Noninvasive in vivo imaging of monocyte trafficking to atherosclerotic lesions.

BACKGROUND: Monocytes play a key role in atherogenesis, but their participation has been discerned largely via ex vivo analyses of atherosclerotic lesions. We sought to establish a noninvasive technique to determine monocyte trafficking to atherosclerotic lesions in live animals. METHODS AND RESULTS: Using a micro-single-photon emission computed tomography small-animal imaging system and a Food and Drug Administration-approved radiotracer ([indium 111] oxyquinoline, (111)In-oxine), we demonstrate here that monocyte recruitment to atherosclerotic lesions can be visualized in a noninvasive, dynamic, and 3-dimensional fashion in live animals. We show in vivo that monocytes are recruited avidly to plaques within days of adoptive transfer. Using micro-single-photon emission computed tomography imaging as a screening tool, we were able to investigate modulatory effects on monocyte recruitment in live animals. We found that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors rapidly and substantially reduce monocyte recruitment to existing atherosclerotic lesions, as imaged here in vivo. CONCLUSIONS: This novel approach to track monocytes to atherosclerotic plaques in vivo should have broad applications and create new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.

SPARC is a VCAM-1 counter-ligand that mediates leukocyte transmigration.

VCAM-1 is a cell surface molecule, which has been shown to mediate leukocyte adhesion to the endothelium and subsequent transmigration. Although VCAM-1 regulates adhesion through its interaction with VLA-4, VLA-4 does not play a role in VCAM-1-dependent diapedesis, an observation suggesting the presence of a second ligand for VCAM-1. We now report a novel interaction between VCAM-1 and secreted protein acidic and rich in cysteine (SPARC), which induces actin cytoskeletal rearrangement and intercellular gaps, physiological processes known to be important for leukocyte transmigration. The binding of leukocyte-derived SPARC to VCAM-1 was demonstrated to be necessary for leukocyte transmigration through endothelial monolayers (diapedesis) in vitro, and furthermore, SPARC null mice have abnormalities in leukocyte recruitment to the inflamed peritoneum in vivo. These findings provide new insight into the mechanisms of transendothelial leukocyte migration and suggest a potential, targetable interaction for therapeutic intervention.

In vivo imaging of activated endothelium using an anti-VCAM-1 magnetooptical probe.

It has been suggested that vascular cell adhesion molecule-1 (VCAM-1) could serve as an early marker for inflammation of the endothelium. The ability to noninvasively image VCAM-1 could thus be a useful tool to diagnose a number of inflammatory diseases at early stages. Here we demonstrate that magnetooptical nanoparticles conjugated to anti-VCAM-1 antibodies can be used to specifically detect VCAM-1 expression on endothelial cells in culture and in vivo. Elevated VCAM-1 expression was detected on cultured murine heart endothelial cells by both fluorescence and magnetic resonance, while only basal expression levels were detected on murine dermal endothelial cells. Intravital microscopy of a murine inflammatory model injected with the VCAM-1 targeted nanoparticles revealed specific labeling of the activated endothelium, with labeling kinetics yielding a maximum vessel wall signal 6 h after injection. In contrast, nontargeted nanoparticles did not exhibit any specific labeling of the endothelium. These studies suggest that the developed nanoparticle would be useful for MR and optical detection of activated endothelium.

Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.

BACKGROUND: We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction. METHODS AND RESULTS: We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone. CONCLUSIONS: This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.

Bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells do not contribute to vasculogenesis in Lewis lung carcinoma.

The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, and vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, and vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development. In the present study, we explored the recruitment behavior of bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin(-)c-kit(+)Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.

Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle.

