Published online before print June 30, 2003, doi: 10.1161/01.CIR.0000080326.15367.0C
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2003;108:336.)
© 2003 American Heart Association, Inc.
Basic Science Reports |
Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to 
vß3
From the Department of Neurosurgery (D.B.E., J.E.C., M.E.S.) and the Cardiovascular Division (H.L.P., A.L.K., S.K., J.S., J.R.L.), University of Virginia School of Medicine, Charlottesville.
Correspondence to Jonathan R. Lindner, MD, Box 800158, Cardiovascular Division, University of Virginia Medical Center, Charlottesville, VA 22908. E-mail jlindner{at}virginia.edu
Received February 18, 2003; revision received April 9, 2003; accepted April 9, 2003.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrast-enhanced ultrasound (CEU) with microbubbles targeted to
v-integrins expressed on the neovascular endothelium could be used to image angiogenesis.
Methods and Results— Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to vß3 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular v-integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2±1 versus 35±14 mm2, P<0.001). Tumor blood volume increased by 
35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, vß3-targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from vß3-targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7±0.4 versus 3.3±1.0 relative units, P<0.05). CEU signal from vß3-targeted microbubbles was greatest at the periphery of tumors, where v-integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86).
Conclusions— CEU with microbubbles targeted to vß3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications.
Key Words: angiogenesis • cancer • ultrasound
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The growth of new blood vessels plays an important role in many disease processes, including chronic ischemic cardiovascular disease, wound healing, and cancer. For tumors, angiogenesis appears to be a critical determinant of growth, invasion, and metastatic potential.1–3 Noninvasive imaging techniques to assess tumor angiogenesis are currently being developed to diagnose primary and metastatic disease and to assess prognosis. Most new methods for evaluating angiogenesis rely on the detection of abnormal perfusion, microvascular blood volume, or vascular permeability. An alternate strategy is to detect abnormal vascular endothelial cell phenotype. Detection of
v-integrin expression in tumor neovessels may be particularly advantageous, since these integrins appear to play a functional role in angiogenesis4,5 and have been implicated as a marker of metastatic potential and poor prognosis in certain tumors.6 Assessing tumor vascular phenotype may also provide important information on susceptibility to novel antiangiogenic tumoricidal therapies.
Contrast-enhanced ultrasound (CEU) with targeted microbubble contrast agents has recently been used to noninvasively assess expression of endothelial cell adhesion molecules in vivo.7,8 Microbubbles targeted to v-integrins by conjugation of the disintegrin echistatin to their shell surface have recently been developed that adhere to the endothelial surface of FGF-2–treated microvessels and to matrigel neovessels.9 In this study, we hypothesized that microbubbles targeted to vß3 could be used to noninvasively evaluate tumor angiogenesis with ultrasound. To test our hypothesis, targeted imaging of a malignant glioma model in rats was performed and compared with data on tumor blood flow and neovascular blood volume derived from established techniques for CEU perfusion imaging.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Microbubble Preparation
For targeted microbubbles, biotinylated microbubbles were first prepared by sonication of an aqueous dispersion of decafluorobutane gas, distearoylphosphatidylcholine (Avanti Polar Lipids), polyethyleneglycol-(PEG-) stearate (Sigma), and distearoyl-phosphatidylethanolamine-PEG-biotin (prepared from components from Avanti Polar Lipids, Shearwater Polymers). Microbubbles were combined with streptavidin (Sigma), washed, and combined with biotinylated echistatin (Sigma).9 Control lipid microbubbles without echistatin were also prepared. For confocal microscopy, microbubbles were fluorescently labeled by dioctadecyl tetramethylindocarbocyanine (DiI) (Molecular Probes).9 For perfusion imaging, nontargeted, lipid-shelled microbubbles (MP1950) were prepared.10 Microbubble size distribution was determined by electrozone sensing (Coulter Multisizer IIe, Beckman-Coulter).
Glioblastoma Tumor Model
Tumors were created by intracerebral implantation of human glioma cells in athymic rats (Hilltop Lab Animals, Inc, Scottdale, Pa). Eighteen rats were anesthetized with ketamine (40 mg/kg) and xylazine (8 mg/kg). Under sterile conditions, a small skin incision was made and a 2-mm burr hole was made in the parietal bone of the skull. In 14 of these rats, 1x105 U87MG cultured human glioma cells embedded in absorbable gelatin matrix were stereotactically implanted through the burr hole into the cerebral hemisphere at a depth of 4 to 6 mm. Gelatin matrix alone was implanted into 4 control rats. The incision was closed and animals were studied at either 14 (n=5) or 28 (n=9) days after U87MG cell implantation and at 28 days for control rats.
