Markers of apoptosis in cardiovascular tissues focus on Annexin V

Abstract

In the last decade, apoptosis (or programmed cell death) has become appreciated as an important process in the development of the cardiovascular system. Moreover, apoptosis contributes to the adaptation of the system to the environment. We are at the beginning of understanding its relevance to cardiovascular physiology and pathology. This understanding forms the key to implement apoptosis in diagnosis and therapy of cardiovascular diseases. New avenues for pharmacological intervention are expected to arise from the synergy of our knowledge about the molecular mechanisms of apoptosis, and how apoptosis integrates in the complex environment of the cardiovascular tissue. The latter strongly depends on techniques to measure apoptosis. Currently, we are facing a relative paucity in available techniques, covering both specificity and sensitivity, and furthermore allowing quantitative analysis, preferably in combination with morphology. This field, however, is rapidly evolving and is fed by the expanding knowledge about the molecular mechanisms of apoptosis. In this paper we will briefly review the available techniques to detect and/or quantify apoptosis. These methods are based on the analysis of cellular morphology, either by light- or electron microscopy, DNA fragmentation (TdT-mediated X-dUTP nick end labeling or in situ nick end labeling), or cytoplasmic and membrane changes. Furthermore, the advantages and limitations of these techniques for their use in cardiovascular research will be outlined. In the text we will refer to available reviews and protocols which discuss the techniques in more detail. The main part of this article will, however, focus on a recently introduced technique, the Annexin V-based apoptosis detection assay. The principle, characteristics, pro's and contra's of this new apoptosis detection assay will be discussed.

Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes

“…Although cytochrome c has been shown to be released in adult cardiomyocytes subjected to oxidative stress, 31 the activity of executioner caspases was not measured and apoptotic cells were defined as cells exposing phosphatidylserine in the outer outleaf of the cytosolic membrane, an early apoptotic event not directly related to the execution phase of the apoptotic process. 32 Finally, release of cytochrome c has been detected ex vivo in isolated whole rat hearts subjected to ischemia-reperfusion injury. 8 However, although processing of caspase-8 and caspase-9 was detected in tissue slices immunostained with specific antibodies against the cleaved forms, addition of z-LEHD.fmk, which was used as an inhibitor of caspase-9, did not clearly change the number of Tdtmediated dUTP nick end-labeling (TUNEL)-positive cardiomyocytes.…”

Defective apoptosis of peripheral-blood lymphocytes in hyper-IgD and periodic fever syndrome

“…At 0, 5, and 24 hours cells were stained with annexin-V-FITC and PI (Apoptest-FITC; VPS Diagnostics, Hoeven, The Netherlands) and analyzed by flow cytometry (Coulter XL; Beckman Coulter, Fullerton, CA) The lymphocyte population was selected based on forward and side scatter and staining with labeled antibodies to CD14, CD3, and CD19. 3 At 2 hours cells were permeabilized and the profluorescent caspase-3/7 substrate was added (Apo-One Homogenous Caspase-3/7 Assay; Promega, Madison, WI). Fluorescence was measured using a fluorometer (POLARstar Galaxy; BMG Labtech, Offenburg, Germany) at excitation 485 nm and emission 520 nm.…”

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes

“…Inhibition of the ceramide pathway in mammalian cells leads to cellular proliferation [36], and this effect appears to be conserved in urodeles. In NRVMs exposed to H 2 O 2 , cell surface levels of annexin V, an indicator of early apoptosis [37], were significantly reduced following a 12 h pretreatment with A1-CM compared to NRVMs that did not receive A1-CM (Figure 2(c–e)). To test whether EVs in A1-CM mediate cytoprotection, we exposed A1 cells to 40 µM GW4869, or DMSO vehicle control, and applied the resulting CM to NRVMs prior to oxidative stress.…”