ABSTRACT
Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.
Subject(s)
Diabetes Mellitus, Type 1 , Metal Nanoparticles , Humans , Autoantigens , Proinsulin/genetics , Gold , Injections, Intradermal , Single-Cell Gene Expression Analysis , Peptides/genetics , Receptors, Antigen, T-Cell/geneticsABSTRACT
Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics.
Subject(s)
Antigens/metabolism , Gold/chemistry , Metal Nanoparticles/chemistry , Peptides/pharmacology , Amino Acid Sequence , Animals , Cell Proliferation , Dendritic Cells/drug effects , Injections, Intradermal , Mice, Inbred C57BL , Mice, Transgenic , Needles , Peptides/chemistry , Peptides/pharmacokinetics , Phenotype , Skin/drug effects , Solubility , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Molecular imaging offers great potential for noninvasive visualization and quantitation of the cellular and molecular components involved in atherosclerotic plaque stability. In this chapter, we review emerging molecular imaging modalities and approaches for quantitative, noninvasive detection of early biological processes in atherogenesis, including vascular endothelial permeability, endothelial adhesion molecule up-regulation, and macrophage accumulation, with special emphasis on mouse models. We also highlight a number of targeted imaging nanomaterials for assessment of advanced atherosclerotic plaques, including extracellular matrix degradation, proteolytic enzyme activity, and activated platelets using mouse models of atherosclerosis. The potential for clinical translation of molecular imaging nanomaterials for assessment of atherosclerotic plaque biology, together with multimodal approaches is also discussed.
Subject(s)
Arteries/metabolism , Atherosclerosis/metabolism , Molecular Imaging/methods , Plaque, Atherosclerotic , Animals , Arteries/pathology , Atherosclerosis/pathology , Biomarkers/metabolism , Blood Platelets/metabolism , Blood Platelets/pathology , Contrast Media , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Macrophages/metabolism , Macrophages/pathology , Magnetic Resonance Imaging , MiceABSTRACT
BACKGROUND: Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. METHODS AND RESULTS: A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. CONCLUSIONS: These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.
Subject(s)
Aortic Aneurysm/epidemiology , Aortic Dissection/epidemiology , Disease Susceptibility/epidemiology , Endothelium, Vascular/metabolism , Muscle, Smooth, Vascular/metabolism , Reactive Oxygen Species/metabolism , Aortic Dissection/etiology , Aortic Dissection/metabolism , Angiotensin II/adverse effects , Animals , Aortic Aneurysm/etiology , Aortic Aneurysm/metabolism , Cyclophilins/genetics , Cyclophilins/metabolism , Disease Models, Animal , Disease Susceptibility/etiology , Disease Susceptibility/metabolism , Male , Matrix Metalloproteinases/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Signal Transduction , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Molecular imaging is emerging as a key experimental tool for the identification of inflammatory cellular and molecular processes involved in the development of cardiovascular disease. This review summarises current molecular imaging approaches for the detection of vascular inflammation using a range of nano- and micro-sized contrast agents. We highlight strategies for detection of cell adhesion molecules, which are key regulators of endothelial activation and leukocyte recruitment in atherogenesis and ischaemia-reperfusion in jury. In particular, we address the properties of targeted microparticles of iron oxide (MPIO) for MRI detection of endothelial cell-specific activation of adhesion molecules in experimental models of atherosclerosis, acute vascular inflammation and ischaemia-reperfusion injury, which are otherwise undetectable by conventional imaging modalities. The ability of targeted MPIO to detect endothelial activation could enable early subclinical disease detection and development of novel therapeutic strategies. We discuss opportunities for further development and potential translation of targeted MPIO for clinical imaging of cardiovascular disease.
