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Vascular imaging of angiogenesis in mouse models of disease requires multi modal imaging hardware capable of targeting both structure and function at different physical scales. The three dimensional (3D) structure and function vascular information allows for accurate differentiation between biological processes. For example, image analysis of vessel development in angiogenesis vs. arteriogenesis enables more accurate detection of biological variation between subjects and more robust and reliable diagnosis of disease. In the recent years a number of micro imaging modalities have emerged in the field as preferred means for this purpose. They provide 3D volumetric data suitable for analysis, quantification, validation, and visualization of results in animal models. This review highlights the capabilities of microCT, ultrasound and microPET for multimodal imaging of angiogenesis and molecular vascular targets in a mouse model of tumor angiogenesis. The basic principles of the imaging modalities are described and experimental results are presented.
Angiogenesis is the formation of capillaries from existing vessels1, which occurs during embryonic development and in adults in either a physiological2-4 or pathological5 situation. Formation of angiogenic vessels is regulated by growth factors, integrins, membrane-bound proteinases and the composition of the extracellular matrix (ECM) 6-8. Each of these factors stimulate extracellular and intracellular signaling cues that work in concert to promote angiogenic processes that regulate endothelial cell branching, sprouting, lumen formation and proliferation6. Anti-angiogenic factors are produced during the angiogenic process to maintain the balance of pro-angiogenic molecules and their effects on endothelial cells.
The vascular endothelium is normally maintained in a differentiated, quiescent state. Pro-angiogenic factors destabilize the quiescent endothelium into migratory, proliferative endothelial cells which are attenuated by anti-angiogenic factors. The pro- and anti-angiogenic molecules have cell survival and death functions that are tightly controlled to maintain a balance 8-11.
Many negative regulators of angiogenesis are cleavage products of ECM proteins that are not inhibitory in their normal intact conformation, such as matrix proteins collagen XVIII and IV and hemostatic proteins such as plasminogen12-18. The breakdown of these proteins destabilizes the extracellular matrix, which leads to collapse and regression of capillary tubes. In other cases an extracellular protein may possess angiogenic properties, while its cleavage product is anti-angiogenic. This is the case for plasminogen activator inhibitor-1(PAI-1), which has been shown to be pro-angiogenic 19-21 while a cleaved PAI-1, rPAI-123, is a potent inhibitor of angiogenesis. In arterial endothelial cells, it inhibits pro-angiogenic fibroblast growth factor-2 (FGF-2) signaling pathways and functions22, 23. In mouse models of vascular disease, rPAI-123 inhibits angiogenic vessels to result in reduced disease progression.
The complexity of the physiological processes involved in angiogenesis at the cellular level is enormous. In many cases a single imaging modality can not provide enough details and information about the underlined interactions. For example, functional imaging modalities lack information about structure. It is impossible to determine the size of vessels from imaging modalities such as SPECT and PET or ultrasound. A multimodal imaging approach where functional information acquired from molecular imaging modalities is complemented with structural details is often required for successful imaging studies and verification of results.
Like normal tissue, tumors require nutrients in order to grow and survive. The neo-vasculature of tumors provides a means of supplying oxygen and nutrients to the tumor once it reaches a certain size. The tumor cells secrete specific molecules that promote recruitment of host vessels to the tumor, a process called angiogenesis. The very subtle stage of tumor induced angiogenesis is now the primary focus of study in the vascular biology field. The following sections review our modalities of choice for vascular imaging of models of disease implanted in mice; describe their current state, and present examples of their application for imaging of angiogenesis.
MicroPET, microCT, ultrasound and confocal imaging have become valuable tools for studying the tumor vasculature in small animal models. Each of these modalities provides unique advantages over the other. This review will discuss each in the context of tumor angiogenesis with emphasis on distinct advantages and disadvantages each brings to the imaging field.
Micro Computed Tomography (MicroCT) is a structural imaging modality that provides differentiation of contrast-enhanced tissues or structures with high attenuation from non-enhanced soft tissues24, 25. Traditional MicroCT imaging applications include screening for anatomical abnormalities26-31 as well as detection and quantification of changes in live animals32, 33 or tissue samples removed from sacrificed animals34. Most current in vivo MicroCT scanners have resolutions ranging from 100 to 30 μm, while ex vivo scanners have resolutions from 30 to 1 μm. The imaging is a two-step procedure. In the first step, 2D X-ray projections of the imaged object at different orientations of the x-ray source or object are acquired by the x-ray detector. In the second step the acquired projections are first corrected for various image artifacts and/or distortions due to sensor nonlinearity and then used to reconstruct the final volumetric data representing the scanned object in 3D. Depending on the parameters of the imaging protocol, a scan may take from several minutes to hours. During image acquisition, X-rays emitted by the source are attenuated as they pass through the imaged object. This proportionally reduces the original intensity of the X-rays due to absorption or scatter of photons24, 25. Objects with low attenuation properties, like soft tissue, allow most of the X-rays to pass through the object unabsorbed and arrive at the detector on the other end. However, if the imaged object has high attenuating properties, such as bone for example, fewer X-rays will have the kinetic energy to arrive at the detector.