Contrast-Enhanced Ultrasound Imaging
For contrast administration, a jugular vein was cannulated with PE50 tubing. CEU of the brain was performed in the mid-coronal plane using gray-scale pulse-inversion imaging (HDI 5000, Philips Ultrasound) and a linear-array transducer (L7–4). Transmission frequency of 3.3 MHz and a mechanical index of 0.8 were used. The acoustic focus was placed at the mid-brain level. Gain settings were optimized and kept constant throughout the experiments.
Targeted Imaging
Data were acquired 10 minutes after bolus intravenous injection of 1x108 vß3-targeted or control microbubbles, performed in random order. The initial frame, containing signal from both retained and any freely circulating microbubbles,10 was captured. Microbubbles were then destroyed with continuous (
30 Hz) high-power imaging for 3 seconds. Several frames subsequently obtained at a pulsing interval (PI) of 20 seconds, containing contrast-enhanced signal only from freely circulating microbubbles, were averaged and digitally subtracted from the initial frame to derive the signal from retained microbubbles alone.10 Since the number of microbubbles retained depends on both binding kinetics and regional microbubble influx, the image representing retained microbubbles was normalized to parametric blood flow data (below) to derive a parametric color-coded image uniquely reflecting the microbubble retention fraction.
Perfusion Imaging
Microvascular perfusion was measured by means of intermittent imaging at a mechanical index of 0.9 to 1.0 during a continuous intravenous infusion of MP1950 (10 to 12.5 µL/min), as previously described.11,12 Acoustic intensity (AI) was measured within regions of interest determined from review of corresponding histologic sections. PI versus AI data were fit to the function y=A (1-e-ßt), where y is AI at the pulsing interval t, A is plateau intensity or microvascular blood volume, and ß is the rate constant reflecting microvascular red blood cell (RBC) velocity.12 Blood flow was determined by the product of A and ß. Parametric images of A, ß, and Axß were derived by fitting the 1-exponential function to data on a pixel-by-pixel basis.
Histology and Immunohistochemistry
Immunostaining was performed on paraffin-embedded sections. For detecting v-integrin expression, an affinity-purified rabbit polyclonal antibody against rat v subunit (AB1930, Chemicon International) was used as a primary antibody. A biotinylated goat anti-rat antibody (Vector laboratories) was used as a secondary antibody. Staining was performed using a peroxidase kit (ABC Vectastain Elite, Vector Laboratories) and 3,3'-diaminobenzidine chromogen (DAKO). Slides were counterstained with hematoxylin. For control purposes, staining was also performed with secondary antibody alone, which did not demonstrate any nonspecific staining. Hematoxylin and eosin staining was also performed on paraffin-embedded sections, and mean tumor size was determined by the largest area in the coronal plane. These sections were used to register imaging data for performing regional analysis of perfusion and targeted microbubble signal.
Confocal Microscopy
Fluorescent confocal microscopy was performed in 4 rats 28 days after U87MG implantation. A single intravenous injection of either 1x108 di-I–labeled control or vß3-targeted microbubbles was performed in 2 animals for each agent. After 10 minutes, 1 mg fluorescein Lycopersiconesculentum lectin (Vector laboratories) in saline was infused. Perfusion fixation was performed with 4% paraformaldehyde in PBS, and thick sections of the brain were examined with a confocal laser scanning microscope (LSM510, Carl Zeiss Inc) (x20 objective) and dual fluorescence overlays. Observations were made in 15 to 20 optical fields throughout the tumor and contralateral normal hemisphere.