Subject(s)
Cardiovascular Diseases/diagnosis , Contrast Media , Molecular Imaging/methods , Animals , Cardiovascular Diseases/pathology , Cell Adhesion Molecules/metabolism , Contrast Media/administration & dosage , Endothelium, Vascular/pathology , Ferric Compounds , Humans , Inflammation/diagnosis , Inflammation/pathology , Magnetic Resonance Imaging/methods , Microspheres , Nanoparticles , Particle SizeABSTRACT
OBJECTIVE: Endothelial cell activation is an important mediator of monocyte recruitment to sites of vascular inflammation. We hypothesized that high-affinity dual-ligand microparticles of iron oxide (MPIO), targeted to P-selectin and vascular cell adhesion molecule-1 (PV-MPIO), would identify activated endothelial cells during atherosclerosis progression. METHODS AND RESULTS: In vivo magnetic resonance imaging in apolipoprotein E-deficient mice showed rapid binding of PV-MPIO to the aortic root, which was maximal 30 minutes post-MPIO injection and maintained at 60 minutes. Minimal binding was observed for control IgG-MPIO. Intensely low magnetic resonance signal areas, corresponding to PV-MPIO binding, were detected early (14 weeks), during foam cell formation. Contrast effects increased at 20 weeks during fibrofatty lesion development (P<0.05), but reduced by 30 weeks (P<0.01). Across all lesion severities, magnetic resonance imaging contrast effects correlated with lesion macrophage area quantified by immunohistochemistry (R=0.53; P<0.01). Near-infrared fluorescently labeled PV-MPIO were shown, by flow cytometry, to bind only activated endothelial cells and not to macrophages. Using en face immunofluorescence, we further demonstrate selective PV-MPIO accumulation at atherosclerosis-susceptible sites, with minimal binding to atherosclerosis-spared regions. CONCLUSIONS: This high-affinity leukocyte-mimetic magnetic resonance imaging agent reveals endothelial activation. PV-MPIO demonstrate exceptionally rapid in vivo steady state accumulation, providing conspicuous magnetic resonance contrast effects that can be objectively quantified. In atherosclerosis progression, PV-MPIO tracked closely with the burden and distribution of plaque macrophages, not merely plaque size. On a biocompatible platform, this approach has potential for quantitative magnetic resonance imaging of inflammatory disease activity.
Subject(s)
Aorta/pathology , Aortic Diseases/diagnosis , Atherosclerosis/diagnosis , Biomimetic Materials , Contrast Media , Endothelium, Vascular/pathology , Leukocytes/pathology , Macrophages/pathology , Magnetic Resonance Imaging , Animals , Antibodies , Antibody Affinity , Aorta/immunology , Aorta/metabolism , Aortic Diseases/genetics , Aortic Diseases/immunology , Aortic Diseases/metabolism , Aortic Diseases/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/immunology , Atherosclerosis/metabolism , Atherosclerosis/pathology , Binding Sites, Antibody , Contrast Media/pharmacokinetics , Disease Models, Animal , Disease Progression , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Female , Ferric Compounds , Flow Cytometry , Humans , Immunohistochemistry , Leukocytes/immunology , Leukocytes/metabolism , Ligands , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , P-Selectin/immunology , P-Selectin/metabolism , Particle Size , Spectroscopy, Near-Infrared , Time Factors , Vascular Cell Adhesion Molecule-1/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Molecular imaging of disease states can enhance diagnosis allowing for accurate and more effective treatment. By specifically targeting molecules differentially expressed in disease states, researchers and clinicians have a means of disease characterization at a cellular or tissue level. Targeted micron-sized particles of iron oxide (MPIO) have been used as molecule-specific contrast agents for use with magnetic resonance imaging (MRI), and early evidence suggests they may be suitable for use with other imaging modalities. Targeting of MPIO to markers of disease is commonly achieved through the covalent attachment of antibodies to the surface of the particles, providing an imaging agent that is both highly specific and which binds with high affinity. When comparing micron-sized particles with nanometre-sized particles, the former provide substantial signal dropout in MRI and confer the sensitivity to detect low levels of target. Furthermore, larger particles appear to bind to targets more potently than smaller particles. Animal models have also demonstrated favorable blood clearance characteristics of MPIO, which are important in achieving favorable signal over background and to attain clearance and disposal. Although the current generation of commercially available MPIO are not suitable for administration into humans, future work may focus on the development of biodegradable and nonimmunogenic MPIO that may allow the use of these imaging agents in a clinical setting.
