Niko Deckers

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis.

In vitro measurement of cell death with the annexin A5 affinity assay

One of the hallmarks of cell death is the cell surface–expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5–negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive–and propidium iodide–negative, and secondary necrotic cells are annexin A5–positive and propidium iodide–positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.

Site-specific 68Ga-labeled Annexin A5 as a PET imaging agent for apoptosis

PURPOSE Two variants of Annexin A5 (Cys2-AnxA5 and Cys165-AnxA5) were labelled with Gallium-68 in order to evaluate their biological properties. PROCEDURES Biodistribution and pharmacokinetics of the radiotracers were studied with μPET in healthy mice and in a mouse model of hepatic apoptosis. μPET imaging after IV injection of the tracers in combination with μMRI was performed in Daudi tumor bearing mice before and after treatment with a combination of chemotherapy and radiotherapy. RESULTS The biodistribution data indicated a fast urinary clearance with only minor hepatobilliary clearance, although a high retention in the kidneys was observed. Animals treated with anti-Fas showed a 3 to 8 times higher liver uptake as compared to healthy animals. Tumor uptake of (68)Ga-Cys2-AnxA5 and (68)Ga-Cys165-AnxA5 was low but significantly increased after therapy. CONCLUSION Both (68)Ga-Cys2-AnxA5 and (68)Ga-Cys165-AnxA5 show a clear binding to apoptotic cells and are promising tracers for rapid evaluation of cancer therapy.

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

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 αvβ3 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.

In Vivo Molecular Imaging of Apoptosisand Necrosis in Atherosclerotic PlaquesUsing MicroSPECT-CT and MicroPET-CT Imaging

PurposeThe 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).ProceduresCollar-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.ResultsSPECT 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.ConclusionBoth 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.

Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells

Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo.

Transfection efficiency of lipoplexes for site-directed delivery

Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (ρ) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.