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Cardiovascular Research 1998 38(3):802-813; doi:10.1016/S0008-6363(98)00055-8
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Molecular cloning of genes differentially regulated by TNF-{alpha} in bovine aortic endothelial cells, fibroblasts and smooth muscle cells1

Jie Lina,2,2, Birgit Liliensieka, Michael Kanitza, Ulrike Schimanskib, Hubert Böhrerc, Rüdiger Waldherrd, Eike Martinc, Günter Kauffmannb, Reinhard Zieglera and Peter P. Nawrotha,*

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.

Received 25 June 1997; accepted 2 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Tumor necrosis factor-{alpha} (TNF-{alpha}) is a pleiotropic cytokine binding to and thereby stimulating vascular cells. TNF-{alpha} 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-{alpha}, less is known about gene suppression by TNF-{alpha} 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-{alpha}. The gene expression of 6 isolated cDNAs after TNF-{alpha} was investigated by dot blots and nuclear run-on analysis in BAEC. The investigated genes were partially or completely sequenced. Differential expression after TNF-{alpha} 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-{alpha} 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-{alpha} concentrations.

KEYWORDS Experimental; Vascular; Cellular; Molecular biology; Atherosclerosis; Cytokines; Gene expression; Endothelial function; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The endothelium forms the interface between circulating blood and extravascular tissues. It plays a central role in physiologic processes such as embryogenesis, angiogenesis, nutrient uptake and maintenance of vascular integrity. The endothelium has also been implicated in diseases such as arteriosclerosis [1, 2], tumor growth [3], metastasis [4], thrombotic and inflammatory diseases [5].

A pleiotropic cytokine previously shown to alter the endothelial cell phenotype is tumor necrosis factor-{alpha} (TNF-{alpha}) [6, 7]. The change of the endothelial cell phenotype in cytokine mediated diseases involves upregulation of genes coding for leukocyte adhesion molecules [8, 9], activators of coagulation [10], or inhibitors of fibrinolysis [11]. Thus the endothelium must adapt its phenotype in response to signals released by other cells. Genes upregulated by TNF-{alpha} have extensively been studied [12–15], while suppression of gene transcription has received less attention. Examples of genes relevant in cardiovascular diseases and downregulated in endothelial cells by TNF-{alpha} are thrombomodulin [16], the constitutive nitric oxide synthase [17, 18]and vascular endothelial cell growth factor receptors [19]. These results show that the TNF-{alpha} mediated change in the endothelial cell phenotype occurs not only by up-, but also by downregulation of gene expression.

Induction of tissue factor, leukocyte adhesion molecules and others occur at the transcriptional level within minutes after TNF-{alpha} stimulation. Similar the repression of thrombomodulin or the constitutive nitric oxide synthase occurs very rapidly after TNF-{alpha} stimulation. Therefore, we have recently initiated systematic studies in cultured bovine aortic endothelial cells (BAEC) to identify genes still downregulated by TNF-{alpha} after at least 6 h. This study was not addressed to genes for which mRNA expression already returns to baseline values after 6 h TNF-{alpha} stimulation of BAEC. We have identified 6 intermediate response genes, 3 of these genes are known: connective tissue growth factor (CTGF; A1), fibronectin (A8) and the mitochondrial genome (B1). The sequences of the remaining 3 genes (C1, C5, C7) have not been reported previously. The expression of these genes was investigated after TNF-{alpha} stimulation of vascular cells, namely BAEC, fibroblasts and vascular smooth muscle cells (SMC). RNA transcripts of the new gene C7 were detected by in situ hybridization in human aortic aneurysms in comparison to controls. Our data provide new insights into the TNF-{alpha} mediated intermediate response on endothelial cells and open possibilities for the study of cellular interactions in the vascular wall in situations where high local TNF-{alpha} concentrations are present.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Cells were cultured in Dulbecco's minimal essential medium (DMEM; Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS; Gibco BRL, Eggenstein, Germany), 600 µg/ml glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C, 5% CO2 in 150 cm2 flasks (Nunc, Wiesbaden, Germany).

Bovine aortic endothelial cells (BAEC) were isolated as described previously [7, 20], characterized by the expression of von Willebrand factor, thrombomodulin, and morphologic features. Experiments were performed with cells between passages 5–10 that had been confluent for 2 days.

Bovine fibroblasts were prepared from foreskin and identified by immunohistochemical staining with a monoclonal antibody against fibronectin (Sigma-Aldrich, Deisenhofen, Germany). Cells for experiments were used between passage 3–5. Vascular smooth muscle cells (SMC) were isolated from the media of a bovine aorta as described [21]and characterized by immunohistochemical staining with a monoclonal antibody against {alpha}-actin (ProGen, Heidelberg, Germany).