Endothelial vascular adhesion molecule-1 (VCAM-1) is a critical component of the leukocyte-endothelial adhesion cascade, and its strict temporal and spatial regulation make it an ideal target for imaging and therapy. The goal of this study was to develop novel VCAM-1-targeted imaging agents detectable by MRI and fluorescence imaging using phage display-derived peptide sequences and multimodal nanoparticles (NPs). We hypothesized that VCAM-1-mediated cell internalization of phage display-selected peptides could be harnessed as an amplification strategy to chaperone and trap imaging agents inside VCAM-1-expressing cells, thus improving target-to-background ratios. To accomplish our goal, iterative phage display was performed on murine endothelium under physiological flow conditions to identify a family of VCAM-1-mediated cell-internalizing peptides. One specific sequence, containing the VHSPNKK motif that has homology to the alpha-chain of very late antigen (a known ligand for VCAM-1), was shown to bind VCAM-1 and block leukocyte-endothelial interactions. Compared with VCAM-1 monoclonal antibody, the peptide showed 12-fold higher target-to-background ratios. A VHSPNKK-modified magnetofluorescent NP (VNP) showed high affinity for endothelial cells expressing VCAM-1 but surprisingly low affinity for macrophages. In contrast, a control NP without VCAM-1-targeting sequences showed no affinity for endothelial cells. In vivo, VNP successfully identified VCAM-1-expressing endothelial cells in a murine tumor necrosis factor-alpha-induced inflammatory model and colocalized with VCAM-1-expressing cells in atherosclerotic lesions present in cholesterol-fed apolipoprotein E apoE-/- mice. These results indicate that: (1) small peptide sequences can significantly alter targeting of NPs, (2) the used amplification strategy of internalization results in high target-to-background ratios, and (3) this technology is useful for in vivo imaging of endothelial markers.

Murine neuronal progenitor cells are preferentially recruited to tumor vasculature via alpha4-integrin and SDF-1alpha-dependent mechanisms.

Recent studies have described neuronal progenitor cell recruitment to tumors in vivo, however, the mechanisms mediating this recruitment are not yet understood. When C17.2 murine neuronal progenitors stably expressing luciferase (C17.2-luc) were adoptively transferred into mice carrying subcutaneous Lewis lung carcinomas they accumulated at 1% injected dose/g of tumor tissue. C17.2-luc demonstrated significantly greater accumulation and transmigration on tumor-derived endothelium (TEC) than on normal endothelium under physiologically relevant flow conditions. Function blocking of alpha4-integrin reduced recruitment of C17.2-luc cells to normal endothelium but not to TEC, however, function blocking of SDF-1alpha reduced overall accumulation of C17.2-luc on TEC and specifically reduced transendothelial migration. Together, these data suggest that recruitment of C17.2-luc cells to TEC is mediated via SDF-1alpha/CXCR4 activation that results in modification of alpha4-integrin and results in improved recruitment of C17.2-luc cells.

In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors.

Magnetic resonance imaging (MRI) allows noninvasive and three-dimensional visualization of whole organisms over time, and, therefore, would be ideally suited to monitor cell trafficking in vivo. Until now, systemically injected cells had been difficult to visualize by MRI because of relatively inefficient labeling methods. We developed a novel, biocompatible, and physiologically inert nanoparticle (highly derivatized cross-linked iron oxide nanoparticle; CLIO-HD) for highly efficient intracellular labeling of a variety of cell types that now allows in vivo MRI tracking of systemically injected cells at near single-cell resolution. CD8+ cytotoxic T lymphocytes labeled with CLIO-HD were detectable via MRI with a detection threshold of 2 cells/voxel in vitro and approximately 3 cells/voxel in vivo in live mice. Using B16-OVA melanoma and CLIO-HD-labeled OVA-specific CD8+ T cells, we have demonstrated for the first time high resolution imaging of T-cell recruitment to intact tumors in vivo. We have revealed the extensive three-dimensional spatial heterogeneity of T-cell recruitment to target tumors and demonstrated a temporal regulation of T-cell recruitment within the tumor. Significantly, our data indicate that serial administrations of CD8+ T cells appear to home to different intratumoral locations, and may, therefore, provide a more effective treatment regimen than a single bolus administration. Together, these results demonstrate that CLIO-HD is uniquely suited for quantitative repetitive MRI of adoptively transferred cells and that this approach may be particularly useful for evaluating novel cell-based therapies in vivo.

