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Quantification of Endothelial αvβ3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies

      Abstract

      Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvβ3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvβ3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

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      References

        • Alonso A.
        • Della Martina A.
        • Stroick M.
        • Fatar M.
        • Griebe M.
        • Pochon S.
        • Schneider M.
        • Hennerici M.
        • Allemann E.
        • Meairs S.
        Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound.
        Stroke. 2007; 38: 1508-1514
        • Anderson C.R.
        • Hu X.
        • Zhang H.
        • Tlaxca J.
        • Decleves A.E.
        • Houghtaling R.
        • Sharma K.
        • Lawrence M.
        • Ferrara K.W.
        • Rychak J.J.
        Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent.
        Invest Radiol. 2011; 46: 215-224
        • Antonov A.S.
        • Kolodgie F.D.
        • Munn D.H.
        • Gerrity R.G.
        Regulation of macrophage foam cell formation by αvβ3 integrin: Potential role in human atherosclerosis.
        Am J Pathol. 2004; 165: 247-258
        • Barger A.C.
        • Beeuwkes 3rd, R.
        • Lainey L.L.
        • Silverman K.J.
        Hypothesis: Vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis.
        N Engl J Med. 1984; 310: 175-177
        • Bettinger T.
        • Bussat P.
        • Tardy I.
        • Pochon S.
        • Hyvelin J.M.
        • Emmel P.
        • Henrioud S.
        • Biolluz N.
        • Willmann J.K.
        • Schneider M.
        • Tranquart F.
        Ultrasound molecular imaging contrast agent binding to both e- and p-selectin in different species.
        Invest Radiol. 2012; 47: 516-523
        • Boyle J.J.
        Macrophage activation in atherosclerosis: Pathogenesis and pharmacology of plaque rupture.
        Curr Vasc Pharmacol. 2005; 3: 63-68
        • Brooks P.C.
        • Clark R.A.
        • Cheresh D.A.
        Requirement of vascular integrin alpha v beta 3 for angiogenesis.
        Science. 1994; 264: 569-571
        • Daeichin V.
        • Akkus Z.
        • Skachkov I.
        • Kooiman K.
        • Needles A.
        • Sluimer J.
        • Janssen B.
        • Daemen M.J.
        • van der Steen A.F.
        • de Jong N.
        • Bosch J.G.
        Quantification of bound microbubbles in ultrasound molecular imaging.
        IEEE Trans Ultrason Ferroelectr Freq Control. 2015; 62: 1190-1200
        • Daeichin V.
        • Bosch J.G.
        • Needles A.
        • Foster F.S.
        • van der Steen A.
        • de Jong N.
        Subharmonic, non-linear fundamental and ultraharmonic imaging of microbubble contrast at high frequencies.
        Ultrasound Med Biol. 2015; 41: 486-497
        • Davidson B.P.
        • Chadderdon S.M.
        • Belcik J.T.
        • Gupta S.
        • Lindner J.R.
        Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression.
        J Am Soc Echocardiog. 2014; 27: 786-793.e2
        • Dayton P.A.
        • Ferrara K.W.
        Targeted imaging using ultrasound.
        J Magn Reson Imaging. 2002; 16: 362-377
        • Demos S.M.
        • Alkan-Onyuksel H.
        • Kane B.J.
        • Ramani K.
        • Nagaraj A.
        • Greene R.
        • Klegerman M.
        • McPherson D.D.
        In vivo targeting of acoustically reflective liposomes for intravascular and transvascular ultrasonic enhancement.
        J Am Coll Cardiol. 1999; 33: 867-875
        • Deshpande N.
        • Ren Y.
        • Foygel K.
        • Rosenberg J.
        • Willmann J.K.
        Tumor angiogenic marker expression levels during tumor growth: Longitudinal assessment with molecularly targeted microbubbles and us imaging.
        Radiology. 2011; 258: 804-811
        • Ellegala D.B.
        Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to v 3.
        Circulation. 2003; 108: 336-341
        • Greis C.
        Ultrasound contrast agents as markers of vascularity and microcirculation.