Human recombinant TNF-{alpha} (1x108 U/mg; Knoll AG, Ludwigshafen, Germany) was added in aliquots, diluted in DMEM containing 10% FCS, to 2 days confluent cells. Unless not otherwise indicated, 1 nM TNF-{alpha} was incubated for 16 h. TNF-{alpha} activity was confirmed by its capacity of inducing tissue factor expression in BAEC and in human umbilical vein endothelial cells [10]. Cycloheximide (Sigma-Aldrich, Deisenhofen, Germany) was added at 10 µg/ml 30 min before the addition of TNF-{alpha} [12].

2.2 RNA preparation
Total RNA was extracted using a procedure of guanidinium thiocyanate lysis followed by ultracentrifugation through CsCl, as described [22]. Cultured cells were washed 3 times with ice cold phosphate buffered saline (PBS) before guanidinium thiocyanate solution was added. Tissues from various adult bovine organs were homogenized in the same lysis buffer, using a homogenizer (Ultra-turrax, Wheaton, Milleville, USA).

2.3 Construction of a subtractive cDNA library
BAEC used for RNA extraction were incubated with either medium alone or 1 nM TNF-{alpha} for 6 h. Poly(A)+ RNA was obtained after two rounds of adsorption to oligo-dT-cellulose (Pharmacia, Freiburg, Germany) [22]. For the synthesis of the first strand cDNA, 10 µg poly(A)+ RNA from BAEC incubated with medium alone were used and processed by using a cDNA synthesis kit (Stratagene, Heidelberg, Germany) according to the manufacturer's instruction with minor modification. Briefly, a fifty-base oligo-d(T) primer-adapter with an Xho I site was used as primer. The final reaction volume was 100 µl containing 10 µl of 10x first strand buffer, 0.01 M dithiothreitol (DTT), 0.6 mM dATP/dGTP/dTTP, 0.3 mM 5-Methyl-dCTP, 0.56 µg/ml primer, 0.2 U/ml RNase inhibitor, 1.0 µl [{alpha}-P32]dATP (specific activity{approx}800 Ci/mmol; Amersham Buchler), and 5 µl (20 U/µl) M-MuLV reverse transcriptase (Gibco BRL, Eggenstein, Germany). After 1 h incubation at 37°C, the same volume of 0.2 M NaOH was added to the reaction and incubated at 70°C for 20 min to hydrolyse the mRNA template. The labeled cDNA was applied to a Sephadex G-50 column (Pharmacia, Freiburg, Germany) and precipitated with ethanol. The integrity of the cDNA was confirmed by an alkaline gel followed by autoradiography. Subtractive hybridization was carried out according to the method of Davis et al. [23]. Precipitated cDNA was suspended in 50 µg (10 µl) poly (A)+ RNA isolated from BAEC incubated with 1 nM TNF-{alpha} for 6 h as described above. The hybridization reaction was carried out in a sealed glass capillary (50 µl; siliconized) in 0.5 M phosphate buffer, 5 mM EDTA, 0.5% SDS, with a final volume of 12 µl. The reaction was incubated for 20 h at 65°C in a shaking water bath. Hydroxyapatite chromatography was then used to fractionate the mixture in 0.12 M phosphate buffer, 0.1% SDS. Single-stranded material (5.1% of labeled cDNA) was eluted at 60°C and double-stranded materials (94.9% of labeled cDNA) at 98°C. Second strand cDNA was synthesized with DNA polymerase I (Klenow fragment; Stratagene, Heidelberg, Germany) and the hairpin loop was cleaved with 120 U/ml nuclease S1 (Stratagene, Heidelberg, Germany) at 37°C for 30 min [24]. An EcoR I adaptor (Stratagene, Heidelberg, Germany) was used to prepare the adhesive 5'-ends. Xho I adapter-primer ends were digested with Xho I and cDNAs above 300 bp were selected by Sephacryl S-400 column chromatography. The cDNA was ligated into lambda ZAP II vector arms (Stratagene, Heidelberg, Germany) with known orientation, and the ligation mixture packaged (Stratagene, Heidelberg, Germany) according to the manufacturers instruction.

