CD40 ligation induces tissue factor expression in human vascular smooth muscle cells - PubMed (original) (raw)

CD40 ligation induces tissue factor expression in human vascular smooth muscle cells

U Schönbeck et al. Am J Pathol. 2000 Jan.

Abstract

Tissue factor (TF) instigates the extrinsic pathway of blood coagulation. Plaque disruption and exposure of circulating factor VII/VIIa to subendothelial procoagulants such as TF leads to intravascular thrombosis, a frequent cause of acute atherosclerotic events. Although the expression of TF in the intima of human atherosclerotic lesions is well established, little is known about the mechanisms of TF regulation in vascular smooth muscle cells (SMC). We demonstrate here that TF colocalizes with the receptor CD40 on lesional SMC within atherosclerotic lesions in situ. In cultured vascular SMC, ligation of CD40 with native CD40 ligand (CD40L) derived from activated T lymphocytes or recombinant human CD40L (rCD40L) induced the transient expression of TF on the cell surface (as determined by FACS analysis) in a concentration- and time-dependent manner and enhanced total cell-associated TF (as determined by ELISA). CD40L-induced TF on vascular SMC is functional and activates coagulation. In accordance with the increased TF activity, stimulation of vascular SMC with rCD40L did not affect either protein expression or activity of tissue factor pathway inhibitors. In summary, these findings demonstrate the potential of the CD40/CD40L signaling pathway to augment the procoagulant activity in human vascular SMC. Because TF and CD40 colocalize on lesional SMC in human atheroma, CD40/CD40L signaling may contribute to the TF expression and hence to increased thrombogenicity of plaques during the inflammatory responses of atherogenesis and arterial injury.

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Figures

Figure 1.

Figure 1.

Tissue factor colocalizes with CD40-positive smooth muscle cells in human atherosclerotic lesions. Frozen sections of human carotid lesions were analyzed for α-actin (top panels: left, ×100; right, ×400), CD40 (left middle panel, green, ×400) and TF (lower left panel, red, ×400) staining within human atherosclerotic lesions, employing immunhistochemistry (for SMC) and immunofluorescence double labeling (for CD40 and TF). The lumen of the artery is at the top of each photomicrograph. Staining employing control IgG is shown for CD40 (right middle panel, ×400) and TF (right lower panel, ×400). Analysis of atheroma from six different donors showed similar results.

Figure 2.

Figure 2.

CD40L induces tissue factor in human vascular smooth muscle cells in vitro. A: FACS analysis for human TF on vascular SMC, cultured 24 hours before the experiment in IT medium and subsequently for the indicated times with cell-membrane preparations of PMA-activated (50 ng/ml, 12 hours) T lymphocytes (left panels) or recombinant CD40L (10 μg/ml rCD40L; right panels) in the absence or presence of the α-CD40L mAb (α-CD40L, 10 μg/ml). Staining performed with control IgG is shown as lines. B: Lysates of human vascular SMC, previously cultured 24 hours in IT medium, then incubated for 24 hours with the respective concentrations of recombinant human CD40 ligand (rCD40L) in the absence or presence of the α-CD40L mAb (α-CD40L, 10 μg/ml), were analyzed for TF protein expression by ELISA (upper panel). TF activity was analyzed in membrane-preparations of human vascular SMC (lower panel), as described in Materials and Methods. Data shown are representative of three experiments performed with cells of different origins.

Figure 3.

Figure 3.

CD40L does not affect expression of TFPI in human vascular SMC. Human vascular SMC, cultured 24 hours before the experiment in IT medium, were incubated for 24 hours with the respective concentrations of rCD40L in the absence or presence of the anti-CD40L mAb (α-CD40L, 10 μg/ml) and analyzed for TFPI expression by ELISA (A) as well as TFPI-activity assay (B). Recombinant TFPI-1 was applied for control purposes. Data shown are representative of three and seven experiments, respectively, performed with cells of different origins.

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