        Clin Hemorheol Microcirc. 2009; 43: 1-9
        • Hamilton A.J.
        • Huang S.L.
        • Warnick D.
        • Rabbat M.
        • Kane B.
        • Nagaraj A.
        • Klegerman M.
        • McPherson D.D.
        Intravascular ultrasound molecular imaging of atheroma components in vivo.
        J Am Coll Cardiol. 2004; 43: 453-460
        • Haubner R.
        Alphavbeta3-integrin imaging: A new approach to characterise angiogenesis?.
        Eur J Nucl Med Mol Imaging. 2006; 1: 54-63
        • Hauff P.
        • Reinhardt M.
        • Briel A.
        • Debus N.
        • Schirner M.
        Molecular targeting of lymph nodes with l-selectin ligand-specific us contrast agent: A feasibility study in mice and dogs.
        Radiology. 2004; 231: 667-673
        • Hellings W.E.
        • Peeters W.
        • Moll F.L.
        • Piers S.R.
        • van Setten J.
        • Van der Spek P.J.
        • de Vries J.P.
        • Seldenrijk K.A.
        • De Bruin P.C.
        • Vink A.
        • Velema E.
        • de Kleijn D.P.
        • Pasterkamp G.
        Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: A prognostic study.
        Circulation. 2010; 121: 1941-1950
        • Hoshiga M.
        • Alpers C.E.
        • Smith L.L.
        • Giachelli C.M.
        • Schwartz S.M.
        Alpha-v beta-3 integrin expression in normal and atherosclerotic artery.
        Circ Res. 1995; 77: 1129-1135
        • Hynes R.O.
        Integrins: A family of cell surface receptors.
        Cell. 1987; 48: 549-554
        • Kaufmann B.A.
        Ultrasound molecular imaging of atherosclerosis.
        Cardiovasc Res. 2009; 83: 617-625
        • Kaufmann B.A.
        • Lindner J.R.
        Molecular imaging with targeted contrast ultrasound.
        Curr Opin Biotechnol. 2007; 18: 11-16
        • Khanicheh E.
        • Mitterhuber M.
        • Xu L.
        • Haeuselmann S.P.
        • Kuster G.M.
        • Kaufmann B.A.
        Noninvasive ultrasound molecular imaging of the effect of statins on endothelial inflammatory phenotype in early atherosclerosis.
        PloS One. 2013; 8: e58761
        • Khanicheh E.
        • Qi Y.
        • Xie A.
        • Mitterhuber M.
        • Xu L.
        • Mochizuki M.
        • Daali Y.
        • Jaquet V.
        • Krause K.H.
        • Ruggeri Z.M.
        • Kuster G.M.
        • Lindner J.R.
        • Kaufmann B.A.
        Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties.
        Arterioscl Thromb Vasc Biol. 2013; 33: 2187-2192
        • Klibanov A.L.
        Microbubble contrast agents: Targeted ultrasound imaging and ultrasound-assisted drug-delivery applications.
        Invest Radiol. 2006; 41: 354-362
        • Laitinen I.
        • Saraste A.
        • Weidl E.
        • Poethko T.
        • Weber A.W.
        • Nekolla S.G.
        • Leppänen P.
        • Ylä-Herttuala S.
        • Hölzlwimmer G.
        • Walch A.
        • Esposito I.
        • Wester H.-J.
        • Knuuti J.
        • Schwaiger M.
        Evaluation of αvβ3 integrin-targeted positron emission tomography tracer 18F-galacto-RGD for imaging of vascular inflammation in atherosclerotic mice.
        Circ Cardiovasc Imaging. 2009; 2: 331-338
        • Langheinrich A.C.
        • Kampschulte M.
        • Buch T.
        • Bohle R.M.
        Vasa vasorum and atherosclerosis— Quid novi?.
        Thromb Haemost. 2007; 97: 873-879
        • Lanza G.M.
        • Wallace K.D.
        • Scott M.J.
        • Cacheris W.P.
        • Abendschein D.R.
        • Christy D.H.
        • Sharkey A.M.
        • Miller J.G.
        • Gaffney P.J.