2.4 Differential plaque hybridization
The lambda cDNA library was plated with XL1-blue host cells (Stratagene, Heidelberg, Germany) in 150 mm diameter dishes at a density of 5,000 plaque forming units per dish. A duplicate set of nitrocellulose filters (Amersham Buchler, Braunschweig, Germany) was taken from each dish. 1 µg poly(A)+ RNA isolated from BAEC untreated or treated with 1 nM TNF-{alpha} for 6 h by two rounds of adsorption to oligo-dT-cellulose (Pharmacia, Heidelberg, Germany) [22]was used to synthesize cDNA probes of up to 2x109 counts per min/µg specific-activity, using M-MuLV reverse transcriptase (Gibco BRL, Eggenstein, Germany) and [{alpha}-P32]dCTP (specific activity{approx}3,000 Ci/mmol; Amersham Buchler, Braunschweig, Germany). Prehybridization was performed in 5xSSPE (1xSSPE contains 0.18 M NaCl, 10 mM Na3PO4, 1 mM EDTA, pH 7.7), 0.5% sodium dodecyl sulfate (SDS), 5x Denhardt's solution [22], 0.02 mg/ml salmon sperm DNA (Sigma-Aldrich, Deisenhofen, Germany), and 50% formamide (vol/vol; Gibco BRL, Eggenstein, Deutschland) at 42°C for 2 h. Filters were hybridized in the same solution for 40 h with 2x106 counts per min/ml labeled cDNA. Washing was performed once in 2xSSC (1xSSC is 150 mM NaCl, 15 mM sodium citrate), 0.1% SDS at room temperature for 15 min and then in 0.1xSSC, 0.5% SDS at 42°C for 30 min. Filters were exposed to Agfa Curix RP 1000 G X-ray films with intensifying screen at –80°C for 2 days. Plaques preferentially hybridized with the cDNA probe of unstimulated cells were selected. After a second round screening, single plaques were purified.

2.5 Cross-screening to determine related cDNA sequences
For cross-hybridization analyses, lambda phage clones, eluted in phage dilution buffer [22], were transferred to 200x200 mm2 dishes containing XL1-blue host cells (Stratagene, Heidelberg, Germany) in a grid array and incubated at 37°C for 6–8 h. The plaques were lifted with positively charged nylon filters (Hybond N+; Amersham Buchler, Braunschweig, Germany). Up to 10 filters were obtained from each dish. The inserts of the lambda recombinant clones to be used as probes were subcloned into pBluescript SK phagemids (Stratagene, Heidelberg, Germany) by an in vivo excision procedure with helper phage R 408 (Stratagene, Heidelberg, Germany). The plasmid DNAs were [{alpha}-P32]-labeled using a random primer labeling system (Promega, Heidelberg, Germany) and [{alpha}-P32]dCTP (specific activity{approx}3,000 Ci/mmol; Amersham Buchler, Braunschweig, Germany). The hybridization was carried out under the same condition as described above. Afterwards the filters were extensively washed with 0.1xSSC, 0.5% SDS at 68°C for 1 h and exposed to X-ray films at –80°C overnight.

2.6 Sequencing of TNF-{alpha} suppressed cDNAs
Double-stranded cDNAs in pBluescript SK, obtained in cross-screening, were used for dideoxy sequencing [25], using modified DNA polymerase I (Sequenase 2.0; Amersham Buchler, Braunschweig, Germany) and [{alpha}-S35]dATP (specific activity{approx}1,000 Ci/mmol, Amersham Buchler, Braunschweig, Germany). All clones selected as independent in cross-screening were partially sequenced from both ends, using –40 and Reverse Primers for the pBluescript SK vector (Stratagene, Heidelberg, Germany). Searches of the GenBank and the EMBL data bases were performed with the BLASTN of HUSAR sequence analysis program package (Version 4.0). For each clone at least 200 base pairs from both ends were used searching for similarities to previously published genes. The full length sequence of the C7 cDNA was obtained by automatic sequencing (MWG-Biotech, Ebersberg, Germany) and was compared to published sequences as described above.

2.7 Dot blot analysis
Filters for dot hybridization were prepared essentially as described by Kafatos et al. [26]. Linearized plasmid pBluescript SK containing each cDNA insert (1 µg) was denatured in 0.4 M NaOH at room temperature for 30 min and chilled on ice. The solution was then brought to 10xSSC (ice cold) and blotted onto Hybond N membrane (Amersham Buchler, Braunschweig, Germany), using a 96-well manifold apparatus (Bio-Rad Laboratories, München, Germany). Poly(A)+ RNA was isolated from total RNA with a mRNA purification kit (Qiagen, Hilden, Germany) and 0.5 µg were used to synthesize radioactive labeled cDNA probes using M-MuL V reverse transcriptase (Gibco BRL, Eggenstein, Germany) and [{alpha}-P32]dCTP (specific activity{approx}3,000 Ci/mmol; Amersham Buchler, Braunschweig, Germany). Hybridization was carried out as described above. The filters were washed in 0.1xSSC, 0.5% SDS at 68°C for 30 min and exposed to X-ray films at –80°C. The experiments have been repeated at least twice.