Statistical Methods
Data are expressed as mean±1 SD. Comparisons of continuous variables were made by means of the unpaired Student’s t test. Bonferroni’s correction was applied for multiple comparisons. Correlations between targeted contrast ultrasound signal and blood volume were made by linear regression analysis. Differences were considered significant at P<0.05 (2-sided).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Tumor Histology
All control rats and 12 of 14 injected with U87MG cells survived to their assigned follow-up period. Tumors were detected by histology in all animals undergoing U87MG implantation (Figure 1A). Mean tumor size measured in the coronal plane was substantially greater at 28 versus 14 days (35±14 versus 2±1 mm2, P<0.001). Immunohistology demonstrated
v-integrin expression on the microvascular endothelium of tumor neovessels (Figure 1B), the density of which was greatest at the outer margins of the tumor. Microvascular endothelial v-integrin staining was also observed heterogeneously in nonneoplastic tissue immediately adjacent to the tumors (Figure 1B) but not in control regions contralateral to the tumors or in regions injected with gelatin matrix vehicle in the control rats.
|
Tumor Perfusion by CEU
Illustrated in Figure 2 are CEU images and corresponding pulsing interval versus acoustic intensity data demonstrating microvascular perfusion abnormalities in a rat 28 days after U87MG implantation. In this example, the glioma was characterized by slower blood velocity but slightly greater microvascular blood volume compared with normal parenchyma from the contralateral hemisphere. A gradient of microvascular blood velocity from peripheral to central portions of the tumor can be discerned. The results for all rats are depicted in Figure 3, where data from tumors or from regions of gelatin vehicle implantation are normalized to the contralateral normal hemisphere. In control animals, no abnormalities in microvascular blood volume, blood velocity, or blood flow were seen in regions of gelatin vehicle implantation. In tumors, normalized microvascular blood volume was significantly lower than control regions at 14 days but increased by day 28. Microvascular blood velocity progressively decreased with tumor growth. At day 28, mean blood velocity (ß) was lower in the central compared with outer half of the tumor (0.14±0.02 versus 0.28±0.10 s-1, P<0.05). Normalized blood flow also tended to decrease with tumor growth, and was lower in the inner compared with outer half of the tumor (0.45±0.19 versus 0.70±0.10, P<0.05) at day 28. In nonneoplastic parenchyma immediately surrounding the tumor, increased microvascular blood flow was often observed with abundant high-velocity vessels (Figure 2B). However, blood flow in these regions was sometimes reduced when tumor size was large, possibly from tumor compression (Figure 4B).
|
|
P<0.05 compared with tumor data at 14 days.
|
Targeted CEU Imaging of v-Integrin Expression
The mean diameter of control and vß3-targeted microbubbles was not significantly different (3.3±0.4 versus 3.1±0.4 µm). On confocal microscopy, microbubble retention was observed within tumor microvessels at 28 days for vß3-targeted (range, 7 to 20 per 10 optical fields) but not control microbubbles (
1 per 10 optical fields) (Figure 4A). Retention of targeted microbubbles was most frequently detected in small (<15 µm diameter) neovessels, although retention was occasionally seen in larger vessels at the tumor margins. Microbubbles were not observed in the extravascular space and were rarely observed in contralateral control regions (1 per 20 optical fields).
Contrast ultrasound performed 10 minutes after injection of vß3-targeted microbubbles demonstrated intense signal enhancement within the tumors and lower intensity enhancement in surrounding nonneoplastic brain parenchyma (Figure 4B). Signal enhancement with targeted microbubbles was also seen in the few cases of metastatic seeding of the meninges (Figure 4B). Results from all animals are depicted in Figure 5. For control microbubbles, signal enhancement in tumors or in regions of gelatin matrix implantation relative to the contralateral normal hemisphere was low. Signal enhancement was also low for vß3-targeted microbubbles in control regions implanted with gelatin matrix. The relative signal from vß3-targeted microbubbles in tumors was much greater than in control regions and was higher for tumors at 28 compared with 14 days. The relative signal was higher for the outer versus the inner half of the tumor at 28 days, although this difference reached only borderline statistical significance (3.3±1.0 versus 2.4±0.4, P=0.06). The CEU signal from vß3-targeted microbubbles correlated well with microvascular blood volume within the tumors derived from contrast ultrasound perfusion imaging (Figure 6).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In this study, we have demonstrated for the first time that tumor angiogenesis can be assessed with ultrasound imaging and microbubbles targeted to
