(Top) Black blood image of the thoracic aorta (arrow) and segmentation of the vessel wall (outlined in yellow) is shown for the week 0 image. The color-coded overlay of signal enhancement (%) shows patchy areas of high angiogenesis. On the week 1 image, the signal enhancement has clearly decreased due to the antiangiogenic effect of targeted fumagillin treatment. (Bottom) The level of signal enhancement gradually increases at weeks 2 and 3 after fumagillin treatment, until week 4, when the level of enhancement is practically identical to the week 0 image.

(Top) Serial imaging of angiogenesis in untreated (triangles) and fumagillin-treated animals remaining on high-cholesterol diet (squares) or switched to normal chow (circles). Fumagillin treatment at week 0 reduced angiogenesis compared with untreated control animals (*p < 0.05), but withdrawing the high-cholesterol feed had no effect. (Bottom) Aortic angiogenesis in rabbits without treatment (triangles), with 30 µg/kg fumagillin (circles), or with 90 µg/kg fumagillin (squares). Fumagillin treatment at 30 versus 90 µg/kg produced identical responses. *p < 0.05.

(Top) Cardiac magnetic resonance enhancement in untreated (triangles), atorvastatin-treated (circles) and fumagillin-treated animals (squares) during 8 weeks of follow-up imaging. Untreated and statin-treated animals showed a constant level of angiogenesis in the aortic wall. Animals treated with targeted fumagillin nanoparticles at 0 and 4 weeks showed decreased angiogenesis (*p < 0.05) after each dose, which returned to baseline levels within 4 weeks. (Bottom) Enhancement in rabbits receiving atorvastatin alone (triangles) or in conjunction with 1 (squares) or 2 (circles) doses of targeted fumagillin nanoparticles. The combination of 2 fumagillin doses and statin produced a sustained decrease in angiogenesis (*p < 0.05).

Blood samples drawn at the end of the 8-week study were assessed for liver function enzymes to assess the chronic effects of the high-lipid diet and experimental treatments. All values tended to be within normal ranges and identical for all treatment groups. Alkaline phosphate (AlkP), however, was elevated in all groups, and alanine aminotransferase (ALT) was increased in animals receiving 2 doses of targeted fumagillin nanoparticles and oral statin (Fum 2x + Statin) (*p < 0.05). AST = aspartate aminotransferase; Fum = fumagillin; GGT = gamma glutamyltransferase; NS = not significant.

Routine light microscopy and fluorescence microscopy of β₃-targeted rhodamine nanoparticles and fluorescein isothiocyanate (FITC)-labeled lectin, a vascular endothelial marker, enables localization of nanoparticle binding with respect to normal and pathological vascular structures. (A) Hematoxylin and eosin staining of aorta shows small plaque, media and adventia (x10). (B) High-power fluorescent image demonstrates colocalization of rhodamine-labeled β₃-targeted nanoparticles (C, x60) with FITC-labeled lectin (D, x60), indicating that the nanoparticles are vascularly constrained to the neovessels of the adventitia. No rhodamine-labeled β₃-targeted nanoparticles were detected in the lumen plaque (not shown).

Cardiac magnetic resonance (CMR) molecular imaging of angiogenesis with β₃-targeted paramagnetic nanoparticles and histological measurement of microvessel density was performed on a separate cohort of atherosclerotic rabbits. Aortic sections were stained for β₃-integrin (LM-609) and platelet/endothelial cell adhesion molecule (PECAM), a general vascular marker. The number of microvessels expressing both _vβ₃-integrin and PECAM were counted in 3 independent full-transverse sections per animal. The number of angiogenic microvessels per high-powered field was correlated in a logarithmic fashion (R² = 0.84) to the CMR signal enhancement observed after injection of β₃-targeted particles.