Online Supplementary Material

Detailed Methods

Mouse Embryonic Endothelial Cells

10cm culture plates were coated with 0.1% gelatin (Sigma). Embryonic endothelial cells (Wild-type Eng+/+, heterozygous Eng+/- and homozygous null Eng-/-), were derived from mouse embryos and yolk sacs isolated at embryonic stage (E) 9.0 and selectively immortalized as previously described[1]. Cells were grown in media (MCDB131 + 15% FCS (Wisent Bioproducts, Montreal, Quebec, Canada), 1% Penicillin/Streptomycin (Invitrogen, Burlington, Canada), 1% L-glutamine (Invitrogen)) and passaged every 2-3 days. Cells were washed with PBS and incubated with 0.25% Trypsin (Invitrogen) to detach cells. After neutralization with media, the entire solution was spun at 1200 rpm for 5 minutes. A second rinsing was conducted with PBS and the pellet was re-suspended in media. Cells were counted with a hemocytometer in a 1:4 mixture of Trypan Blue (Invitrogen) and plated at 700,000 cells/60 cm 2. 15 μg/mL EGF Endothelial Mitogen (Biomedical Technologies, Stoughton, MA, USA) was added to each plate with mixing.

Flow Cytometry

Flow cytometry was performed to confirm the presence (or absence) of endoglin on the surface of Eng+/+, Eng+/- and Eng-/- MEEC. Cells were detached with brief trypsinization, neutralized with cell media and washed in chilled FACs buffer (Ca2+-Mg2+-free phosphate-buffered saline (PBS, Invitrogen) plus 10 mM Hepes (Invitrogen) and 10% FBS). Cells (1x105) were blocked for 20 minutes on ice with anti-mouse CD16/CD32 purified antibody (eBioscience, 14-0161-82, San Diego, California, USA) before a 30 minute incubation with saturating amounts of the biotinylated antibody to mouse endoglin (clone MJ7/18[2], produced in the in house hybridoma facility, Sunnybrook HSC, Toronto, Canada) or rat isotype IgG2 control (anti-mouse IgG2a Biotin, eBioscience, 13-4321-85), then washed again. Streptavidin-FITC (0.25 μg/1x105 cells for 30 minutes, eBioscience, 111-4317-87) was employed as a secondary antibody, followed by additional washings in FACs buffer. The fluorescent samples were run on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) modified with a digital DXP upgrade from Cytek Development (Fremont, CA, USA). 20,000 events were collected using Flow Jo Collectors’ edition software and data was analyzed with Flow Jo analysis software. A primary gate was established around the principal population established by forward and side-scatter channels and the level of FITC expression was visualized in one-dimensional histograms.

Animals

PCR to verify embryo genotype Genotype was verified using PCR. Tail tissue from embryos were collected and processed according to the “Genomic DNA from tissue” protocols for the NucleoSpin Tisssue XS kit (Macherey-Nagel, Mountain View, CA). To determine protein and DNA yield and purity, a measure of the eluent absorbance was performed at 260 nm (A260) and 280 nm (A280). Any great deviations in A260 eluent concentrations were diluted in H2O, and PCR mix including primers (10x PCR buffer II without MgCl2, 50 mM MgCl2, 20 pmol/μl ME1R, 20 pmol/μl MEZR, 20 pmol/μl ME1F, 2.5 mM dNTPs, sterile ddH20 (54%), 5U/μL Taq DNA polymerase and 50 ng/uL DNA) was prepared. PCR was performed on a PTC- +/+ 200 Peltier Thermal Cycler. A 300 bp band is expected for Eng and an additional 476 bp band is expected for Eng+/- (double band indicated +/- genotype)[3]. 2

Parallel plate flow chamber microbubble binding experiments

Cells Endoglin targeted microbubble binding was assessed in cell culture experiments using parallel plate flow chambers (acquired from Dr. H.E. Achneck, Duke University Medical Center, Durham, NC, USA[4]). Eng+/+, Eng+/- and Eng-/- mouse embryonic endothelial cells were plated (500,000 cells) on sterile, unfrosted glass slides (75 x 25 x 1mm (Fisher Scientific, Ottawa, Ontario, Canada)) coated with 0.1% gelatin (Sigma-Aldrich, Oakville, Ontario, Canada), and allowed to grow for 48 hours to reach confluency.