In molecular imaging of vessels in mouse models of disease, we are interested in the vascular structures whose attenuation properties differ very little from the surrounding soft tissue. Consequently, vascular MicroCT requires the use of contrast agents such as Fenestra, Microfil, and Bismuth. The contrast agents either circulate in the blood pool or are used to replace it. An example of a three-dimensional (3D) MicroCT volume showing the vasculature of a tumor sample is shown in Figure 1 below.
MicroCT has emerged as an important structural imaging modality for vascular applications and angiogenesis due to its high spatial resolution34. It provides a high resolution 3D representation of vascular structures that directly reflects the level of angiogenesis or inhibition/development of neo-vasculature and provides a quantitative means for assessment of tumor growth over time. MicroCT data can be used to obtain measures such as vascular volume, tumor volume35, vascular surface area36, vessel density37, and branching patterns38, 39 which can be then combined/fused with functional information available from molecular imaging techniques such as PET, SPECT, MRI, or Ultrasound. Volume rendering showing the neo-vasculature and volume of tumors can be obtained from the microCT images, as demonstrated in Figure 2 which compares the vasculature of mice treated with saline or the angiogenesis inhibitor, rPAI-123.
Ultrasound is another modality for imaging of angiogenesis in vivo24, 25, 40. A transmitter produces s train of short pulses of high frequency oscillations, which are transformed by a transducer into high frequency mechanical oscillations. The vibrational energy of the mechanical oscillations is directed into the scanned object and swept back and forth. The return of an echo depends mainly on the type of scanned object/tissue and penetration depth. Echoes are detected by the transducer and transformed into electrical signals that are processed by a receiver to create an image.
High frequency micro-ultrasound imaging41 is particularly useful for obtaining repeated, non-invasive in vivo measurements to assess growth and to insure tumors are at the same size at initiation of treatment. In-vivo microCT has a limited application for tumor measurements because of the typically lower resolution of in-vivo scanners i.e. 50 microns. In addition, it gives a significant dose of radiation to the animal that may interfere with the study. Ex-vivo microCT has a much higher resolution, but requires samples from sacrificed animals. Therefore, it is not possible to image the same animal over time. Unlike microCT, ultrasound is non invasive, does not interfere with the study by using radiation and can be utilized to measure tumor size and volume repeatedly. Such measurements are important for every study in order to determine animals with relatively similar tumor measurements, which can be then divided into groups. MRI can be used to measure tumor volume but in most cases it is cost prohibitive comparing to ultrasound.
Other applications of ultrasound include spectral and power doppler ultrasounds42-44 used to measure blood flow velocities. Ultrasound contrast agents for angiogenesis have also been developed45. They are gas-filled microbubbles that are administered intravenously to the systemic circulation. Their size is in the range of 1 to 5 μm, which allows circulation in the capillary network. Microbubbles are strongly echogenic due to their large acoustic impedance mismatch with the surroundings. The response of an ultrasound contrast agent to an acoustic field depends on the applied mechanical index, which is proportional to the peak pressure of the acoustic wave divided by the square root of its frequency. At a low mechanical index (MI<0.1), the particles show Rayleigh scattering of the impinging wave, which increases with the sixth power of the particle radius and is dampened with increasing shell stiffness and gas density of the interior phase. At intermediate mechanical index (0.1<MI<0.5), microbubbles start to resonate in the sound field, producing also higher harmonics, which can be exploited using higher harmonic imaging. Increasing the mechanical index further (i.e. MI>0.5) also allows to disrupt the agents and release the trapped interior phase. That event produces not only a strong characteristic signal, but can also be used for therapeutic application, such as drug delivery.
Microbubbles can be used to image blood perfusion in organs and measure blood flow rate in the heart, in tumors, or other organs46. Targeted ligands that bind to specific receptors expressed on the vascular endothelium can be conjugated to microbubbles, enabling the microbubble complex to accumulate selectively in areas of interest, such as neo-vasculature or diseased/abnormal tissues. That form of molecular imaging is known as targeted contrast-enhanced ultrasound. It generates a strong signal if the targeted microbubbles bind to the receptors expressed on the area of interest. Furthermore, targeted contrast-enhanced ultrasound has many other applications in both delivery of therapeutics and medical diagnostics. The inhibition of tumor angiogenesis induced by rPAI-123 and imaged with ultrasound is shown in Figure 3.
Micro positron emission tomography (microPET) is another imaging modality that allows for visualization and quantitative assessment of angiogenesis in vivo47-49. MicroPET provides 3D image volumes representing the temporal and spatial distribution of a contrast agent (radioisotope) localized at a target50. The radioactive agent decays by positron emission. A positron travels a very short distance in tissue (<2mm) before colliding with an ordinary atomic electron. The two annihilate and a pair of high energy photons comes into existence. The photons travel in opposite directions at the speed of light. Detection of many such pairs leads to localization of their origin and ultimately, the creation of an image24, 25.
