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Open Access

Apoptosis of vascular cells in culture and reactive oxygen species generation following exposure to plasma of septic patients

  • MA Pedro1, 3,
  • M Janiszewski2, 3,
  • TA Janiszewski1,
  • E Silva3,
  • E Knobel3 and
  • FRM Laurindo1
Critical Care20015(Suppl 3):P22

Published: 26 June 2001


Reactive Oxygen SpeciesNADPHNADHVascular Smooth Muscle CellNADPH Oxidase

The inflammatory response that occurs in sepsis has been related to the presence of cytokines, and also other soluble factors such as fas and fas-l, which can induce apoptotic cell death of endothelial cells. Also, it can result in endothelial dysfunction, with microvascular thrombosis and activation of the coagulation cascade. These events involve the transduction of extracellular stimuli by reactive oxygen species (ROS; O2•-, H2O2) that result in activation of intracellular signaling pathways like MAPKinases. We sought to evaluate the effect of soluble factors present in plasma of septic patients in rabbit endothelial (REC) and vascular smooth muscle cells (RASM) in culture, by assaying for apoptosis with a TUNEL detection method. Also, we assessed NAD(P)H oxidase production of ROS in these plasma samples and in REC and RASM homogenates after incubation with plasma, as assessed with the lucigenin 5 μM chemiluminescence technique.

Septic plasma showed ROS generation when incubated with NADPH but not with NADH (51.44 ± 21.18 and 8.90 ± 4.04 versus 7.77 ± 2.60 and 1.8 ± 0.99 cpm × 103/mg/min, NADPH and NADH versus controls, respectively; n = 5-3). When incubated with homogenates of REC or RASM in the presence of both NADH or NADPH, septic plasma caused a 2- to 3-fold increase in ROS generation versus healthy control plasma (n = 5-3). Thus, septic plasma lead to apoptosis of REC and RASM, abrogated by a SOD mimic and NAD(P)H oxidase inhibitor DPI. Intrinsic NADPH oxidase ROS production was detected in the septic plasma. It also enhanced the NAD(P)H oxidase ROS production in REC and RASM homogenates. These data suggest that in sepsis there is a possible link between ROS production and vascular cell apoptosis.






P versus control

REC + healthy control plasma (n = 3)

7.0 ± 1.5


REC + septic plasma (n = 3)

42.8 ± 11.7


RASM + healthy control plasma (n = 3)

0.7 ± 0.7


RASM + septic plasma (n = 3)

33.0 ± 13.3


REC + septic plasma + SOD mimic 20 μm (n = 3)

6.7 ± 1.8


RASM + septic plasma+ SOD mimic 20 μm (n = 3)

3.0 ± 3.0


REC + septic plasma + DPI 20 μm (n = 3)

6.0 ± 1.7


RASM + septic plasma + DPI 20 μm (n = 3)

1.0 ± 0.6


Authors’ Affiliations

Heart Institute, Canada
Emergency Medicine Department, School of Medicine, University of São Paulo, Brazil
Intensive Care Unit, Hospital Israelita Albert Einstein, Brazil


© The Author(s) 2001