        • Wickline S.A.
        A novel site-targeted ultrasonic contrast agent with broad biomedical application.
        Circulation. 1996; 94: 3334-3340
        • 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: 455-460
        • Lindner J.R.
        Microbubbles in medical imaging: Current applications and future directions.
        Nat Rev Drug Discov. 2004; 3: 527-533
        • Lindner J.R.
        Contrast ultrasound molecular imaging of inflammation in cardiovascular disease.
        Cardiovasc Res. 2010; 26: 116
        • 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: 2107-2112
        • Liu Y.
        • Davidson B.P.
        • Yue Q.
        • Belcik T.
        • Xie A.
        • Inaba Y.
        • McCarty O.J.
        • Tormoen G.W.
        • Zhao Y.
        • Ruggeri Z.M.
        • Kaufmann B.A.
        • Lindner J.R.
        Molecular imaging of inflammation and platelet adhesion in advanced atherosclerosis: Effects of antioxidant therapy with NADPH oxidase inhibition.
        Circ Cardiovasc Imaging. 2013; 6: 74-82
        • Michel J.B.
        • Virmani R.
        • Arbustini E.
        • Pasterkamp G.
        Intraplaque haemorrhages as the trigger of plaque vulnerability.
        Eur Heart J. 2011; 32: 1977-1985
        • Moreno P.R.
        • Purushothaman K.R.
        • Fuster V.
        • Echeverri D.
        • Truszczynska H.
        • Sharma S.K.
        • Badimon J.J.
        • O'Connor W.N.
        Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: Implications for plaque vulnerability.
        Circulation. 2004; 110: 2032-2038
        • Moreno P.R.
        • Purushothaman K.R.
        • Sirol M.
        • Levy A.P.
        • Fuster V.
        Neovascularization in human atherosclerosis.
        Circulation. 2006; 113: 2245-2252
        • Muchir A.
        • Shan J.
        • Bonne G.
        • Lehnart S.E.
        • Worman H.J.
        Inhibition of extracellular signal-regulated kinase signaling to prevent cardiomyopathy caused by mutation in the gene encoding a-type lamins.
        Hum Mol Genet. 2009; 18: 241-247
        • Naghavi M.
        • Libby P.
        • Falk E.
        • Casscells S.W.
        • Litovsky S.
        • Rumberger J.
        • Badimon J.J.
        • Stefanadis C.
        • Moreno P.
        • Pasterkamp G.
        • Fayad Z.
        • Stone P.H.
        • Waxman S.
        • Raggi P.
        • Madjid M.
        • Zarrabi A.
        • Burke A.
        • Yuan C.
        • Fitzgerald P.J.
        • Siscovick D.S.
        • de Korte C.L.
        • Aikawa M.
        • Airaksinen K.E.
        • Assmann G.
        • Becker C.R.
        • Chesebro J.H.
        • Farb A.
        • Galis Z.S.
        • Jackson C.
        • Jang I.K.
        • Koenig W.
        • Lodder R.A.
        • March K.
        • Demirovic J.
        • Navab M.
        • Priori S.G.
        • Rekhter M.D.
        • Bahr R.
        • Grundy S.M.
        • Mehran R.
        • Colombo A.
        • Boerwinkle E.
        • Ballantyne C.
        • Insull Jr., W.
        • Schwartz R.S.
        • Vogel R.
        • Serruys P.W.
        • Hansson G.K.
        • Faxon D.P.
        • Kaul S.
        • Drexler H.
        • Greenland P.
        • Muller J.E.
        • Virmani R.
        • Ridker P.M.
        • Zipes D.P.
        • Shah P.K.
        • Willerson J.T.
        From vulnerable plaque to vulnerable patient: A call for new definitions and risk assessment strategies. Part II.
        Circulation. 2003; 108: 1772-1778
        • Needles A.
        • Arditi M.
        • Rognin N.G.
        • Mehi J.
        • Coulthard T.
        • Bilan-Tracey C.
        • Gaud E.
        • Frinking P.
        • Hirson D.
        • Foster F.S.
