Imaging apoptosis for detecting plaque instability: rendering death a brighter facade

The relatively poor correlation between the risk of atherosclerotic plaque rupture and the degree of luminal obstruction before this event implies a strong imperative for in vivo detection of the processes underlying progressive plaque destabilization. In addition to the morphologic characteristics, apoptosis and inflammation comprise two important indicators of plaque instability. Apoptotic macrophage death results in enlargement of the plaque necrotic core and positive vascular remodelling, whereas apoptosis of the smooth muscle cells leads to attenuation of the fibrous cap. Imaging of apoptotic cells with annexin A5 provides an opportunity for the non-invasive assessment of cell death, and hence plaque vulnerability. The clinical detection of apoptosis could therefore promote the development of novel intervention strategies.     

Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis

Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer and myocardial infarction. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim, we synthesized two types of lipid-based bimodal contrast agents that enable the detection of apoptotic cells with both MRI and optical techniques. MR contrast was provided either by entrapment of iron oxide particles within pegylated micelles or by incorporation of Gd-DTPA-bis(stearylamide) (Gd-DTPA-BSA) lipids within the lipid bilayer of pegylated liposomes. The resulting contrast agents were approximately 10 and 100 nm in diameter, respectively. Additional fluorescent lipids were incorporated in the lipid (bi)layer of the contrast agents to allow parallel detection with optical methods. Multiple human recombinant annexin A5 molecules were covalently coupled to introduce specificity for apoptotic cells. Both annexin A5-conjugated contrast agents were shown to significantly increase the relaxation rates of apoptotic cell pellets compared to untreated control cells and apoptotic cells that were treated with nonfunctionalized nanoparticles. Increased relaxation rates were confirmed to originate from association of the contrast agents to apoptotic cells by confocal microscopy. The targeted nanoparticles presented in this study, which differ both in size and in magnetic properties, may have applications for the in vivo detection of apoptosis.     

Caspase sensitive gold nanoparticle for apoptosis imaging in live cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.     

Synergistic enhancement of iron oxide nanoparticle and gadolinium for dual-contrast MRI

PurposeThe use of MR contrast agents allows accurate diagnosis by exerting an influence on the longitudinal (T₁) or transverse (T₂) relaxation time of the surrounding tissue. In this study, we combined the use of iron oxide (IO) particles and nonspecific extracellular gadolinium chelate (Gd) in order to further improve the sensitivity and specificity of lesion detection.ProceduresWith a 7-Tesla scanner, pre-contrasted, IO-enhanced and dual contrast agent enhanced MRIs were performed in phantom, normal animals, and animal models of lymph node tumor metastases and orthotopic brain tumor. For the dual-contrast (DC) MRI, we focused on the evaluation of T₂ weighted DC MRI with IO administered first, then followed by the injection of a bolus of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA).ResultsBased on the C/N ratios and MRI relaxometry, the synergistic effect of coordinated administration of Gd-DTPA and IO was observed and confirmed in phantom, normal liver and tumor models. At 30 min after administration of Feridex, Gd-DTPA further decreased T₂ relaxation in liver immediately after the injection. Additional administration of Gd-DTPA also immediately increased the signal contrast between tumor and brain parenchyma and maximized the C/N ratio to ?4.12 ± 0.71. Dual contrast MRI also enhanced the delineation of tumor borders and small lesions.ConclusionsDC-MRI will be helpful to improve diagnostic accuracy and decrease the threshold size for lesion detection.     

