Concentration dependent dual effect of thrombin in endothelial cells via Par-1 and Pi3 Kinase

Abstract

Disruption of endothelial barrier is a critical pathophysiological factor in inflammation. Thrombin exerts a variety of cellular effects including inflammation and apoptosis through activation of the protease activated receptors (PARs). The activation of PAR-1 by thrombin is known to have a bimodal effect in endothelial cell permeability with a low concentration (pM levels) eliciting a barrier protective and a high concentration (nM levels) eliciting a barrier disruptive response. It is not known whether this PAR-1-dependent activity of thrombin is a unique phenomenon specific for the in vitro assay or it is part of a general anti-inflammatory effect of low concentrations of thrombin that may have a physiological relevance. Here, we report that low concentrations of thrombin or of PAR-1 agonist peptide induced significant anti-inflammatory activities. However, relatively high concentration of thrombin or of PAR-1 agonist peptide showed pro-inflammatory activities. By using function-blocking anti-PAR-1 antibodies and PI3 kinase inhibitor, we show that the direct anti-inflammatory effects of low concentrations of thrombin are dependent on the activation of PAR-1 and PI3 kinase. These results suggest a role for cross communication between PAR-1 activation and PI3 kinase pathway in mediating the cytoprotective effects of low concentrations of thrombin in the cytokine-stimulated endothelial cells. J. Cell. Physiol. 219: 744-751, 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1. The barrier permeability effect of thrombin on endothelial cells

(A) Effect of indicated concentrations of thrombin on permeability in primary HUVECs were monitored as described under “Materials and Methods”. (B) The same as (A) except that permeability was monitored at an optimal concentration of thrombin (50 pM) in either the absence or presence of blocking (B) or non-blocking (NB) anti-PAR-1 antibodies. (C and D) The same as (A and B) except that the cell type was primary HPAECs.

Figure 2. Analysis of the TNF-α–mediated adhesion of leukocytes to endothelial cells

TNF-α-mediated (10 ng/mL for 4 hours) adhesion of freshly isolated neutrophils to primary HUVECs was analyzed after treating monolayers with indicated concentrations of thrombin in the absence (A) or presence of blocking or nonblocking anti-PAR-1 antibodies (B) as described under “Materials and Methods”. (C and D) The same as (A and B) except that the cell type was primary HPAECs.

Figure 3. Analysis of the TNF-α-mediated transendothelial migration (TEM) of leukocytes through endothelial cells

TNF-α-mediated (10 ng/mL for 4 hours) TEM of freshly isolated neutrophils to primary HUVECs was analyzed after treating monolayers with indicated concentrations of thrombin in the absence (A) or presence of function-blocking or non-blocking anti-PAR-1 antibodies (B) as described under “Materials and Methods”. (C and D) The same as (A and B) except that the cell type was primary HPAECs.

Figure 4. Analysis of the expression of TNF-α-mediated cell adhesion molecules (CAM) on endothelial cells and the inhibitory effect of low concentrations of thrombin

Confluent monolayers of HUVECs were treated with different concentrations of thrombin for 4 hours followed by analysis of the expression of VCAM-1 (whiter bar), ICAM-1 (grey bar) and E-selectin (black bar) in response to TNF-α for 4 hours by an ELISA in the absence (A) or presence of blocking or non-blocking ant-PAR-1 antibodies (B) as described under “Materials and Methods”. (C and D) The same as (A and B) except that cell type was primary HPAECs.

Figure 5. The effect of PAR-1 agonist peptide on barrier permeability in endothelial cells

(A) The effect of increasing concentrations of PAR-1 agonist peptide, TFLLRN, (x-axis) on the permeability in HUVECs was monitored as described under “Materials and Methods”. (B) The same as (A) except that the cell type was primary HPAECs.

Figure 6. The effect of PI3 kinase inhibitor (LY-294002) on antiinflammatory activities of low concentrations of thrombin in endothelial cells

(A) Endothelial cells were preincubated with LY-294002 (10 μM) followed by monitoring the permeability in the presence of50 pM thrombin in HUVECs (white bar) or 75 pM on HPAECs (Black bar) as described under “Materials and Methods”. (B) The same as (A) except that neutrophil adhesion to endothelial cells was monitored as described under “Materials and Methods”. (C) The same as (A) except that TEM was measured as described under “Materials and Methods”.