The main advantage of microPET over other imaging modalities is its high sensitivity. Very low concentrations of a radioisotope are sufficient to delineate a lesion based on its physiological characteristics. Furthermore, because there is limited background radiation, microPET inherently has a high signal to noise ratio. Due to the small size of the label (a radioactive atom vs. a microbubble 2-6μm) the contrast agent is readily available in the capillary network and can be used in combination with any other type of targeting agents such as metabolic substrates, small organic molecules, peptides, macromolecules, antibodies, nanoparticles, microbubbles, etc., to image any type of target.
Kinetic models are often used to describe the dynamics involved in the utilization of the radioisotopes51-54. The models consist of compartments and attempt to capture the spatial and temporal distributions of the isotopes that result from the complex physiological events at the cellular level. They are aimed at understanding and quantifying the physiological interactions of the radioactively labeled compound in a particular micro-environment. The resulting kinetic models describe the tracer behavior and provide a means for quantitative analysis of microPET data.
Recently antibody targeting of receptors for angiogenic growth factors55-57 and integrins associated with angiogenesis58-60 have received considerable attention as potential therapeutics in the treatment of cancer. The added advantage of antibody therapy is that the antibody can be conjugated to a radioactive molecule for imaging of the tumor vasculature. MicroPET has become a favorite imaging modality for antibody targeted imaging and therapy. In many cases copper-64 (Cu-64) is the preferred isotope due to its longer half life (12.7h)61, 62 and reduced background53.
One scheme for imaging angiogenesis is to Cu-64 label an antibody specific for an epitope on vascular endothelial growth factor receptor-2 (VEGFR-2), which is expressed on angiogenic vessels. The labeled antibody is administering by intravenous injection into the vasculature of a tumor-bearing mouse as illustrated in Figure 4. Kinetic modeling of the acquired microPET data can be used to determine the concentration of antigen-bound antibody in the tumor vasculature and provide a sense of the expression level of the imaging target as well as wash in/out rates51, 63, 64.
Confocal microscopy is one approach to optical molecular imaging that provides reconstruction of 3D images. In this technique, serial optical sections can be collected from specimens thereby allowing reconstruction of their third dimension by stacking the images on top of each other (commonly referred to as z-stacks). It is not a medical imaging modality as the ones described above, but employs targeted molecular imaging of specific biological molecules65-67. Targeted molecules are probed by using a fluorophore-tagged antibody specific for vascular targets found on endothelial or smooth muscle cells. Another alternative is using two sets of antibodies: primary and secondary. The primary antibody probes the target of interest, while a fluorophore-labeled secondary antibody amplifies the interaction of the primary antibody with the target molecule. The advantage of this technique is that the signal can be amplified and co-localized molecules can be detected through the use of secondary antibodies labeled with different fluophores that can be detected at different wave lengths. This technique can be applied to cultured cells or tissue sections.
Confocal microscopy plays a very important role in correlation and validation of results. In vascular imaging experiments collected Z-stacks can be thresholded to segment out the signal of the fluorophore bound to the specific target expressed on the blood vessel. Given the total volume of the z-stack, VZ, and the volume of the bound stain, Vs, the density of the imaged target can be measured as the ratio of the volume of the bound stain over the volume of the stack: VS/VZ. This is illustrated in Figure 5.
Functional imaging modalities such as microPET reveal valuable insights into biochemical, physiological, and pharmacological processes in vivo. Their major limitation, however, is the lack of high spatial resolution and detailed anatomical or vascular information. This problem can be easily alleviated by using complementary information provided by a structural imaging modality such as microCT and techniques from image registration.
Image registration can be rigid or nonrigid. Rigid registration deals with the alignment of rigid objects or objects that can not change shape or deform locally, such as bone fragments. Nonrigid registration, on the other hand, deals with the alignment of volumes of deformable or nonrigid objects and is very important for vascular analysis of mouse models of disease. Many algorithms for both rigid and nonrigid registration have been developed68-70. The accuracy of any algorithm for image registration can be quantified as the difference between true and estimated positions of all image points. The smaller the difference, the more accurate the registration, but determining the position of the same point in two volumes of a nonrigid object may not always be an easy task71. Unless a point is unique and can be detected in the two volumes, such a measure can not be computed because an image point from one volume may not exist in another. Despite such difficulties, as illustrated in Figure 6, functional activity from microPET can be overlaid with structural information from microCT for more detailed analysis and validation of results.
The field of angiogenesis is a rapidly growing area in which multimodal molecular imaging efforts are transforming the established understanding of the field. Vascular imaging techniques, as the ones described above, provide a valuable combination of structural and functional information that has the potential to advance the angiogenesis related research. The molecular imaging of vessels in mouse models of disease is extremely important to our understanding of the mechanisms that contribute to the progression of diseases such as cancer and ischemic diseases. Utilization of complimentary information provided by small animal imaging modalities provides the necessary basis for successful analysis and evaluation of imaging studies.
This study was supported by Dartmouth-Philips Research Consortium (MJM-K).
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