v-integrins. These data combined with CEU-derived regional tissue blood velocity, volume, and flow are likely to provide important physiological insights into angiogenesis associated with neoplastic and chronic ischemic cardiovascular diseases. The growth of most solid tumors is highly dependent on local vascular proliferation. The ability to image angiogenesis may, therefore, provide a sensitive means to diagnose malignancies and to predict their rapid expansion. Assessing angiogenic phenotype may also provide important information on metastatic potential since abnormalities of neovascular architecture and permeability are thought to facilitate tumor cell shedding into the circulation.3 Vascular density has been shown to correlate with the likelihood of metastasis and the overall prognosis for patients with several types of cancers.3,13,14
In this study, we examined malignant gliomas, which are highly dependent on angiogenesis for growth and invasion.15 Angiogenesis was assessed by using two different CEU techniques. Molecular characterization was performed with a novel microbubble contrast agent targeted to vß3. This integrin is highly expressed by angiogenic endothelium and is thought to play a functional role in matrix remodeling, and anchorage-dependent endothelial migration and proliferation.4,5,16 Vascular expression of vß3 has also been shown to predict metastasis and poor survival.6
Microbubbles were targeted by conjugation of echistatin to the shell surface. This peptide derived from the venom of the viper Echis carinatus bears the RGD motif and has enhanced binding affinity for vß3 over nonactivated 2bß3.17 Echistatin-bearing microbubbles have been shown to adhere to the endothelial surface of microvessels treated with sustained-release FGF-2 and produce strong ultrasound signal enhancement in subcutaneous matrigel models of angiogenesis in mice.9 Although targeting v-integrins in tumors has been explored with magnetic resonance and positron emission tracers,18,19 we believe that microbubbles are ideal for detecting vascular endothelial phenotype alone since they are not diffusible and are confined to the vascular compartment.
The functional aspect of angiogenesis was also evaluated in this study by quantifying microvascular perfusion with nontargeted microbubbles. Perfusion patterns on CEU have been used previously to differentiate malignant from benign tumors in the liver20 and to enhance sensitivity of ultrasound for diagnosing breast and prostate tumors.21,22 However, correlation between CEU data and tumor vascularity on histology has been met with mixed results.23–25 One reason for discordance is that the extent of tumor angiogenesis may not necessarily be reflected by perfusion due to low functional efficiency of tumor neovessels. In other words, the percentage of vessels with flow at any given point of time may be low.26 Another more practical reason has been the use of high frame rate, high power imaging techniques that preferentially detect flow in large rather than small vessels. We instead used imaging techniques and off-line processing that provides information on microvascular perfusion11 and that has been shown to be accurate for assessing malignant neovascularization.25
The results from molecular and perfusion imaging at different time intervals in this study are congruent with the angiogenic progression for malignant gliomas. Intravital microscopy of gliomas has revealed an initial avascular slow growth phase in the first week followed by vascular budding in adjacent tissues and early development of a disordered tumor microcirculation with low functional efficiency in the second week.26,27 Accordingly, we found that microvascular blood volume in tumors at 14 days after U87MG implantation was lower than in normal brain parenchyma. Yet, early neovessel development was detected by enhanced signal from vß3-targeted microbubbles. By 28 days, when glioma neovascularization is advanced,26,27 microvascular blood volume by CEU had increased and was generally slightly greater than normal tissue. A strong signal from vß3-targeted microbubbles was obtained at this time and was greatest at the outer margins of the tumor, where angiogenic activity is the greatest26 and where v-integrin staining was most prominent on immunohistology. Despite an increase in microvascular blood volume from 14 to 28 days, mean blood flow for the entire tumor progressively decreased due to a reduction over time in RBC velocity in the glioma microcirculation. The radial gradient in RBC velocity in gliomas at 28 days has been similarly been observed with microscopy.26 These spatial heterogeneities in perfusion have been attributed to altered fractal dimensions, increased interstitial pressures, and abnormal vasoregulatory and hemorheologic properties and are responsible in part for predictable spatial heterogeneities in pH and oxygen that influence tumor growth and necrosis.28,29
At both 14 and 28 days after U87MG implantation, vß3-targeted microbubbles produced signal enhancement in nonneoplastic brain parenchyma surrounding the tumors. Expression of v-integrins on the endothelium of microvessels was found occasionally in these regions, consistent with vascular remodeling required for microvascular sprouting from adjacent normal tissue.30 Although signal-enhancement from vß3-targeted microbubbles in adjacent nonneoplastic parenchyma overestimates tumor size, it may also enhance sensitivity for detecting very small tumors or metastases.