Subject(s)
Endothelial Cells/chemistry , Magnetic Resonance Imaging/methods , Microspheres , Molecular Imaging/methods , Animals , Biomarkers/analysis , Endothelial Cells/cytology , Endothelial Cells/metabolism , Ferrosoferric Oxide/chemistry , Humans , Particle SizeABSTRACT
Metastasis to the brain is a leading cause of cancer mortality. The current diagnostic method of gadolinium-enhanced MRI is sensitive only to larger tumors, when therapeutic options are limited. Earlier detection of brain metastases is critical for improved treatment. We have developed a targeted MRI contrast agent based on microparticles of iron oxide that enables imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Our objectives here were to determine whether VCAM-1 is up-regulated on vessels associated with brain metastases, and if so, whether VCAM-1-targeted MRI enables early detection of these tumors. Early up-regulation of cerebrovascular VCAM-1 expression was evident on tumor-associated vessels in two separate murine models of brain metastasis. Metastases were detectable in vivo using VCAM-1-targeted MRI 5 d after induction (<1,000 cells). At clinical imaging resolutions, this finding is likely to translate to detection at tumor volumes two to three orders of magnitude smaller (0.3-3 × 10(5) cells) than those volumes detectable clinically (10(7)-10(8) cells). VCAM-1 expression detected by MRI increased significantly (P < 0.0001) with tumor progression, and tumors showed no gadolinium enhancement. Importantly, expression of VCAM-1 was shown in human brain tissue containing both established metastases and micrometastases. Translation of this approach to the clinic could increase therapeutic options and change clinical management in a substantial number of cancer patients.
Subject(s)
Brain Neoplasms/secondary , Magnetic Resonance Imaging/methods , Animals , Brain Neoplasms/diagnosis , Brain Neoplasms/metabolism , Disease Progression , Humans , Immunohistochemistry , Mice , Sensitivity and Specificity , Up-Regulation , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Diagnosis of multiple sclerosis (MS) currently requires lesion identification by gadolinium (Gd)-enhanced or T(2)-weighted magnetic resonance imaging (MRI). However, these methods only identify late-stage pathology associated with blood-brain barrier breakdown. There is a growing belief that more widespread, but currently undetectable, pathology is present in the MS brain. We have previously demonstrated that an anti-VCAM-1 antibody conjugated to microparticles of iron oxide (VCAM-MPIO) enables in vivo detection of VCAM-1 by MRI. Here, in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we have shown that presymptomatic lesions can be quantified using VCAM-MPIO when they are undetectable by Gd-enhancing MRI. Moreover, in symptomatic animals VCAM-MPIO binding was present in all regions showing Gd-DTPA enhancement and also in areas of no Gd-DTPA enhancement, which were confirmed histologically to be regions of leukocyte infiltration. VCAM-MPIO binding correlated significantly with increasing disability. Negligible MPIO-induced contrast was found in either EAE animals injected with an equivalent nontargeted contrast agent (IgG-MPIO) or in control animals injected with the VCAM-MPIO. These findings describe a highly sensitive molecular imaging tool that may enable detection of currently invisible pathology in MS, thus accelerating diagnosis, guiding treatment, and enabling quantitative disease assessment.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/diagnosis , Encephalomyelitis, Autoimmune, Experimental/metabolism , Magnetic Resonance Imaging/methods , Multiple Sclerosis/diagnosis , Multiple Sclerosis/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Brain/metabolism , Brain/pathology , Contrast Media , Disease Models, Animal , Disease Progression , Endothelium/metabolism , Endothelium/pathology , Female , Ferric Compounds , Gadolinium DTPA , Humans , Immunohistochemistry , Mice , Translational Research, BiomedicalABSTRACT
OBJECTIVES: Optical coherence tomography (OCT) is a high resolution imaging technique used to assess superficial atherosclerotic plaque morphology. Utility of OCT may be enhanced by contrast agents targeting molecular mediators of inflammation. METHODS AND RESULTS: Microparticles of iron oxide (MPIO; 1 and 4.5 µm diameter) in suspension were visualized and accurately quantified using a clinical optical coherence tomography system. Bound to PECAM-1 on a plane of cultured endothelial cells under static conditions, 1 µm MPIO were also readily detected by OCT. To design a molecular contrast probe that would bind activated endothelium under conditions of shear stress, we quantified the expression (basal vs. TNF-activated; molecules µm(-2)) of VCAM-1 (not detected vs. 16 ± 1); PECAM-1 (132 ± 6 vs. 198 ± 10) and E-selectin (not detected vs. 46 ± 0.6) using quantitative flow cytometry. We then compared the retention of antibody-conjugated MPIO targeting each of these molecules plus a combined VCAM-1 and E-selectin (E+V) probe across a range of physiologically relevant shear stresses. E+V MPIO were consistently retained with highest efficiency (P < 0.001) and at a density that provided conspicuous contrast effects on OCT pullback. CONCLUSION: Microparticles of iron oxide were detectable using a clinical OCT system. Assessment of binding under flow conditions recommended an approach that targeted both E-selectin and VCAM-1. Bound to HUVEC under conditions of flow, targeted 1 µm E+V MPIO were readily identified on OCT pullback. Molecular imaging with OCT may be feasible in vivo using antibody targeted MPIO.
