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The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal-epithelial-specific Dsc2 knockdown (KD) (Dsc2ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell-cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.

The single pass, transmembrane proteins of the cadherin family have been appreciated as important proteins that regulate intercellular adhesion. In addition to this critical function, cadherins contribute to important signalling events that control cellular homeostasis. Many examples exist of classical, desmosomal and atypical cadherins participating in the regulation of signalling events that control homeostatic functions in cells. Much of the work on cadherin mediated signalling focuses on classical cadherins or on specific disease states such as pemphigus vulgaris. Cadherin mediated signalling has been shown to play critical roles during development, in proliferation, apoptosis, disease pathobiology and beyond. It is becoming increasingly clear that cadherins operate through a range of molecular mechanisms. The diversity of pathways and cellular functions regulated by cadherins suggests that we have only scratched the surface in terms of the roles that these versatile proteins play in signalling and cellular function.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin β1 and β4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs. NWO ZonMw [MKMD 40-42600-98-13007]; FCT [SFRH/BPD/70277/2010] info:eu-repo/semantics/publishedVersion

Blood-derived monocytes remove apoptotic cells and terminate inflammation in settings as diverse as atherosclerosis and Alzheimer’s disease. They express high levels of the proresolving receptor ALX/FPR2, which is activated by the protein annexin A1 (ANXA1), found in high abundance in inflammatory exudates. Using primary human blood monocytes from healthy donors, we identified ANXA1 as a potent CD14+CD16− monocyte chemoattractant, acting via ALX/FPR2. Downstream signaling pathway analysis revealed the p38 MAPK-mediated activation of a calcium independent phospholipase A2 with resultant synthesis of lysophosphatidic acid (LPA) driving chemotaxis through LPA receptor 2 and actin cytoskeletal mobilization. In vivo experiments confirmed ANXA1 as an independent phospholipase A2–dependent monocyte recruiter; congruently, monocyte recruitment was significantly impaired during ongoing zymosan-induced inflammation in AnxA1−/− or alx/fpr2/3−/− mice. Using a dorsal air-pouch model, passive transfer of apoptotic neutrophils between AnxA1−/− and wild-type mice identified effete neutrophils as the primary source of soluble ANXA1 in inflammatory resolution. Together, these data elucidate a novel proresolving network centered on ANXA1 and LPA generation and identify previously unappreciated determinants of ANXA1 and ALX/FPR2 signaling in monocytes.

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 99mTc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following 124I-Hypericin (124I-Hyp) injection to visualize necrosis. RESULTS: SPECT depicted increased 99mTc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of 124I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, 124I-Hyp was able to target necrosis in the atherosclerotic plaque. CONCLUSION: Both 99mTc-AnxAF568 and 124I-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.

Cardiovascular disease (CVD) is still the leading cause of death in the Western World. Adverse outcomes of CVD include stroke, myocardial infarction, and heart failure. Atherosclerosis is considered to be the major cause of CVD and is estimated to cause half of all deaths in developed countries. Atherosclerotic lesions of the vessel wall may obstruct blood flow mechanically through stenosis, but rupture of atherosclerotic plaques causing formation of occlusive thrombi is far more prevalent. Unfortunately, conventional diagnostic tools fail to assess whether a plaque is vulnerable to rupture. Research over the past decade identified the biological processes that are implicated in the course towards plaque rupture, like cell death and inflammation. Knowledge about plaque biology propelled the development of imaging techniques that target biologic processes in order to predict the vulnerable plaque. This paper discusses novel and existing molecular imaging targets and addresses advantages and disadvantages of these targets and respective imaging techniques in respect of clinical application and socio-economic impact.

OBJECTIVE: To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice. METHODS: Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry. RESULTS: Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were

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.

Cardiovascular disease (CVD) is still the leading cause of death in the Western World. Adverse outcomes of CVD include stroke, myocardial infarction, and heart failure. Atherosclerosis is considered to be the major cause of CVD and is estimated to cause half of all deaths in developed countries. Atherosclerotic lesions of the vessel wall may obstruct blood flow mechanically through stenosis, but rupture of atherosclerotic plaques causing formation of occlusive thrombi is far more prevalent. Unfortunately, conventional diagnostic tools fail to assess whether a plaque is vulnerable to rupture. Research over the past decade identified the biological processes that are implicated in the course toward plaque rupture, like cell death and inflammation. Knowledge about plaque biology propelled the development of imaging techniques that target biologic processes in order to predict the vulnerable plaque. This paper discusses novel and existing molecular imaging targets and addresses advantages and disadvantages of these targets and respective imaging techniques with regard to clinical application and socioeconomic impact.

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

Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase 'eat me' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp-annexin A5, RGD-anxA5) that has gained the function to interact with alpha(v)beta(3) receptors of the phagocyte. We describe design and characterization of RGD-anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD-anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD-anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD-anxA5 is a promising therapeutic agent. Cell Death and Differentiation (2013) 20, 49-56; doi:10.1038/cdd.2012.107; published online 7 September 2012

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 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.

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.