Several limitations of the study should be noted. Although the signal from vß3-targeted microbubbles correlated closely with the extent of angiogenesis, gliomas were evaluated early during rapid growth phase when vascular v-integrin expression is likely to be greatest. Whether vß3-integrin imaging will be useful later in the angiogenic progression needs to be determined. The ligand echistatin can also bind other endothelial ligands such as 5ß1, the expression of which is also increased in neovascular endothelium. However, any strategy that uses small peptides for targeting vß3 is likely to be limited by some degree of binding to other RGD-binding integrins. Although the number of animals was small, we believed the marked differences in targeted microbubble signal did not justify continued investigation. A new method for deriving retention fraction was developed since the number of retained microbubbles is dependent not only on binding kinetics but also on regional blood flow, which determines microbubble influx. This method assumes that binding efficiency is not influenced by blood flow or microvascular velocity. This assumption is, however, based on our in vivo and flow chamber observations that targeted microbubble retention is influenced mostly by ligand density and is relatively independent of shear stresses at or below physiological values.7 The image processing algorithms used relied on digital subtraction of log-compressed rather than linear acoustic intensity data. Use of log-compressed data for perfusion assessment has been validated previously against radiolabeled microsphere measurements12 and is relatively accurate since received signal intensities are still relatively low where log and linear data are similar. Finally, we did not directly correlate microbubble density on confocal microscopy to signal intensity because of the highly deformable nature of the thick sections which precluded estimation of tissue volume. The purpose of confocal microscopy was instead to make qualitative descriptions of the location of microbubble retention and to exclude aggregation or extravasation.
We conclude that microbubbles targeted to v-integrins can be used to image and spatially assess angiogenic responses that occur early in the development of malignant gliomas. Further investigation will be needed to determine whether targeted imaging of angiogenic phenotype in different tumor types provides additional diagnostic and prognostic information to imaging methods already used in the clinical setting.
|
Acknowledgments |
|---|
This study was supported by grants R01-HL48890, R01-HL65704, and K08-HL03810 from the National Institutes of Health, Bethesda, Md; a Grant-in-Aid from the American Heart Association Mid-Atlantic Affiliate, Baltimore, Md; and a grant from the Pratt Foundation, Charlottesville, Va. Dr Leong-Poi is a recipient of a Fellowship Award from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada (H.L.P.). The authors are grateful to Dr Scott Vandenberg, Dr Maria-Beatrice Lopes, and John Sanders for their assistance with histopathology and Gerard Redpath for cell cultures.
|
Footnotes |
|---|
*Drs Ellegala and Leong-Poi contributed equally to this work.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971; 285: 1182–1186.[Medline] [Order article via Infotrieve]
2. Weidner N, Semple JP, Welch WR, et al. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med. 1991; 324: 1–8.[Abstract]
3. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med. 1998; 49: 407–424.[CrossRef][Medline] [Order article via Infotrieve]
4. Brooks PC, Clark RAF, Cheresh DA. Requirement of vascular integrin vß3 for angiogenesis. Science. 1994; 264: 569–571.
5. Friedlander M, Brooks PC, Shaffer R, et al. Definition of two angiogenic pathways by distinct v integrins. Science. 1995; 270: 1500–1503.
6. Gasparinin G, Brooks PC, Biganzoli E, et al. Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer. Clin Cancer Res. 1998; 4: 2625–2634.[Abstract]
7. Lindner JR, Song J, Christiansen JP, et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation. 2001; 104: 2107–2112.
8. Demos SM, Alkan-Onyuksel H, Kane BJ, et al. In vivo targeting of acoustically active reflective liposomes for intravascular and transvascular ultrasonic enhancement. J Am Coll Cardiol. 1999; 33: 867–875.
9. Leong-Poi H, Christiansen J, Klibanov AL, et al. Non-invasive assessment of angiogenesis by ultrasound and microbubbles targeted to v-integrins. Circulation. 2003; 107: 455–460.
10. Lindner JR, Song J, Xu F, et al. Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes. Circulation. 2000; 102: 2745–2750.
11. Dawson D, Vincent MA, Clarke A, et al. Assessment of capillary recruitment in skeletal muscle using contrast-enhanced ultrasound. Am J Physiol. 2002; 282: E714–E720.
12. Wei K, Jayaweera AR, Firoozan S, et al. Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion. Circulation. 1998; 97: 473–483.