2.8 Nuclear run-on transcription assay
Nuclear run-on transcription assays were performed essentially as described previously [27–29]. When used, cycloheximide was added 30 min before TNF-{alpha} to the BAEC. In brief, about 1x107 nuclei were isolated from BAEC and run-on reactions were performed in 0.7 M KCl, 50 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 0.25 mM DTT, 1 mM EDTA in the presence of 250 µCi [{alpha}-P32]UTP (specific activity{approx}3,000 Ci/mmol, Amersham Buchler, Braunschweig, Germany) and incubated for 30 min at 30°C. The synthesized mRNA was treated with DNAse I (Promega, Heidelberg, Germany), afterwards with proteinase K (Sigma-Aldrich, Deisenhofen, Germany) and extracted with 0.45 µm Millipore filters (type HA, Millipore, Eschborn, Germany). RNA was collected by EtOH precipitation and 1x106 counts per min/ml hybridization solution were added to dot blot filters (described in differential plaque hybridization). The experiments have been repeated 3 times.

2.9 Northern blot analysis
12.5 µg total RNA per lane was separated on 1.1% agarose gels containing 6.4% formaldehyde. The integrity of RNA was checked by ethidium bromide staining of the 18 S and 28 S ribosomal RNA. After electrophoresis RNA was transferred overnight by capillary blotting in 20xSSC to Hybond N nylon membrane (Amersham Buchler, Braunschweig, Germany). Hybridization was performed using the same conditions as described for differential plaque hybridization and 2-3x106 counts per min labeled probe per ml hybridization solution. Selected cDNAs were labeled with the random primer labeling system (Promega, Heidelberg, Germany) as described for cross-hybridization. Filters were washed 15 min at room temperature in 1xSSC, 0.1% SDS and 20 min at 68°C in 0.1xSSC, 0.5% SDS before exposure to X-ray films overnight at –80°C. The filters were stripped and rehybridized against human glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH; ATCC Rockville, USA) to standardize the amount of RNA loaded. The density of autoradiographic signals was quantitated using a GS-700 Imaging Densitometer (Bio-Rad, München, Germany). Relative densitometry units were obtained, dividing the densitometry values for the studied gene by densitometry signals of GAPDH.

2.10 In situ hybridization
Non radioactive in situ hybridization was performed as described previously [30–32]. Briefly, antisense and sense single-strand cRNAs were synthesized from the bovine C7 cDNA fragment (1010 bp) cloned into pBluescript SK, as described in 2.5. For the in vitro transcription an RNA transcription kit (Stratagene, Heidelberg, Germany) and digoxygenin-labeled UTP (Boehringer, Mannheim, Germany) was used. The antisense and sense probes have been characterized by agarose gel electrophoresis and Northern blots. The antisense riboprobe recognized the correct 3.8 kb band, while the sense probe gave no signal.

Human control vessels and aortic aneurysms were directly frozen in liquid nitrogen using isopentan. The tissue sections were fixed in 4% paraformaldehyde in PBS, pH 7.4, and acetylated for 15 min at room temperature in freshly prepared 0.25% acetic anhydride in 0.1 M triethenolamine HCl plus 0.9% NaCl, pH 8.0, after washing three times with PBS. Afterwards, the glass slides were dehydrated with 10, 25, 50, 75, 95 and 100% ethanol. The sections were prehybridized for 2 h at room temperature with hybridization solution containing 4xSSC, 5% dextran sulfate, 100 mM Dithiothreitol, 500 µg/ml denatured salmon sperm DNA (Sigma-Aldrich, Deisenhofen, Germany), 250 µg/ml yeast transfer RNA (Boehringer, Mannheim, Germany), 1xDenhardt's solution [22], and 50% (vol/vol) formamide. After prehybridization, hybridization solution plus 5–10 ng cRNA probe per µl solution was applied to each section, followed by incubation in a moist chamber for 20 h at 37°C. Samples were washed 10 min in 2xSSC at 37°C, 20 min in 1xSSC at 37°C, 1 h in 0.5xSSC plus 50% dimethylformamide at 42°C and 10 min in 0.1xSSC at 37°C. For immunological detection the DIG nucleic acid detection kit (Boehringer, Mannheim, Germany) was used according to the manufacturer's instructions. The sections were counterstained with Sigma Fast Red (Deisenhofen, Germany) and embedded with Kaiser's glycerol gelatin (Merck, Darmstadt, Germany).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Subtractive hybridization and cDNA library screening for TNF-{alpha} suppressed genes
To isolate genes suppressed by a 6 h incubation of BAEC with 1 nM TNF-{alpha}, a subtractive cDNA library was constructed into the lambda ZAP II vector. The first strand cDNA was synthesized from poly(A)+ RNA isolated from unstimulated endothelial cells. It was hybridized with a 10-fold excess of poly(A)+ RNA obtained from BAEC incubated with TNF-{alpha} for 6 h. After selection by hydroxyapatite chromatography the cDNAs coding for genes suppressed in BAEC treated for 6 h with 1 nM TNF-{alpha}, was 20 times enriched. The size of the original library was about 23,000 plaque forming units with most of the inserts ranging from 0.5 to 2.5 kb, as determined by DNA agarose gel electrophoresis (data not shown). The library was amplified once.

