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

Abstract

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 g/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 Ϸ53% (3.3Ϯ0.14 versus 6.97Ϯ0.42 g/mL) and mRNA levels decreased Ϸ70% 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 Ϸ98% 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 Ϸ73% (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 g/mL). HTG-VLDL decreased promoter activity Ϸ52% to 57% in the ptPA222/luc-transfected cells compared with NTG-VLDL-treated or buffer control cells.

Cell Biology and Lipoproteins in Atherosclerosis

“…Besides, in vitro studies reveal that LDL and hypertriglyceridemic-VLDL (HTG-VLDL) decrease TPA antigen and mRNA expression and suppresses TPA-mediated plasmin generation in cultured EC model systems [171]. Studies on the promotor region demonstrate that effect of HTG-VLDL is mediated by repression of TPA gene expression [172].…”

Elevated expression and release of tissue-type, but not urokinase-type, plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes

“…The gels were photographed and the intensity of the individual t-PA, u-PA and GADPH mRNA bands measured by laser densitometric scanning, using a Molecular Dynamics Personal Densitometer. Changes in t-PA and u-PA mRNA levels were expressed as a relative ratio of t-PA or u-PA mRNA/ GAPDH mRNA [34].…”

Polyphenolics Increase t‐PA and u‐PA Gene Transcription in Cultured Human Endothelial Cells

“…A 2220 and 2368 base pair (bp) segment of the promoter and 5' flanking region of the human t-PA and u-PA genes, respectively, was amplified by PCR, by using upstream and downstream primers and the PCR-amplified PA promoter fragments purified and ligated into the promoterless and enhancerless firefly luciferase reporter gene (luc, pGL2-basic expression vector) to generate the pt-PA222/luc and pu-PA236/luc promoter constructs, as we have previously described in detail Tabengwa et al, 2000b). Sequencing was carried out on both strands by the University of Alabama at Birmingham Automated Sequencing Core Facility for sequence and orientation verification.…”

“…Semiconfluent cultured HUVECs were transiently transfected with either the pt-PA222/luc or pu-PA236/luc promoter constructs and cotransfected with a thymidine kinase promoter-driven Renilla luciferase construct (pRL-TK, internal control), as we have previously described in detail Tabengwa et al, 2000b). Transiently transfected cultured HUVECs were incubated in the absence or presence of 10 M of each of the phenolics in serum-free M199 that contained 0.25% BSA for 1 hr at 37°C, were rinsed once with serum-free M199/BSA, and then were incubated for an additional 16 hr at 37°C in the absence of phenolics in complete 10% serum-containing culture medium.…”