13. Weidner N, Carroll PR, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993; 143: 401–409.[Abstract]
14. Angeletti CA, Lucchi M, Fontanini G. et al. Prognostic significance of tumoral angiogenesis in completely resected late stage lung carcinoma (stage IIIA-N2). Cancer. 1996; 78: 409–415.[CrossRef][Medline] [Order article via Infotrieve]
15. Millauer B, Shawver LK, Plate KH, et al. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature. 1994; 367: 576–579.[CrossRef][Medline] [Order article via Infotrieve]
16. Meredith JE, Fazeli B, Schwartz MA. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993; 4: 953–961.[Abstract]
17. Wierzbicka-Patynowski I, Niewiarowski S, Marcinkiewicz, et al. Structural requirements of echistatin for the recognition of vß3 and 5ß1 integrins. J Biol Chem. 1999; 274: 37809–37814.
18. Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in vivo by vß3-targeted magnetic resonance imaging. Nat Med. 1998; 4: 623–626.[CrossRef][Medline]
[Order article via Infotrieve]
19. Haubner R, Wester HJ, Weber WA, et al. Non-invasive imaging of vß3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res. 2001; 61: 1781–1785.
20. Burns PN, Wilson SR, Simpson DH. Pulse inversion imaging of liver blood flow: improved method for US characterizing focal masses with microbubble contrast. Invest Radiol. 2000; 35; 58–71.[Medline] [Order article via Infotrieve]
21. Kedar RP, Cosgrove D, McCready VR, et al. Microbubble contrast agent for color Doppler US: effect on breast masses: work in progress. Radiology. 1996; 198: 679–686.
22. Halpern EJ, Rosenberg M, Gomella LG. Prostate cancer: contrast-enhanced US for detection. Radiology. 2001; 219: 219–225.
23. Yang WT, Tse GMK, Lam PKW, et al. Correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvessel density for the quantitation of angiogenesis. J Ultrasound Med. 2002; 21: 1227–1235.
24. Iordanescu I, Becker C, Zetter B, et al. Tumor vascularity: evaluation in a murine model with contrast-enhanced color Doppler US: effect of angiogenesis inhibitors. Radiology. 2002; 222: 460–467.
25. Forsberg F, Dicker AP, Thakur ML, et al. Comparing contrast-enhanced ultrasound to immunohistochemical markers of angiogenesis in a human melanoma xenograft model: preliminary results. Ultrasound Med Biol. 2002; 28: 445–451.[CrossRef][Medline] [Order article via Infotrieve]
26. Vajkoczy P, Schilling L, Ullrich A, et al. Characterization of angiogenesis and microcirculation of high-grade glioma: an intravital multifluorescence microscopic approach in the athymic nude mouse. J Cereb Blood Flow Metab. 1998; 18: 510–520.[CrossRef][Medline] [Order article via Infotrieve]
27. Vajkoczy P, Ullrich A, Menger MD. Intravital fluorescence videomicroscopy to study tumor angiogenesis and microcirculation. Neoplasia. 2000; 2: 53–61.[CrossRef][Medline] [Order article via Infotrieve]
28. Jain RK. Delivery of molecular and cellular medicine to solid tumors. Microcirculation. 1997; 4: 1–23.[Medline] [Order article via Infotrieve]
29. Endrich B, Reinhold HS, Gross JF, et al. Tissue perfusion inhomogeneity during early tumor growth in rats. J Natl Cancer Inst. 1979; 62: 387–395.[Medline] [Order article via Infotrieve]
30. Wei L, Erinjeri J, Rovainen CM, et al. Collateral growth and angiogenesis around cortical stroke. Stroke. 2001; 32: 2179–2184.
