<em>In Vivo</em> Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

University of Cincinnati College of Medicine

Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl₃ vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A.

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is "flipped" to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7-8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints.

Saposin C coupled lipid nanovesicles specifically target arthritic mouse joints for optical imaging of disease severity.

Objective: A fundamental metric in the diagnosis of arthropathies is the pattern of joint involvement, including differences in proximal vs. distal joints and patterns of symmetrical or asymmetrical disease. The basis for joint selectivity among arthritides and/or within a defined disease such as rheumatoid arthritis remains enigmatic. Coagulation and fibrinolytic activity is observed in both experimental animals with inflammatory joint disease and patients with inflammatory arthritis. However, the contribution of specific hemostatic factors to joint disease is not fully defined. We sought to determine the contribution of the fibrinolytic protease, plasmin(ogen), to TNFα-driven arthritis in distinct joints of mice. Methods: The impact of plasminogen and/or fibrinogen genetic deficiencies on arthritis progression was evaluated in Tg197 mice genetically-predisposed to spontaneous, non-abating, and erosive polyarthritis due to exuberant human TNFα expression. Results: Elimination of plasminogen in Tg197 mice significantly exacerbated the incidence and severity of arthritis within the paw joints, but simultaneously and dramatically diminished the entire spectrum of pathologies within the knee joints of the same animals. These opposing outcomes were both mechanistically linked to fibrin(ogen) in that superimposing fibrinogen deficiency reversed both the pro-arthritic phenotype in the paws and arthritis resistance in the knees of plasminogen-deficient mice. Intriguingly, the diminution in disease severity in the knees, but not the paws, was associated with a plasminogen-dependent reduction in matrix metalloproteinase-9 activity. Conclusions: Plasminogen is a key molecular determinant of inflammatory joint disease capable of simultaneously driving or ameliorating arthritis pathogenesis in distinct anatomical locations of the same subject. © 2014 American College of Rheumatology.

Malinda Frederick

Division of Rheumatology,
Cincinnati Children's Hospital Medical Center,
Division of Rheumatology, Cincinnati Children's Hospital Medical Center

University of Cincinnati College of Medicine

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Malinda Frederick

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Rheumatology,Cincinnati Children's Hospital Medical Center,Division of Rheumatology, Cincinnati Children's Hospital Medical Center

University of Cincinnati College of Medicine

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Division of Rheumatology,
Cincinnati Children's Hospital Medical Center,
Division of Rheumatology, Cincinnati Children's Hospital Medical Center