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Angiogenesis plays an important role in the pathophysiology of atherosclerosis and after myocardial infarction. Furthermore, angiogenesis has been the focus of many therapeutic strategies. In view of that, a direct and clear understanding of the role of these pathways in the living subject is needed. Molecular Imaging has emerged as a powerful tool to study biological processes non-invasively. In this review, evidence will be presented and discussed on the feasibility of different molecular imaging strategies to study the involvement of angiogenic pathways in the assessment of the atherosclerotic disease and as a tool to assess angiogenic therapy. Focus will be placed on those imaging modalities with the potential to be translated to clinical use.
Angiogenesis constitutes the formation and/or recruitment of new blood vessels1, a process that may have different significance depending on the field under study2. In Oncology, angiogenesis is associated with providing more nutrients to the tumor and thus tumor growth3, 4, what have led to development of therapies targeted to inhibit angiogenic pathways and reducing tumor size5–8. In Cardiovascular Diseases the formation of new blood vessels can have different significances and can be have either a positive or negative connotation, depending on the pathophysiological background where angiogenesis takes place.
Atherosclerosis (ASD) is a complex disease that affects vascular beds throughout the body and patho-physiologically is characterized by the accumulation of cholesterol, different mediators of inflammation, and ultimately fibrosis of the vessel wall and affected myocardium9. Furthermore, there is recent evidence on the presence of angiogenesis on both the atherosclerotic plaque as well as of the vasa-vasorum network10, 11, and the important role it plays in the initiation, development and progression of atherosclerotic vascular lesions10. The neo-vascularization described in early atherosclerosis, similar to that seen in cancer12, is mostly composed of immature vessels that area characterized by increased vascular permeability13, 14. Clinically accepted therapies for reduction of high cholesterol and ASD (e.g. statin and angiotensin converting enzyme inhibitor-ACEI-therapies) that have been shown to decrease cardiovascular mortality and disease progression, have also been shown to reduce the level of neovascularization in early atherosclerosis15, 16, what suggest an association between neo-vascularization and ASD.
Myocardial infarction (MI) is caused by sudden occlusion of a coronary artery, interrupting blood flow to the downstream myocardium, leading to tissue anoxia and cell death9. As a biological response to this insult, a number of biological pathways (e.g. pro-inflammatory and pro-angiogenic mechanisms) are activated9, 17. The activation of these mechanisms has been associated to myocardial remodeling, ultimately determining how the myocardial tissue adjusts to the new tissue “microenvironment”18–20. Angiogenesis has also been shown to be part of the remodeling process21, and has been observed mainly on the “border zone” (zone between normal and infarcted myocardium), suggesting that it can be part of the tissue response to the ischemic stimuli. Integrins and vascular endothelial growth factor (VEGF) have been associated to the angiogenic response after MI22, 23.
Integrins are a family of proteins expressed by endothelial cells to interact with their extracellular microenvironment24. Integrins are composed by two subunits, α and β. The first and most largely studied integrin is the vitronectin-type αv β3 integrin25. Each individual integrin subunit consists of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail.
Integrins have many different functions that are carried by different portions of the protein. Integrins are the main receptors for extracellular matrix (ECM) proteins, such as fibronectin, laminins, or collagens, and may play a fundamental role in tissue remodeling26. Besides promoting physical adhesion, integrin ligation also initiates signaling that induces cell spreading, migration, survival, proliferation, and differentiation26.
Vascular endothelial growth factor (VEGF)27 is the most prominent member of a family of growth factors, and is composed of seven protein sub-types: VEGF-A, VEGFB, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor28. VEGF-A is a dimmer with at least seven homodimeric isoforms (121, 145, 148, 165, 183, 189, or 206 amino acids). VEGF is a family of proteins that have a similar background, with same modifications, which may be responsible for the specific proteins and ligand specificity29.
