PECAM-1 phosphorylation and tissue factor expression in HUVECs exposed to uniform and disturbed pulsatile flow and chemical stimuli

Abstract

We analyzed the relationship between platelet endothelial cell adhesion molecule-1 (PECAM-1) activation and tissue factor (TF) RNA expression in human umbilical vein endothelial cells (HUVECs) exposed to mechanical and chemical stimulation. Fifty percent confluent and 100% confluent HUVEC cultures were exposed to pulsatile forward flow, as a model for uniform flow, or pulsatile to-and-fro flow, as a model for disturbed flow, using a parallel-plate flow chamber system for up to 4 hours in the presence or absence of 4 U/mL thrombin. Protein lysates were immunoprecipitated for PECAM-1 and then immunoblotted with anti-phospho-tyrosine antibody. TF RNA expression was determined using quantitative reverse transcription polymerase chain reaction. HUVECs exposed to disturbed flow induced higher TF expression at 4 hours than HUVECs exposed to uniform flow in sparse cultures (16.8 ± 5.8 vs 5.1 ± 1.2; P < .05). HUVECs exposed to disturbed flow and thrombin induced higher TF RNA expression at 4 hours than cultures exposed to uniform flow and thrombin in both confluent (47.0 ± 6.0 vs 30.2 ± 4.9; P < .05) and sparse (72.3 ± 10.7 vs 49.8 ± 4.7; P < .05) cultures. In confluent HUVEC cultures, PECAM-1 is minimally phosphorylated by disturbed and uniform flow, while in sparse HUVEC cultures, PECAM-1 phosphorylation at 15 minutes is greater in both disturbed and uniform flow (2.0 ± 0.2 and 2.1 ± 0.4 respectively; P < .05). Thrombin treatment of static HUVECs exhibited greater PECAM-1 phosphorylation at 15 minutes in confluent compared with sparse cultures (3.0 ± 0.5 vs 2.3 ± 0.1; P < .05). PECAM-1 phosphorylation of HUVECs exposed to both flow and thrombin is significantly higher in sparse cultures compared with either flow or thrombin stimulation alone but was suppressed in confluent cultures. The significantly higher TF RNA expression induced by disturbed flow and cell confluence indicates that suppression of PECAM-1 phosphorylation may be an important contributory mechanical signal pathway that promotes TF expression when HUVECs are exposed to disturbed flow.