Subject(s)
Coronary Vessels/metabolism , Ferric Compounds/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Molecular Imaging/methods , Molecular Probes , Tomography, Optical Coherence , Animals , Antibodies, Monoclonal/metabolism , Arterioles/immunology , Arterioles/metabolism , Biomarkers/metabolism , Cells, Cultured , Coronary Vessels/immunology , E-Selectin/immunology , E-Selectin/metabolism , Flow Cytometry , Human Umbilical Vein Endothelial Cells/immunology , Humans , Immunohistochemistry , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Ligands , Male , Microscopy, Fluorescence , Microscopy, Video , Particle Size , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein Binding , Rats , Rats, Wistar , Research Design , Stress, Mechanical , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Inflammation is a key component of many neurological diseases, yet our understanding of the contribution of these processes to tissue damage remains poor. For many such diseases, magnetic resonance imaging (MRI) has become the method of choice for clinical diagnosis. However, many of the MRI parameters that enable disease detection, such as passive contrast enhancement across a compromised blood-brain barrier, are weighted towards late-stage disease. Moreover, whilst these methods may report on disease severity, they are not able to provide information on either disease activity or the underlying molecular processes. There is a need, therefore, to develop methods that enable earlier disease detection, potentially long before clinical symptoms become apparent, together with identification of specific molecular processes that may guide specific therapy. This chapter describes the methodology for the synthesis and validation of two novel, functional MRI-detectable probes, based on microparticles of iron oxide (MPIO), which target endothelial adhesion molecules. These contrast agents enable the detection of acute brain inflammation in vivo, at a time when pathology is undetectable by conventional MRI. Such molecular MRI methods are opening new vistas for the acute diagnosis of CNS disease, together with the possibility for individually tailored therapy and earlier, more sensitive assessment of the efficacy of novel therapies.
Subject(s)
Central Nervous System/pathology , Inflammation/diagnosis , Magnetic Resonance Imaging/methods , Animals , Antibodies/metabolism , Central Nervous System/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Ferric Compounds/metabolism , Lewis X Antigen/metabolism , Mice , Rats , Sialyl Lewis X Antigen , Statistics as Topic , Tumor Necrosis Factor-alpha/pharmacology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
For molecular magnetic resonance imaging (mMRI), microparticles of iron oxide (MPIO) create potent hypointense contrast effects that extend a distance far exceeding their physical size. The potency of the contrast effects derive from their high iron content and are significantly greater than that of ultra-small particles of iron oxide (USPIO), commonly used for MRI. Due to their size and incompressible nature, MPIO are less susceptible to nonspecific vascular egress or uptake by endothelial cells. Therefore, MPIO may be useful contrast agents for detection of endovascular molecular targets by MRI. This Chapter describes the methodology of a novel, functional MPIO probe targeting vascular cell adhesion molecule-1 (VCAM-1), for detection of acute brain inflammation in vivo, at a time when pathology is undetectable by conventional MRI. Protocols are included for conjugation of MPIO to mouse monoclonal antibodies against VCAM-1 (VCAM-MPIO), the validation of VCAM-MPIO binding specificity to activated endothelial cells in vitro, and the application of VCAM-MPIO for in vivo targeted MRI of acute brain inflammation in mice. This functional molecular imaging tool may potentially accelerate accurate diagnosis of early cerebral vascular inflammation by MRI, and guide specific therapy.
Subject(s)
Encephalitis/diagnosis , Ferric Compounds , Magnetic Resonance Imaging/methods , Animals , Mice , Particle Size , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Contrast agents are widely used with magnetic resonance imaging (MRI) to increase the contrast between regions of interest and the background signal, thus providing better quality information. Such agents can work in one of two ways, either to specifically enhance the signal that is produced or to localize in a specific cell type of tissue. Commonly used image contrast agents are typically based on gadolinium complexes or super-paramagnetic iron oxide, the latter of which is used for imaging lymph nodes. When blood-brain barrier (BBB) breakdown is a feature of central nervous system (CNS) pathology, intravenously administered contrast agent enters into the CNS and alters contrast on MR scans. However, BBB breakdown reflects downstream or end-stage pathology. The initial recruitment of leukocytes to sites of disease such as multiple sclerosis (MS), ischemic lesions, or tumours takes place across an intact, but activated, brain endothelium. Molecular imaging affords the ability to obtain a "non-invasive biopsy" to reveal the presence of brain pathology in the absence of significant structural changes. We have developed smart contrast agents that target and reversibly adhere to sites of disease and have been used to reveal activated brain endothelium when images obtained by conventional MRI look normal. Indeed, our selectively targeted micro-particles of iron oxide have revealed the early presence of cerebral malaria pathology and ongoing MS-like plaques in clinically relevant models of disease.