Similar to many other proteins, VEGF has specific receptors to which it binds to elucidate specific actions. The angiogenic properties of VEGF are mainly mediated via two endothelium-specific receptor tyrosine kinases, Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2)30. VEGF-A isoforms bind to both VEGFR-1 and VEGFR-2, which are mostly present in vascular endothelial cells. VEGFR-1 plays a major role in physiologic and developmental angiogenesis and its function varies with the stages of development, the states of physiologic and pathologic conditions, while VEGFR-2 is the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF28.
For more than a decade, significant interest has been placed on therapeutic alternatives (e.g. trans-myocardial laser revascularization) to restore blood flow to affected areas of the heart31. Gene therapy with many different growth factors (e.g. fibroblast growth factor, vascular endothelial growth factor-VEGF-) has also been developed in an attempt to provide the “roadblocks” and create new blood vessels in the myocardium32, 33. In the last few years, stem cell therapies are being developed with the goal of creating new vessels34, and/or provide the necessary structure that can sustain a body’s generated pro-angiogenic response.
Despite significant advances in the understanding of the benefits of these therapies, numerous questions remain. Issues like the bio-availability of these genes in vivo, the kinetics of these growth factors in the living subjects are among some of the questions that remain to be answered.
In summary, angiogenesis plays a major role in the pathophysiology of different cardiovascular diseases, and is also the goal of different therapeutic approaches. Integrins and VEGF have been associated with, and used as therapeutic strategies for ASD. To better understand and assess the involvement of angiogenic pathways it becomes imperative to be able to monitor and quantify the angiogenic response in the living subject.
Over the last few years, significant efforts have been placed in developing novel strategies to image and quantify the angiogenic response, whether is to study disease or assess the response to therapy. Development of angiogenesis-targeted molecular imaging probes could serve as a new tool to better understand the role and expression profile of angiogenic-related molecules in many angiogenesis-related diseases, as well as for the assessment of angiogenic therapies.
As previously mentioned, integrins are transmembrane protein that play an important role in angiogenesis35. Because of that, they have become interesting targets to develop strategies to image the angiogenic stimuli, and through that approach be able to study the patho-physiological states where angiogenesis is present. Due to the transmembrane characteristic of integrins, specially the ανβ3 integrin, imaging approaches to image integrins have focused on the development of ligands that will bind to the extracellular portion of the integrin molecule. In other words, an exogenously administered ligand will attach (in a specific manner) angiogenic receptor expressed in the cell surface, which will retain the ligand in the area of interest, and that can be imaged and monitored non-invasively (Figure 1).
With these premises, a number of investigators have developed a number of imaging probes to image integrin expression in angiogenesis. Harris et al reported the high affinity and selectivity of 111In-RP748 for the activated conformation of the vitronectin-type ανβ3 integrin receptor36. Using 111In-RP748 as the imaging probe, Meoli et al demonstrated the feasibility of imaging the activation of the vitronectin-type ανβ3 integrin after MI (Figure 2)37 and showed that the expression of ανβ3 integrin after MI occurs in the area of the MI, but not in remote zones (Figure 2)37. This constitutes the first report of non-invasive monitoring of angiogenesis in the myocardium. Investigators went further and showed that in areas where angiogenesis was increased (increased uptake of 111In-RP748), there was also increased uptake of 99Tc-59-21 (hypoxia marker), establishing a relationship between hypoxia and angiogenesis in the myocardium38. As the imaging modality, investigators used single photon emission computed tomography, a modality commonly used in the clinics. Studies like those from Meoli et al are critical as they set the basis for future non-invasive approaches to study pathophysiology of angiogenesis expression in the myocardium, and at the same time bringing these studies closer to the clinics.
These imaging strategies have also been used to assess angiogenesis in other vascular territories. Hua et al, using a 99Tc-labeled peptide that binds to the ανβ3 integrin, described the kinetics of ανβ3 integrin in a murine hind-limb ischemia model39. Similarly, Lee et al. used a 125I-labeled RGD compound to track the expression of ανβ3 integrins after peripheral ischemia40. Furthermore using Ultrasound as the imaging modality and microbubbles labeled with ανβ1 integrin, Leong-Poi et al, confirmed previous observations and showed that integrin expression is upregulated in a model of hind-limb ischemia41. In addition to their biological significance, these studies showed how these different imaging modalities can be used to study biology non-invasively across different vascular beds.