Murine Lewis lung carcinoma-derived endothelium expresses markers of endothelial activation and requires tumor-specific extracellular matrix in vitro.

The purpose of the study was to identify characteristics specific to tumor-derived endothelium that may be important in tumor biology, or for the development of targeted therapeutics or imaging agents. Normal C57Bl/6 murine heart or lung endothelium, or C57Bl/6 murine Lewis lung carcinoma tumor-derived endothelium was isolated from excised tissues using specific antibodies. The endothelium was cultured using either native fibronectin, or the oncofetal form of fibronectin. Cell surface adhesion molecule expression was analyzed by flow cytometry, and the cellular distribution of specific molecules was examined using indirect immunofluorescence staining. Oncofetal fibronectin was critical for maintaining the phenotype of tumor-derived endothelium, which demonstrated an elongated morphology in vitro, with few cell-cell contacts. They expressed high levels of CD31, CD102, and vascular endothelial cadherin, and constitutively expressed CD62E, CD54, and CD106, indicating an "activated" phenotype. Moreover, they expressed significantly greater levels of Sca-1 and Flk-1 than normal murine endothelium. Cellular distribution of CD31, beta-catenin, and CD106 in tumor-derived endothelium was not continuous at cell borders, as observed in cultures of murine heart endothelium. In conclusion, Lewis lung carcinoma-derived tumor endothelium exhibits a specific phenotype in vitro, distinct from normal endothelium, and could be used as an in vitro tool for developing targeted agents.

Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment.

Chemokines and adhesion molecules play a critical role in the recruitment of leukocytes into specific organ sites. Little is known, however, regarding the repertoire of chemokines and adhesion molecules expressed within different vascular beds. In this study, we compare adhesion molecule expression, chemokine induction, and T-cell subset-endothelial interactions under defined flow conditions on resting and tumor necrosis factor (TNF)-alpha-activated murine lung endothelial cells (MLECs) and heart endothelial cells (MHECs). Our study revealed that only MHECs exhibited high constitutive VCAM-1 expression. Exposure to TNF-alpha up-regulated adhesion molecule expression and chemokine production in both MLECs and MHECs. However, high levels of Regulated on Activation Normal T cell Expressed And Secreted (RANTES) expression were detected only in TNF-alpha-activated MHECs. TNF-alpha-stimulated MLECs and MHECs both supported T-helper cell interactions under defined flow conditions. Most T cells instantaneously arrested on MHECs but exhibited a rolling phenotype on MLECs. Blocking studies revealed that T-cell arrest on MHECs was mediated by constitutive VCAM-1 and TNF-alpha-induced RANTES. These findings are consistent with the hypothesis that functional heterogeneity of endothelial cells from different sites exists and some of it is retained in vitro. Furthermore, these results provide an insight into the molecular mechanisms that may mediate T-helper cell recruitment to these organs.

Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro.

Recent evidence has suggested a role for neutrophil proteases during certain inflammatory responses. We demonstrated previously that neutrophil proteases can degrade components of the adherens junctions during neutrophil-endothelial adhesion. We tested the hypothesis that degradation of VE-cadherin at lateral junctions by elastase or MMP-9 facilitates neutrophil transendothelial migration. Neutrophils from MMP-9 or elastase null mice and strain-matched control mice expressed high levels of LFA-1, Mac-1, and L-selectin on their cell surface. Under flow conditions, wild-type and deficient neutrophils rolled, arrested, and transmigrated activated murine endothelium. There was no difference in the total numbers of interacting neutrophils or in the percentage of transmigrated cells. In addition, deficient neutrophils remained capable of degrading murine endothelial VE-cadherin. These results indicate that although neutrophil proteases may play a role in the acute inflammatory response, neutrophil elastase or MMP-9 is not essential for neutrophil transendothelial migration in this murine system.