Apparatus and Set up We modified a previously published protocol[4] to assemble an air pressure driven flow apparatus (see Supplementary Materials FIGURE 1). All flow loop and chamber parts were sterilized by autoclaving at 121oC prior to experimentation. 250 mL media bottles were used as reservoirs for circulating running fluid within the loop, with three holes drilled onto the bottle caps in order to insert tubes for inlet, outlet and ventilation (using a 20 μm syringe filter). 14 L/S Pharmed BPT soft tubing (Cole Parmer, Montreal, Quebec, Canada) was inserted into each of these holes to ensure a tight seal. Outlet tubing was connected to 13 L/S tubing that was mounted on the peristaltic pump (MasterFlex L/S, Cole Parmer) to drive movement of fluid. A custom-made glass flow dampener (diameter 35 mm) was used to pressurize air within the loop and exert steady laminar flow at a rate of 4 mL/s across cells. The outgoing end of the chamber was connected to two 4-way stopcocks (B. Braun Medical Inc., Mississauga, Ontario, Canada) followed by 14 L/S C Flex tube leading directly to the reservoir. After assembling the loop, 50 mL of 10% FBS solution (in PBS Ca+ Mg+) was allowed to circulate at 4 mL/min to remove air bubbles from within the system. Assembled flow loops were maintained within a 37oC incubator (Darwin Chambers, St. Louis, MO, USA) throughout the duration of the experiment.

After priming the system, cell seeded slides were inserted into parallel plate flow chambers manufactured and provided by Dr. Achneck [4]. Flow chambers were assembled, primed with 10% FBS solution and integrated into the flow loop system according to previously described methods [4]. Cells were exposed to flowing 10% FBS solution until immediately before microbubbles exposure to ensure sufficient blocking of unspecific binding.

Microbubble Binding, Imaging and Analysis Prior to addition of microbubbles solution, outlet 14 L/S tube was pulled up to the extent that the seal was intact but it was no longer in contact with fluid in the reservoir. Remaining liquid in the outlet tube and dampener was emptied by pumping air into the dampener using the peristaltic pump. Microbubbles (1x107 MB/mL PBS) were drawn across the cells for 5 minutes at a flow rate of 4 mL/minute, corresponding to an approximate shear stress of 2 dynes/cm 2, using an adjustable peristaltic pump (MasterFlex L/S, Cole Parmer, Montreal, Quebec, Canada) to simulate in vivo flow conditions. For a Newtonian fluid flowing through a parallel plate flow chamber with a rectangular geometry, the steady laminar shear stress at the wall is described as[5]:

Q =  ·w·h 2  = 6·µ·Q 6·µ w·h2

Where  is wall shear stress (hydrodynamic force per unit of surface area exposed to a flow, acting tangen tially on the cells, dynes/cm2), µ is viscosity of the perfusate (g cm-1 s-1), Q is flow rate (mL/s), w is chann el width (cm) and h is height (cm). A typical value of viscosity for medium used is 0.9 cP (0.009 g cm -1 s- 3

1)[4] or 1x10-3kg m-1 s-1 for most aqueous fluids (including buffers) at room temperature[6]. We assumed a viscosity of 1 cP = 0.01 g/cm3 for PBS[7].

Flow chambers were inverted throughout this procedure to maximize bubble-cell interaction. Following microbubble exposure, chambers were fixed to their upright position and rinsed with PBS for 8 minutes. Cells and bubbles were immediately imaged within the chambers using bright field and phase contrast imaging at a magnification of 40X (Nikon TE300, Nikon, Mississauga, Ontario, Canada). Slides were +/+ +/- -/- +/+ +/- prepared in triplicate for each cell and bubble type (MBE: n = 3, n = 3, n = 3; MBC: n = 3, n = 3, -/- +/+ +/- -/- n = 3; MBU: n = 3, n = 3, n = 3), with 10 randomly selected fields of view recorded for each slide.

