A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI.
ABSTRACT: Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-alpha, but not IL-1beta or lymphotoxin-alpha (LT-alpha), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-alpha injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies.
Project description: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.
Project description:IL-17 is argued to play an important role in the multiple sclerosis-like disease experimental autoimmune encephalitis (EAE). We investigated the therapeutic effects of anti-IL-17A in a chronic relapsing EAE ABH mouse model using conventional scoring, quantitative behavioral outcomes, and a novel vascular cell adhesion molecule 1 (VCAM-1)-targeted magnetic resonance imaging (MRI) contrast agent [anti-VCAM-microparticles of iron oxide (MPIO)] to identify conventionally undetectable neuropathology. Mice were administered prophylactic or treatment regimens of anti-IL-17A or IgG and two injections of anti-VCAM-MPIO before undergoing T2*-weighted three-dimensional and gadolinium-diethylenetriamine pentaacetic acid T1-weighted MRI. Rotarod, inverted screen, and open field motor function tests were performed, conventional clinical scores calculated, and central IL-17A mRNA expression quantified during acute disease, remission, and relapse. Prophylactic anti-IL-17A prevents acute disease and relapse and is associated with reduced clinical and functional severity. Treatment regimens delay relapse, improve functional scores, and are associated with reduced VCAM-MPIO lesions during remission. No significant alteration was detectable in levels of gadolinium-diethylenetriamine pentaacetic acid- or VCAM-MPIO-positive lesions during relapse. Prophylactic and treatment anti-IL-17A were therapeutically effective in chronic relapsing EAE, improving clinical and quantifiable functional outcomes. IL-17A expression seems significant during acute disease but less important chronically. Disease-related immunoneuropathology is more sensitively detected using VCAM-MPIO MRI, which may, therefore, be used to monitor therapy meaningfully.
Project description:<h4>Rationale and objective</h4>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.<h4>Methodology and findings</h4>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.<h4>Conclusions</h4>(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.
Project description:Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule ?(v)?(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for ?(v)?(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 ?m MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with ?(v)?(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to ?(v)?(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.
Project description: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.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.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.
Project description:<h4>Background</h4>Molecular MRI is an evolving field of research with strong translational potential. Selection of the appropriate MRI sequence, field strength and contrast agent depend largely on the application. The primary aims of the current study were to: 1) assess the sensitivity of different MRI sequences for detection of iron oxide particles in mouse brain; 2) determine the effect of magnetic field strength on detection of iron oxide particles in vivo; and 3) compare the sensitivity of targeted microparticles of iron oxide (MPIO) or ultra-small superparamagnetic iron oxide (USPIO) for detection of vascular cell adhesion molecule-1 (VCAM-1) in vivo.<h4>Methods</h4>Mice were injected intrastriatally with interleukin 1? to induce VCAM-1 expression on the cerebral vasculature. Subsequently, animals were injected intravenously with either VCAM-MPIO or VCAM-USPIO and imaged 1 or 13 hours post-injection, respectively. MRI was performed at 4.7, 7.0, or 9.4 T, using three different <i>T</i><sub>2</sub>*-weighted sequences: single gradient echo 3D (GE3D), multi-gradient echo 3D (MGE3D) and balanced steady-state free precession 3D (bSSFP3D).<h4>Results</h4>MGE3D yielded the highest signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for the detection of iron oxide particles. All sequences showed a significant increase in SNR and CNR from 4.7 to 7.0 T, but no further improvement at 9.4 T. However, whilst targeted MPIO enabled sensitive detection of VCAM-1 expression on the cerebral vasculature, the long half-life (16.5 h vs 1.2 min) and lower relaxivity per particle (1.29×10<sup>-14</sup> vs 1.18×10<sup>-9</sup> Hz L/particle) of USPIO vs. MPIO rendered them impractical for molecular MRI.<h4>Conclusion</h4>These findings demonstrate clear advantages of MPIO compared to USPIO for molecularly-targeted MRI, and indicate that the MGE3D sequence is optimal for MPIO detection. Moreover, higher field strengths (7.0/9.4 T) showed enhanced sensitivity over lower field strengths (4.7 T). With the development of biodegradable MPIO, these agents hold promise for clinical translation.
Project description:Atherosclerotic plaque rupture leads to acute thrombus formation and may trigger serious clinical events such as myocardial infarction or stroke. Therefore, it would be valuable to identify atherothrombosis and vulnerable plaques before the onset of such clinical events. We sought to determine whether the noninvasive in vivo visualization of activated platelets was effective when using a target-specific MRI contrast agent to identify thrombi, hallmarks of vulnerable or high-risk atherosclerotic plaques.Inflammatory thrombi were induced in mice via topical application of arachidonic acid on the carotid. Thrombus formation was imaged with intravital fluorescence microscopy and molecular MRI. To accomplish the latter, a paramagnetic contrast agent (P975) that targets the glycoprotein alpha(IIb)beta(3), expressed on activated platelets, was investigated. The specificity of P975 for activated platelets was studied in vitro. In vivo, high spatial-resolution MRI was performed at baseline and longitudinally over 2 hours after injecting P975 or a nonspecific agent. The contralateral carotid, a sham surgery group, and a competitive inhibition experiment served as controls. P975 showed a good affinity for activated platelets, with an IC(50) (concentration of dose that produces 50% inhibition) value of 2.6 micromol/L. In thrombosed animals, P975 produced an immediate and sustained increase in MRI signal, whereas none of the control groups revealed any significant increase in MRI signal 2 hours after injection. More important, the competitive inhibition experiment with an alpha(IIb)beta(3) antagonist suppressed the MRI signal enhancement, which is indicative for the specificity of P975 for the activated platelets.P975 allowed in vivo target-specific noninvasive MRI of activated platelets.
Project description: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.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.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.
Project description:Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.
Project description: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 ?m 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 ?m 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 ?m(-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 ?m E+V MPIO were readily identified on OCT pullback. Molecular imaging with OCT may be feasible in vivo using antibody targeted MPIO.