Subject(s)
Brain/pathology , Contrast Media , Ferric Compounds , Magnetic Resonance Imaging , Animals , Brain/blood supply , Brain/metabolism , Cell Line , Contrast Media/administration & dosage , Contrast Media/metabolism , Ferric Compounds/administration & dosage , Ferric Compounds/metabolism , Male , Mice , Particle Size , Rats , Rats, WistarABSTRACT
RATIONALE AND OBJECTIVE: Vascular cell adhesion molecule-1 (VCAM-1) is upregulated in ischemia reperfusion injury (IRI), persisting after restoration of blood flow. We hypothesized that microparticles of iron oxide targeting VCAM-1 (VCAM-MPIO) would depict "ischemic memory" and enable in vivo assessment of VCAM-1 expression. METHODOLOGY AND FINDINGS: Mice subject to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO (4.5 mg iron/kg body weight). Contrast agent bound rapidly (<30 minutes) in IRI-kidneys and appeared as intensely low signal areas by MRI in vivo. Automated segmentation and quantification yielded MPIO contrast volumes of 5991±354×10(6) µm(3) in IRI vs. 87±7×10(6) µm(3) in kidneys with no surgical intervention (P<0.001); 90±8×10(6) µm(3) in IRI kidneys exposed to control (IgG-MPIO) and 625±80×10(6) µm(3), in IRI kidneys pre-treated with a blocking dose of VCAM-1 antibody (P<0.001). In keeping with quantitative MRI data, VCAM-1 mRNA expression in IRI was 65-fold higher than in kidneys without surgical intervention (3.06±0.63 vs. 0.05±0.02, P<0.001). Indeed VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R(2)=0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. Serial imaging showed VCAM-MPIO accumulation at target within 30 minutes, persisting for ≥90 minutes, while unbound VCAM-MPIO was cleared rapidly from blood, with sequestration by mac-3 positive Kupffer cells in the liver and monocyte/macrophages in the spleen. CONCLUSIONS: (1) VCAM-MPIO detected VCAM-1 expression and defined its 3-dimensional distribution, revealing "ischemic memory" in renal IRI; (2) automated volumetric quantification of VCAM-MPIO accurately reflected tissue levels of VCAM-1 mRNA; and (3) VCAM-MPIO bound rapidly to target with active sequestration of unbound MPIO in the liver and spleen.
Subject(s)
Gene Expression , Kidney/diagnostic imaging , Magnetic Resonance Imaging/methods , Reperfusion Injury/metabolism , Vascular Cell Adhesion Molecule-1/analysis , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Contrast Media/analysis , Contrast Media/metabolism , Disease Models, Animal , Ferric Compounds/analysis , Ferric Compounds/metabolism , Humans , Kidney/blood supply , Kidney/metabolism , Male , Mice , Mice, Inbred C57BL , Radiography , Reperfusion Injury/diagnostic imaging , Reperfusion Injury/genetics , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
The pathogenesis of stroke is multifactorial, and inflammation is thought to have a critical function in lesion progression at early time points. Detection of inflammatory processes associated with cerebral ischemia would be greatly beneficial in both designing individual therapeutic strategies and monitoring outcome. We have recently developed a new approach to imaging components of the inflammatory response, namely endovascular adhesion molecule expression on the brain endothelium. In this study, we show specific imaging of vascular cell adhesion molecule (VCAM)-1 expression in a mouse model of middle cerebral artery occlusion (MCAO), and a reduction in this inflammatory response, associated with improved behavioral outcome, as a result of preconditioning. The spatial extent of VCAM-1 expression is considerably greater than the detectable lesion using diffusion-weighted imaging (25% versus 3% total brain volume), which is generally taken to reflect the core of the lesion at early time points. Thus, VCAM-1 imaging seems to reveal both core and penumbral regions, and our data implicate VCAM-1 upregulation and associated inflammatory processes in the progression of penumbral tissue to infarction. Our findings indicate that such molecular magnetic resonance imaging (MRI) approaches could be important clinical tools for patient evaluation, acute monitoring of therapy, and design of specific treatment strategies.