The Kruskal-Wallis Anova and Mood’s median test were used to analyze significant differences in cell attachment rates (number of MB/cell) for each microbubble type (MBE, MBC, and MBU) and cell genotype (Eng+/+, Eng+/- and Eng-/-). Mann-Whitney tests were performed to ascertain significant differences between individual means. A false discovery rate[8] and experiment wise error rate[9] were employed to adjust for multiple comparisons.

Ultrasound Molecular Imaging

Experimental Preparation Wild-type CD-1 male and female mus musculus (Charles River Laboratories, St-Constant, Quebec, Canada) were mated to produce staged embryos, with embryonic day (E) 0.5 defined as noon of the day a vaginal plug was observed. Prior to the experiments, dissection plates were created using a 1:8 volume ratio of curing agent to base (Sylgard 184 Silicone Elastomer Kit, Dow Corning, Midland, Michigan, USA) and set overnight. Glass needles (1x90 mm glass capillaries with filament) were pulled (PN-30, Narishige, East Meadow, New York, USA) and clear ultrasound gel (Aquasonic, Fairfield, New Jersey, USA) was centrifuged at 140g for twenty minutes and drawn into 30 mL syringes for easy application. Additional syringes were filled with phosphate buffered saline (PBS). Female luers (Cole Parmer, Montreal, Quebec, Canada) were attached to 400 mm pieces of tubing (VWR, Mississauga, Ontario, Canada). Finally, embryo media (89% Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Sigma, Oakville, Ontario, Canada), 9% Fetal Bovine Serum, 1% 1M Hepes, 1% Penicillin-Streptomycin (10,000 units Pen., 10,000 µg Strep.) (Gibco, Burlington, Ontario, Canada)) was prepared and kept at 4oC. Before experimentation, the imaging platform (Integrated Rail System, VisualSonics Inc., Toronto, Canada) was arranged beneath the surgical microscope (Stereomaster, Fisher Scientific, Ottawa, Ontario, Canada). Aliquots of embryo media were kept on ice. Syringes of ultrasound gel and PBS were preheated in a water-bath maintained at 45oC.

Western Blots

Methods used to obtain cell and tissue proteins and to perform western blot for endoglin have been described previously[3, 10].

Protein Extraction – cells Cells from passages 16 (Eng +/+), 18 (Eng +/-) and 21 (Eng -/-) were plated at 1,000,000 cells/10 cm dish and allowed to grow until confluent (~ 2 days). After washing with PBS, the dishes were placed on ice to prevent protein degradation while cells were collected. An aliquot of lysis buffer (500 μL 10x TNE (100mM Tris; 2.0M NaCl; 10mM EDTA; pH 7.4), 50 μL Protein inhibitor cocktail (Fermentas, Ottawa, Canada, R1321), 500μL 10% Triton x-100 (Sigma), 500 μL 0.1 M NaPPi (Sigma), 250 μL 0.5M NaF

(Sigma), 50 μL 0.1M Na3VO4 (Sigma), 3 mL ddH2O) was added to each plate. Cells were gently removed and mixed vigorously. Samples were then rotated for 30 minutes at 4 oC and spun at 13 000 rpm for 30 minutes at 4oC. The cell lysate supernatant was transferred to fresh eppendorf tubes and the pellets were discarded. 4

Protein Extraction – brains 50mg of tissue was used for protein extraction. Lysis buffer (1/2 tablet of protease-inhibitor per 10 mL lysis buffer, Fermentas) was added to each sample at a 6x dilution factor (by μL volume) for embryos. Tissue was homogenized on ice and then rotated for 30 minutes at 4oC. Samples were subsequently centrifuged at 13,000 g for 30 minutes at 4oC, the supernatant was transferred to ice and pellets were discarded.

