Overview of Imaging Atherosclerosis
Robert J. Gropler, David K. Glover, Albert J. Sinusas, Heinrich Taegtmeyer in Cardiovascular Molecular Imaging, 2007
Apoptosis is believed to play a role in the formation of necrotic debris that constitutes necrotic cores often found in unstable vulnerable plaques (5). There is data to suggest that apoptosis of macrophages contributes to the size of a necrotic core (128). Furthermore, there is evidence that apoptosis of SMCs is associated with a thin fibrous cap (129). Recently, Narula et al. used radiolabeled annexin V to target apoptotic cells in a rabbit model of atherosclerosis using gamma camera methods. Annexin V has a high affinity for phosphatidylserine (PS), which becomes exposed on the surface of apoptotic cells. In this study, the investigators measured the in vivo aortic uptake of radiolabeled annexin V. They showed that there was an almost 10-fold higher uptake in the aorta of atherosclerotic rabbits compared to control rabbits (Fig. 4) (130). Given that apoptosis is a potential determinant of plaque instability, imaging atherosclerosis using annexin V may turn out to be useful in assessing plaque vulnerability. Quite interestingly, these methods are now being used in humans to study usefulness in assessment of carotid atherosclerosis (Fig. 5). Although preliminary, the results are suggesting that imaging carotid atherosclerosis with radiolabeled annexin V may discern carotid plaque features indicative of stability or rather lack thereof (131).
The Etiology of the Antiphospholipid Syndrome
Howard J.A. Carp in Recurrent Pregnancy Loss, 2020
Furthermore, aPL may influence the placental circulation by attacking certain placental epitopes such as Annexin A5, a potent anticoagulant protein. Annexin V, found on the apical surface of placental syncytiotrophoblast, forms a protective shield on the phospholipid surface, blocking phospholipids from becoming available for coagulation reactions. The annexin-V shield could be damaged by either binding to anti-annexin-V or preventing its binding to the PL membrane, or by blocking autoantibodies against annexin-V/PL [36]. Anti-annexin-V autoantibodies have been detected in patients with systemic lupus erythematosus (SLE) and APS associated with pregnancy loss, while reduced levels of annexin-V have been observed on the placental villi of women with aPL, recurrent pregnancy loss, and a thrombogenic background [37].
Apoptosis and Cell Death
John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie in Basic Sciences Endocrine Surgery Rhinology, 2018
The ultimate fate of apoptotic cells in vivo is to be phagocytosed by macrophages or neighbouring cells acting as semiprofessional phagocytes. The membrane of apoptotic cells is altered to prevent leakage of intracellular material until phagocytosis, a process involving protein cross-linking by tissue transglutaminase. The lipid components of the membrane are also altered, in that phosphatidyl serine, normally only present on the inner membrane leaflet, is flipped into the outer leaflet and exposed on the cell surface, enabling macrophages to identify and engulf the dying cell. The engulfment of the cell prior to loss of plasma membrane integrity is key to the process of eliminating excess or damaged cells without eliciting an inflammatory response. If this does not occur, the plasma membrane becomes permeable, and the cell is said to undergo ‘secondary necrosis’, with the leakage of damage-associated molecular pattern molecules (DAMPs) able to elicit a non-infectious immune response. It is exposure of phosphatidyl serine on the outer leaflet that is recognized in the Annexin V assay. Annexin V is an anticoagulant protein from human placenta that binds to phosphatidyl serine lipids.6 Labelled Annexin V binding in conjunction with propidium iodide staining of unfixed cells distinguishes early apoptosis, as cells that are not permeable to propidium iodide but are Annexin V positive. However, once the plasma membrane becomes permeable, it can no longer distinguish apoptosis and secondary necrosis, because Annexin V then has access to the inner plasma membrane leaflet. For use in fixed cells or in vivo, the cells are labelled before fixation.