Subject(s)
Endothelium, Vascular/metabolism , Gene Expression Regulation , Infarction, Middle Cerebral Artery/metabolism , Magnetic Resonance Imaging , Vascular Cell Adhesion Molecule-1/biosynthesis , Animals , Disease Models, Animal , Endothelium, Vascular/diagnostic imaging , Humans , Infarction, Middle Cerebral Artery/diagnostic imaging , Inflammation/diagnostic imaging , Inflammation/metabolism , Ischemic Preconditioning/methods , Mice , Monitoring, Physiologic/methods , RadiographyABSTRACT
The rapidly evolving field of molecular imaging promises important advances in the diagnosis, characterization and pharmacological treatment of vascular disease. Magnetic resonance imaging (MRI) provides a modality that is well suited to vascular imaging as it can provide anatomical, structural and functional data on the arterial wall. Its capabilities are further enhanced by the use of a range of increasingly sophisticated contrast agents that target specific molecules, cells and biological processes. This article will discuss one such approach, using microparticles of iron oxide (MPIO). MPIO have been shown to create highly conspicuous contrast effects on T(2)(*)-weighted MR images. We have developed a range of novel ligand-conjugated MPIO for molecular MRI of endothelial adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and P-selectin expressed in vascular inflammation, as well as activated platelet thrombosis. This review discusses the application of ligand-targeted MPIO for in vivo molecular MRI in a diverse range of vascular disease models including acute vascular inflammation, atherosclerosis, thrombosis, ischemia-reperfusion injury and ischemic stroke. The exceptionally conspicuous contrast effects of ligand-conjugated MPIO provide a versatile and sensitive tool for quantitative vascular molecular imaging that could refine diagnosis and measure response to treatment. The potential for clinical translation of this new class of molecular contrast agent for clinical imaging of vascular syndromes is discussed.
Subject(s)
Atherosclerosis/diagnosis , Ferric Compounds , Nanoparticles , Thrombosis/diagnosis , Vasculitis/diagnosis , Diagnostic Imaging/methods , Humans , Particle SizeABSTRACT
Rapid advances in the field of nanotechnology promise revolutionary improvements in the diagnosis and therapy of neuroinflammatory disorders. An array of iron oxide nano- and microparticle agents have been developed for in vivo molecular magnetic resonance imaging (mMRI) of cerebrovascular endothelial targets, such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and the glycoprotein receptor GP IIb/IIIa expressed on activated platelets. Molecular markers of glioma cells, such as matrix metalloproteinase-2 (MMP-2), and markers for brain tumor angiogenesis, such as alpha (v) beta (3) integrin (alpha(v)beta(3)), have also been successfully targeted using nanoparticle imaging probes. This chapter provides an overview of targeted, iron oxide nano- and microparticles that have been applied for in vivo mMRI of the brain in experimental models of multiple sclerosis (MS), brain ischemia, cerebral malaria (CM), brain cancer, and Alzheimer's disease. The potential of targeted nanoparticle agents for application in clinical imaging is also discussed, including multimodal and therapeutic approaches.