This article has been cited by other articles:
 |
|
 |
|
  H. Leong-Poi Molecular imaging using contrast-enhanced ultrasound: evaluation of angiogenesis and cell therapy Cardiovasc Res, November 1, 2009; 84(2): 190 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kobulnik, M. A. Kuliszewski, D. J. Stewart, J. R. Lindner, and H. Leong-Poi Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection. J. Am. Coll. Cardiol., October 27, 2009; 54(18): 1735 - 1742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Saraste, S. G. Nekolla, and M. Schwaiger Cardiovascular molecular imaging: an overview Cardiovasc Res, September 1, 2009; 83(4): 643 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Zannetti, S. Del Vecchio, F. Iommelli, A. Del Gatto, S. De Luca, L. Zaccaro, A. Papaccioli, J. Sommella, M. Panico, A. Speranza, et al. Imaging of {alpha}v{beta}3 Expression by a Bifunctional Chimeric RGD Peptide not Cross-Reacting with {alpha}v{beta}5 Clin. Cancer Res., August 15, 2009; 15(16): 5224 - 5233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Turkbey, H. Kobayashi, M. Ogawa, M. Bernardo, and P. L. Choyke Imaging of Tumor Angiogenesis: Functional or Targeted? Am. J. Roentgenol., August 1, 2009; 193(2): 304 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. TILKI, M. SEITZ, B. B. SINGER, S. IRMAK, C. G. STIEF, O. REICH, and S. ERGUN Molecular Imaging of Tumor Blood Vessels in Prostate Cancer Anticancer Res, May 1, 2009; 29(5): 1823 - 1829. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Willmann, Z. Cheng, C. Davis, A. M. Lutz, M. L. Schipper, C. H. Nielsen, and S. S. Gambhir Targeted Microbubbles for Imaging Tumor Angiogenesis: Assessment of Whole-Body Biodistribution with Dynamic Micro-PET in Mice Radiology, October 1, 2008; 249(1): 212 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Willmann, A. M. Lutz, R. Paulmurugan, M. R. Patel, P. Chu, J. Rosenberg, and S. S. Gambhir Dual-targeted Contrast Agent for US Assessment of Tumor Angiogenesis in Vivo Radiology, September 1, 2008; 248(3): 936 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kaul Myocardial Contrast Echocardiography: A 25-Year Retrospective Circulation, July 15, 2008; 118(3): 291 - 308. [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Lee, A. Lyshchik, J. Huamani, D. E. Hallahan, and A. C. Fleischer Relationship Between Retention of a Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)-Targeted Ultrasonographic Contrast Agent and the Level of VEGFR2 Expression in an In Vivo Breast Cancer Model J. Ultrasound Med., June 1, 2008; 27(6): 855 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Cai and X. Chen Multimodality Molecular Imaging of Tumor Angiogenesis J. Nucl. Med., June 1, 2008; 49(Suppl_2): 113S - 128S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Vicenzini, R. Delfini, F. Magri, F. Puccinelli, M. Altieri, A. Santoro, M. F. Giannoni, L. Bozzao, V. Di Piero, and G. L. Lenzi Semiquantitative Human Cerebral Perfusion Assessment With Ultrasound in Brain Space-Occupying Lesions: Preliminary Data J. Ultrasound Med., May 1, 2008; 27(5): 685 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Kruskal Can Contrast-enhanced US with Targeted Microbubbles Monitor the Response to Antiangiogenic Therapies? Radiology, February 1, 2008; 246(2): 339 - 340. [Full Text] [PDF]
|
|
|
|
|
|
J. K. Willmann, R. Paulmurugan, K. Chen, O. Gheysens, M. Rodriguez-Porcel, A. M. Lutz, I. Y. Chen, X. Chen, and S. S. Gambhir US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice Radiology, February 1, 2008; 246(2): 508 - 518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Palmowski, J. Huppert, G. Ladewig, P. Hauff, M. Reinhardt, M. M. Mueller, E. C. Woenne, J. W. Jenne, M. Maurer, G. W. Kauffmann, et al. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects Mol. Cancer Ther., January 1, 2008; 7(1): 101 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lyshchik, A. C. Fleischer, J. Huamani, D. E. Hallahan, M. Brissova, and J. C. Gore Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 Expression Using Targeted Contrast-Enhanced High-Frequency Ultrasonography J. Ultrasound Med., November 1, 2007; 26(11): 1575 - 1586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hricak, P. L. Choyke, S. C. Eberhardt, S. A. Leibel, and P. T. Scardino Imaging Prostate Cancer: A Multidisciplinary Perspective Radiology, April 1, 2007; 243(1): 28 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Cai, J. Rao, S. S. Gambhir, and X. Chen How molecular imaging is speeding up antiangiogenic drug development. Mol. Cancer Ther., November 1, 2006; 5(11): 2624 - 2633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Cai, Y. Wu, K. Chen, Q. Cao, D. A. Tice, and X. Chen In vitro and In vivo Characterization of 64Cu-Labeled AbegrinTM, a Humanized Monoclonal Antibody against Integrin {alpha}v{beta}3 Cancer Res., October 1, 2006; 66(19): 9673 - 9681. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Vicenzini, M. C. Ricciardi, F. Puccinelli, and G. L. Lenzi Cerebral Perfusion in a High-Grade Glioma Evaluated With Sonographic Contrast Pulse Sequencing Technology. J. Ultrasound Med., September 1, 2006; 25(9): 1215 - 1218. [Full Text] [PDF]