Approximately 50 000 recombinant phage plaques were screened by differential hybridization. Initially, 600 differential signals were detected. A second screening of the positive plaques revealed that 398 (about 67%) of the isolated phages consistently showed differential signals. To exclude sequence-related cDNA clones, cross-hybridization was performed by labeling the inserts of randomly picked clones to hybridize against all other isolated phages. Those showing cross-hybridization were considered to be derived from an identical mRNA [28]. After successive rounds of cross-hybridization, 49 clones were considered as unique sequences. Based on the density of the signals and the degree of suppression, 6 clones were further analyzed.

3.2 Sequences of TNF-{alpha} suppressed genes
The isolated cDNAs were partially sequenced and at least 200 base pairs were analyzed from each end of the cDNA. The sequence data were screened for similarity with previously identified genes. This led to the identification of three clones: connective tissue growth factor (CTGF; A1), fibronectin (A8) and the mitochondrial genome (B1). The sequences of the clones C1, C5 and C7 did not show apparent identity with genes published in the data bases searched. The full length cDNA sequence of the gene C7 was obtained and the sequence data were submitted to GenBank (accession number AF031163). Analysis of the 1010 bp C7 sequence revealed the cloned cDNA to code for the 3' untranslated region of a new gene.

3.3 Time course and dose dependence of TNF-{alpha} mediated gene suppression on mRNA level
Dot hybridization was used to determine the time dependence of gene suppression. Poly (A)+ RNA from BAEC incubated with 1 nM TNF-{alpha} for different periods of time was labeled by reverse transcription, and hybridized to the isolated cDNAs in the vector pBluescript SK. GAPDH and pBluescript SK served as controls. As shown in Fig. 1A, the suppression of the analyzed genes after TNF-{alpha} stimulation was significant after at least 4 h. The mRNA of the mitochondrial genome (B1) and C5 reached a minimum level at 4 h after addition of TNF-{alpha}, and maintained at a level that was lower than in unstimulated cells until 16 h. The suppression of fibronectin (A8) and C1 was maximal at 8 h and stayed at a lower level than in unstimulated cells until 16 h. By contrast, the expression of CTGF (A1) was significantly suppressed after 8 h and reached a minimum at 16 h, the longest time point analyzed. The maximal suppression for C7 was detected between 4 and 8 h after TNF-{alpha} addition and till 16 h the mRNA expression did not recover.


Figure 1
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  		Fig. 1 Time course and dose response of TNF-{alpha} mediated gene suppression in cultured BAEC. [{alpha}-P32]-labeled cDNAs from confluent BAEC treated with 1 nM TNF-{alpha} for 0 to 16 h (A), or BAEC treated with different concentrations of TNF-{alpha} for 16 h (B) were hybridized against the pBluescript SK plasmids containing inserts of the cDNA clones studied. The cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to standardize the hybridized cDNAs and the plasmid pBluescript SK (vector) served as background control.

 
Suppression of gene expression was also dependent on the dose of TNF-{alpha} (Fig. 1B). The effect of TNF-{alpha} was half-maximal at about 200 pM, corresponding to the affinity of the TNF-receptors. Heat inactivated TNF-{alpha} had no effect on cultured BAEC.

