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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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Microbubbles-echistatin

MBE
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: January 11, 2012.

Chemical name:Microbubbles-echistatin
Abbreviated name:MBE
Synonym:
Agent category:Polypeptide
Target:Integrin αvβ3
Target category:Receptor
Method of detection:Ultrasound
Source of signal:Microbubbles
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about echistatin.

Background

[PubMed]

Ultrasound is the most widely used imaging modality (1) and is expanding its role in noninvasive molecular imaging with ligand-carrying microbubbles (2). Microbubbles are comprised of spherical cavities filled by a gas encapsulated in a shell. The shells are made of phospholipids, surfactant, denatured human serum albumin or synthetic polymer. Ligands and antibodies can be incorporated into the shell surface of microbubbles. Microbubbles are usually 2 to 8 μm in size. They provide a strongly reflective interface and resonate to ultrasound waves. They are used as ultrasound contrast agents in imaging of inflammation, angiogenesis, intravascular thrombus, and tumors (3-5). They are also potentially used for drug and gene delivery (6).

Angiogenesis is the formation of new blood capillaries and vessels from existing vessels (7-9). It involves the degradation of the basal membrane surrounding the parental vasculature, and the proliferation of endothelial and smooth muscle cells to form new vessels, resulting in an improvement in tissue perfusion and function. The major stimuli for angiogenesis include ischemia, hypoxia, inflammation, and shear stress (10). A large number of endogenous angiogenic factors are involved in angiogenesis (11), including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), and transforming growth factor (TGF-β). Extracellular matrix and proteolytic enzymes as well as integrins are also involved during angiogenesis (12). There are also endogenous inhibitors of angiogenesis such as, angiostatin, endostatin and platelet factor-4 (13, 14).

Tumor angiogenesis represents a continuous and important process in tumor development in which the tumor attempts to gain an independent blood supply (15). This process is driven by the tumor's chronic overproduction of pro-angiogenic factors, which bind to receptors on nearby vessel endothelial cells. Angiogenesis is essential for the growth of solid tumors and their metastases. Imaging angiogenesis may be useful for monitoring angiogenic treatments of tumors and cardiovascular diseases (9, 16, 17). The αvβ3 integrin is strongly expressed on tumor cells and activated endothelial cells. In contrast, expression of αvβ3 integrin is weak on resting endothelial cells and most normal tissues. A tripeptide sequence consisting of Arg-Gly-Asp (RGD) is identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins including αvβ3.

Echistatin is a 5.4 kDa snake venom disintegrin that binds to αvβ3 integrin with high affinity (18). Microbubbles were targeted by conjugation of echistatin to the shell surface and used to image angiogenesis with contrast-enhanced ultrasound (CEU) (19). Microbubbles-echistatin may provide a noninvasive method for assessing therapeutic angiogenesis (20).

Synthesis

[PubMed]

For targeted microbubbles, biotinylated microbubbles were first prepared by sonication of an aqueous dispersion of decafluorobutane gas, distearoylphosphatidylcholine, polyethyleneglycol-(PEG-) stearate, and distearoylphosphatidylethanolamine-PEG-biotin. Microbubbles were combined with streptavidin, washed, and combined with biotinylated echistatin (MBE) or monoclonal antibody (mAb R3-34) against αv integrin (MBα). Control lipid microbubbles (MBC) were also prepared. For confocal microscopy, microbubbles were fluorescently labeled by dioctadecyl tetramethylindocarbocyanine or dioctadecyl oxacarbocyanine. For perfusion imaging, nontargeted lipid-shelled microbubbles (MP1950) were also prepared. Microbubble concentration and size distribution were determined by electrozone sensing. The microbubbles are about 2-4 microns in diameter. A ligand to microbubble ratio was estimated to be about 60,000 by flow cytometry (19-21).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

No publication is currently available.

