Combining nanotechnology with current biomedical knowledge for the vascular imaging and treatment of atherosclerosis

Abstract

Activation of vasa vasorum (the microvessels supplying the major arteries) at specific sites in the adventitia initiates their proliferation or ‘angiogenesis’ concomitant with development of atherosclerotic plaques. Haemorrhagic, leaky blood vessels from unstable plaques proliferate abnormally, are of relatively large calibre but are immature neovessels poorly invested with smooth muscle cells and possess structural weaknesses which may contribute to instability of the plaque by facilitation of inflammatory cell infiltration and haemorrhagic complications. Weak neovascular beds in plaque intima as well as activated adventitial blood vessels are potential targets for molecular imaging and targeted drug therapy, however, the majority of tested, currently available imaging and therapeutic agents have been unsuccessful because of their limited capacity to reach and remain stably within the target tissue or cells in vivo. Nanoparticle technology together with magnetic resonance imaging has allowed the possibility of imaging of neovessels in coronary or carotid plaques, and infusion of nanoparticle suspensions using infusion catheters or implant-based drug delivery represents a novel and potentially much more efficient option for treatment. This review will describe the importance of angiogenesis in mediation of plaque growth and development of plaque instability and go on to investigate the possibility of future design of superparamagnetic/perfluorocarbon-derived nanoparticles for imaging of the vasculature in this disease or which could be directed to the adventitial vasa vasorum or indeed intimal microvessels and which can release active payloads directed against primary key external mitogens and intracellular signalling molecules in endothelial cells responsible for their activation with a view to inhibition of angiogenesis.

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Introduction

According to a World Health Organization Fact Sheet (EURO/03/06) cardiovascular disease (CVD) is the number one killer in Europe, with heart disease and stroke being the major cause of death in all 53 Member States. Figures show that 34 421 (23% of all non-communicable diseases) of Europeans died from CVD in 2005. The report also highlighted the fact that there is approximately a 10-fold difference in premature CVD mortality between Western Europe and countries in Central and Eastern Europe (i.e. there is a higher occurrence of CVD amongst the poor and vulnerable). The problem for the European Union is that there is a direct correlation between the premature death rate and the viability of countries’ economies. Although improvements in understanding have helped to reduce the number of Western Europeans dying from CVD and related diseases further advances will require a clearer understanding of the pathobiological mechanisms responsible for the development of stroke, atherosclerosis and myocardial infarction. Approximately 75% of acute coronary events and 60% of symptomatic carotid artery disease are associated with disruption of atherosclerotic plaques.¹ As early as 1971, Folkman² introduced the concept of angiogenesis as a necessity for tumour growth. Its importance in other major pathological conditions, including, atherosclerosis, myocardial infarction and stroke, was later realised.^3,4

Atherosclerosis and neovessel formation

Atherosclerosis is primarily a chronic inflammatory disorder; however, angiogenesis plays an important and complex role in development of unstable lesions.⁴ During plaque development many pro-angiogenic pathways are re-actived and this leads to formation of immature blood vessels prone to rupture. Infiltration of microvessels into the media, intima and plaques originates predominantly from proliferating vasa vasorum. Intima and media of coronary atherosclerotic vessels are infiltrated with a tumour-like mass of microvessels prone to leak.⁵ However, chronic minimal injury also leads to intraluminal endothelial dysfunction and pro-inflammatory intracellular signalling pathways are recruited which lead to transcriptional up-regulation of expression of cytokines, adhesion molecules and chemoattractantproteins.

Plaque angiogenesis is now accepted to have a fundamental role in the pathophysiological development of atherosclerosis, providing nutrients to the developing and expanding intima and also potentially creating an unstable haemorrhagic environment prone to rupture. The expression of intimal neovessels is directly related to the stage of plaque development, the presence of symptomatic disease and the risk of plaque rupture. In atherosclerosis, intimal neovascularisation arises most frequently from the dense network of vessels in the adventitia, adjacent to a plaque, rather than from the main artery lumen. New blood vessels may have an active role in plaque metabolic activity and actively promote its growth beyond the critical limits of diffusion from the artery lumen. A strong correlation between areas of increased vascularity and intraplaque haemorrhage was first demonstrated by histological staining with anti-CD34 in symptomatic patients following endarterectomy.⁶ The irregular nature of blood vessel formation has been likened to tumour angiogenesis, and hence the factors responsible for their growth may be different from those seen during normal wound healing. Our previous studies and those of others have suggested that haemorrhagic, leaky blood vessels from unstable carotid plaques proliferate abnormally. These relatively large calibre but immature neovessels are poorly invested with smooth muscle cells and possess structural weaknesses which may contribute to instability of the plaque by facilitation of inflammatory cell infiltration and haemorrhagic complications.⁷ In a study of coronary artery atherogenesis, from patients subjected to heart transplant, lesions with the highest neovessels content were demonstrated to be of type VI and associated with the highest rate of thrombotic episodes.⁸

