Background— Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrast-enhanced ultrasound (CEU) with microbubbles targeted to α _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 α _v β ₃ by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular α _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 mm ² , 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, α _v β ₃ -targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from α _v β ₃ -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 α _v β ₃ -targeted microbubbles was greatest at the periphery of tumors, where α _v -integrin expression was most prominent, and correlated well with tumor microvascular blood volume ( r =0.86). Conclusions— CEU with microbubbles targeted to α _v β ₃ 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.

Background Routine methods capable of assessing tissue inflammation noninvasively are currently not available. We hypothesized that tissue retention of microbubbles targeted to the endothelial cell adhesion molecule P-selectin would provide a means to assess inflammation with ultrasound imaging. Methods and Results Phospholipid microbubbles targeted to P-selectin (MB _p ) were created by conjugating monoclonal antibodies against murine P-selectin to the lipid shell. The microvascular behaviors of MB _p and control microbubbles without antibody (MB) or with isotype control antibody (MB _iso ) were assessed by intravital microscopy of cremasteric venules of control and tumor necrosis factor (TNF)-α–stimulated wild-type mice. Retention of all microbubbles increased ( P <0.05) with TNF-α treatment because of increased attachment to activated leukocytes. Extensive attachment of MB _p directly to the venular endothelium or to adherent platelet-leukocyte aggregates was observed in TNF-α–stimulated mice, resulting in 4-fold greater ( P <0.01) retention of MB _p than either MB _iso or MB. Enhanced retention of MB _p was completely abolished in TNF-α–stimulated P-selectin–deficient mice. The ultrasound signal from microbubbles retained in inflamed tissue was assessed by contrast-enhanced renal ultrasound imaging of the kidneys of mice undergoing ischemia-reperfusion injury. In wild-type mice, this signal was significantly higher ( P <0.05) for MB _p (12±2 U) than either MB _iso (6±3 U) or MB (5±3 U). In P-selectin–deficient mice, the signal for MB _p was equivalent to that from control microbubbles. Conclusions Microvascular retention of microbubbles targeted to P-selectin produces strong signal enhancement on ultrasound imaging of inflamed tissue. These results suggest that site-targeted microbubbles may be used to assess inflammation, tissue injury, and other endothelial responses noninvasively with ultrasound.

Background— The ability to image vascular inflammatory responses may allow early diagnosis and treatment of atherosclerosis. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression with contrast-enhanced ultrasound (CEU) could be used for this purpose. Methods and Results— Attachment of VCAM-1–targeted and control microbubbles to cultured endothelial cells was assessed in a flow chamber at variable shear stress (0.5 to 12.0 dynes/cm ² ). Microbubble attachment to aortic plaque was determined by en face microscopy of the thoracic aorta 10 minutes after intravenous injection in wild-type or apolipoprotein E–deficient mice on either chow or hypercholesterolemic diet. CEU molecular imaging of the thoracic aorta 10 minutes after intravenous microbubble injection was performed for the same animal groups. VCAM-1–targeted but not control microbubbles attached to cultured endothelial cells, although firm attachment at the highest shear rates (8 to 12 dynes/cm ² ) occurred only in pulsatile flow conditions. Aortic attachment of microbubbles and targeted CEU signal was very low in control wild-type mice on chow diet. Aortic attachment of microbubbles and CEU signal for VCAM-1–targeted microbubbles differed between treatment groups according to extent of VCAM-1–positive plaque formation (median CEU videointensity, 1.8 [95% CI, 1.2 to 1.7], 3.7 [95% CI, 2.9 to 7.3], 6.8 [95% CI, 3.9 to 7.6], and 11.2 [95% CI, 8.5 to 16.0] for wild-type mice on chow and hypercholesterolemic diet and for apolipoprotein E–deficient mice on chow and hypercholesterolemic diet, respectively; P <0.001). Conclusions— CEU molecular imaging of VCAM-1 is capable of rapidly quantifying vascular inflammatory changes that occur in different stages of atherosclerosis. This method may be potentially useful for early risk stratification according to inflammatory phenotype.

Background—Lipid microbubbles used for perfusion imaging with ultrasound are retained within inflamed tissue because of complement-mediated attachment to leukocytes within venules. We hypothesized that incorporation of phosphatidylserine (PS) into the microbubble shell may enhance these interactions by amplifying complement activation and thereby allow ultrasound imaging of inflammation.Methods and Results—In 6 mice, intravital microscopy of tissue necrosis factor-α–treated cremaster muscle was performed to assess the microvascular behavior of fluorescein-labeled lipid microbubbles with and without PS in the shell. Ten minutes after intravenous injection, microbubble attachment to leukocytes within inflamed venules was greater for PS-containing than for standard lipid microbubbles (20±4 versus 10±3 per 20 optical fields,P<0.05). The ultrasound signal from retained microbubbles was assessed in the kidneys of 6 mice undergoing renal ischemia-reperfusion injury and in 6 control kidneys. The signal from retained microbubbles in control kidneys was low (<2.5 video intensity units) for both agents. After ischemia-reperfusion, the signal from retained microbubbles was 2-fold higher for PS-containing than for standard lipid microbubbles (18±6 versus 8±2 video intensity units,P<0.05). An excellent relation was found between the ultrasound signal from retained microbubbles and the degree of renal inflammation, assessed by tissue myeloperoxidase activity.Conclusions—We conclude that noninvasive assessment of inflammation is possible by ultrasound imaging of microbubbles targeted to activated leukocytes by the presence of PS in the lipid shell.