In order to understand the role of the different biological pathways in the angiogenic stimuli, it is imperative to be able to image the different angiogenic pathways involved. As mentioned before, VEGF/VEGFR is the main angiogenic pathway and has been strongly related to the angiogenesis described in ASD and after MI.
Using the imaging approach depicted in Figure 1, Cai et al have developed an imaging probe (64Cu-DOTA-VEGF121) for positron emission tomography (PET) that binds selectively to the VEGFRs, and have shown the feasibility of VEGFR imaging in a cancer model42. More recently, our laboratory has translated this approach to monitor the expression of VEGFRs after MI43. In a rodent model of MI (permanent coronary artery ligation), we showed that MI is associated with increased uptake of this probe-as a surrogate of increased VEGFR expression (Figure 3)-. In these studies we followed the binding of 64Cu-DOTA-VEGF121 for 4 weeks, and showed that there is an early expression of VEGFRs after MI (peak binding occurred during the first week), and then the expression decreased reaching levels not different from baseline by the fourth week. This is the first study that describes non-invasively the expression of VEGFR over time after MI and because it uses a clinically applicable imaging modality (PET) constitutes an important step towards the application of these modalities in the clinics.
As with integrins, imaging of VEGFRs has also been performed in the peripheral vasculature. Lu et al, using a 111In-VEGF121 labeled molecule, showed that ischemic tissue had increased tracer uptake-as a surrogate of VEGFR expression- compared to sham-operated or contralateral muscle, and that this uptake was increased as early as 3 hours44. More recently, our laboratory has used the 64Cu-DOTA-VEGF121 probe to monitor VEGFR expression in a murine model of hind-limb ischemia45. Furthermore, using VEGFR-1 and -2 mutants we demonstrated that VEGFR-2 is the main VEGF receptor activated after hind-limb ischemia, and showed that exercise training lead to increased VEGFR-2 expression and improved exercise tolerance in these animals.
When put together, the above mentioned studies on integrin- and VEGF-imaging, these studies are of critical importance for a number of reasons: on one side, they serve as proof of concept for imaging angiogenic biological pathways across different vascular territories. Furthermore, they provide important information on the biology that underlies these pathophysiological states in a non-invasive manner. Lastly, and because most of these studies have been performed using clinical imaging modalities, they open the door for the transition of these modalities to the clinics.
As mentioned above different therapeutic strategies are targeted to the expression of VEGF, as a mediator for the angiogenic stimuli. Until recently, most of the studies used to assess the effect of these therapeutic strategies have been based on histology or assessment of organ function (e.g. using Echocardiography for left ventricular ejection fraction-LVEF-). While the assessment of LVEF is important and is routinely used clinically, by itself it does not provide information on the actual expression of the growth factor. Recent developments in molecular biology now permit the non-invasive imaging of trans-gene expression46, 47. Wu et al demonstrated the feasibility of monitoring VEGF gene therapy, in a rodent model of MI, using PET48. In these studies, the investigators used a dual cassette strategy, where one gene cassette delivered the therapeutic gene (VEGF), while in the other one a reporter gene (thymidine kinase, a PET reporter gene) is used to report the expression of the therapeutic gene (Figure 5). The authors demonstrated a good correlation between the reporter gene signal and the VEGF trans-gene expression, enabling them to use the reporter gene to monitor VEGF expression in the living subject (Figure 6). Wagner et al use a similar strategy to monitor the trans-gene expression of therapeutic VEGF in a large animal model of MI49, bringing these strategies closer to clinical applications.