Spectroscopy and Protein Measurement Protein content was quantified by the Bradford method (Bio-Rad). Supernatant samples were diluted at a ratio of 1:4 in cold ddH2O and quantified against a standard curve prepared using BSA (0, 0.08, 0.17, 0.35, 0.70, 1.41, 2.82 mg/mL, Sigma, Oakville, Canada).

Sample Preparation Both reducing (RB) (62mg/mL DL-Dithiothreitol, Sigma) and non-reducing buffer (NRB) mixtures (Invitrogen) were prepared for each sample, with a 3:1 ratio of supernatant to buffer and mixed vigorously. The final solution was boiled for 5 minutes and stored at -20oC. Remaining supernatant was transferred to -80oC.

Blotting 30 μg protein samples were fractionated by SDS-PAGE (Novex, Invitrogen, Burlington, Canada, NP0001) and transferred onto polyvinylidene fluoride (PVDF) membrane (GE Amersham Hybond-P, GE Healthcare, Baie d’Urfe, Quebec, Canada)[3]. After rinsing, the membrane was blocked in a 5% milk (Bio-Rad, Mississauga, Canada, 170-6404) solution for 1 hr and washed (TBST-T: 0.02M Tris base, 0.137M NaCl, 0,1% Tween 20 in H2O) before primary antibody incubation (in 5% Milk). Membranes were probed with rat anti-mouse CD105 UNLB Clone MJ7/18 (1:1000 cells, 1:500 embryos; Southern Biotech, 1860-01, Birmingham, Alabama, USA) or goat anti-mouse polyclonal platelet endothelial cell adhesion molecule-1 (PECAM-1) (1:1000 cells, 1:500 embryos; Santa Cruz Biotechnology, sc-1506 Dallas, Texas) overnight at 4oC. Membranes were washed (TBST-T) and incubated in the dark with appropriate secondary antibodies (1:10000, goat anti-rat HRP (GE Healthcare, NA935) or donkey anti- goat HRP (Santa Cruz Biotechnology, sc-2020)) prepared in 3% milk solution. Membranes were thoroughly washed (3x, 10 minutes) with TBST-T. Additional probing was performed for the control β- actin (1:10,000 Sigma), with secondary sheep anti-mouse HRP antibodies (1:10,000, GE Healthcare, RPN4301, Baie d’Urfe, Quebec, Canada).

Detection The presence of the mouse endoglin dimer as a band at 170 kDa in non-reducing conditions, and the PEC AM-1 monomer at 130 kDa in reducing conditions, were revealed using a chemiluminescence assay (Perk in Elmer, Woodbridge, Ontario, Canada)[3]. Radiographs (Konica Minolta SRX-101A, Mississauga, Ont ario, Canada) were digitized (Scanjet HP G4050) using Adobe Photoshop 3.0. AlphaEase FC (Alpha Inno tech, Toronto, Canada) was used to quantify band densities. Expression of β-actin (42 kDa band) was me asured as an internal loading control. Endoglin protein levels were subsequently normalized to PECAM- 1 and β-actin expression levels.

For endothelial cell extracts, densitometry measurements were tabulated and the ratios of endoglin:β-actin (E:β) and endoglin:PECAM-1 (E:P) for each sample were computed and normalized to the average Eng+/+ MEEC ratio for the E:β or E:P groups. Results were analyzed with a one-way analysis of variances where equal variance may not be assumed. Multiple comparisons were accounted for with a Bonferroni post- hoc test. Likewise, ratios for E:β and E:P for embryo western blots were normalized to embryo Eng+/+ values for each category. Two tail sample t-tests were conducted to assess statistical differences between 5 genotypes, with results plotted as a function of mean + s.d. Equal variance may be assumed.