Annexin 1 protects against apoptosis induced by serum deprivation in transformed rat retinal ganglion cells

To investigate whether Annexin 1 can protect a retinal ganglion cells line (RGC-5) from apoptosis as induced by serum deprivation. Annexin 1 location in RGC-5 cells was determined using an indirect immunofluorescent assay. Expression of Annexin 1 in RGC-5 cultures deprived of serum for 0, 2 days was semi-quantified by western blot and RT-PCR. Effects of varying concentrations of the Annexin 1 peptide fragment, Ac2-26, on the survival of the RGC-5 cells was determined, and apoptotic cells were quantified by flow cytometry. Immunoblot and RT-PCR analysis was preformed to identify caspase 3, bax and bcl-2 in RGC extracts. Annexin 1 was localized in the cytoplasm of RGC-5 cells and the expression of Annexin 1, caspase 3 and bax was upregulated in serum-deprived RGC-5 cells. Ac2-26 attenuated the apoptosis resulting from serum deprivation of RGC-5 in a concentration-dependent manner, decreased caspase 3 and bax levels and produced an increase of bcl-2 in cell lysates. Annexin 1, in specific the peptide fragment Ac2-26, may play an important role in decreasing apoptosis in serum-deprived RGC-5 cells.     

Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.

BACKGROUND:Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-alpha act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. METHODS:The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. RESULTS:By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. CONCLUSIONS:This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity.     

Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1.     

A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI

A novel polyacrylamide superparamagnetic iron oxide nanoparticle platform is described which has been synthetically prepared such that multiple crystals of iron oxide are encapsulated within a single polyacrylamide matrix (PolyAcrylamide Magnetic [PAM] nanoparticles). This formulation provides for an extremely large T2 and T2* relaxivity of between 620 and 1140 sec(-1) mM(-1). Administration of PAM nanoparticles into rats bearing orthotopic 9L gliomas allowed quantitative pharmacokinetic analysis of the uptake of nanoparticles in the vasculature, brain, and glioma. Addition of polyethylene glycol of varying sizes (0.6, 2, and 10 kDa) to the surface of the PAM nanoparticles resulted in an increase in plasma half-life and affected tumor uptake and retention of the nanoparticles as quantified by changes in tissue contrast using MRI. The flexible formulation of these nanoparticles suggests that future modifications could be accomplished allowing for their use as a targeted molecular imaging contrast agent and/or therapeutic platform for multiple indications.     

Annexin VI: an intracellular target for ATP

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.     

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x(L)/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose-response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment.     

Annexin II is a novel receptor for Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa (P. aeruginosa) are critical in ventilated and poly-traumatized patients. Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P. aeruginosa is urgently required. Here, we aimed to identify novel receptors that are involved in internalization of P. aeruginosa into mammalian epithelial cells. Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P. aeruginosa. The significance of the interaction of annexin II with P. aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P. aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P. aeruginosa into human epithelial cells.     

Annexin 1 cleavage in activated neutrophils: a pivotal role for proteinase 3

Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery.     

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V,caspase cleavage and dUTP incorporation assays

H. P. Dong, A. Holth, M. G. Ruud, E. Emilsen, B. Risberg and B. Davidson Measurement of apoptosis in cytological specimens by flow cytometry: comparison of annexin V, caspase cleavage and dUTP incorporation assays Objective: To compare the performance of different assays for measuring apoptosis in cytological specimens. Methods: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase‐3 and ‐8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. Results: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V‐positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). Conclusions: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.     

Identification of the First Fungal Annexin: Analysis of Annexin Gene Duplications and Implications for Eukaryotic Evolution

Annexin homologues have been found in animals, plants, and distinct protist lineages. We report the identification of the first fungal annexin, encoded by the anx14 gene of the filamentous ascomycete Neurospora crassa. Annexins have a complex evolutionary history and exhibit a large number of gene duplications and gene losses in various taxa, including the complete loss of annexin sequences from another ascomycete, the budding yeast Saccharomyces cerevisiae. Surprisingly, the N. crassa annexin homologue is most closely related to the annexin homologue of the slime mold Dictyostelium discoideum, suggesting a phylogenetic link between cellular slime molds and true fungi. Both of these annexin homologues are closely related to the family of annexin homologues present in animals, an observation consistent with the existence of the animal–fungal clade. These data further suggest that the gene duplications that generated the family of annexin sequences present in animals, fungi, and slime molds began prior to the divergence of these taxa. Received: 10 December 1997 / Accepted: 17 April 1998     

