Insulin-like growth factor binding proteins and angiogenesis: from cancer to cardiovascular disease

Angiogenesis is a tightly regulated activity that is vital during embryonic development and for normal physiological repair processes and reproduction in healthy adults. Pathological angiogenesis is a driving force behind a variety of diseases including cancer and retinopathies, and inhibition of angiogenesis is a therapeutic option that has been the subject of much research, with several inhibitory agents now available for medical therapy. Conversely, therapeutic angiogenesis has been mooted as having significant potential in the treatment of ischemic conditions such as angina pectoris and peripheral arterial disease, but so far there has been less translation from lab to bedside.The insulin-like growth factor binding proteins (IGFBP) are a family of seven proteins essential for the binding and transport of the insulin-like growth factors (IGF). It is being increasingly recognised that IGFBPs have a significant role beyond simply modulating IGF activity, with evidence of both IGF dependent and independent actions through a variety of mechanisms. Moreover, the action of the IGFBPs can be stimulatory or inhibitory depending on the cell type and environment. Specifically the IGFBPs have been heavily implicated in angiogenesis, both pathological and physiological, and they have significant promise as targeted cell therapy agents for both pathological angiogenesis inhibition and therapeutic angiogenesis following ischemic injury. In this short review we will explore the current understanding of the individual impact of each IGFBP on angiogenesis, and the pathways through which these effects occur.     

Synthesis and application of cNGR-containing imaging agents for detection of angiogenesis

Angiogenesis is a multi-step process regulated by pro- and anti-angiogenic factors. Inhibition of angiogenesis is a potential anti cancer treatment strategy that is now investigated clinically. In addition, advances in the understanding of the angiogenic process have led to the development of new angiogenesis therapies for ischemic heart disease.Currently, researchers search for objective measures that indicate pharmacological responses to pro- and anti-angiogenic drugs and therefore, there is a great interest in techniques to visualize angiogenesis noninvasively. As CD13 is selectively expressed in angiogenic blood vessels, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in animal models and patients. Here, an overview on the currently used CD13 targeted molecular imaging probes for noninvasive visualization of angiogenesis is given.     

Molecular imaging with targeted contrast ultrasound

Molecular imaging with contrast ultrasound relies on the detection of targeted microbubbles or other acoustically active nanoparticles. These microbubbles are retained in diseased tissue where they produce an acoustic signal because of their resonant properties in the ultrasound field. Targeting is accomplished either through manipulating the chemical properties of the microbubble shell or through conjugation of disease-specific ligands for the targeted molecule to the microbubble surface. As microbubbles cannot leave the intravascular space, the disease process must be characterized by molecular changes in the vascular compartment to be imaged. Inflammation, angiogenesis and thrombus formation are central pathophysiologic processes in many disease states and produce phenotypic changes in the vascular compartment. Thus, targeted contrast ultrasound in the future could aid in the diagnosis of such diverse diseases as atherosclerosis, transplant rejection and tumor-related angiogenesis.     

Targeted molecular imaging of angiogenesis in PET and SPECT: a review

Over the past few decades, there have been significant advancements in the imaging techniques of positron emission tomography (PET) and single photon emission tomography (SPECT). These changes have allowed for the targeted imaging of cellular processes and the development of hybrid imaging systems (e.g., SPECT/CT and PET/CT), which provide both functional and structural images of biological systems. One area that has garnered particular attention is angiogenesis as it relates to ischemic heart disease and limb ischemia. Though the aforementioned techniques have benefits and consequences, they enable scientists and clinicians to identify regions that are vulnerable to or have been exposed to ischemic injury via non-invasive means. This literature review highlights the advancements in molecular imaging techniques and specific probes as they pertain to the process of angiogenesis in cardiovascular disease.     

Generation of mice with a conditional and reporter allele for Tmem100

Activin receptor‐like kinase 1 (ACVRL1; ALK1) is predominantly expressed in arterial endothelial cells and plays an important role in angiogenesis. ACVRL1 mutations cause hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder for which the underlying mechanism is poorly understood. We have found that expression of transmembrane protein 100 (Tmem100) is downregulated in the lung of Acvrl1‐deficient mice; however, its function is unknown. To elucidate the role of Tmem100 in vivo, we generated a conditional knockout allele for Tmem100 in which exon3, containing the entire coding sequence, was flanked by loxP sequences. The targeted allele also possessed a lacZ reporter cassette in intron2 for visualization of Tmem100 expression. We found that Tmem100 was predominantly expressed in arterial endothelial cells of developing embryos. The conditional and reporter allele will be a useful resource to investigate the in vivo role of Tmem100, especially in angiogenesis. genesis 48:673–378, 2010. © 2010 Wiley‐Liss, Inc.     