Most of the current imaging strategies to image angiogenesis are based on monitoring the binding of labeled probes on the cell surface receptors (Figure 1), and use that as a surrogate of receptor expression, and most importantly, of a certain pathway activation. Use of cell surface receptors as surrogates of pathway activation is important as in many cases, as integrins and VEGF receptors are an integral part of the activation of that pathway, and in many cases mediate function. From the imaging perspective, exogenously administered labeled probes do not have to cross the cell membrane, what may facilitate the interaction between the ligand and the recipient (i.e. receptor)46, 47.
On the other side, cell surface receptors may not be representative of the complete intra-cellular angiogenic story. For example, VEGF activates a number of downstream pathways, whose action may remain intracellular and determine cell surface actions, beyond those related to VEGFRs50. In that case, imaging cell surface receptors may not be sufficient to accurately study a given angiogenic pathway. From the signal-acquisition perspective, receptors can only bind to a certain number of molecules (ligands), what may limit the signal that remains in the area of interest46, 47.
The studies by Wu et al and others elegantly illustrate how genetic therapies can be monitored non-invasively. This strategy ensures that the delivered therapeutic gene (VEGF in this case) actually gets transcribed and translated in the protein of interest. When assessing gene therapy effects, caution should be exercised, as increased trans-gene expression of a therapeutic gene is the first step, but may only be a part of the biologic response in the living subject. Other pathways, may act by deactivating of further activating the angiogenic signal. From the imaging perspective, these strategies use an enzyme-based approach, which results in increased signal (one enzymatic molecule can interact with a large number of administered probes, amplifying the signal)46, 47. On the other side, for the enzyme to interact with the exogenously administered probe, the latter has to cross the cell membrane, what may limit the protein-probe interaction. Future, advances in reporter gene design and development of novel and more sensitive probes may result in improved signal-to-noise ratios.
Clinical imaging of angiogenic pathway activation will be important in many aspects. Angiogenesis has been shown to be involved in the pathogenesis of atherosclerosis. Imaging of VEGF and related pathways can provide a non-invasive assessment of the activity of atherosclerosis in the heart. However, current limitations in spatial resolution and sensitivity of clinical systems may preclude an accurate study of angiogenic pathways in the myocardial microvasculature. With the current imaging technology, clinical imaging of angiogenesis appears more plausible for imaging of angiogenesis in the peripheral vascular territories (e.g. carotid artery, femoral artery).
Imaging of VEGF and related pathways will likely also play a clinical role in post MI-myocardial remodeling, what determines patient outcomes. Patients are routinely placed on a number of therapies to modulate LV remodeling (e.g. statins, ACEIs). Currently, the best way to assess the effect of remodeling and which therapies are appropriate is to measure left ventricular size, left ventricular ejection fraction, and monitor patient outcomes. However, we are not able to monitor the biology of LV remodeling, such as the development of neo-vascularization, and other factors that can be involved in such response. Using molecular imaging, we will be able to monitor the post-MI state and assess the therapeutic effect of different therapies on LV remodeling.
Another area where imaging of angiogenesis will also be very important will be on the assessment of genetic therapies. Whether it is to evaluate gene or cell therapy, molecular imaging of angiogenesis (using strategies as in Figure 1) will provide invaluable information on the effect of these therapies (using imaging strategies as in Figure 5) and will likely lead to improvement of different strategies.
Angiogenesis plays an important role in the pathophysiology of many pathophysiological states. Furthermore, it is the target of many therapeutic strategies. Thus, it is imperative that investigators have ways to assess the presence of absence of angiogenesis, as well as methods for gene expression and product (e.g. formation of new blood vessels) quantification.
Molecular Imaging is a powerful emerging tool for the quantification of biological processes, with perturbation of the tissue under study. We have presented evidence that different “biochemical signatures” of angiogenesis can already be detected non-invasively.
In the future, we anticipate that novel probes targeting novel pathways will be developed and will help broaden the spectrum of pathways that can be studied non-invasively. That, in conjunction with advances in imaging hardware will be critical for the advancement of the field of non-invasive biological imaging.