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Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos

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Abstract

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng +/+), heterozygous (Eng +/−) and null (Eng −/−) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng +/− and Eng +/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p < 0.05) and embryonic studies (p < 0.001); MBE binding was approximately twofold higher in Eng +/+ cells and embryos compared with their Eng +/− counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

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Abbreviations

BPM:

Beats per minute

CI:

Confidence interval

CMPR:

Contrast mean power ratio

E:

Embryonic day

E:β:

Endoglin:β-actin ratio

Eng:

Endoglin

Eng +/+ :

Endoglin wild-type

Eng +/− :

Endoglin heterozygous

Eng −/− :

Endoglin homozygous null

E:P:

Endoglin:PECAM-1 ratio

HR:

Heart rate

MBC :

Rat isotype IgG2 control antibody-targeted microbubbles

MBE :

Endoglin-targeted microbubbles

MBU :

Untargeted microbubbles

MFI:

Median fluorescence intensity

ROI:

Region of interest

TGFβ:

Transforming growth factor β

US:

Ultrasound

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Acknowledgments

The authors would like to thank John M. Hudson for assistance with microbubble handling and Valentin Sotov for assistance with MEEC Western blotting. This work was supported by the Terry Fox Programme of the National Cancer Institute of Canada.

Conflict of interest

F.S. Foster acknowledges his role as consultant to VisualSonics Inc.

Ethical standard

The experimental procedures performed in this study were approved by the Animal Care Committee at Sunnybrook Research Institute (Toronto, ON, Canada).

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Authors and Affiliations

  1. Department of Medical Biophysics, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Avenue, S640, Toronto, Ontario, M4N 3M5, Canada

    J. M. Denbeigh, B. A. Nixon, P. A. Marsden, M. C. Puri & F. S. Foster

  2. Department of Medicine and Pathobiology, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, M5B 1W8, Canada

    J. J. Y. Lee & P. A. Marsden

  3. Anesthesia Research, Keenan Research Centre, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, M5B 1T8, Canada

    M. Jerkic

  4. Department of Immunology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8, Canada

    M. Jerkic & M. Letarte

  5. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada

    M. C. Puri

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  1. J. M. Denbeigh
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  2. B. A. Nixon
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  3. J. J. Y. Lee
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  4. M. Jerkic
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  6. M. Letarte
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  8. F. S. Foster
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Correspondence to J. M. Denbeigh.

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Denbeigh, J.M., Nixon, B.A., Lee, J.J.Y. et al. Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos. Angiogenesis 18, 69–81 (2015). https://doi.org/10.1007/s10456-014-9447-0

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