Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to α_v-integrins

Background - Noninvasive methods for characterizing neovessel formation during angiogenesis are currently lacking. We hypothesized that angiogenesis could be imaged with the use of contrast-enhanced ultrasound (CEU) with microbubbles targeted to α_v-integrins. Methods and Results - Microbubbles targeted to α_v-integrins were prepared by conjugating echistatin (MB_E) or monoclonal antibody against murine α_v (MBα) to their surface. Control microbubbles (MB_c) were also prepared. The microvascular behavior of these microbubbles was assessed by intravital microscopy of the cremaster muscle in mice treated for 4 days with sustained-release FGF-2. Microvascular retention was much greater (P<0.01) for MB_E (11±6 mm^-3) and MBα (10±7 mm^-3) than that for MB_c (1±1 mm^-3). Retained MB_E and MBα attached directly to the microvascular endothelial surface. Microbubble retention in 4 control mice was minimal. Subcutaneous matrigel plugs enriched with FGF-2 were created in 12 mice and studied 10 days later. Neovessels within the matrigel stained positive for α_v-integrins. CEU demonstrated greater (P<0.01) acoustic intensity for MB_E (16.0±5.9 U) and MBα (17.0+-5.5 U) compared with MB_c (5.8±2.6 U). The signal from targeted microbubbles (MB_E and MBα) correlated well (r=0.90) with the matrigel blood volume determined by CEU perfusion imaging. Conclusions - CEU with microbubbles targeted for α_v-integrins may provide a noninvasive method for assessing therapeutic angiogenesis.