Animal Studies

Rodents

[PubMed]

The effect of bFGF on angiogenesis was assessed with microbubbles targeted to αvβ3 integrin. FGF-2 pellets or control pellets were implanted intrascrotally in mice for 4 days (19). FGF-2 stimulation of cremaster microvessel formation was confirmed by fluorescent and intravital microscopy as compared to control mice. Microvascular retention was significantly greater for MBE and MBα than that for MBc. Retained MBE and MBα attached directly to the microvascular endothelial surface. Microbubble retention in control mice was minimal. Matrigel plugs enriched with FGF-2 were subcutaneously implanted in mice for 10 days. Neovessels within the matrigel were positive for αv-integrins by immunostaining. CEU showed significantly greater acoustic intensity for MBE (16.0±5.9 U) and MBα (17.0±5.5 U) compared with MBc (5.8±2.6 U). The signal from targeted microbubbles (MBE and MBα) correlated well (r=0.90) with the matrigel blood volume determined by CEU perfusion imaging. CEU with microbubbles targeted for αv-integrins may provide a noninvasive method for assessing therapeutic angiogenesis.

Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells (20). On day 14 or day 28 after implantation, CEU was performed with MBE and MBc. CEU perfusion imaging with MBc was used to derive tumor microvascular blood volume and blood velocity. Vascular αv-integrin was expressed on the microvascular endothelial cells of tumor neovessels as assessed by immunohistochemistry. Tumor size increased significantly from 14 to 28 days (2±1 versus 35±14 mm2). Tumor blood volume increased by about 35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, MBE (7 to 20 per 10 optical fields) but not MBc (<1 per 10 optical fields) were retained preferentially within the tumor microcirculation. CEU signal from MBE in tumors increased significantly from 14 to 28 days (1.7±0.4 versus 3.3±1.0 relative units). CEU signal from control microbubbles increased non-significantly from 1.1±0.2 to 1.2±0.3 relative units from 14 days to 28 days. CEU signal from MBE was greatest at the periphery of tumors, where αvβ3 -integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86). CEU with MBE can non-invasively detect early tumor angiogenesis. This technique, in conjunction with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications.

Other Non-Primate Mammal Studies

[PubMed]

No publication is currently available.

Non-Human Primate Studies

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

K08 HL03810, R01 HL48890, R01 HL65704

References

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Dijkmans P.A., Juffermans L.J., Musters R.J., van Wamel A., ten Cate F.J., van Gilst W., Visser C.A., de Jong N., Kamp O. Microbubbles and ultrasound: from diagnosis to therapy. Eur J Echocardiogr. 2004;5(4):245–56. [PubMed: 15219539]
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Battegay E.J. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J Mol Med. 1995;73(7):333–46. [PubMed: 8520966]
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Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med. 2003;3(7):643–51. [PubMed: 14601638]
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Sinusas A.J. Imaging of angiogenesis. J Nucl Cardiol. 2004;11(5):617–33. [PubMed: 15472646]
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Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9(6):653–60. [PubMed: 12778163]
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Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6(4):389–95. [PubMed: 10742145]
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Folkman J. Endogenous angiogenesis inhibitors. Apmis. 2004;112(7-8):496–507. [PubMed: 15563312]
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O'Reilly M.S., Holmgren L., Shing Y., Chen C., Rosenthal R.A., Cao Y., Moses M., Lane W.S., Sage E.H., Folkman J. Angiostatin: a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth. Cold Spring Harb Symp Quant Biol. 1994;59:471–82. [PubMed: 7587101]
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Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1(1):27–31. [PubMed: 7584949]
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Carmeliet P. Manipulating angiogenesis in medicine. J Intern Med. 2004;255(5):538–61. [PubMed: 15078497]
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Miller J.C., Pien H.H., Sahani D., Sorensen A.G., Thrall J.H. Imaging angiogenesis: applications and potential for drug development. J Natl Cancer Inst. 2005;97(3):172–87. [PubMed: 15687360]
18.
McLane M.A., Sanchez E.E., Wong A., Paquette-Straub C., Perez J.C. Disintegrins. Curr Drug Targets Cardiovasc Haematol Disord. 2004;4(4):327–55. [PubMed: 15578957]
19.
Leong-Poi H., Christiansen J., Klibanov A.L., Kaul S., Lindner J.R. Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)-integrins. Circulation. 2003;107(3):455–60. [PubMed: 12551871]
20.
Ellegala D.B., Leong-Poi H., Carpenter J.E., Klibanov A.L., Kaul S., Shaffrey M.E., Sklenar J., Lindner J.R. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3. Circulation. 2003;108(3):336–41. [PubMed: 12835208]
21.
Lindner J.R., Song J., Christiansen J., Klibanov A.L., Xu F., Ley K. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation. 2001;104(17):2107–12. [PubMed: 11673354]

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