PECAM-1 phosphorylation and tissue factor expression in HUVECs exposed to uniform and disturbed pulsatile flow and
Takeshi Moriguchi1, Bauer E Sumpio1
1Department of Surgery, Yale University School of Medicine, New Haven, Conn; Veterans Administration Connecticut, West Haven, Conn.
Introduction:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