These processes are in part initiated by hypoxia generated in the plaque, the specific action of growth factors, particularly vascular EC growth factor (VEGF) and basic fibroblastgrowth factor (FGF-2), secreted by vascular and inflammatory cells and other factors such as hemodynamic stress. Immature neovessels may contribute to instability of the plaque by facilitation of inflammatory cell infiltration and haemorrhagic complications. Intraplaque haemorrhage results in rapid expansion of the plaque necrotic core, due to the fact that red blood cell membranes are a rich source of free cholesterol and phospholipids and the process occurring in association with excessive macrophageinfiltration. The size of the necrotic core directly correlates with the risk of plaque rupture. Furthermore, intraplaque haemorrhage and plaque rupture were found to be proportional to neovessel density in coronary atheroma. Adventitial vessels in unstable plaques contain perivascular smooth muscle cells; however, after plaque rupture, the fibrous cap is disrupted with a luminal thrombus and the newer branches of vasa vasorum close to the necrotic core consist almost entirely of a single layer of EC overlying a ruptured, leaky basement membrane, and associated with remnants of red blood cells.¹ Defects are thought to be caused by proteolytic damage from on-going inflammation and release of signalling molecules affecting cell–cell contact.

There is promotion of wound healing through enhanced expression of a variety of growth factors in the neointima of the plaque. The sub endothelial world becomes extremely heterogeneous in composition. All the above support the hypothesis that angiogenesis plays a major role in the development of coronary and carotid artery symptomatic disease.^9,10 Therefore, inhibition of angiogenesis might be an important target for prevention of development of active, unstable plaque lesions (Fig. 1 shows a high grade neointimal plaque lesion rich in microvessels). Identification of pathophysiological changes related to angiogenesis may lead in future to the design of novel therapeutic agents. A number of candidate molecules and targets have already been identified. In the first place, important components of the plaque which are thrombogenic include fibrinogen, fibrin, FDP, and thrombin. Beyond the activity of thrombin in generating a fibrin clot and activating platelets, thrombin affects endothelial cell migration and angiogenesis.¹¹ Other pro-angiogenic factors are found in these plaques i.e.: vascular endothelial cellgrowth factor (VEGF), placental growth factor (PLGF), basic fibroblastgrowth factor (FGF-2), transforming growth factor-β (TGF-β), matrix metalloproteinases (MMPs), nitric oxide (NO), platelet-derived growth factor (PDGF), interleukin-8 (IL-8), and platelet activating factor (PAF). As the thickness of the intima/media increases, the diffusion capacity of oxygen and nutrients from the lumen is exceeded. An angiogenic response is stimulated by hypoxia and ischemia. Regulation of angiogenesis by hypoxia and the role of the hypoxia-inducible factor (HIF) system has been shown to be a major inducer of VEGF genetranscription.¹²

Fig. 1 A, High grade VI unstable carotid plaque removed from a patient following endarterectomy, B, low power microscopic view showing rupture sites as holes within the intimal plaque region (arrows), and an area rich in neovessels (boxed). C and D, CD105 positive neovessels (CD105 is a marker of active endothelial cells) in the intimal tissue from a complicated atherosclerotic plaque. Only selected microvessels are CD105 positive in these lesions suggesting a dynamic feature of angiogenesis within the plaque architecture.

An increasing number of angiogenic therapeutic targets have been proposed in order to facilitate modulation of neovascularisation and its consequences in diseases such as cancer and macular degeneration. A complete knowledge of the mechanisms responsible for initiation of adventitial vessel proliferation, their extension into the intimal regions and possible de novo synthesis of neovessels following differentiation of bone-marrow-derived stem cells is required in order to contemplate potential single or combinational anti-angiogenic therapies. Novel technologies employing laser-capture microdissection, RNA/DNA amplification and global genomic analysis can be used to identify optimal targets both extra and intracellularly following isolation of individual primarily activated vessels.¹³