|
|
|
|
|
|
F. A. Jaffer, P. Libby, and R. Weissleder Molecular and Cellular Imaging of Atherosclerosis: Emerging Applications J. Am. Coll. Cardiol., April 4, 2006; 47(7): 1328 - 1338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Provenzale Good Things Come in Small Packages: A Review of the Proceedings of the 2005 Academy of Molecular Imaging Meeting Am. J. Roentgenol., February 1, 2006; 186(2): 291 - 296. [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, Z. Xiong, Y. Wu, W. Cai, J. R. Tseng, S. S. Gambhir, and X. Chen Quantitative PET Imaging of Tumor Integrin {alpha}v{beta}3 Expression with 18F-FRGD2 J. Nucl. Med., January 1, 2006; 47(1): 113 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wu, X. Zhang, Z. Xiong, Z. Cheng, D. R. Fisher, S. Liu, S. S. Gambhir, and X. Chen microPET Imaging of Glioma Integrin {alpha}v{beta}3 Expression Using 64Cu-Labeled Tetrameric RGD Peptide J. Nucl. Med., October 1, 2005; 46(10): 1707 - 1718. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Leong-Poi, J. Christiansen, P. Heppner, C. W. Lewis, A. L. Klibanov, S. Kaul, and J. R. Lindner Assessment of Endogenous and Therapeutic Arteriogenesis by Contrast Ultrasound Molecular Imaging of Integrin Expression Circulation, June 21, 2005; 111(24): 3248 - 3254. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Achilefu, S. Bloch, M. A. Markiewicz, T. Zhong, Y. Ye, R. B. Dorshow, B. Chance, and K. Liang Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression PNAS, May 31, 2005; 102(22): 7976 - 7981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Abramowicz Ultrasonographic Contrast Media: Has the Time Come in Obstetrics and Gynecology? J. Ultrasound Med., April 1, 2005; 24(4): 517 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Lee, K.-H. Jung, S.-H. Song, D. H. Kim, B. C. Lee, H. J. Sung, Y.-M. Han, Y. S. Choe, D. Y. Chi, and B.-T. Kim Radiolabeled RGD Uptake and {alpha}v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs J. Nucl. Med., March 1, 2005; 46(3): 472 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Miller, H. H. Pien, D. Sahani, A. G. Sorensen, and J. H. Thrall Imaging Angiogenesis: Applications and Potential for Drug Development J Natl Cancer Inst, February 2, 2005; 97(3): 172 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.A Dijkmans, L.J.M Juffermans, R.J.P Musters, A van Wamel, F.J ten Cate, W van Gilst, C.A Visser, N de Jong, and O Kamp Microbubbles and ultrasound: from diagnosis to therapy Eur J Echocardiogr, August 1, 2004; 5(4): 245 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Lindner and J. Sklenar Placing faith in numbers: quantification of perfusion with myocardial contrast echocardiography J. Am. Coll. Cardiol., May 19, 2004; 43(10): 1814 - 1816. [Full Text] [PDF]
|
|
|
|
|
|
J. N. Kirkpatrick, T. Wong, J. E. Bednarz, K. T. Spencer, L. Sugeng, R. P. Ward, J. M. DeCara, L. Weinert, T. Krausz, and R. M. Lang Differential diagnosis of cardiac masses using contrast echocardiographic perfusion imaging J. Am. Coll. Cardiol., April 21, 2004; 43(8): 1412 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Foster and I. L. Gerber Masses of the heart: perfusing the "good" from the bad J. Am. Coll. Cardiol., April 21, 2004; 43(8): 1420 - 1422. [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Jaffer and R. Weissleder Seeing Within: Molecular Imaging of the Cardiovascular System Circ. Res., March 5, 2004; 94(4): 433 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kaul and J. R. Lindner Visualizing coronary atherosclerosis in vivo: thinking big, imaging small J. Am. Coll. Cardiol., February 4, 2004; 43(3): 461 - 463. [Full Text] [PDF]
|
|
|
|
 |
|
 H-D Liang and M J K Blomley The role of ultrasound in molecular imaging Br. J. Radiol., December 1, 2003; 76(suppl_2): S140 - S150. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||