        Nonlinear contrast imaging with an array-based micro-ultrasound system.
        Ultrasound Med Biol. 2010; 36: 2097-2106
        • Ni M.
        • Wang Y.
        • Zhang M.
        • Zhang P.-F.
        • Ding S.-F.
        • Liu C.X.
        • Liu X.L.
        • Zhao Y.X.
        • Zhang Y.
        Atherosclerotic plaque disruption induced by stress and lipopolysaccharide in apolipoprotein e knockout mice.
        Am J Physiol Heart Circ Physiol. 2009; 296: H1598-H1606
        • Palmowski M.
        • Huppert J.
        • Ladewig G.
        • Hauff P.
        • Reinhardt M.
        • Mueller M.M.
        • Woenne E.C.
        • Jenne J.W.
        • Maurer M.
        • Kauffmann G.W.
        • Semmler W.
        • Kiessling F.
        Molecular profiling of angiogenesis with targeted ultrasound imaging: Early assessment of antiangiogenic therapy effects.
        Mol Cancer Ther. 2008; 7: 101-109
        • Petronzelli F.
        • Pelliccia A.
        • Anastasi A.M.
        • Lindstedt R.
        • Manganello S.
        • Ferrari L.E.
        • Albertoni C.
        • Leoni B.
        • Rosi A.
        • D'Alessio V.
        • Deiana K.
        • Paganelli G.
        • De Santis R.
        Therapeutic use of avidin is not hampered by antiavidin antibodies in humans.
        Cancer Biother Radiopharm. 2010; 25: 563-570
        • Purushothaman K.R.
        • Fuster V.
        • O'Connor W.N.
        • Moreno P.R.
        Neovascularization is the most powerful independent predictor for progression to disruption in high-risk atherosclerotic plaques.
        J Am Coll Cardiol. 2003; 41: 352-353
        • Rademakers T.
        • Douma K.
        • Hackeng T.M.
        • Post M.J.
        • Sluimer J.C.
        • Daemen M.J.
        • Biessen E.A.
        • Heeneman S.
        • van Zandvoort M.A.
        Plaque-associated vasa vasorum in aged apolipoprotein e–deficient mice exhibit proatherogenic functional features in vivo.
        Arterioscler Thromb Vasc Biol. 2013; 33: 249-256
        • Redgrave J.N.
        • Lovett J.K.
        • Gallagher P.J.
        • Rothwell P.M.
        Histological assessment of 526 symptomatic carotid plaques in relation to the nature and timing of ischemic symptoms: The Oxford Plaque study.
        Circulation. 2006; 113: 2320-2328
        • Rigatelli G.
        • Zanchetta M.
        Endovascular therapies for noncoronary atherosclerosis in the elderly: Aortoiliac and femorotibial lesions.
        Am J Geriatr Cardiol. 2005; 14: 195-199
        • Shelton S.E.
        • Lindsey B.D.
        • Tsuruta J.K.
        • Foster F.S.
        • Dayton P.A.
        Molecular acoustic angiography: A new technique for high-resolution superharmonic ultrasound molecular imaging.
        Ultrasound Med Biol. 2016; 42: 769-781
        • Shim C.Y.
        • Liu Y.N.
        • Atkinson T.
        • Xie A.
        • Foster T.
        • Davidson B.P.
        • Treible M.
        • Qi Y.
        • Lopez J.A.
        • Munday A.
        • Ruggeri Z.
        • Lindner J.R.
        Molecular imaging of platelet–endothelial interactions and endothelial von Willebrand factor in early and mid-stage atherosclerosis.
        Circ Cardiovasc Imaging. 2015; 8: e002765
        • Sluimer J.C.
        • Gasc J.M.
        • van Wanroij J.L.
        • Kisters N.
        • Groeneweg M.
        • Sollewijn Gelpke M.D.
        • Cleutjens J.P.
        • van den Akker L.H.
        • Corvol P.
        • Wouters B.G.
        • Daemen M.J.
        • Bijnens A.P.
        Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis.
        J Am Coll Cardiol. 2008; 51: 1258-1265
        • Staub D.
        • Patel M.B.