Development of contrast agents targeted to macrophage scavenger receptors for MRI of vascular inflammation

Atherosclerosis is a leading cause of death in the U.S. Because there is a potential to prevent coronary and arterial disease through early diagnosis, there is a need for methods to image arteries in the subclinical stage as well as clinical stage using various noninvasive techniques, including magnetic resonance imaging (MRI). We describe a development of a novel MRI contrast agent targeted to plaques that will allow imaging of lesion formation. The contrast agent is directed to macrophages, one of the earliest components of developing plaques. Macrophages are labeled through the macrophage scavenger receptor A, a macrophage specific cell surface protein, using an MRI contrast agent derived from scavenger receptor ligands. We have synthesized and characterized these contrast agents with a range of relaxivities. In vitro studies show that the targeted contrast agent accumulates in macrophages, and solution studies indicate that micromolar concentrations are sufficient to produce contrast in an MR image. Cell toxicity and initial biodistribution studies indicate low toxicity, no detectable retention in normal blood vessels, and rapid clearance from blood. The promising performance of this contrast agent targeted toward vascular inflammation opens doors to tracking of other inflammatory diseases such as tumor immunotherapy and transplant acceptance using MRI.     

Current research progress in targeted anti‐angiogenesis therapy for osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumour with a peak in incidence during adolescence. Delayed patient presentation and diagnosis is common with approximately 15% of OS patients presenting with metastatic disease at initial diagnosis. With the introduction of neoadjuvant chemotherapy in the 1970s, disease prognosis improved from 17% to 60%‐70% 5‐year survival, but outcomes have not significantly improved since then. Novel and innovative therapeutic strategies are urgently needed as an adjunct to conventional treatment modalities to improve outcomes for OS patients. Angiogenesis is crucial for tumour growth, metastasis and invasion, and its prevention will ultimately inhibit tumour growth and metastasis. Dysregulation of angiogenesis in bone microenvironment involving osteoblasts and osteoclasts might contribute to OS development. This review summarizes existing knowledge regarding pre‐clinical and developmental research of targeted anti‐angiogenic therapy for OS with the aim of highlighting the limitations associated with this application. Targeted anti‐angiogenic therapies include monoclonal antibody to VEGF (bevacizumab), tyrosine kinase inhibitors (Sorafenib, Apatinib, Pazopanib and Regorafenib) and human recombinant endostatin (Endostar). However, considering the safety and efficacy of these targeted anti‐angiogenesis therapies in clinical trials cannot be guaranteed at this point, further research is needed to completely understand and characterize targeted anti‐angiogenesis therapy in OS.     

Radiotracer breast cancer imaging: Beyond FDG and MIBI

Radiotracer imaging with MIBI and FDG have shown the benefit of the functional imaging of breast cancer. Newer radiopharmaceuticals targeted to particular aspects of breast cancer biology will likely play an important role in directing more specific and individualized breast cancer treatment. Future studies will need to test the ability of SPECT and PET imaging to detect breast cancer, but also to assess target expression, identify resistance factors, and measure early response to treatment. This will require protocols designed to test the predictive capability of imaging in the setting of a therapy trial, a new paradigm for breast cancer imaging, for which radiotracer imaging is ideally suited.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.     

Targeted contrast agents for magnetic resonance imaging and ultrasound

The development of contrast agents that can be localized to a particular tissue or cellular epitope will potentially allow the noninvasive visualization and characterization of a variety of disease states. Recent advances have been made in the field of molecular imaging with magnetic resonance imaging and ultrasound and varied approaches have been devised to overcome the high background tissue signal. The types of agents and applications developed include gadolinium-conjugated targeting molecules for imaging of fibrin, superparamagnetic iron oxide particles for stem-cell tracking, multimodal perfluorocarbon nanoparticles for visualization of angiogenesis, liposomes for targeting atheroma components, and microbubbles for imaging transplant rejection.     