3.4 Nuclear run-on transcription assay
To determine whether TNF-{alpha} downregulation of the gene expression occurred at the transcriptional level, we performed a nuclear run-on analysis. For this purpose, we isolated nuclei from BAEC untreated or treated with 1 nM TNF-{alpha} for 0.5 to 4 h. A plasmid containing cDNA for bovine vimentin, which is not TNF-{alpha} responsive, showed strong equivalent hybridization in all experimental groups. As shown in Fig. 2A, two genes (CTGF; A1, and C7) were suppressed at the transcriptional level. The transcriptional suppression of CTGF (A1) and C7 was significant already within 30 min after TNF-{alpha} stimulation. Afterwards the CTGF (A1) expression was continuously downregulated to a weak signal above background until 4 h, the longest time point analyzed. Transcriptional suppression of these genes occurred also in the presence of cycloheximide (Fig. 2B), indicating that protein synthesis was not involved. The other genes tested in nuclear run-on analysis did not reveal TNF-{alpha} mediated suppression (data not shown). Hence, suppression of these transcripts does not occur at the level of transcription.


Figure 2
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  		Fig. 2 Genes suppressed at the transcriptional level (Nuclear run-on analysis). Nuclei were isolated from untreated or 1 nM TNF-{alpha} treated BAEC at the indicated time points (A). BAEC were pretreated for 30 min with cycloheximide (CHX, 10 µg/ml) and further incubated for 2 h ± addition of 1 nM TNF-{alpha} (B). The newly transcribed mRNAs were hybridized against the pBluescript SK plasmids containing inserts of the cDNA clones studied. The cDNA for vimentin was used to standardize the signals and the plasmid vector (vector) served as background control. Only the genes suppressed at the transcriptional level are shown in this figure.

 
3.5 Expression of TNF-{alpha} suppressed genes in different adult organs
Organ specific expression of the 6 TNF-{alpha} suppressed genes was studied by dot hybridization using cDNA probes derived from mRNA isolated from bovine organs. Expression in these organs was compared with the expression in cultured BAEC, fibroblasts and SMC (Fig. 3). The gene A1, coding for CTGF, was most abundant in endothelial cells and also expressed in highly vascularized tissues (liver, lung and kidney). It was almost undetectable in skeletal and cardiac muscles. Fibronectin (A8) was more prominently expressed in SMC, and tissues like liver, lung and aorta than in cultured BAEC. The mitochondrial genome (B1) was most abundant in BAEC and also highly expressed in SMC, fibroblasts and lung. C1 was expressed ubiquitous in all cultured cells and tissues tested, with exception of the aorta. C5 Showed the highest expression in kidney and was undetectable in SMC and spleen tissue. Low amounts of C7 mRNA transcripts were detected in cell extracts of BAEC, heart, skeletal muscle, aorta, testis and ovary. No C7 was detected in a whole liver extract.


Figure 3
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  		Fig. 3 Expression of TNF-{alpha} suppressed genes in cultured cells of the vessel wall and adult bovine organs. [{alpha}-P32]-labeled cDNAs from cultured bovine aortic endothelial cells (BAEC), vascular smooth muscle cells (SMC), and fibroblasts (Fb), as well as cDNAs derived from extracts of bovine organs were used to hybridize against the pBluescript SK plasmids containing inserts of the cDNAs studied. The cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for standardization and the plasmid vector (vector) served as background control.

 
3.6 TNF-a mediated gene suppression in different cell types of the vessel wall
BAEC, SMC and fibroblasts were incubated for 16 h with 1 nM TNF-{alpha} to compare whether there are cell type specific responses to TNF-{alpha} with respect to the 6 genes studied. Northern blot analysis showed suppression of all 6 genes in cultured BAEC (Fig. 4). The genes CTGF (A1) and mitochondrial genome (B1) were suppressed in all cell types tested. C1 and C7 showed no significant downregulation in fibroblasts and SMC, whereas these genes are significant downregulated by TNF-{alpha} in BAEC. Fibronectin (A8) was suppressed in BAEC and fibroblasts and not detectable in SMC. C5 was downregulated in BAEC and in fibroblasts. Therefore, the response of cultured vascular cells to TNF-{alpha} is not uniform.


Figure 4
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  		Fig. 4 TNF-{alpha} mediated gene suppression in different cell types of the vessel wall. Bovine aortic endothelial cells (BAEC, column and lane 1, 2), fibroblasts (Fb, column and lane 3, 4) and vascular smooth muscle cells (SMC, column and lane 5, 6) were analysed by Northern blots before (Control, light shading) and after a 16 h incubation with 1 nM TNF-{alpha} (TNF, dark shading). The Northern blot signals were quantitated by densitometry, where GAPDH served as normalization standard. The calculated relative densitometry units are shown as bar graphs and the hybridization signals are depicted below. The size of the mRNAs was determined based on 18 S and 28 S ribosomal RNA. The molecular weight of the band recognized by the different cDNA clones is placed in brackets next to the obtained signal.

