Molecular imaging of angiogenic therapy in peripheral vascular disease with alphanubeta3-integrin-targeted nanoparticles

Abstract

Noninvasive molecular imaging of angiogenesis could play a critical role in the clinical management of peripheral vascular disease patients. The alpha(nu)beta(3)-integrin, a well-established biomarker of neovascular proliferation, is an ideal target for molecular imaging of angiogenesis. This study investigates whether MR molecular imaging with alpha(nu)beta(3)-integrin-targeted perfluorocarbon nanoparticles can detect the neovascular response to angiogenic therapy. Hypercholesterolemic rabbits underwent femoral artery ligation followed by no treatment or angiogenic therapy with dietary L-arginine. MR molecular imaging performed 10 days after vessel ligation revealed increased signal enhancement in L-arginine-treated animals compared to controls. Furthermore, specifically targeted nanoparticles produced two times higher MRI signal enhancement compared to nontargeted particles, demonstrating improved identification of angiogenic vasculature with biomarker targeting. X-ray angiography performed 40 days postligation revealed that L-arginine treatment increased the development of collateral vessels. Histologic staining of muscle capillaries revealed a denser pattern of microvasculature in L-arginine-treated animals, confirming the MR and X-ray imaging results. The clinical application of noninvasive molecular imaging of angiogenesis could lead to earlier and more accurate detection of therapeutic response in peripheral vascular disease patients, enabling individualized optimization for a variety of treatment strategies.

FIG. 1

Angiogenesis is seen as highly diffuse enhancement throughout the ligated (right) leg with only slight enhancement of the control (left) leg. The animal receiving tap water (left panel) shows more enhancement in the ligated leg compared to the control leg. The L-arginine treated rabbit (right panel) shows a more dense distribution of angiogenesis in the ligated limb, while the control limb appears similar to the untreated animal.

FIG. 2

Molecular imaging of angiogenesis with α_vβ₃-integrin-targeted nanoparticles allows specific detection of effective neovascular therapy. To represent the overall image contrast, the contrast index was calculated as the product of the area and magnitude of the signal enhancement. Two hours after injection of targeted nanoparticles in L-arginine treated rabbits, 104% higher signal is observed in the ischemic (right) limb compared to the control (left) limb, indicating early detection of therapeutic response. Untreated animals show only 49% more signal in the ischemic vs. control limbs after targeted nanoparticle injection.

FIG. 3

Specific imaging of angiogenesis with α_vβ₃-integrin-targeted nanoparticles produced 104% higher signal in the ischemic limb (right) compared to the control limb (left) in L-arginine treated rabbits. Non-targeted nanoparticles, however, produce only ~50% higher enhancement in the ischemic limb compared to the control limb, representing nonspecific entrapment of nanoparticles in the highly permeable angiogenic vasculature (* p < 0.05 vs. left leg, # p < 0.05 vs. right leg of all other groups).

FIG. 4

Histology of muscle samples from the ischemic limb of animals treated with tap water or L-arginine. LEFT: H&E staining showed areas of intramuscular hemorrhage (White Arrows) in tap water animals that was not observed in the L-arginine treated group. MIDDLE and RIGHT: Staining for microvasculature (Black Arrows) showed a greater number of capillaries in L-arginine treated animals compared to tap water treatment. These results support the MRI findings that L-arginine treatment augments angiogenic response to ischemia and suggests that persistent hemorrhage occurs in the untreated animals.

FIG. 5

X-ray angiography forty days after ligation of the femoral artery reveals impact of high cholesterol diet and L-arginine treatment on angiogenesis. A) Examples of X-ray angiograms showing ligation of right femoral artery (arrows) and higher density of collateral vessels in L-arginine treated rabbit. B) The high cholesterol diet significantly impedes revascularization in the ischemic (right) hindlimb (* p < 0.05), but L-arginine therapy restores angiogenic response to ischemia.