Generation of transgenic mice overexpressing EfnB2 in endothelial cells

Genetic studies have shown that ephrin-B2 and its cognate EphB4 receptor are necessary for normal embryonic angiogenesis. Moreover, there is overwhelming evidence that ephrin-B2 is involved in tumor vascularization, yet its role in adult angiogenesis has been difficult to track genetically. Here, we report the generation of transgenic mice that over-express EfnB2 specifically in endothelial cells (ECs). We show that exogenous expression of EfnB2 under the control of the Tie2 promoter/enhancer regions in ECs does not affect viability or growth of the transgenic animals. We further show that targeted expression of EfnB2 in ECs is not sufficient to rescue severe cardiovascular defects at mid-gestation stages but rescues early embryonic lethality associated with loss-of-function mutation in EfnB2. This mouse model will be useful to study the role of ephrin-B2 in physiological and pathological angiogenesis.     

Assessing responses to cancer therapy using molecular imaging

Tumor responses to therapy in the clinic are still evaluated primarily from non-invasive imaging measurements of reductions in tumor size. This approach, however, lacks sensitivity and can only give a delayed indication of a positive response to treatment. Major advances in our understanding of the molecular mechanisms responsible for cancer, combined with new targeted clinical imaging technologies designed to detect the molecular correlates of disease progression and response to treatment, are set to revolutionize our approach to the detection and treatment of the disease. We describe here the imaging technologies available to image tumor cell proliferation and migration, metabolism, receptor and gene expression, apoptosis and tumor angiogenesis and vascular function, and show how measurements of these parameters can be used to give early indications of positive responses to treatment or to detect drug resistance and/or disease recurrence. Special emphasis has been placed on those applications that are already used in the clinic and those that are likely to translate into clinical application in the near future or whose use in preclinical studies is likely to facilitate translation of new treatments into the clinic.     

Betaglycan knock‐down causes embryonic angiogenesis defects in zebrafish

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF‐β) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF‐β receptor type III, is a TGF‐β coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF‐β‐binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time‐lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583–603, 2015. © 2015 Wiley Periodicals, Inc.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

Neovascularization and Hematopoietic Stem Cells

Vasculogenesis and angiogenesis are the major forms of blood vessel formation. Angiogenesis is the process where new vessels grow from pre-existing blood vessels, and is very important in the functional recovery of pathological conditions, such as wound healing and ischemic heart diseases. The development of better animal model and imaging technologies in past decades has greatly enriched our understanding on vasculogenesis and angiogenesis processes. Hypoxia turned out to be an important driving force for angiogenesis in various ischemic conditions. It stimulates expression of many growth factors like vascular endothelial growth factor, platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor, which play critical role in induction of angiogenesis. Other cellular components like monocytes, T cells, neutrophils, and platelets also play significant role in induction and regulation of angiogenesis. Various stem/progenitor cells also being recruited to the ischemic sites play crucial role in the angiogenesis process. Pre-clinical studies showed that stem/progenitor cells with/without combination of growth factors induce neovascularization in the ischemic tissues in various animal models. In this review, we will discuss about the fundamental factors that regulate the angiogenesis process and the use of stem cells as therapeutic regime for the treatment of ischemic diseases.     

Ultrasound Molecular Imaging with Targeted Microbubbles for Cancer Diagnostics: From Bench to Bedside

Background

Ultrasound plays an important role in cancer diagnosis. B-mode imaging and contrast-enhanced ultrasound are routinely used to detect cancerous lesions in breast and liver. The use of ultrasound contrast agents (UCAs) such as microbubbles (MBs), which can be functionalized with targeting ligands, has further enabled ultrasound molecular imaging (USMI) of specific molecular markers in pre-clinical and the first clinical studies. As targeted MBs have a diameter of 1–4 μm, they are limited to the blood vasculature upon intravenous injection, and can bind to markers of the vascular endothelium. USMI with targeted MBs was applied for imaging of markers of inflammation, angiogenesis, and the tumor endothelium.