Data Analysis and Mathematical Methods

Analysis for Parallel Plate Flow Chamber Experiments

For each cell and bubble type, the number of attached microbubbles per cell was calculated within each field of view. This was achieved by dividing the number of attached microbubbles counted in-focus in brightfield images by the number of cells identified within phase images. Since the data were not drawn from a normally distributed population (Shapiro-Wilks tests), a non-parametric analysis was used. Both the Kruskal-Wallis Anova and Mood’s median test were performed on cell attachment rates of MBs, +/+ +/- -/- sorted according to contrast agent (MBE, MBC, and MBU) and cell genotype (Eng , Eng and Eng ). Mann-Whitney tests were conducted to ascertain significant differences between individual groups. p values were tested using the false discovery rate (cutoff = 0.05* i/n; i = sample number, n= total number of samples[8]) and the more conservative experiment wise error rate[9] (cutoff = 0.05/(n-i+1)) to account and adjust for multiple comparisons. Results are presented as box and whisker plots (minimum; first quartile; median; third quartile; maximum).

Analysis for Molecular Imaging

Time-intensity plots were generated for 1.5 mm2 regions of interest (ROIs) in the embryonic left and right brain hemispheres and the ratio of the average signal intensity of the ‘pre-destruction’ to ‘post- destruction’ sequences was used to produce a measure of the molecular signal called the contrast mean power ratio (CMPR). A linear mixed model was performed in PASW Statistics 18 (IBM Corporation, Armonk, NY, USA) to ascertain whether there was any significant difference between endoglin targeted and control microbubble binding to Eng+/+ or Eng+/- embryos. A number of experimental parameters were tested, with average CMPR defined as the dependent variable and the quantitative explanatory variable heart rate (HR) as a covariate (CF). Categorical explanatory factors microbubble type, litter, embryonic stage and genotype were defined as fixed (FF), while genotype nested in litter (litter|genotype) and litter (1|litter) were classified as random effects (RE).

The linear mixed model may be summarized as:

R = intercept + litter FF + stage FF + genotype FF + MB FF + heart rate CF + (1|litter) RE + (litter|genotype) RE

Statistical analysis revealed that the random effects (litter|genotype) and (1|litter) do not account for a meaningful amount of the variance estimates. Likewise, type III tests of the fixed factors indicate that stage and heart rate are not significant effects and may be removed from analysis. Main effects were compared with Bonferroni adjustments made for multiple comparisons. Plots of the estimated CMPR means (mean + 95% Confidence Interval (CI)) are presented (Origin 9, Northampton, MA, USA) for each microbubble and embryo type. Statistical analyses were performed in PASW Statistics 18 (IBM, Armonk, NY, USA). Embryos were excluded from analysis if there was profuse bleeding during injection. 6

Supplemental Figures and Figure Legends

TABLE 1 Summary of results and statistical analysis for MB injected embryos.

Microbubble type Genotype CMPR Mean 95% Confidence Interval

+/+ MBE Eng 9.71 9.05, 10.38 Eng+/- 5.51 4.87, 6.15

+/+ MBC Eng 1.42 0.41, 2.43 Eng+/- 1.46 0.45, 2.47

+/+ MBU Eng 1.70 0.65, 2.75 Eng+/- 1.76 0.67, 2.84

Linear Mixed Model: Between-subject effects

df F p value

Genotype 1 12.75 <0.001 Microbubble type 2 147.65 <0.001

Genotype * Microbubble type 2 18.29 <0.001

Table 1 Summary of results and statistical analysis for microbubble injected embryos. Summary of linear mixed model analysis for microbubble binding in embryos, with a Bonferroni correction for multiple comparisons. Genotype, microbubble type and the combined interaction (genotype * microbubble type) were found to be significant factors in determining CMPR. df = degrees of freedom 7

TABLE 2 Summary of statistical analysis for microbubble adhesion in vitro.

