Near Infrared Fluorescence (NIRF) Molecular Imaging of Oxidized LDL with an Autoantibody in Experimental Atherosclerosis.

We aimed to develop a quantitative antibody-based near infrared fluorescence (NIRF) approach for the imaging of oxidized LDL in atherosclerosis. LO1, a well- characterized monoclonal autoantibody that reacts with malondialdehyde-conjugated LDL, was labeled with a NIRF dye to yield LO1-750. LO1-750 specifically identified necrotic core in ex vivo human coronary lesions. Injection of LO1-750 into high fat (HF) fed atherosclerotic Ldlr(-/-) mice led to specific focal localization within the aortic arch and its branches, as detected by fluorescence molecular tomography (FMT) combined with micro-computed tomography (CT). Ex vivo confocal microscopy confirmed LO1-750 subendothelial localization of LO1-750 at sites of atherosclerosis, in the vicinity of macrophages. When compared with a NIRF reporter of MMP activity (MMPSense-645-FAST), both probes produced statistically significant increases in NIRF signal in the Ldlr(-/-) model in relation to duration of HF diet. Upon withdrawing the HF diet, the reduction in oxLDL accumulation, as demonstrated with LO1-750, was less marked than the effect seen on MMP activity. In the rabbit, in vivo injected LO1-750 localization was successfully imaged ex vivo in aortic lesions with a customised intra-arterial NIRF detection catheter. A partially humanized chimeric LO1-Fab-Cys localized similarly to the parent antibody in murine atheroma showing promise for future translation.

Evaluation of methods for sizing and counting of ultrasound contrast agents.

A precise, accurate and well documented method for the sizing and counting of microbubbles is essential for all aspects of quantitative microbubble-enhanced ultrasound imaging. The efficacy of (a) electro-impedance volumetric zone sensing (ES) also called a Coulter counter/multisizer; (b) optical microscopy (OM); and (c) laser diffraction (LD), for the sizing and counting of microbubbles was assessed. Microspheres with certified mean diameter and number concentration were used to assess sizing and counting reproducibility (precision) and reliability (accuracy) of ES, OM and LD. SonoVue™ was repeatedly (n = 3) sized and counted to validate ES, OM and LD sizing and counting efficacy. Statistical analyses of intra-method variability for the SonoVue™ mean diameter showed that the best microbubble sizing reproducibility was obtained using OM with a mean diameter sizing variability of 1.1%, compared with a variability of 4.3% for ES and 7.1% for LD. The best microbubble counting reproducibility was obtained using ES with a number concentration variability of 8.3%, compared with a variability of 22.4% for OM and 32% for LD. This study showed that no method is fully suited to both sizing and counting of microbubbles.

Celecoxib activates PI-3K/Akt and mitochondrial redox signaling to enhance heme oxygenase-1-mediated anti-inflammatory activity in vascular endothelium.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) provide important control of pain and inflammation, they have been overshadowed by concerns regarding atherothrombotic complications. However, celecoxib seems to have a relatively good cardiovascular profile and may improve endothelial function in coronary heart disease. This led us to the hypothesis that celecoxib induces the vasculoprotective enzyme heme oxygenase-1 (HO-1). In human umbilical vein and aortic endothelial cells, 24-48 h treatment with celecoxib induced HO-1 mRNA and protein expression and increased HO-1 enzyme activity. This effect was not seen with rofecoxib or indomethacin. Supplementation of culture medium with iloprost or prostaglandin E(2) failed to reverse celecoxib-mediated HO-1 induction, indicating a cyclooxygenase-independent mechanism. Rather, this action of celecoxib involved generation of mitochondria-derived reactive oxygen species, Akt phosphorylation, and nuclear translocation of the transcription factor Nrf2, with N-acetylcysteine, PI-3K antagonist LY290042, and dominant-negative Akt abrogating the effects. Furthermore, celecoxib-induced HO-1 was inhibited by dominant-negative Nrf2. The functional significance of HO-1 induction was revealed by celecoxib-mediated inhibition of VCAM-1 expression, a response reversed by the HO-1 antagonist zinc protoporphyrin. HO-1 induction provides a molecular mechanism for clinical observations indicating relative freedom from atherothrombotic complications in patients taking celecoxib compared to other NSAIDs with comparable anti-inflammatory activity.

Sphingosine 1-phosphate enhances Fc gamma receptor-mediated neutrophil activation and recruitment under flow conditions.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that is released by platelets and endothelial cells and has been implicated in diverse biological functions. We hypothesized that S1P may influence immune complex-mediated polymorphonuclear neutrophil activation. Using flow cytometry and fluorescence spectrometry, we found that exogenous addition of S1P led to an enhanced polymorphonuclear neutrophil Fcgamma receptor-mediated rise in intracellular Ca(2+) and reactive oxygen species generation in a pertussis toxin-independent manner, while having only a small effect by itself. Thus, S1P amplifies a positive feedback loop where Fcgamma receptor-mediated rises in Ca(2+) and reactive oxygen species are interdependent, with reactive oxygen species acting to increase tyrosine phosphorylation and activity of upstream signaling intermediates. S1P augmentation of Fcgamma receptor signaling translates to downstream functional consequences, including shape change and recruitment to endothelial surfaces coated with suboptimal levels of immune complexes. Taken together, S1P from activated platelets or endothelial cells may serve to amplify leukocyte recruitment and tissue injury at sites of immune complex deposition in vasculitis.

Enhanced recruitment of Th2 and CLA-negative lymphocytes by the S128R polymorphism of E-selectin.

E-selectin is a cytokine-inducible endothelial cell adhesion molecule that binds a restricted population of T lymphocytes consisting of Th1 memory cells bearing the cutaneous lymphocyte Ag (CLA). A serine to arginine (S128R) polymorphism in E-selectin has been reported at increased frequency in patients with systemic lupus erythematosus and atherosclerosis. Here we tested the hypothesis that the S128R substitution may contribute to increased vascular disease by altering the number and/or phenotype of lymphocytes interacting with E-selectin under shear flow. We observed that CHO cell monolayers transfected with S128R recruited significantly greater numbers of unfractionated lymphocytes than monolayers expressing an equivalent density of wild-type (WT) E-selectin. Depletion of the CLA(+) subpopulation or generation of CLA(-) lymphoblasts abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R. Generation of Th subsets revealed preferential interaction of Th0 and Th2, but not Th1, cells with S128R compared with WT. However, only T cells of a memory phenotype interacted with S128R, since neither monolayer supported rolling of CD45RA(+) cells. Our results demonstrate that the S128R polymorphism extends the range of lymphocytes recruited by E-selectin, which may provide a mechanistic link between this polymorphism and vascular inflammatory disease.