Plexiform-like lesions and increased tissue factor expression in a rat model of severe pulmonary arterial hypertension

Severe pulmonary arterial hypertension (PAH) occurs in idiopathic form and in association with diverse diseases. The pathological hallmarks are distal smooth muscle hypertrophy, obliteration of small pulmonary arteriole lumens, and disorganized cellular proliferation in plexiform lesions. In situ thrombosis is also observed. A detailed understanding of the disease progression has been hampered by the absence of an animal model bearing all the pathological features of human disease. To create a model with these characteristics, we gave young (200-g) rats monocrotaline 1 wk following left pneumonectomy; controls with vehicle treatment or sham operation were also studied. In experimental rats, pulmonary arteries had distal smooth muscle hypertrophy and proliferative perivascular lesions. The lesions had a plexiform appearance, occurred early in disease development, and were composed of cells expressing endothelial antigens. Three-dimensional microangiography revealed severe vascular pruning and disorganized vascular networks. We found that expression of tissue factor (TF), the membrane glycoprotein that initiates coagulation, facilitates angiogenesis, and mediates arterial injury in the systemic circulation, was increased in the pulmonary arterioles and plexiform-like lesions of the rats. TF was also heavily expressed in the vessels and plexiform lesions of humans with pulmonary arterial hypertension. We conclude that plexiform-like lesions can be reproduced in rats, and this model will facilitate experiments to address controversies about the role of these lesions in PAH. Increased TF expression may contribute to the prothrombotic diathesis and vascular cell proliferation typical of human disease.     

Assessment of molecular imaging of angiogenesis with three-dimensional ultrasonography

Molecular imaging (MI) with ultrasonography relies on microbubble contrast agents (MCAs) adhering to a ligand-specific target for applications such as characterizing tumor angiogenesis. It is projected that ultrasonic (US) MI can provide information about tumor therapeutic response before the detection of phenotypic changes. One of the limitations of preclinical US MI is that it lacks a comprehensive field of view. We attempted to improve targeted MCA visualization and quantification by performing three-dimensional (3D) MI of tumors expressing αvβ3 integrin. Volumetric acquisitions were obtained with a Siemens Sequoia system in cadence pulse sequencing mode by mechanically stepping the transducer elevationally across the tumor in 800-micron increments. MI was performed on rat fibrosarcoma tumors (n = 8) of similar sizes using MCAs conjugated with a cyclic RGD peptide targeted to αvβ3 integrin. US MI and immunohistochemical analyses show high microbubble targeting variability, suggesting that individual two-dimensional (2D) acquisitions risk misrepresenting more complex heterogeneous tissues. In 2D serial studies, where it may be challenging to image the same plane repeatedly, misalignments as small as 800 microns can introduce substantial error. 3D MI, including volumetric analysis of inter- and intra-animal targeting, provides a thorough way of characterizing angiogenesis and will be a more robust assessment technique for the future of MI.     

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.     

Targeted imaging of hypoxia-induced integrin activation in myocardium early after infarction.

The alphavbeta3-integrin is expressed in angiogenic vessels in response to hypoxia and represents a potential novel target for imaging myocardial angiogenesis. This study evaluated the feasibility of noninvasively tracking hypoxia-induced alphavbeta3-integrin activation within the myocardium as a marker of angiogenesis early after myocardial infarction. Acute myocardial infarction was produced by coronary artery occlusion in rodent and canine studies. A novel (111)In-labeled radiotracer targeted at the alphavbeta3-integrin ((111)In-RP748) was used to localize regions of hypoxia-induced angiogenesis early after infarction. In rodent studies, the specificity of (111)In-RP748 for alphavbeta3-integrin was confirmed with a negative control compound ((111)In-RP790), and regional uptake of these compounds correlated with (201)Tl perfusion and a (99m)Tc-labeled nitroimidazole (BRU59-21), which was used as a quantitative marker of myocardial hypoxia. The ex vivo analysis demonstrated that only (111)In-RP748 was selectively retained in infarcted regions with reduced (201)Tl perfusion and correlated with uptake of BRU59-21. In canine studies, myocardial uptake of (111)In-RP748 was assessed using in vivo single-photon-emission computed tomography (SPECT), ex vivo planar imaging, and gamma well counting of myocardial tissue and correlated with (99m)Tc-labeled 2-methoxy-2-methyl-propyl-isonitrile ((99m)Tc-sestamibi) perfusion. Dual-radiotracer in vivo SPECT imaging of (111)In-RP748 and (99m)Tc-sestamibi provided visualization of (111)In-RP748 uptake within the infarct region, which was confirmed by ex vivo planar imaging of excised myocardial slices. Myocardial (111)In-RP748 retention was associated with histological evidence of alphavbeta3-integrin expression/activation in the infarct region. (111)In-RP748 imaging provides a novel noninvasive approach for evaluation of hypoxia-induced alphavbeta3-integrin activation in myocardium early after infarction and may prove useful for directing and evaluating angiogenic therapies in patients with ischemic heart disease.