MOLECULAR-CLONING OF GENES DIFFERENTIALLY REGULATED BY TNF-ALPHA IN BOVINE AORTIC ENDOTHELIAL-CELLS, FIBROBLASTS AND SMOOTH-MUSCLE CELLS

Objective: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cy tokine binding to and thereby stimulating vascular cells. TNF-alpha me diated intermediate stimulation of vascular cells is believed to play a pivotal role in the development of arteriosclerosis. While extensive information has recently become available on gene induction by TNF-al pha, less is known about gene suppression by TNF-alpha in vascular cel ls. Endothelial cells are the first cell layer within the vessel wall interacting with circulating, cytokine releasing cells. Therefore, the y were selected as target for these study. Methods: A differential scr eening approach has been used to isolate cDNAs whose abundance was sup pressed by incubating bovine aortic endothelial cells (BAEC) for 6 h w ith 1 nM TNF-alpha. The gene expression of 6 isolated cDNAs after TNF- alpha was investigated by dot blots and nuclear run-on analysis in BAE C. The investigated genes were partially or completely sequenced. Diff erential expression after TNF-alpha stimulation of BAEC, bovine fibrob lasts and vascular smooth muscle cells (SMC) was studied by Northern b lots. RNA transcripts of the clone C7 in aortic aneurysms were examine d by in situ hybridization. Results: 49 independent cDNAs were isolate d by the differential screening approach and 6 clones were further ana lyzed. These genes were downregulated in a time and dose dependent man ner in BAEC. Sequence analysis revealed that 3 cDNAs encoded previousl y unidentified genes (C1, C5, C7), while 3 encoded known genes: connec tive tissue growth factor (CTGF; A1), fibronectin (A8) and the mitocho ndrial genome (B1). A1 and B1 were suppressed in BAEC, fibroblasts and SMC, whereas A8, C1, C5 and C7 were not uniformly downregulated in th e investigated cells. C7 RNA transcripts were exclusively induced in t he endothelium of an uninflamed aortic aneurysm. The transcripts were undetectable in an inflamed aortic aneurysm and control vessels. Concl usions: Gene suppression is a prominent feature of the intermediate ef fect of TNF-alpha on endothelial cells. Differences in the expression of the tested genes in endothelial cells, fibroblasts and vascular smo oth muscle cells open possibilities for the study of cellular interact ions in the vascular wall in disease situations with high local TNF-al pha concentrations. (C) 1998 Elsevier Science B.V. All rights reserved .