Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to αv-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 alpha(v)-integrins. Methods and Results-Microbubbles targeted to alpha(v)-integrins were prepared by conjugating echistatin (MBE) or monoclonal antibody against murine alpha(v) (MBalpha) to their surface. Control microbubbles (MBc) 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 MBE (11 +/- 6 mm(-3)) and MBα (10 +/- 7 mm(-3)) than that for MBc (1 +/- 1 mm(-3)). Retained MBE 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 MBE (16.0 +/- 5.9 U) and MBalpha (17.0 +/- 5.5 U) compared with MBc (5.8 +/- 2.6 U). The signal from targeted microbubbles (MBE and MBalpha) correlated well (r=0.90) with the matrigel blood volume determined by CEU perfusion imaging. Conclusions-CEU with microbubbles targeted for alpha(v)-integrins may provide a noninvasive method for assessing therapeutic angiogenesis.