These studies demonstrate that molecular imaging with targeted paramagnetic nanoparticles can be utilized to specifically detect angiogenesis in skeletal muscle. Within 30 minutes, αv
-integrin-targeted PFC nanoparticles produced higher enhancement in the ligated limb than the control leg. Two hours after nanoparticle injection, the pro-angiogenic effects of L-arginine were apparent as more than two times higher signal enhancement was observed in the ischemic leg compared to the control limb. On the other hand, untreated animals showed only 50% higher enhancement in the ischemic leg. The relative contributions of specific targeting and passive accumulation of the nanoparticles was demonstrated with a non-targeted formulation. The non-targeted agent produced only half the enhancement compared targeted nanoparticles in L-arginine treated animals, suggesting that the immature and tortuous vasculature entraps this particulate contrast agent, but that active biomarker targeting improves particle deposition and detection of angiogenic therapy. Previous studies on cholesterol fed rabbits have demonstrated that the circulating concentration of targeted and nontargeted nanoparticles 2 hours after injection is identical, resulting in approximately 40 μM Gd3+
in the blood pool (38
). For the eventual clinical application of this targeted contrast agent, a number of inter-related parameters would need to be optimized to maximize the specific vs. nonspecific image enhancement, including nanoparticle dose, post-injection imaging timepoint and nanoparticle relaxivity (39
). Histology of muscle samples and X-ray angiography performed 40 days post-surgery corroborated that L-arginine effectively increased the angiogenic response to hindlimb ischemia. Furthermore, angiography demonstrated that the high-cholesterol diet impeded normal revascularization of the ischemic hindlimb following femoral artery ligation.
The histological results demonstrated an increased number of microvessels in the L-arginine treated animal compared to the control animal, but the capillary density was not quantified in this study. Previous studies, however, have measured the effects of L-arginine treatment on capillary density after femoral artery ligation. One study found that capillary density in the ischemic limb was 30% higher in L-arginine treated rabbits compared to controls (18
). It was also revealed that the capillary density in the control leg was identical for both treatment groups, suggesting that L-arginine treatment does not cause widespread neovascular proliferation in normal tissues. Another study on a rat model of hindlimb ischemia reported that cholesterol feeding reduced the capillary density in the ischemic limb by 30%, while L-arginine treatment restored the capillary density to the levels measured in control animals (7
). The results of these previous studies combined with the results reported in this study, support our conclusions that cholesterol feeding diminishes the angiogenic response to hindlimb ischemia and that L-arginine treatment can normalize neovascular proliferation in this animal model. Furthermore, histological staining of the αv
-integrin itself was not performed in these experiments because a range of different cell types, including macrophages, platelets, lymphocytes and smooth muscle cells (29
), express the αv
-integrin. Previous reports have demonstrated that the size of αv
-targeted nanoparticles confines them to the vasculature (28
) where they can only interact with endothelial cells. Therefore, histological staining of an endothelial marker, such as CD31, is more likely to reflect possible sites of nanoparticle binding than staining for the targeted integrin.
The adverse effects of a high-cholesterol diet on revascularization following ischemic injury have been demonstrated in previous studies on genetically modified rabbits and mice. The Watanabe rabbit is genetically predisposed to atherosclerosis as a result of abnormally high levels of LDL cholesterol in the blood. This strain displays reduced capillary development and increased muscle necrosis following femoral artery ligation compared to normal New Zealand White rabbits (8
). Similarly, the ApoE-/-
mouse is genetically modified to be deficient in apolipoprotein E, inhibiting the ability of the liver to clear lipids from the blood and causing increased plasma cholesterol levels. After ligation of the femoral artery, ApoE-/-
mice showed impaired capillary proliferation and reduced vascular endothelial growth factor (VEGF) expression compared with C57 control mice, which could be mitigated with adenoviral VEGF gene transfer therapy (6
). These studies demonstrate the mechanisms and consequences of hyperlipidemia in revascularization following ischemic insult, as well as effective therapies to counteract the impaired angiogenesis despite continued atherosclerosis and elevated blood cholesterol. In the current study, MRI enhancement in untreated animals was identical for targeted and nontargeted nanoparticles. This finding agrees with the x-ray angiography results () as well as a previous study (7
) showing that cholesterol feeding significantly impairs angiogenesis in response to hindlimb ischemia. While the MRI experiments demonstrated that hypercholesterolemia inhibits revascularization, imaging was only performed at a single timepoint and could not determine if this resulted from decreased magnitude or delayed response or both. Further MRI studies with αv
-integrin-targeted PFC nanoparticles could serially monitor angiogenesis in control diet and cholesterol-fed animals in order to determine the magnitude and temporal development of angiogenesis in this hindlimb ischemia model.
