Jennifer R. Allport

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 &agr;-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-&agr;–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.

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.

In vivo imaging of gene and cell therapies

Molecular imaging can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to commonly used clinical imaging, it sets forth to probe the molecular abnormalities that are the basis of disease, rather than imaging the end effects of these molecular alterations. Development of new imaging technologies requires a multidisciplinary collaboration between biologists, chemists, physicists, and imaging scientists to create novel agents, signal amplification strategies, and imaging techniques that successfully address these questions. In this article we attempt to present some of the recent developments and show how molecular imaging can be used, at least experimentally, to assess specific molecular targets for gene- and cell-based therapies. In particular, we place emphasis on the development and use of experimental small-animal models, which are particularly inclined toward this approach, primarily in combination with magnetic resonance (MR), radionuclide, and optical imaging. In the future, specific imaging of disease targets will allow earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy.

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, 111In-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.

CD11b/CD18-coated microspheres attach to E-selectin under flow.

Neutrophils can attach to E‐selectin under flow. Proposed ligands for E‐selectin carry SLex‐type glycans. The leukocyte β2 integrins are glycosylated with SLex. Thus, we speculated that β2 integrins could support attachment to E‐selectin. To test this hypothesis, we coated 10–diameter microspheres with purified CD11b/CD18 (αMβ2) and investigated the adhesion of the resulting αMβ2 microspheres to E‐selectin. Under in vitro flow conditions, the αMβ2 microspheres attached to Chinese hamster ovary cells expressing E‐selectin (CHO‐E) and 4‐h interleukin‐1 β‐activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E‐selectin. microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18‐E‐selectin bond has sufficient biophysical properties to mediate attachment of neutro‐phil‐sized particles to E‐selectin under flow. J. Leukoc. Biol. 67: 196–205; 2000.

Murine neuronal progenitor cells are preferentially recruited to tumor vasculature via a4-integrin and SDF-1a 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 a4-integrin reduced recruitment of C17.2-luc cells to normal endothelium but not to TEC, however, function blocking of SDF-1a 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- 1a/CXCR4 activation that results in modification of a4-integrin and results in improved recruitment of C17.2-luc cells.

Endothelial-Dependent Mechanisms of Monocyte Recruitment

There is convincing evidence that the vascular endothelial cell lining is a major site of pathophysiologically relevant change in the blood vessel wall that occurs in response to injury, infection, mechanical perturbations, hypercholesterolemia, or other stimuli. Recent studies suggest a general dysfunction of endothelial cells in many diseases. For example, systemic sclerosis (scleroderma) is a connective tissue disease that is characterized by progressive fibrosis (excess collagen deposition) that affects small blood vessels resulting in leukocyte infiltration and damage, and often vascular dysfunction (8, 21). Systemic lupus erythematosus (SLE) and Kawasaki disease are of autoimmune origin and patients often have autoantibodies directed to a variety of cell and nuclear components that may produce vascular immune complexes and result in vessel, tissue and organ damage (11, 13). In atherosclerosis, chronic recruitment of mononuclear leukocytes to contribute to disease pathogenesis by producing cytokines and growth factors, which may in turn amplify leukocyte recruitment, induce smooth muscle cell migration into the intima and promote dysregulated growth of smooth muscle (34). In rheumatoid arthritis, a chronic systemic inflammatory disorder occurs that affects many tissues and organs, especially the joints, where the common manifestations are a nonsuppurative synovitis with lymphocytes, plasma cells and macrophages (13). The common manifestation of these diseases is sustained emigration of blood mononuclear leukocytes across vascular endothelium.