Modulation of endothelial monolayer permeability induced by plasma obtained from lipopolysaccharide-stimulated whole blood

Abstract

The aim of this study was to elucidate the time course of the permeability response of endothelial monolayers after exposure to plasma obtained from lipopolysaccharide (LPS)-treated human whole blood; to investigate the role of apoptosis in monolayer permeability, and to inhibit the permeability increase, particularly after addition of the plasma stimulus. Human umbilical vein endothelial cells (HUVEC) were cultured on semiporous membranes and the permeability for albumin was measured after exposure, according to different schedules, to LPS-conditioned plasma. Apoptotic HUVEC were measured by both flow cytometry and ELISA. A variety of agents, including antibodies against cytokines, inhibitors of NF-kappaB, and a caspase inhibitor, were added to HUVEC, either prior to or after the stimulus. A maximum increase of the permeability was achieved after 4-6 h of exposure to LPS-conditioned plasma. This response was not accompanied by an increase in the number of apoptotic HUVEC. Administration of antibodies against both Tumour Necrosis Factor-alpha (TNF-alpha) and Interleukin-1beta (IL-1beta) to HUVEC within 1 h after stimulation significantly reduced the permeability increase. Similarly, pyrollidine di-thiocarbamate (PDTC), but not N-acetylcysteine, could prevent the permeability response, and was still effective when added within 2 h after LPS-conditioned plasma. The TNF-alpha/IL-1beta signal present in LPS-conditioned plasma appears to increase endothelial permeability through intracellular pathways that very likely involve the activation of NF-kappaB. Although poststimulatory inhibition of the permeability response proves to be possible with agents such as PDTC, the window of opportunity appears very small if placed in a clinical perspective.

Fig. 1

Stimulation of endothelial permeability by LPS-conditioned plasma. HUVEC monolayers are incubated with LPS-conditioned plasma and albumin permeability is expressed relative to that induced under reference conditions (▪). Data represent mean ± sem (n = 4) (a) Effect of increasing incubation times. (b) Effect of replacing LPS-conditioned plasma with control plasma after various time periods. (c) Effect of increasing incubation time with control plasma after a 2 h incubation with LPS-conditioned plasma. *P < 0·05 versus incubation with control plasma (CP) alone.

Fig. 2

Endothelial cell death after exposure to LPS-conditioned plasma. Data represent the absorbance (OD) value (n = 2) as a measure of cytoplasmic histone-bound DNA fragments in HUVEC cultures incubated for 6 h with serum-free medium (M199), with 20% control plasma (CP), and with 20% LPS-conditioned plasma (LPSCP). Cells were also pre-incubated with the apoptosis-inhibitor Z-VAD-fmk.

Fig. 3

Stimulation of endothelial permeability by LPS-conditioned plasma: prevention by antibodies against TNF-α and IL-1β. HUVEC monolayers are incubated with LPS-conditioned plasma (LPSCP) for 6 h and at various time points both antibodies are added. Data (mean ± sem, n = 8) depict the permeability relative to that induced by LPSCP (▪) alone (= 100%) and to that induced by control plasma (CP, 0%). *P < 0·05 versus LPSCP.

Fig. 4

Effect of PDTC on endothelial permeability induced by LPS-conditioned plasma. Data (mean ± sem, n = 8) depict the permeability relative to that induced by LPS-conditioned plasma (LPSCP, black bars) alone (100% reflects increase versus incubation with control plasma, CP). (a) PDTC (10–10³ µM) is added to the to the cultures 1 h prior to LPSCP. (b) Effect of administration of 200 µM PDTC at various time points in relation to the addition of LPSCP (at 0 h). *P < 0·05 versus LPSCP.