Nanomedicine is the medical use of molecular-sized particles to deliver drugs, heat, light or other substances to specific cells in the human body. Engineering particles to be used in this way allows detection and/or treatment of diseases or injuries within the targeted cells, thereby minimising the damage to healthy cells in the body. Nanotechnology exploits novel properties of materials when they are reduced to the size of a few hundred to thousand atoms. At this scale (anything from a few nanometres up to a hundred nanometres; a nanometre being one billionth of a metre) materials start to exhibit quite different properties than would normally be expected. For example, gold appears red (the Romans used such nanoparticles of gold to stain glass), while titanium dioxide and zinc oxide (both used in sun-blocks) become transparent instead of white, while retaining their ability to block UV light. These are simple examples, however nanomaterials can have quite different physical (e.g., strength, flexibility, thermal), electronic, magnetic, and optical properties compared with bulk materials. Some nanomaterials such as carbon nanotubes possess several different properties including strength (50–100 times stronger than steel), electronic properties (for example in displays), and biomedical uses (as drug delivery systems). As a result of these new properties, nanomaterials have the potential to impact every technology sector.

For medicine, nanotechnology promises new therapies, more rapid and sensitive diagnostic and investigative tools for normal and diseased tissues, and new materials for tissue engineering.

Targeting therapeutic drugs directly at disease sites would increase effectiveness and reduce side-effects. A range of nanoparticles (around 150 nm in diameter) made of the biodegradable polymer poly((±)-lactic-co-glycolic acid) and poly(ethylene glycol) have been developed which can encapsulate drugs, specific proteins or antibodies or which can be attached to artificial RNA strands known as aptamers. The nanoparticles can be taken up by specific cells where the nanoparticles dissolve to release the protein or drug. This technology has already been used to effectively treat and eliminate tumours in animal models.¹⁴ The treatment of disease depends on the identification of a target and delivery of a therapeutic agent which either causes the function to be restored, switches off inappropriate activity, or in the case of cancer, destroys the cell. However, many pharmaceuticals are limited in their development or application because of poor solubility, poor stability, or side-effects in inappropriate tissues. Manipulating the composition of a drug formulation at the nanoscale can resolve some of these issues. For example, various pharmaceutical companies in collaboration with researchers have shown that drugs can be stabilised at room temperature and ambient moisture when formulated as part of a nanostructured lattice with peptides and sugars. Similarly, in relation to use as possible treatments in disease, the anti-cancer drug paclitaxel was formulated with the human serum protein to create a nanostructure that requires no solvent, showed increased efficacy and consequently decreased side-effects (this is now licensed in the EU).

Although at an early stage of development, nanoshells can be used to encapsulate drugs, protecting them from the environment and offering targeted release (essential for toxic anti-cancer therapies). Nanoshells can be made from polymers (which fuse with cell membranes and release their contents within the cell) or a mix of polymer and gold (which can be induced to melt when irradiated with infrared light thus releasing their contents; Fig. 2.). Other nanomaterials can be used to target or directly treat diseased tissues by physical rather than biochemical means. For example, paramagnetic iron nanoparticles can be made to accumulate in tumour cells through the use of magnetic fields¹⁵ and can be used to destroy tumour cells by heat through the application of alternating magnetic fields (known as magnetic fluid hyperthermia [MFH]).^16,17 In the future functionalising such nanoparticles with targeting biomolecules could enable them to be delivered systemically.

Fig. 2 (a) 10–12 nm gold nanoparticles visualised with transmission electron microscopy (TEM). (b) Modelling gold nanoparticles conjugated to thiolated-organic molecules. (c) Nanoparticles can be modified with different family of molecules in order to design advanced nanosystems. These nanosystems may be used as a carrier for selected drugs or biomarkers .

For molecular imaging, PFC nanoparticles can carry very large payloads of gadolinium to detect pathological biomarkers with magnetic resonance imaging (MRI). Beads can be injected intravenously/intraventricularly followed by needle withdrawal, and the signal is created via the interaction between the water signal (proton density) and the magnetic properties, R₁ relaxation rate and R₂ transverse relaxation rate of the imaged tissues (Fig. 3 shows uptake of nanoparticles by fibroblastsin vitro). A detailed description of this process is beyond the scope of this review but can be found in Waters and Wickline.¹⁸ A variety of different epitopes, including α₅β₃-integrin, tissue factor and fibrin, have been imaged using nanoparticles formulated with appropriate antibodies or peptidomimetics as targeting ligands. Lipophilic drugs can also be incorporated into the outer lipid shell of nanoparticles for targeted delivery. Upon binding to the target cell, the drug is exchanged from the particle surfactant monolayer to the cell membrane through a novel process called ‘contact facilitated drug delivery’. By combining targeted molecular imaging and localised drug delivery, perfluorocarbon (PFC) nanoparticles provide diagnosis and therapy with a single agent.¹⁹

Fig. 3 (a) Human fibroblast cellular uptake of advanced nanosystem nanoparticles visualised with transmission electron microscopy (TEM). (b) Nanoparticles can improve peptide delivery by directly improving pharmacokinetics and biodistribution to increase solubility, stability and efficacy.