Novel approaches to cancer therapy with ibuprofen-loaded Eudragit® RS 100 and/or octadecylamine-modified PLGA nanoparticles by assessment of their effects on apoptosis
Published in Drug Development and Industrial Pharmacy, 2020
Gülsel Yurtdaş-Kırımlıoğlu, Şennur Görgülü, Murat Sami Berkman
Annexin V is a protein that can bind to phospholipids such as phosphatidylserine, one of the membrane lipids known for its translocation on the cell surface in apoptosis. It is used to determine the stages of apoptosis by marking it with a fluorescent substance (FITC) to observe changes in the cell membrane. Since this binding can also be seen in both apoptosis and necrosis, non-vital propidium iodide (PI) is also used simultaneously as a second dye in the analysis and this binding rate of this complex is measured by flow cytometer. When cells are treated simultaneously with Annexin V-FITC (green fluorescence) and PI (red fluorescence), they show positive light scatter in the presence of changes in membrane integrity. If the integrity of the membrane has not changed, both dyes are expressed as negative for intact cells (FITC−/PI−), as both positive for late apoptotic or early necrotic cells (FITC+/PI+). Apoptotic or early apoptotic cells are expressed as FITC positive (FITC+/PI−), dead, and necrotic cells are expressed as PI positive (FITC−/PI+) [33–37].
Development of a PHBV nanoparticle as a peptide vehicle for NOD1 activation
Published in Drug Delivery, 2021
Mauricio Cabaña-Brunod, Pablo A. Herrera, Valeria Márquez-Miranda, Felipe M. Llancalahuen, Yorley Duarte, Danilo González-Nilo, Juan A. Fuentes, Cristián Vilos, Luis Velásquez, Carolina Otero
As different concentrations of PHBV NPs generate a discrete cytotoxic effect on RAW 264.7 cells. The next step was to determine whether these NPs could be triggering some apoptosis process. To this end, we evaluated the integrity of the cell membrane by annexin V, an apoptosis early marker. Annexin V preferentially binds to phospholipids that are translocated from the inside to the outside of the cell membrane in the early stages of apoptosis. Results obtained by flow cytometry indicate no significant changes in apoptosis levels concerning the negative control with only culture medium (except at a concentration of 1000 µg/ml) (Figure 6(A)). Furthermore, at a chosen concentration of 100 µg/ml, empty NPs do not show significant changes in apoptosis levels over time when measured at different times (1, 2, 4, 6, 24, and 48 h) (Figure 6(B)). As a positive control, a concentration of 1 µM H2O2 was used as an oxidizing agent capable of inducing apoptosis by over 50% in RAW 264.7 cells (Piao et al., 2011).
The Safe Soluble Compound Dehydroascorbic Acid Inhibits Various Upstream and Downstream Effectors of PI3K and KRAS Signaling Pathways in Undruggable PIK3CA/KRAS-Mutant Colorectal Cancer Stem-Like Cells
Published in Nutrition and Cancer, 2021
Fahimeh Kalbkhani, Ali Pirnejad, Sohrab Sam, Mohammad Reza Sam
Apoptosis was analyzed by a double-staining method using Annexin-V FLOUS/Propidium iodide (PI) labeling solution according to the manufacturer's instructions. In apoptotic cells, the membrane phospholipid phosphatidylserine, which is normally found in the internal portion of the cell membrane, is translocated to the outer leaflet of the plasma membrane, thereby exposing phosphatidylserine to the external environment. Annexin-V is a calcium-dependent phospholipid binding protein that has an affinity for phosphatidyl serine and is useful in identifying apoptotic cells. PI binds to cellular DNA is useful in identifying necrotic cells. LS174T cells were treated with 100-, 500-, and 1000 μM L-DHA for 48 h. Thereafter, the cells were washed twice with sterile cold PBS buffer and after centrifugation, cell pellets were then resuspended in 100 μl of 1× binding buffer at a density of 5 × 105 cells/ml with FITC-Annexin V. The cells were gently mixed and incubated in the dark at room temperature for 20 min. To differentiate cells with membrane damage, PI solution was added to the cell suspension prior to the flow cytometric analysis using a fluorescence-activated cell sorter (Dako, USA). Early apoptosis was defined as cells positive for Annexin V-FITC only. Late apoptosis was defined as cells positive for Annexin V-FITC and PI, and necrotic cells were defined as cells positive for PI only.
Related Knowledge Centers
- Annexin
- Antibody
- Coagulation
- In Vitro
- Protein
- Apoptosis
- Flow Cytometry
- Phosphatidylserine
- Thrombin
- Phospholipase A1