Subject(s)
Diagnostic Imaging/methods , Drug Delivery Systems/methods , Magnetic Resonance Imaging/methods , Metal Nanoparticles/therapeutic use , Nanotechnology/methods , Neurobiology/methods , Animals , Biomarkers/analysis , Biomarkers/metabolism , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain Diseases/drug therapy , Brain Diseases/therapy , Diagnostic Imaging/trends , Drug Delivery Systems/trends , Humans , Magnetic Resonance Imaging/trends , Nanotechnology/trends , Neurobiology/trends , Proteins/drug effects , Proteins/metabolismABSTRACT
Ruptured atherosclerotic plaques, lined with activated platelets, constitute an attractive target for magnetic resonance imaging (MRI). This study evaluated whether microparticles of iron oxide (MPIO) targeting ligand-induced binding sites (LIBS) on the activated conformation of glycoprotein IIb/IIIa could be used to image platelets. MPIO (size: 1 microm) were conjugated to anti-LIBS or control single-chain antibody. Following guidewire injury to mouse femoral artery, platelet adhesion was present after 24 h. Mice were perfused with anti-LIBS-MPIO (or control MPIO) via the left ventricle and 11.7-tesla MRI was performed on femoral arteries ex vivo. A 3D gradient echo sequence attained an isotropic resolution of 25 microm. MPIO binding, quantified by MRI, was 4-fold higher with anti-LIBS-MPIO in comparison to control MPIO (p < 0.01). In histological sections, low signal zones on MRI and MPIO correlated strongly (R(2) = 0.72; p < 0.001), indicating accurate MR quantification. In conclusion, anti-LIBS-MPIO bind to activated platelets in mouse arteries, providing a basis for the use of function-specific single-chain antibody-MPIO conjugates for molecular MRI, and represent the first molecular imaging of a conformational change in a surface receptor. This presents an opportunity to specifically image activated platelets involved in acute atherothrombosis with MRI.
Subject(s)
Ferric Compounds , Magnetic Resonance Imaging/methods , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Animals , Antibodies , Binding Sites , Binding Sites, Antibody , Blood Platelets , Femoral Artery/injuries , Femoral Artery/ultrastructure , Humans , Male , Mice , Mice, Inbred C57BL , Platelet Activation , Platelet Adhesiveness , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , Protein Conformation , Receptors, Immunologic/metabolismABSTRACT
OBJECTIVE: Microparticles of iron oxide (MPIO) distort magnetic field creating marked contrast effects far exceeding their physical size. We hypothesized that antibody-conjugated MPIO would enable magnetic resonance imaging (MRI) of endothelial cell adhesion molecules in mouse atherosclerosis. METHODS AND RESULTS: MPIO (4.5 microm) were conjugated to monoclonal antibodies against vascular cell adhesion molecule-1 (VCAM-MPIO) or P-selectin (P-selectin-MPIO). In vitro, VCAM-MPIO bound, in dose-dependent manner, to tumor necrosis factor (TNF)-alpha stimulated sEND-1 endothelial cells, as quantified by light microscopy (R2=0.94, P=0.03) and by MRI (R2=0.98, P=0.01). VCAM-MPIO binding was blocked by preincubation with soluble VCAM-1. To mimic leukocyte binding, MPIO targeting both VCAM-1 and P-selectin were administered in apolipoprotein E-/- mice. By light microscopy, dual-targeted MPIO binding to endothelium overlying aortic root atherosclerosis was 5- to 7-fold more than P-selectin-MPIO (P<0.05) or VCAM-MPIO (P<0.01) alone. Dual-targeted MPIO, injected intravenously in vivo bound aortic root endothelium and were quantifiable by MRI ex vivo (3.5-fold increase versus control; P<0.01). MPIO were well-tolerated in vivo, with sequestration in the spleen after 24 hours. CONCLUSIONS: Dual-ligand MPIO bound to endothelium over atherosclerosis in vivo, under flow conditions. MPIO may provide a functional MRI probe for detecting endothelial-specific markers in a range of vascular pathologies.
Subject(s)
Arteriosclerosis/diagnosis , Ferric Compounds , Magnetic Resonance Imaging/methods , P-Selectin/physiology , Vascular Cell Adhesion Molecule-1/physiology , Animals , Apolipoproteins E/genetics , Arteriosclerosis/physiopathology , Cell Adhesion/physiology , Endothelium, Vascular/physiology , Mice , Mice, Knockout , Particle Size , Sensitivity and SpecificityABSTRACT
Multiple sclerosis is a disease of the central nervous system that is associated with leukocyte recruitment and subsequent inflammation, demyelination and axonal loss. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and its ligand, alpha4beta1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the alpha4 subunit of alpha4beta1 substantially reduce clinical relapse in multiple sclerosis. Urgently needed is a molecular imaging technique to accelerate diagnosis, to quantify disease activity and to guide specific therapy. Here we report in vivo detection of VCAM-1 in acute brain inflammation, by magnetic resonance imaging in a mouse model, at a time when pathology is otherwise undetectable. Antibody-conjugated microparticles carrying a large amount of iron oxide provide potent, quantifiable contrast effects that delineate the architecture of activated cerebral blood vessels. Their rapid clearance from blood results in minimal background contrast. This technology is adaptable to monitor the expression of endovascular molecules in vivo in various pathologies.