Angiogenesis, the formation of new capillary blood vessels, has been shown to occur quickly in the rabbit model, within 5-10 days after ligation. In contradistinction, arteriogenesis involves remodeling existing vessels to form large caliber collaterals and occurs much later, about 20-40 days after ligation (23
). Most angiogenic therapies, such as the growth factors VEGF and basic fibroblast growth factor (bFGF), are aimed at augmenting the early stages of vessel development (13
). For instance, L-arginine therapy induces nitric oxide production, leading to vasodilation, upregulation of VEGF and proliferation and migration of endothelial cells. However, the only clinical tools available for monitoring the effects of angiogenic therapies in PVD patients, including X-ray angiography and blood flow measurements, are sensitive only to the development of large caliber vessels that accompany arteriogenesis. Previous publications have reported that X-ray angiography can only distinguish therapeutic response after 20-40 days of treatment (13
) reflecting the time needed to develop large conduit arteries that can be visualized with contrast enhanced X-ray imaging. Measuring blood pressure ratios, such as the ratio of the blood pressure in the ankle and the arm, also requires 40 days post surgery to detect therapeutic response (13
). Despite the vasoactive properties of L-arginine, previous studies have shown no differences in systolic blood pressure or resting blood flow between L-arginine treated and control rabbits for up to 40 days of treatment (18
). Using a molecularly targeted contrast agent, the early signatures of neovascular development can be detected only 10 days after the initiation of therapy. The earlier detection of therapeutic response could prove very useful for monitoring patient response and allow modification of ineffective therapies much earlier than is possible with angiographic techniques.
Small molecule agents, such as radiolabeled peptides, have also been developed to image expression of αv
-integrin in angiogenic vasculature. These small molecules however, are able to penetrate into tissues and bind to extravascular integrins that may be expressed by macrophages, platelets, lymphocytes or smooth muscle cells (29
). For instance, a 99m
Tc-labeled arginine-glycine-aspartate (RGD) peptide yielded only ~50% more signal in the ischemic compared to the sham-operated limb (40
), which may reflect nonvascular binding of the peptide. Similarly, a 123
I-labeled RGD peptide produced 80% higher signal in the ischemic vs. control limbs (41
). PFC nanoparticles, on the other hand, are confined to the vascular space (28
) and may more specifically denote angiogenesis. In the present study, PFC nanoparticles produced more than two times higher signal in the ischemic limb compared to the contralateral control. Larger imaging agents, including proteins and nanoparticles, have also been utilized in PET studies. A 64
Cu labeled VEGF protein displayed 160% higher signal in the ischemic limb compared to the control limb (42
), perhaps as a result of limited extravasation from the vascular space. A nonspecific protein, however, also displayed about 40% higher uptake in the ischemic limb compared to the control limb. PET imaging of a αv
-targeted nanoparticle with a 12 nm diameter showed 4 times higher signal in the ischemic limb compared to the control limb, but the nontargeted agent showed 2 times higher uptake (43
), again demonstrating some level of nonspecific accumulation of the imaging probe in the angiogenic vasculature.
As a molecular imaging modality, MRI offers several advantages over the nuclear imaging methods required to detect radiolabeled tracers. MRI offers higher resolution images without exposure to ionizing radiation. MRI also offers a range of contrast weightings that allow various anatomical images to be registered with maps corresponding to molecular signatures of disease and/or therapy. For instance, displays the image obtained immediately after nanoparticle injection in grayscale to visualize the location of the major vasculature and musculature of both the ischemic and control hindlimbs. Overlaid on this anatomical image, the signal enhancement measured two hours after nanoparticle injection is colorcoded in red to indicate locations of upregulated angiogenesis in the muscle tissue. The combination of high resolution anatomical imaging and mapping of biomarker expression in one imaging session utilizing a single scanner cannot be achieved with other clinical modalities, such as positron emission tomography or ultrasound.
As a blood pool agent, delivery of PFC nanoparticles to the ischemic musculature could be impaired as a result of reduced blood flow. The unaltered flow of blood to the control limb, therefore, could greatly reduce the observed ratio of tissue enhancement in the ischemic and control legs. Using ultrasound imaging, Lindner et al. normalized the image enhancement from integrin-targeted microbubbles based on the ratio of blood flow in the ischemic and contralateral limbs of rats (44
). With this method, they demonstrated approximately five times higher enhancement in the ischemic limb compared to the control limb despite a reduction in blood flow by 25-50%. Assuming a similar blood flow ratio in the rabbit hindlimb ischemia model used in the current study, the corrected tissue enhancement in the ischemic limb would be 3-4 times higher than the control limb utilizing the αv
-integrin-targeted PFC nanoparticles.
In conclusion, this study demonstrates that MRI with targeted paramagnetic nanoparticles can specifically detect angiogenesis and the therapeutic effects of L-arginine in skeletal muscle. Non-targeted nanoparticles produced much lower enhancement compared to the targeted agent. X-ray angiography revealed impaired angiogenesis in the cholesterol-fed animals and confirmed the pro-angiogenic effects of L-arginine, a promoter of nitric oxide production. While current clinical techniques for monitoring PVD patients, such as X-ray angiography and blood flow measurements, are capable of detecting the large caliber vessels that form at the late stages of revascularization, molecular imaging with MRI and targeted contrast agents can map the early signatures of angiogenesis. In clinical practice, earlier detection of therapeutic response could be invaluable for guiding therapeutic interventions, including determining effective drug doses and evaluating new treatment strategies.