Quantitative comparison of in situ methods for detecting apoptosis induced by X-ray irradiation in mouse thymus

Apoptosis, or programmed cell death, is a process fundamental to the homeostasis of multicellular organisms. Therefore, the development of methods for detecting dying and dead cells is of great importance. In the present study, four methodologies for identifying thymocyte apoptosis were evaluated and compared: a classical Haematoxylin and Eosin staining method, two histochemical methods (DNA polymerase-mediated in situ end-labelling and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labelling) and flow cytometry. Aspects important for the quantitation of apoptosis in the mouse thymus after a low dose (below 1 Gy) of X-ray irradiation were emphasized. The nick-end-labelling method had the highest sensitivity among the four methods in detecting apoptotic cells; however, the in situ end-labelling method showed sensitivity similar to nick-end-labelling and possessed better response to incremental increases in radiation. The sensitivity of the Haematoxylin and Eosin staining was lower than the above two methods. Flow cytometry could not detect low-frequency apoptosis after a low radiation dose of below 30 cGy (cGy = 0.01 Gy) and did not respond linearly to increasing radiation. We conclude, therefore, that the in situ end-labelling method is the most adequate of the methods tested, especially for the detection of low-frequency apoptosis This revised version was published online in November 2006 with corrections to the Cover Date.     

Quantitative comparison of in situ methods for detecting apoptosis induced by X-ray irradiation in mouse thymus

Apoptosis, or programmed cell death, is a process fundamental to the homeostasis of multicellular organisms. Therefore, the development of methods for detecting dying and dead cells is of great importance. In the present study, four methodologies for identifying thymocyte apoptosis were evaluated and compared: a classical Haematoxylin and Eosin staining method, two histochemical methods (DNA polymerase-mediated in situ end-labelling and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labelling) and flow cytometry. Aspects important for the quantitation of apoptosis in the mouse thymus after a low dose (below 1 Gy) of X-ray irradiation were emphasized. The nick-end-labelling method had the highest sensitivity among the four methods in detecting apoptotic cells; however, the in situ end-labelling method showed sensitivity similar to nick-end-labelling and possessed better response to incremental increases in radiation. The sensitivity of the Haematoxylin and Eosin staining was lower than the above two methods. Flow cytometry could not detect low-frequency apoptosis after a low radiation dose of below 30 cGy (cGy = 0.01 Gy) and did not respond linearly to increasing radiation. We conclude, therefore, that the in situ end-labelling method is the most adequate of the methods tested, especially for the detection of low-frequency apoptosis     

JCoDA: a tool for detecting evolutionary selection

Background  

The incorporation of annotated sequence information from multiple related species in commonly used databases (Ensembl, Flybase, Saccharomyces Genome Database, Wormbase, etc.) has increased dramatically over the last few years. This influx of information has provided a considerable amount of raw material for evaluation of evolutionary relationships. To aid in the process, we have developed JCoDA (Java Codon Delimited Alignment) as a simple-to-use visualization tool for the detection of site specific and regional positive/negative evolutionary selection amongst homologous coding sequences.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.     

Proboscis profiler: a tool for detecting acanthocephalan morphotypes

Molecular studies conducted over the past 25 years have revealed previously unrecognised diversity in the phylum Acanthocephala. Several nominal species have been shown to represent complexes of morphologically cryptic biological species, a situation potentially confounding the analysis of ecological data. A software tool, ‘Proboscis profiler’, was developed to detect morphological heterogeneity in collections of superficially similar acanthocephalan worms based on the multivariate statistical analysis of proboscis hook dimensions. Proboscis profiler identifies objective, natural groups in a collection of acanthocephalans which may correspond to distinct biological species or populations. Initial trials demonstrate that Proboscis profiler can discriminate biological acanthocephalan species of the Echinorhynchus gadi Zoega in Müller, 1776 complex and differentiate between dorsal and ventral hook rows from the proboscis of E. salmonis Müller, 1784. Proboscis profiler is free software and can be downloaded from .