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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1675.)
© 2000 American Heart Association, Inc.

Thrombosis

Hypertriglyceridemic VLDL Downregulates Tissue Plasminogen Activator Gene Transcription Through cis-Repressive Region(s) in the Tissue Plasminogen Activator Promoter in Cultured Human Endothelial Cells

Edlue M. Tabengwa; Raymond L. Benza; Hernan E. Grenett; Francois M. Booyse

From the Division of Cardiovascular Disease, University of Alabama at Birmingham.

Correspondence to Edlue M. Tabengwa, PhD, 845 19th St South, BBRB 809 (UAB Station), Birmingham AL 35294-2170. E-mail etabengwa{at}cardio.dom.uab.edu

Abstract—The relationship between tissue plasminogen activator (tPA) levels and the potential regulation by hypertriglyceridemic very low density lipoprotein (HTG-VLDL) was examined in a human umbilical vein endothelial cell (HUVEC) culture model system. HUVEC cultures were incubated in the absence/presence of HTG-VLDL or normal (NTG)-VLDL (0 to 50 µg/mL) at 37°C for various times (0 to 24 hours), followed by analyses of tPA antigen (ELISA), mRNA (reverse transcription–polymerase chain reaction), endothelial cell surface–localized plasmin generation assays, and nuclear transcription run-on assays. Secreted tPA antigen levels decreased 53% (3.3±0.14 versus 6.97±0.42 µg/mL) and mRNA levels decreased 70% in HTG-VLDL–treated HUVECs compared with NTG-VLDL–treated and culture medium control cells. Decreased tPA antigen and mRNA expression was associated with a concomitant 98% decrease in tPA-mediated plasmin generation in HTG-VLDL–treated HUVEC cultures. Nuclear transcription run-on assays demonstrated that HTG-VLDL decreased tPA gene transcription 73% (tPA mRNA/GAPDH mRNA) in cultured HUVECs. To identify and localize the repressive element(s) in the tPA promoter responsive to HTG-VLDL, a tPA promoter/luciferase construct (ptPA222/luc) was generated. HUVECs transiently transfected with this construct were incubated in the absence/presence of HTG-VLDL or NTG-VLDL (20 µg/mL). HTG-VLDL decreased promoter activity 52% to 57% in the ptPA222/luc-transfected cells compared with NTG-VLDL–treated or buffer control cells. These results indicate that the 2.2-kb fragment of the promoter and 5' flanking region of the tPA gene contains the repressive sequences that direct the transcriptional downregulation of the tPA promoter. Data from these studies suggest that the repression of tPA gene expression by HTG-VLDL may contribute to the impaired fibrinolysis often associated with hypertriglyceridemia.

Key Words: tissue plasminogen activator • gene regulation • hypertriglyceridemic VLDL • fibrinolysis • tissue plasminogen activator promoter • cis-repressive elements