Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide

Abstract

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.

Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and Function of Brain Microvascular Endothelial Cells

“…1B,C), in accordance with previous demonstrations [27], [34]. LPS induced apoptotic cell death in a time-dependent manner ( P< 0.01), reaching 18.0% ( P< 0.05) after 24 h treatment.…”

“…Necrotic-like cell death induced by the same concentration of LPS was reported in transformed bovine BMEC by Karahashi et al [38], though the extent was over 40% at 24 h and no significant damage was seen at 4 h. Such divergent viability data may not only reflect different reactions to neurotoxins between species and between cell lines and primary cultures [39], but can mostly be due to the fact that this study was performed with medium supplemented with 10% FBS, unlike our own models. In studies performed in similar conditions to ours, namely with no addition of soluble CD14, there is also little LPS-induced LDH release for up to 16 h, being this the maximum time studied [34]. Still, our conditions are suitable for LPS interaction with BMEC given that our medium contains 2% FBS, which has soluble CD14, and that EC also express membrane CD14 in the presence of LPS [8].…”

Self‐Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase‐Cleavable Linker

“…These data suggest that RGDEVD‐DOX is cleaved by caspase‐3 and the caspase‐3 activated prodrug leads to increased expression and activity of caspase‐3 via apoptosis. An earlier report demonstrated caspase‐3 is released from apoptotic cells,13 suggesting that prodrug approaching the tumor after the initial death of targeted cells could be further activated by secreted caspase‐3.…”

Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

“…coli triggers rapid damage and apoptosis in ECs in vivo (39) and in vitro (40). In contrast, LPS from B. henselae is not cytotoxic to ECs (41), which is also reflected by the possibility of long-term cultivation of B. henselae-infected ECs (42).…”