Group Comparison p value Group Comparison p value

+/+ +/- Eng vs. Eng < 0.001 MBE vs. MBC <0.001 +/- -/- +/+ MBE Eng vs. Eng 0.002 Eng MBC vs. MBU <0.001 +/+ -/- Eng vs. Eng < 0.001 MBE vs. MBU <0.001

+/+ +/- Eng vs. Eng < 0.001 MBE vs. MBC 0.130 * +/- -/- +/- MBC Eng vs. Eng < 0.001 Eng MBC vs. MBU <0.001 +/+ -/- Eng vs. Eng 0.003 MBE vs. MBU <0.001

+/+ +/- Eng vs. Eng 0.931 * MBE vs. MBC <0.001 +/- -/- -/- MBU Eng vs. Eng 0.032 * Eng MBC vs. MBU <0.001 +/+ -/- Eng vs. Eng 0.015 * MBE vs. MBU <0.001

* p values were tested using the False Discovery Rate[8] and Experiment Wise Error Rate[9] to account and adjust for multiple comparisons, where those subsequently deemed insignificant are identified with an asterisk.

TABLE 2 Summary of statistical analysis for microbubble adhesion in vitro. Summary of Kruskal- Wallis Anova, with Mann-Whitney tests to compare between groups. At the 0.05 level, the populations were significantly different. A p-value <0.05 was considered to indicate statistical significance between pairings

Fig. 1 Parallel plate flow chamber experimental set-up. Endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) mouse embryonic endothelial cells were cultured on glass slides and mounted in parallel plate flow chambers. Microbubbles (endoglin targeted: MBE, isotype control: MBC or untargeted: 7 MBU) were diluted at 1x10 MB/mL in PBS in the dampner bottle and perfused across the cells at 4mL/min, corresponding to a shear stress of 2 dynes/cm2

Fig.2 Ultrasound Imaging of Exteriorized Living E17.5 Embryos (a) B-mode ultrasound image of an embryo. Prior to injection of targeted microbubbles ( t= 0 seconds). Non-linear contrast image of the embryo before (b) and after (c) injection of targeted microbubbles. (b) Nonlinear contrast image prior to microbubble injection (t = 0 seconds). Only the strongly reflecting interfaces (e.g. bone) are visible, with minimal signal from the soft tissues. (c) Nonlinear contrast image of microbubbles within the embryo at t= 75 seconds after a bolus injection. Contrast is detected throughout the animal, including the heart and brain. (d) Maximum intensity projection ultrasound image. The path of moving bubbles is 8 integrated across consecutive frames (t = 0 to 75 seconds) to form a composite image depicting the vascular architecture. With this method, major vessels and individual chambers of the heart are delineated. Arrowheads: R = ribs, Ht = heart, Br = brain. Scale bar = 3 mm

Supplemental References

1. Pece-Barbara N, Vera S, Kathirkamathamby K et al. (2005) Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor β1 with higher affinity receptors and an activated Alk1 pathway. J.Biol.Chem. 280:27800-27808

2. Ge AZ, Butcher EC (1994) Cloning and expression of a cDNA encoding mouse endoglin, an endothelial cell TGF-beta ligand. Gene 128:201-206

3. Jerkic M, Rivas-Elena JV, Prieto M et al. (2004) Endoglin regulates nitric oxide-dependent vasodilatation. The FASEB journal 18:609-611

4. Lane WO, Jantzen AE, Carlon TA et al. (2012) Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress. Journal of Visualized Experiments 1:e3349

5. Ratner BD, Hoffman AS, Schoen FJ, Lemons JE (November 8, 2012) Biomaterials Science: An Introduction to Material in Medicine. San Diego, California: Academic Press, pp 1573-476

6. Bakker DP, van der Plaats A, Verkerke GJ, Busscher HJ, van der Mei HC (2003) Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces. Appl.Environ.Microbiol. 69:6280

7. Momen-Heravi F, Balaj L, Alian S et al. (2012) Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Frontiers in physiology 3

8. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological):289-300

9. Wright SP (1992) Adjusted p-values for simultaneous inference. Biometrics:1005-1013

10. Jerkic M, Rodriguez-Barbero A, Prieto M et al. (2006) Reduced angiogenic responses in adult Endoglin heterozygous mice. Cardiovasc.Res. 69:845-854