        • Tibrewala A.
        • Ludden D.
        • Johnson M.
        • Espinosa P.
        • Coll B.
        • Jaeger K.A.
        • Feinstein S.B.
        Vasa vasorum and plaque neovascularization on contrast-enhanced carotid ultrasound imaging correlates with cardiovascular disease and past cardiovascular events.
        Stroke. 2010; 41: 41-47
        • Stieger S.M.
        • Dayton P.A.
        • Borden M.A.
        • Caskey C.F.
        • Griffey S.M.
        • Wisner E.R.
        • Ferrara K.W.
        Imaging of angiogenesis using cadence contrast pulse sequencing and targeted contrast agents.
        Contrast Media Mol Imaging. 2008; 3: 9-18
        • Streeter J.E.
        • Herrera-Loeza S.G.
        • Neel N.F.
        • Yeh J.J.
        • Dayton P.A.
        A comparative evaluation of ultrasound molecular imaging, perfusion imaging, and volume measurements in evaluating response to therapy in patient-derived xenografts.
        Technol Cancer Res Treat. 2013; 12: 311-321
        • Tedesco M.M.
        • Terashima M.
        • Blankenberg F.G.
        • Levashova Z.
        • Spin J.M.
        • Backer M.V.
        • Backer J.M.
        • Sho M.
        • Sho E.
        • McConnell M.V.
        • Dalman R.L.
        Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.
        Arterioscler Thromb Vasc Biol. 2009; 29: 1452-1459
        • ten Kate G.L.
        • Renaud G.G.
        • Akkus Z.
        • van den Oord S.C.
        • ten Cate F.J.
        • Shamdasani V.
        • Entrekin R.R.
        • Sijbrands E.J.
        • de Jong N.
        • Bosch J.G.
        • Schinkel A.F.
        • van der Steen A.F.
        Far-wall pseudoenhancement during contrast-enhanced ultrasound of the carotid arteries: Clinical description and in vitro reproduction.
        Ultrasound Med Biol. 2012; 38: 593-600
        • ten Kate G.L.
        • Sijbrands E.J.
        • Valkema R.
        • ten Cate F.J.
        • Feinstein S.B.
        • van der Steen A.F.
        • Daemen M.J.
        • Schinkel A.F.
        Molecular imaging of inflammation and intraplaque vasa vasorum: A step forward to identification of vulnerable plaques?.
        J Nucl Cardiol. 2010; 17: 897-912
        • van den Oord S.C.H.
        • Akkus Z.
        • Bosch J.G.
        • Hoogi A.
        • ten Kate G.L.
        • Renaud G.
        • Sijbrands E.J.G.
        • Verhagen H.J.
        • van der Lugt A.
        • Adam D.
        • de Jong N.
        • van der Steen A.F.W.
        • Schinkel A.F.L.
        Quantitative contrast-enhanced ultrasound of intraplaque neovascularization in patients with carotid atherosclerosis.
        Ultraschall Med. 2015; 36: 154-161
        • van Rooij T.
        • Daeichin V.
        • Skachkov I.
        • de Jong N.
        • Kooiman K.
        Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy.
        Int J Hyperthermia. 2015; 31: 90-106
        • Winter P.M.
        • Morawski A.M.
        • Caruthers S.D.
        • Fuhrhop R.W.
        • Zhang H.
        • Williams T.A.
        • Allen J.S.
        • Lacy E.K.
        • Robertson J.D.
        • Lanza G.M.
        • Wickline S.A.
        Molecular imaging of angiogenesis in early-stage atherosclerosis with αvβ3-integrin–targeted nanoparticles.
        Circulation. 2003; 108: 2270-2274
        • Yan F.
        • Li X.
        • Jin Q.
        • Chen J.
        • Shandas R.
        • Wu J.
        • Li L.
        • Ling T.
        • Yang W.
        • Chen Y.
        • Liu X.
        • Zheng H.
        Ultrasonic imaging of endothelial CD81 expression using CD81-targeted contrast agents in in vitro and in vivo studies.
        Ultrasound Med Biol. 2012; 38: 670-680