© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Molecular cloning of genes differentially regulated by TNF-
in bovine aortic endothelial cells, fibroblasts and smooth muscle cells1
aDepartment of Medicine I, University of Heidelberg, D-69115 Heidelberg, Germany
bDepartment of Radiology, University of Heidelberg, D-69120 Heidelberg, Germany
cDepartment of Anesthesiology, University of Heidelberg, D-69120 Heidelberg, Germany
dDepartment of Pathology, Hebelstraße 8, D-69115 Heidelberg, Germany
* Corresponding author. Tel.: +49 (6221) 568604/568605; Fax: +49 (6221) 564101.
Objective: Tumor necrosis factor-
(TNF-) is a pleiotropic cytokine binding to and thereby stimulating vascular cells. TNF- mediated 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-, less is known about gene suppression by TNF- in vascular cells. Endothelial cells are the first cell layer within the vessel wall interacting with circulating, cytokine releasing cells. Therefore, they were selected as target for these study. Methods: A differential screening approach has been used to isolate cDNAs whose abundance was suppressed by incubating bovine aortic endothelial cells (BAEC) for 6 h with 1 nM TNF-. The gene expression of 6 isolated cDNAs after TNF- was investigated by dot blots and nuclear run-on analysis in BAEC. The investigated genes were partially or completely sequenced. Differential expression after TNF- stimulation of BAEC, bovine fibroblasts and vascular smooth muscle cells (SMC) was studied by Northern blots. RNA transcripts of the clone C7 in aortic aneurysms were examined by in situ hybridization. Results: 49 independent cDNAs were isolated by the differential screening approach and 6 clones were further analyzed. These genes were downregulated in a time and dose dependent manner in BAEC. Sequence analysis revealed that 3 cDNAs encoded previously unidentified genes (C1, C5, C7), while 3 encoded known genes: connective tissue growth factor (CTGF; A1), fibronectin (A8) and the mitochondrial genome (B1). A1 and B1 were suppressed in BAEC, fibroblasts and SMC, whereas A8, C1, C5 and C7 were not uniformly downregulated in the investigated cells. C7 RNA transcripts were exclusively induced in the endothelium of an uninflamed aortic aneurysm. The transcripts were undetectable in an inflamed aortic aneurysm and control vessels. Conclusions: Gene suppression is a prominent feature of the intermediate effect of TNF- on endothelial cells. Differences in the expression of the tested genes in endothelial cells, fibroblasts and vascular smooth muscle cells open possibilities for the study of cellular interactions in the vascular wall in disease situations with high local TNF- concentrations.
KEYWORDS Experimental; Vascular; Cellular; Molecular biology; Atherosclerosis; Cytokines; Gene expression; Endothelial function; Smooth muscle
1 The first two authors contributed equally to the work presented.
2 Current address: Department of Surgery, Harvard Medical School, Boston, USA.
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