has mutated binding sites and serves as a negative control. hPAECs were transfected with either plasmid in a Nucleofector II (program T-23) using the Basic Endothelial Cell Nucleofection kit. After 24 h in starvation media, cells were stimulated, and luciferase production was measured 6 h later in a luminometer using the Dual Luciferase kit (Promega) according to the manufacturer's protocol. Renilla plasmid (Promega) cotransfection was used to control for transfection efficiency.
RhoA and Rac1 pull-down assays
hPAECs were washed three times with ice-cold 1× PBS. Cell lysates were prepared by adding 500 μl of ice-cold magnesium lysis buffer (10 mM Tris HCl, 1.0% SDS, 0.2 mM PMSF, and 100× protease and phosphatase inhibitor cocktails I and II; Millipore) to the cells and scraping into a 1.5-ml microcentrifuge tube on ice before cold centrifugation at 14,000 rpm for 10 min. Supernatants were transferred to fresh microcentrifuge tubes and stored at −80°C.
Active forms of RhoA or Rac1 were precipitated using glutathione beads containing rhotekin or PAK1, respectively, according to the manufacturer's protocol (Millipore). In brief, lysates were incubated with slurry containing the glutathione beads for 1 h at 4°C with constant rotation. At the end of this period, beads were precipitated by centrifuging lysates at 14,000 relative centrifuge force for 20 s. After washing three times with ice-cold buffer, beads were resuspended in Laemmli buffer, boiled, and subjected to Western immunoblot analysis as described above.
Digitally enhanced video differential interference contrast microscopy
48 h after transfection, cells were allowed to attach and spread on glass coverslips in the incubator and viewed on an inverted microscope (Axiovert 35; Carl Zeiss, Inc.) with a Plan Apochromat 100× oil immersion lens (Carl Zeiss, Inc.) and a short distance condenser. The microscope is also equipped with a heated stage, differential interference contrast optics, and epifluorescence. Filters and light paths were controlled with a filter wheel and shutters (Ludl Electronic Products Ltd.). For GFP visualization, a single band excitation filter for FITC was used in combination with Pinkel#1 beam splitter and emission filter (Chroma Technology Corp.). Tissue culture medium without phenol red was kept warm and buffered in a CO2 incubator. The stage temperature was kept at 37°C with an automatic thermostat. Images were collected using a charge-coupled device camera (C2400; B&W) with on-chip integration and a digital image processor (Argus 20; PerkinElmer). Acquired images were assembled in temporal sequences using Openlab software (PerkinElmer) and saved as QuickTime (Apple, Inc.) videos. The distance traveled by the cells was calculated by recording the position of the nucleus in individual frames using ImageJ software and plotting on an x–y axis followed by measurement of distance between each of the individual points. Total distance was obtained from the total sum of these values. Speed was measured by dividing total distance over time (10 h) using the following formula: S = D/t, where S = speed, D = distance and, t = time. A total of four cells was used for each measurement.
Matrigel plug angiogenesis assay in SCID mice
Animal experiments were approved by Stanford University's Institutional Review Board under the guidelines of the American Physiological Society. Matrigel plugs containing hPAECs were prepared as described previously (Skovseth et al., 2007
), and 48 h after transfection, ~1.0 × 106
cells were suspended in chilled 500 μl/plug of Matrigel Growth Factor Reduced solution (BD), to which recombinant BMP-2 diluted to a final concentration of 10 ng/ml or carrier was added. Plugs were implanted in the back of SCID mice (Charles River Laboratories) anesthetized by subcutaneous injection of a ketamine/xylazine cocktail. For each experimental condition, a total of four plugs was implanted in each animal and allowed to remain in place for 14 d. At the end of this period, the animals were humanely killed, and the solid Matrigel plugs were removed and fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Hematoxylin and eosin (H&E) sections were prepared. Unstained tissue samples were double labeled with Alexa Fluor 488–labeled anti–human CD31 and Alexa Fluor 555–labeled anti–mouse CD31 to identify human and murine ECs, respectively, along with DAPI to stain nuclei. 10 images per slide were captured using the 40× objective of an immunofluorescence microscope (DM RA2; Leica) and analyzed via Openlab software. The presence of microvessels (defined as tubes filled with red blood cells) was documented using H&E sections, and the number of human and murine cells per condition was quantified using the aforementioned immunofluorescence techniques.
Values from multiple experiments are expressed as mean ± SEM. Statistical significance was determined using one-way analysis of variance followed by Bonferroni's multiple comparison tests unless stated otherwise. A value of P < 0.05 was considered significant. The number of experiments and the sample number in each group are indicated in the figure legends.
Online supplemental material
Fig. S1 shows semiquantitative RT-PCR data for all 19 known Wnt ligands found in hPAECs at baseline. Fig. S2 shows confocal images of hPAECs stimulated with BMP-2 for 1 h and labeled with a primary antibody specific for β-C and an Alexa Fluor 488–labeled secondary antibody. Fig. S3 shows that hPAECs treated with BMPRII siRNA fail to accumulate β-C when stimulated with BMP-2, an event that correlates with failure to phosphorylate both ERK1/2 and GSK3-β. Fig. S4 shows the loss of BMP-2–mediated β-C accumulation and transcriptional activity in the presence of dominant-negative ERK (ΔERK). Fig. S5 demonstrates both the lack of impact of the Dvl mutant constructs on BMP-mediated β-C accumulation and the peripheral redistribution of Dvl in BMP-2– or Wnt3a-stimulated PAECs. Videos 1 and 2 show hPAECs transfected with WT Dvl at baseline (Video 1) and in the presence of BMP-2 (Video 2). Videos 3 and 4 show hPAECs transfected with ΔDEP exposed to the same conditions as described for Videos 1 and 2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200806049/DC1