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Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.

Annexin A5 (AnxA5) exerts anti-inflammatory, anticoagulant and anti-apoptotic effects through binding cell surface expressed phosphatidylserine. The actions of AnxA5 on atherosclerosis are incompletely understood. We investigated effects of exogenous AnxA5 on plaque morphology and phenotype of advanced atherosclerotic lesions in apoE(-/-) mice. Advanced atherosclerotic lesions were induced in 12 weeks old Western type diet fed apoE(-/-) mice using a collar placement around the carotid artery. After 5 weeks mice were injected either with AnxA5 (n = 8) or vehicle for another 4 weeks. AnxA5 reduced plaque macrophage content both in the intima (59% reduction, P < 0.05) and media (73% reduction, P < 0.01) of advanced atherosclerotic lesions of the carotid artery. These findings corroborated with advanced lesions of the aortic arch, where a 67% reduction in plaque macrophage content was observed with AnxA5 compared to controls (P < 0.01). AnxA5 did not change lesion extension, plaque apoptosis, collagen content, smooth muscle cell content or acellular plaque composition after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions. In vitro, AnxA5 exhibited anti-inflammatory effects in macrophages and a flow chamber based assay demonstrated that AnxA5 significantly inhibited capture, rolling, adhesion as well as transmigration of peripheral blood mononuclear cells on a TNF-α-activated endothelial cell layer. In conclusion, short-term treatment with AnxA5 reduces plaque inflammation of advanced lesions in apoE(-/-) mice likely through interfering with recruitment and activation of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting exposed phosphatidylserine may become a viable strategy to treat patients suffering from advanced atherosclerosis., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Unlabelled: Annexin A1 (AnxA1) is an effector of the resolution of inflammation and is highly effective in terminating acute inflammatory responses. However, its role in chronic settings is less investigated. Because changes in AnxA1 expression within adipose tissue characterize obesity in mice and humans, we queried a possible role for AnxA1 in the pathogenesis of nonalcoholic steatohepatitis (NASH), a disease commonly associated with obesity. NASH was induced in wild-type (WT) and AnxA1 knockout (AnxA1 KO) C57BL/6 mice by feeding a methionine-choline deficient (MCD) diet up to 8 weeks. In MCD-fed WT mice, hepatic AnxA1 increased in parallel with progression of liver injury. This mediator was also detected in liver biopsies from patients with NASH and its degree of expression inversely correlated with the extent of fibrosis. In both humans and rodents, AnxA1 production was selectively localized in liver macrophages. NASH in AnxA1 KO mice was characterized by enhanced lobular inflammation resulting from increased macrophage recruitment and exacerbation of the M1 phenotype. Consistently, in vitro addition of recombinant AnxA1 to macrophages isolated from NASH livers down-modulated M1 polarization through stimulation of interleukin-10 production. Furthermore, the degree of hepatic fibrosis was enhanced in MCD-fed AnxA1 KO mice, an effect associated with augmented liver production of the profibrotic lectin, galectin-3. Accordingly, AnxA1 addition to isolated hepatic macrophages reduced galectin-3 expression., Conclusions: Macrophage-derived AnxA1 plays a functional role in modulating hepatic inflammation and fibrogenesis during NASH progression, suggesting the possible use of AnxA1 analogs for therapeutic control of this disease., (© 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.)

Objective: Annexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size., Procedures: Annexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging., Results: The annexin A5-streptavidin complex (4 ∶ 1, moles:moles, Mw ∼ 200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7 ± 0.5 for Ca2+-binding and an EC50 of 0.9 ± 0.1 mM Ca2+ (EC50 is the Ca2+ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9 ± 0.2, EC50 1.5 ± 0.2 mM Ca2+). Circulation half-life of annexin A5-streptavidin is ± 21 minutes (circulation half-life of annexin A5 is ± 4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding., Conclusion: Increasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.

Purpose: The purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT)., Procedures: Collar-induced carotid atherosclerosis ApoE knockout mice were imaged with (99m)Tc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following (124)I-Hypericin ((124)I-Hyp) injection to visualize necrosis., Results: SPECT depicted increased (99m)Tc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of (124)I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, (124)I-Hyp was able to target necrosis in the atherosclerotic plaque., Conclusion: Both (99m)Tc-AnxAF568 and (124)I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.

