Ultrasound Assessment of Inflammation and Renal Tissue Injury With Microbubbles Targeted to P-Selectin

Abstract

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)-␣-stimulated wild-type mice. Retention of all microbubbles increased (PϽ0.05) with TNF-␣ 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-␣-stimulated mice, resulting in 4-fold greater (PϽ0.01) retention of MB p than either MB iso or MB. Enhanced retention of MB p was completely abolished in TNF-␣-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 (PϽ0.05) for MB p (12Ϯ2 U) than either MB iso (6Ϯ3 U) or MB (5Ϯ3 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.

Deformable gas-filled microbubbles targeted to P-selectin

“…8A). The adhesion of spherical targeted microbubbles in P-selectin deficient mice (P −/− ) was reduced approximately three-fold, similar to results reported previously [3]; targeted adhesion of wrinkled microbubbles in P −/− mouse cremaster dropped nearly seven-fold. We observed no significant difference in adhesion between the wrinkled and spherical microbubbles in the P −/− mice.…”

“…Microbubbles were targeted to P-selectin by attaching biotinylated Rb40.34 to the biotin-PEG-lipid anchor with a streptavidin link [3]. Biotin conjugated microbubbles were washed with degassed DPBS five times in a bucket rotor centrifuge at ∼ 30×g for 4 min to remove excess free unincorporated lipid from the microbubbles, and 3 μg of streptavidin per 10 7 microbubbles were added to the washed dispersion.…”

“…Upon examination with an ultrasound medical imaging system, the adherent microbubbles generate an acoustic response signal that is displayed as video enhancement of the target tissue on the system screen. Targeted ultrasound contrast agents have been used to assess vascular pathology associated with several intravascular markers, including P-selectin [3], ICAM-1 [4,5], GpIIb/IIIa [6,7], the α v integrins [8,9] and other markers of tumor angiogenesis [10]. Successful targeted contrast ultrasound imaging in these systems is possible in part due to extremely high microbubble detection sensitivity of ultrasound imaging, with the capability to visualize individual microbubbles [11].…”

Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects

“…Ultrasound has proven to be highly sensitive for the identification of molecular structures when using targeted contrast agents (10 -13). In this context, antibody-coated, gas-filled microbubbles with diameters of 1 to 5 Am were used as specific contrast agents (11,12) and have been applied to characterize arteriosclerotic disease (14), vascular thrombosis (15), neovasculature (16), lymph nodes (17) as well as cerebral encephalitis and other inflammations (18,19).…”

Molecular Imaging of Cardiovascular Disease

“…Consequently, molecular imaging of contrast ultrasound has involved mainly molecular changes within the vascular compartment. Targeted contrast ultrasound has therefore been applied in angiogenesis, (19,20) vascular infl ammation (21,22) and thrombus formation (23) .…”