Related Experiment Video
Updated: Jul 19, 2026

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12
Sanjeev Kumar1, Sascha Kasseckert, Sawa Kostin
1Department of Clinical Pharmacology, Ruhr-Universität Bochum, Germany.
Objective:
Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study.
Methods:
The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively.
Results:
Apoptosis (29.2+/-1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090+/-52 nmol/l) and acidosis (pHi = 6.52+/-0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH(o) 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%.
Conclusion:
Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.
Related Concept Videos
Caspases
The Intrinsic Apoptotic Pathway
The Extrinsic Apoptotic Pathway
Apoptosis
Overview of Cell Death
Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the 20th century...
Necrosis
Morphological Manifestations of Necrosis
Necrotic cells show different types of morphological appearance depending on the type of tissue and infection. In coagulative necrosis, cells become anucleated and die, but their...