Background: Chronic low back pain (CLBP) is often accompanied by an abnormal motor performance. However, it has not been clarified yet whether these deviations also occur during motor tasks not involving the back and whether the performance is influenced by pain and pain-related cognitions. Therefore, the aim of the present study is to get insight in the contribution of both pain experience and pain-related cognitions to general motor task performance in CLBP., Methods: 13 CLBP patients and 15 healthy subjects performed a hand-function task in three conditions: sitting, lying prone (lying) and lying prone without trunk support (provoking). The last condition was assumed to provoke pain-related cognitions, which was considered successful when a patients' pain expectancy on a numeric rating scale was at least 1 point higher than actual pain experienced. Subjects' performance was expressed in reaction time and movement time. Repeated measures analysis of variance was performed to detect main effect for group and condition. Special interest was given to group*condition interaction, since significant interaction would indicate that patients and healthy subjects performed differently throughout the three conditions., Results: Patients were slower throughout all conditions compared to healthy subjects. With respect to the provoking condition, patients showed deteriorated performance compared to lying while healthy subjects' performance remained equal between these two conditions. Further analysis of patients' data showed that provocation was successful in 54% of the patients. Especially this group showed deteriorated performance in the provoking condition., Conclusion: It can be concluded that CLBP patients in general have worse motor task performance compared to healthy subjects and that provoking pain-related cognitions further worsened performance.

Introduction: Apoptosis is one of the mechanisms behind successful chemotherapy and radiation treatment. Radiolabeled annexin A5 has been demonstrated to be a successful tool in the detection of apoptosis following chemotherapy in vivo., Methods: His-tagged annexin A5 was labeled with [(99m)Tc]-tricarbonyl and evaluated as apoptosis imaging radiotracer in vitro and in vivo. The binding of the radiotracer was evaluated in Colo205 cells stimulated with 5-FU (1 mM) for 4 and 24 h, and confirmed by flow cytometry. Biodistribution and dosimetric studies were performed in healthy nude mice (n=5) via planar scintigraphy. [(99m)Tc]-(CO)(3) His-annexin A5 was also evaluated for in vivo imaging of spontaneous apoptosis in Colo205-bearing mice (n=12)., Results: The labeling procedure yielded a compound with 95-99% radiochemical purity and good in vitro stability. In vitro binding experiments indicated that the radiotracer retained its PS-binding activity. [(99m)Tc]-(CO)(3) His-annexin A5 rapidly cleared from the blood and predominantly accumulated in the kidneys. Absorbed dose (per organ) was found to be 116 ± 64 μGy/MBq for the kidneys and 10.38 ± 0.50 μGy/MBq for the liver. The effective dose was 7.00 ± 0.28 μSv/MBq. Spontaneous apoptosis in Colo205-bearing mice was visualised by [(99m)Tc]-(CO)(3) His-annexin A5 SPECT and correlated well with caspase-3 immunostaining (R=0.867, P<.01)., Conclusion: [(99m)Tc]-(CO)(3) His-annexin A5 may be a useful novel radioligand for the in vivo detection of cell death associated with PS expression. A simple, noninvasive way of detecting apoptosis in vivo could have many applications including a better understanding of the extent and timing of apoptosis in response to cancer therapies and assessment of early tumor response., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Novel nanoporous poly(3,4-ethylenedioxythiophene) (PEDOT) films with basalt-like surface morphology are successfully obtained via a one-step, vapor phase process of oxidative chemical vapor deposition (oCVD) by introducing a new oxidant, CuCl(2). The substrate temperature of the oCVD process is a crucial process parameter for controlling electrical conductivity and conjugation length. Moreover, the surface morphology is also systemically tunable through variations in substrate temperature, a unique advantage of the oCVD process. By increasing the substrate temperature, the surface morphology becomes more porous, with the textured structure on the nanometer scale. The size of nanopores and fibrils appears uniformly over 25 mm x 25 mm areas on the Si wafer substrates. Conformal coverage of PEDOT films grown with the CuCl(2) oxidant (C-PEDOT) is observed on both standard trench structures with high aspect ratio and fragile surfaces with complex topology, such as paper, results which are extremely difficult to achieve with liquid phase based processes. The tunable nanoporosity and its conformal coverage on various complex geometries are highly desirable for many device applications requiring controlled, high interfacial area, such as supercapacitors, Li ion battery electrodes, and sensors. For example, a highly hydrophilic surface with the static water contact angle down to less than 10 degrees is obtained solely by changing surface morphology. By applying fluorinated polymer film onto the nanoporous C-PEDOT via initiative chemical vapor deposition (iCVD), the C-PEDOT surface also shows the contact angle higher than 150 degrees . The hierarchical porous structure of fluorinated polymer coated C-PEDOT on a paper mat shows superhydrophobicity and oil repellency.