Nanotechnology applied to visualisation of unstable plaques and angiogenesis in cardiovascular disease

The majority of potential therapeutic agents have been unsuccessful because of their limited capacity to reach and remain stably within the target tissue or cell in vivo. Infusion of nanoparticle suspensions using infusion catheters or implant-based drug delivery represents a novel and potentially much more efficient option for treatment.²⁰ As mentioned previously, neovascular beds in plaque intima as well as activated adventitial blood vessels are potential targets for molecular imaging and targeted drug therapy. To achieve effective molecular targeting and imaging, the particles must be designed to have a long circulating half-life, to be sensitive and selective to the epitope of interest and produce a prominent contrast to noise ratio enhancement. Obviously they should also be non-toxic. Fibrin-targeted nanoparticles can enhance the contrast in thrombi, as demonstrated in the femoral and carotid arteries of a miniswine model of atherosclerosis (injury-high-cholesterol) using echogenic immunoliposome targeted anti-fibrin antibodies.^21,22 PFC emulsions of 200–300 nm have been used in conjunction with positron emission tomography (PET) and radio-labelled antibodies for imaging of atherosclerotic plaque angiogenesis,²³ and fibrin-specific perfluorocarbon particles were able to deliver effectively plasminogen activator streptokinase in human plasma clots suggesting a role for nano-based targeted thrombolysis in patients with symptomatic cardio–cerebro vascular disease.²⁴ α₅β₃-Integrin is a key endothelial cell receptor which is over-expressed by neovasculature undergoing angiogenesis.²⁵ Furthermore, its specificity and ubiquitous expression has made it a prime target ligand for the visualisation of vascular changes using nuclear imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), and for optical imaging, near infrared fluorescence (NIRF) and MRI in vivo.²⁶ Radiolabelled α₅β₃-targeted probes such as ¹⁸F-galacto-RGD have been successfully used in a clinical setting to detect tumour blood vessels and their density.²⁷ Waters et al.²⁸ showed that specific α₅β₃-integrin-targeted PFC nanoparticles could be used both to detect and quantify neovasculature developing in a rabbit model of aortic valve disease (stenosis) based on measurement of the fluorine signature by MRI demonstrating a potential use of this technology in identifying angiogenesis within atherosclerotic plaques in humans.

When large numbers of paramagnetic gadolinium complexes (>50 000) are incorporated onto emulsion particles, the signal enhancement for each binding site is magnified dramatically compared with conventional contrast reagents.²⁹ Most recently, α₅β₃-targeted fumagilin theranostic paramagnetic nanoparticles, already known to target angiogenic vessels, were delivered to hyperlipidaemic rabbits with and without atorvastatin treatment.³⁰ In these experiments, cardiac magnetic imaging was able to demonstrate a significant reduction in the neovascular signal after nanoparticle treatment which was enhanced in the presence of atorvastatin. These results demonstrated a potential novel strategy for evaluation and treatment of plaque angiogenesis.³⁰ Neovessels in coronary plaques of cholesterol-fed apoE mice have been imaged using vascular cell adhesion molecule-1 peptide sequence (VHSPNKK) bound to cross-linked magneto-fluorescent superparamagnetic iron oxide (CLIO) particles. The particles were shown to selectively bind to aortic plaque vasculature 24 h after injection using MRI and ex vivo MR and corresponded with histology and fluorescent analysis of tissue samples performed after euthanasia, suggesting potential diagnostic and therapeutic applications.³¹Inhibition of angiogenesis both in vitro and in vivo via inhibition of the phospho-inositol-3-kinase–Akt pathway and activation of the caspase-3 apoptotic pathway has been demonstrated using silver nanoparticles prepared from incubation of wet Bacillus licheniformis and silver nitrate solution, showing that even non-modified particles could be useful therapeutic agents.^32,33