 
3.7 Expression of the C7 mRNA in vivo in human advanced aortic aneurysms
Sections for in situ hybridization were obtained from human control vessels and advanced aortic aneurysms. Digoxygenin labeled antisense and sense riboprobes for a non radioactive in situ hybridization were synthesized from the cloned bovine full length C7 cDNA fragment. A healthy control artery, an uninflamed and an inflamed advanced aortic aneurysm were investigated for the expression of the C7 RNA. No C7 RNA transcripts were detected in healthy control arteries (Fig. 5A) and inflamed advanced aortic aneurysm with high amounts of infiltrated inflammatory cells around the vessels (Fig. 5C). By contrast, the expression of the C7 RNA was strongly induced within the endothelial cells in uninflamed advanced aortic aneurysm (Fig. 5B). In vivo C7 transcripts were not detected in other cells of the vessel wall than the endothelial cells, indicating a cell specific overexpression in uninflammed advanced aortic aneurysm. Within advanced aortic aneurysms the endothelial C7 RNA expression is altered by inflammatory cells, since in serial sections of a single atherosclerotic patient C7 expression decreased in areas of inflammatory cell accumulation.


Figure 5
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  		Fig. 5 Expression of the C7 mRNA in vivo in advanced human aortic aneurysms. Sections were obtained from frozen human control vessels and aortic aneurysms. Digoxygenin labeled antisense (AS) and sense (S) riboprobes for a non radioactive in situ hybridization were synthesized from the cloned bovine full length C7 cDNA fragment. The cell specific mRNA expression of C7 was investigated in a healthy control artery (A), in an uninflamed aortic aneurysm (B), and an inflamed aortic aneurysm (C). Pictures of the vessels were taken with a 200xmagnification. The tissues were counter stained with Sigma Fast Red and positive signals are recognizable by a brown colour. C7 sense (S) served as negative control.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Most data available about the TNF-{alpha} effect on cultured endothelial cells refer to the immediate (10 min to 3 h) response. In the present study we asked whether gene suppression is a prominent feature of the intermediate (6 to 16 h) endothelial cell response to TNF-{alpha}. Due to the time point (6 h) selected, we did not find any of the genes known to be suppressed by TNF-{alpha} within 1 h [16–18]. For the construction of the subtractive cDNA library the cDNAs were digested with Xho I to release the Xho I adapters before cloning into the lambda ZAP II phage arms. Therefore, genes like thrombomodulin, the nitric oxide synthase and the vascular endothelial cell growth factor receptor KDR/flk-1 exposing a Xho I restriction site could not be cloned using this strategy. We isolated 49 genes previously not described as downregulated by TNF-{alpha} in cultured endothelial cells and further analyzed 6 of them. This suggests that gene suppression is indeed an important feature of the TNF-{alpha} mediated, intermediate change in the endothelial cell phenotype. Downregulation of the studied genes occurred by two mechanisms: (i) 4 genes are only downregulated in a time and dose dependent manner in dot blots (Fig. 1), indicating posttranscriptional mechanisms and (ii) 2 of the 6 genes analyzed are suppressed independent of protein synthesis at the transcriptional level (Fig. 2). Primary response genes, such as the 2 genes suppressed at the transcriptional level, have in other systems been shown to encode paracrine factors, initiating a cascade of molecular events, finally resulting in alterations of the cellular phenotype [12, 29, 33]. Future studies will be required to learn more about the biological properties of proteins encoded by the cDNAs with previously unpublished sequences.

Some data are available with respect to the functional significance of gene suppression by TNF-{alpha}: One example is the TNF-{alpha} mediated increase in vascular permeability [34, 35], which is a hallmark of inflammatory reactions. In vitro studies showed that fibronectin is decreased in the extracellular matrix after endothelial cells have been exposed to TNF-{alpha} [36]. A suppression of fibronectin (A8) by TNF-{alpha} in cultured BAEC could not be detected by nuclear run-on analysis at the transcriptional level in BAEC. Therefore, we conclude that fibronectin (A8) downregulation occurs at least in part by TNF-{alpha} mediated destabilization of the mRNA [37]. Addition of fibronectin to the culture medium prevents the TNF-{alpha} induced enhancement of the monolayer permeability [36], suggesting that TNF-{alpha} mediated suppression of fibronectin in endothelial cells is an important event in the cytokine mediated capillary leak syndrome. This is further supported by the finding, that synthetic fibronectin peptides are capable of inhibiting infiltration of inflammatory cells in transforming growth factor-β1 knock out mice [38].

