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

Toshikazu Shioiri (1, 2) , Masashi Muroi (1) , Fumihiko Hatao (2) , Masato Nishida (2) , Toshihisa Ogawa (2) , Yoshikazu Mimura (3) , Yasuyuki Seto (2) , Michio Kaminishi (4) , Ken-Ichi Tanamoto (1)

1 Musashino University
2 Department of Metabolic Care and Gastrointestinal Surgery
3 UTokyo - The University of Tokyo
4 Showa General Hospital

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of sever 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 hr 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.

Mots clés: Apoptosis; Toll-like receptor; Sepsis; Endotoxin; Infection; LPS; HUVEC; CHX; LBP; LDH; TLR; HE-SFM; FBS; sCD14; sMD-2; PBS; MTS

Toshikazu Shioiri, Masashi Muroi, Fumihiko Hatao, Masato Nishida, Toshihisa Ogawa, et al.. Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide. Biochimica et Biophysica Acta - Molecular Basis of Disease, 2009, 1792 (10), pp.1011. ⟨10.1016/j.bbadis.2009.06.006⟩. ⟨hal-00562909⟩