Angiogenesis is associated with the growth and malignant transformation of tumours, which form new blood vessels to supply increasing amounts of blood to their core. As such, anticancer therapies are often directed at inhibition of the angiogenic process. On the flip side, conditions such as peripheral arterial disease and coronary artery disease exhibit an insufficient angiogenic response. Here, genetic or stem cell therapies are employed to stimulate angiogenesis.
"The motivation for assessing angiogenesis is to monitor naturally-occurring angiogenic response in ischemic heart disease or cancers, as well as to non-invasively monitor both pro-angiogenic and anti-angiogenic therapies," Dobrucki, from Yale University School of Medicine (New Haven, CT), explained.
Work in progress
The development of strategies for monitoring the angiogenic process is progressing in two directions, says Dobrucki. Firstly, there's the identification of targets with biosignatures unique to angiogenesis. The most promising targets have been identified as vascular endothelial growth factor (VEGF), VEGF receptors and αvβ3-integrin, which is expressed on the surface of proliferating endothelial cells. Attention has thus focused on creating molecular imaging probes for these specific targets.
Of equal importance is the development of high-resolution, high-sensitivity imaging techniques. Radiotracer-based systems, such as SPECT and PET, offer sensitivity in the picomolar range, spatial resolution in the millimetre range, and benefit from the availability of targeted probes and instrumentation. Such systems have already progressed to clinical use.
Early studies investigating SPECT and PET imaging of angiogenesis employed radiolabelled monoclonal antibodies, such as anti-VEGF antibodies labelled with radioiodine. However, despite their high selectivity, the slow clearance rate of antibodies limits their clinical application. Alternative probes followed, including radiolabelled VEGF isoforms and probes based on RGD peptides, which are highly selective for αvβ3-integrin.
Currently, the use of radiolabelled nanoparticles for molecular imaging of angiogenesis is attracting great interest. RGD nanoprobes labelled with PET and SPECT radioisotopes, for example, have been successfully used in preclinical imaging of peripheral and myocardial angiogenesis.
Ultrasonic approach
Ultrasound is a widely available, relatively inexpensive technology, with high temporal resolution (enabling real-time imaging) and a spatial resolution of 0.05–0.5 mm. Targeted ultrasound imaging of angiogenesis offers the potential for high-throughput screening of molecular and pathophysiologic processes. Disadvantages include limited penetration depth in certain tissues, such as lung and bone.
Molecular ultrasound imaging requires the use of contrast agents such as microbubbles. Targeting is achieved either by altering the bubble shells or conjugating ligands to their surfaces, facilitating attachment to specific angiogenesis-associated cells. Researchers have reported successful ultrasound imaging using αvβ3-integrin targeted contrasts. Studies included the evaluation of integrin expression in glioma models of tumour angiogenesis, and tracking of peripheral angiogenesis in a rodent model of hind-limb ischemia.
More recently, microbubbles targeted to growth factor receptors expressed during vascular remodelling have been tested for imaging tumour and peripheral angiogenesis. In vivo ultrasound images of angiosarcoma and malignant glioma tumours exhibited significantly higher average image intensity using targeted microbubbles than with control microbubbles.
The feasibility of targeting ultrasound contrasts to disease-related markers, including angiogenesis, has been established and the technique is now applied as a high-throughput research tool. The authors note, however, that while initial steps are being made towards developing targeted agents for clinical applications, the clinical potential of this technology is contingent on further refinement of microbubble chemistry.
MRI matters
Dobrucki and co-authors also described the application of MRI, which offers high spatial (0.02–0.1 mm) and temporal resolution, as well as excellent soft-tissue contrast. In addition, it allows co-registration of anatomic and functional information using a single imaging modality. They note that though MRI has a relatively low sensitivity for detecting targeted agents, this limitation might be overcome using signal amplification techniques.
The first example of targeted MR imaging of angiogenesis employed gadolinium-containing paramagnetic liposomes conjugated to an αvβ3-integrin antibody to image rabbit carcinomas. Other early work included the use of magnetic nanoparticles to image squamous cell carcinomas, and imaging of melanoma tumour xenografts in mice with αvβ3-integrin-targeted paramagnetic nanoparticles.
The recent introduction of superparamagnetic iron oxide (SPIO) nanoparticles as MR contrast agents has increased the technique's sensitivity compared with gadolinium-based contrasts. In one study, RGD-conjugated SPIO nanoparticles targeted to αvβ3-integrin successfully imaged tumour vasculature using a clinical 1.5T MRI scanner.
Researchers have also addressed the quantification of MR images, by examining differences in signal intensity between baseline images and those acquired after injection of molecular probes. The authors point out, however, that despite progress in quantifying the uptake of targeted agents, many sources of potential error remain. As such, most molecular MRI studies are still qualitative or semi-quantitative, with quantitative accuracy and reproducibility requiring further validation.
Despite the wide array of available imaging modalities, the authors conclude that the optimal approach may well lie in the development of dedicated hybrid imaging systems – some of which are already commercially available – that combine anatomical imaging with targeted functional imaging using radiolabelled agents. They note that the application of nanotechnology to angiogenesis imaging is an exciting new area of research, with further studies needed to assess its clinical feasibility.
"SPECT and PET provide the best sensitivity and generate images that can be quantified; however, they lack anatomical information," Dobrucki explained. "Ultrasound and MRI, on the other hand, provide anatomical information but lack sensitivity; and targeted ultrasound and MRI agents are not widely available."
Dobrucki emphasizes that future progress in non-invasive angiogenesis imaging also relies on the discovery of more specific biological targets, such as matrix metalloproteinases, and the development of new targeted probes. "Effort is going into developing multimodal, multifunctional probes," he told medicalphysicsweb. "Possible functions include targeting epitopes of interest, such as receptor proteins and transporters, as well as targeted local delivery of drugs, the process of which can also be non-invasively monitored."