It is important to clarify at this point that studies employing the use of magnetic nanoparticles for either imaging or treatment of unstable atherosclerosis are still at the experimental stage. Targeting immature vulnerable vasculature is only one method for identifying plaques at risk of thrombosis, and studies using animal models have also demonstrated the potential of nanoparticle-based imaging of macrophages, whose concentration is highest in high-risk regions, for identification of unstable lesions. For example, Lipinski et al.³⁴ showed that gadolinium-containing lipid-based nanoparticles labelled with macrophage-specific antibodies directed against CD36 could be detected in human aortic specimens using cardiac magnetic imaging 24 h after incubation, whilst Sigovan et al.³⁵ used ultra-small superparamagnetic iron oxide (USPIO; P904) for successful quantification of inflammation following co-localisation with macrophages in a rabbit model of induced aortic atherosclerosis, imaged by magnetic resonance angiography. Similarly, tissue factor is a prothrombotic molecule which becomes exposed during plaque rupture and represents a key molecule associated with plaque destabilisation. Ultra-small iron oxide particles, prepared as a liquid perfluorocarbon contrast agent with high gadolinium payload (92 400 per bead), were conjugated to anti-tissue factor antibodies and following injection at picomolar concentrations, MRI at 1.5 T was able to quantify expression in smooth muscle cell monolayer cultures.³⁶

One important consideration of the use of metal or carbon-based nanoparticles for the imaging and/or treatment of human disease is the potential side-effects, in particular, toxicity. Deposition of carbon nanotubes in the lung has been shown to induce inflammation and fibrosis (reviewed in detail by Simeonova and Erdely³⁷), and hence carbon uptake within atherosclerotic vessels may also induce an inflammatory response increasing the risk of plaque rupture. Increased activation of endothelial cells as measured by expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intra cellular adhesion molecule-1 (ICAM-1), occurs following their exposure to aluminananoparticles with a concomitant increase in macrophage adhesion which is a critical early step in the development of arterial plaques as well as more specifically up-regulation of the inflammatory response.³⁸ Imaging of plaques with iron-based nanoparticles such as USPIO as described above and their potential use in future therapies must take into account the potential effects of macrophage storage and release of these particles which could result in the generation of superoxidases and oxidised low-density lipoproteins contributing to plaque instability.^39,40

Summary and concluding remarks

These contrast reagents are an exciting development in imaging of atherosclerosis and represent a potential technology in association with MRI capable of visualising plaque features associated with instability as well as identifying total plaque burden. As mentioned earlier, a number of markers of angiogenesis have been identified which might lend themselves to this new technology and help in identifying unstable haemorrhagic regions of plaques. The α₅β₃-integrin is a membrane-bound adhesion molecule widely expressed, particularly by active EC (not quiescent ones) and smooth muscle cells associated with new blood vessel formation. Winter et al.⁴¹ demonstrated that these integrin-targeted nanoparticles could detect and characterise angiogenesis patterns associated with atherosclerosis in the vasa vasorum of coronary arteries from cholesterol-fed rabbits. The same group also demonstrated that non-invasive MRI imaging was sufficient to visualise and track the particles.⁴² Most recently, Hofmann et al.⁴³ showed that using magnetic nanoparticles enhanced the transduction efficiency of endothelial cell-targeted lentiviral vectors allowing direct targeting by magnetic force even in perfused vessels in a mouse model of perfusion injury, thus intravascular gene targeting could be used in combination with re-positioning of transduced cells using nanoparticle-based technology, thereby combining gene and cell-based therapies.

Future treatments and/or preventative measures may involve synthesis of superparamagnetic nanoparticles multiply/quadruple labelled with antibodies to CD105 or integrins which bind selectively with active microvessels, siRNA directed to key early mitogenic activators and/or intracellular signalling molecules (yet to be defined as part of a blue print for EC activation), green fluorescent protein (GFP) to enable near infrared in vivo optical imaging (NIRF) and confirm probe delivery, and myristoylated polyarginine peptides (MPAP) to allow membrane translocation of the siRNA.⁴⁴ T2 relaxation times can be monitored to examine probe delivery. Whilst this type of combination therapy might be some years away, current studies should aim to form the platform by identifying specific activatory signals of vasa vasorum and intracellularsignal transduction consequences (i.e. a blue print for activation) using currently available in vivo models combined with nanotechnology. Specifically directed siRNA expressed in adenoviral vectors and targeted with nanoparticles might be one method to prime the cells against self or external activation and by prevention of angiogenesis, significantly reduce the rate of formation of arterial plaque.

Nanoparticles could then be used for effective delivery of drugs to target sites with a preferential aim to prevent initial development of neointimal vascular sites by blocking adventitial blood vessel activation in patients with low grade plaques. With this in mind, novel specific markers of plaque angiogenesis such as CD105,⁴⁵ modified C-reactive protein⁴⁶ and VEGF-R2⁴⁷ might represent (1) future imaging targets able to identify patients developing unstable plaque regions susceptible to rupture, and (2) prevent or slow down development of atheroma thereby improving treatment and survival rates of patients with a history of development of myocardial infarction or ischaemic stroke.⁴⁸

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