The early developing stage of arteriosclerosis is accompanied with high local TNF-{alpha} concentrations within the vessel wall in foam cells. In advanced arteriosclerotic lesions TNF-{alpha} is decreased and vascular smooth muscle cells proliferate. The most abundant clone isolated in our screening is connective tissue growth factor (CTGF; A1). We found CTGF (A1) to be expressed in endothelial cells, fibroblasts and vascular smooth muscle cells. Furthermore, this gene is downregulated by TNF-{alpha} in all cell types tested (Fig. 4). CTGF is a secreted growth factor for mesenchymal cells [39, 40]mediating its signal at least in part by binding to the platelet-derived growth factor receptor. CTGF was found to be expressed in high levels in advanced arteriosclerotic lesions and therefore, it is a potential participant in the development of arteriosclerotic lesions [41]. However, in our study we found CTGF (A1) to be suppressed by TNF-{alpha} in BAEC, fibroblasts and smooth muscle cells, indicating that TNF-{alpha} is not the only mediator relevant for arteriosclerosis. TGF-β has recently been shown to induce CTGF in smooth muscle cells and fibroblasts [41, 42], consistent with the hypothesis that TGF-β controlled gene expression is important in the pathogenesis of vascular disease.

The disease activity of the cloned new gene C7 in vascular diseases was investigated by in situ hybridization in advanced aortic aneurysms. The C7 RNA was found to be overexpressed exclusively in the endothelium of human vessels in uninflammed advanced aortic aneurysm (Fig. 5B) and undetectable in control arteries (Fig. 5A), indicating the gene potentially participating in the development of aortic aneurysms. C7 RNA transcripts were undetectable in endothelial cells in inflamed advanced aortic aneurysm with large amounts of infiltrated inflammatory cells within the vessels (Fig. 5C). Downregulation of the endothelial cell specific gene C7 was shown in vivo in inflamed advanced aortic aneurysm accompanied by high local TNF-{alpha} concentrations and in vitro by TNF-{alpha} in endothelial cells. Therefore, downregulation of the C7 cDNA occurs in endothelial cells in vitro and in vivo.

C7 expression was detected in RNA extracts of the bovine aorta using the method of dot hybridization (Fig. 3) whereas no transcripts were found within normal human arteries by in situ hybridization (Fig. 5A). The observed difference in the C7 expression is due to the sensitivities of the methods used because in whole tissue extracts of human control arteries C7 was detected by dot hybridization (data not shown). It is possible that the bovine probe is not sensitive enough to give above background signals in human tissue sections expressing only low amounts of C7 using the technique of in situ hybridization.

It remains unclear whether the C7 signal in whole extracts of skeletal or cardiac muscle is indicative of a strong endothelial C7 signal or other cells than endothelial cells. The negativity of liver extracts despite the high vascularization suggests the C7 signal in skeletal and cardiac muscle to be derived from non vascular cells, since the liver contains not fever endothelial cells than heart or skeletal muscle. In addition C7 mRNA was found in cultured smooth muscle cells and fibroblasts (Fig. 4). Only a careful organ survey using in situ hybridization can clarify this point.

Suppression of CTGF (A1; resulting in decreased proliferation of fibroblasts and smooth muscle cells), suppression of fibronectin (A8; enhancing the capillary permeability, an important initial step in angiogenesis), and low expression of C7 (overexpression was detected in advanced aortic aneurysm) can all be interpreted as protective for the vascular system. Our hypothesis is that cytokine mediated enhanced capillary permeability, and suppression of growth factors for subendothelial cell layers prevent hypoxia in proliferating tissue, as it occurs in TNF-{alpha} mediated tissue remodelling. However more detailed studies are required to confirm this hypothesis. The genes isolated might provide new tools for the investigation of vascular disease. In this respect differences in the cellular response of endothelial cells, fibroblasts and vascular smooth muscle cells (Fig. 4) open additional ways for the study of the differential TNF-{alpha} signalling in various cells of the vascular wall.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft No. 138/3-1, Mildred Scheel Stiftung (P. Nawroth) and the Sonderforschungsbereich 405 (Deutsche Forschungsgemeinschaft, P. Nawroth). P. Nawroth performed this work as a Heisenberg scholar (DFG) and the tenure as a Schilling-professor. U. Schimanski was supported by the faculty of Medicine, University of Heidelberg, D-69120 Heidelberg, Germany. We thank Katsumi Nakagawa for his helpful comments.


   Notes
 
1 The first two authors contributed equally to the work presented. Back

2 Current address: Department of Surgery, Harvard Medical School, Boston, USA. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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