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* Fraunhofer Institute of Toxicology and Aerosol Research, Department of Molecular Toxicology and Pharmacokinetics, Hannover, Germany; and
Klinik für Thorax-, Herz und Gefäss-Chirurgie, Medical School of Hannover, Hannover, Germany
1Correspondence: Fraunhofer Institute of Toxicology and Aerosol Research, Department of Molecular Toxicology and Pharmacokinetics, Nicolai-Fuchs-Str. 1, D-30659 Hannover, Germany. email: Borlak{at}ita.fhg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, GATA-2, and the silencer Oct-1 in conjunction with the gene
expression of all major cytochrome P450 genes and of eNOS in cultures
of endothelial cells of the rat. The purity of cultured endothelial
cells was also confirmed by flow cytometry measurements of PECAM-1, a
surface antigen of endothelial cells. Taken collectively, the gene
expression and flow cytometry studies provide strong evidence for
c/EBP, GATA-2, and Oct-1 to play a key role in the cellular
dedifferentiation of endothelial cells; gene expression of eight
individual CYP genes in conjunction with protein activity could be
significantly increased upon treatment with Aroclor 1254, a
well-documented chemical inducer of a battery of genes. Nevertheless,
the gene expression of c/EBP, GATA-2, and most of the CYP genes was
dramatically reduced (up to 90%) in cell cultures lacking PECAM-1
expression; in strong contrast, expression of the silencer Oct-1 was
massively increased (~14-fold). We thus conclude activation of the
silencer Oct-1 to be strongly correlated with loss of PECAM-1 and eNOS
gene expression, e.g., loss of cellular differentiation and endothelial
function; in conjunction, gene expression of all major P450 isoforms
was dramatically reduced in cultures of dedifferentiated endothelial
cells. This process of cellular dedifferentiation and endothelial
dysfunction was accompanied by down-regulation of endothelial specific
transcription factors.—Thum, T., Haverich, A., Borlak, J. Cellular dedifferentiation of endothelium is linked to activation and
silencing of certain nuclear transcription factors: implications for
endothelial dysfunction and vascular biology.
Key Words: endothelial cells • hepatocytes • gene expression • PECAM-1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The importance of
drug biotransformation in endothelial cells is now being recognized,
and several investigators report cytochrome P450-dependent drug
oxidation activity in endothelial cells; induction of endothelial CYP
monooxygenases can be achieved upon treatment with various chemical
agents (2
3
4
5
6
7)
.
The cell function of endothelium is complex and includes secretion of
vasorelaxing and constricting factors, such as prostacyclin,
endothelial-derived relaxing factor (EDRF), endothelial-derived
hyperpolarizing factor (EDHF), endothelial-derived contracting factor
(EDCF), or endothelin (8
, 9)
, coagulation factors such as
factor VIII antigen, von Willebrand’s factor, and plasminogen
activator, matrix factors including collagen, glycosaminoglycans,
fibronectin (10)
, and metalloproteinases, as well as
secretion of heparane and growth factors that regulate smooth muscle
cell proliferation (11)
. Endothelial cells are also well
characterized for their production of NO, which is the predominant form
of EDRF (8)
, and are well known for their ability to
metabolize plasma lipids, nucleotides, serotonine, catecholamines,
bradykinin, and angiotensin-1 (12)
.
Another important feature of endothelial cells is their antithrombotic
surface and their interaction with leukocytes, the secretion of
chemotactic molecules, and the presence of adhesion molecules including
platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular
cell adhesion molecule-1 (ICAM-1), ICAM-2, ELAM-1, vascular cell
adhesion molecule-1 (VCAM-1), and GMP-140 (13
, 14)
.
PECAM-1 is also expressed in human platelets, leukocytes, and
intercellular junctions of endothelial cells (13)
and is
an important cell adhesion molecule. This particular antigen is of
critical importance for cell migration, inflammatory processes, wound
healing, and angiogenesis (15)
; PECAM-1 expression is
regulated by several transcription factors including the c/EBP, NF
B,
GATA-2, and the CREB binding protein (16
17
18)
. There is
additional evidence for the activator protein 2 (AP-2) and the
platelet-activating factor to positively regulate gene expression of
PECAM-1 (16
, 21)
. In contrast, interferon 
, tumor
necrosis factor (19)
, and thrombospondin-1
(20)
reduce gene expression of PECAM-1.
The molecular mechanisms leading to endothelial dysfunction are ill
understood, and transcription factors are likely to play a key role in
this process. From a clinical point of view, endothelial dysfunction is
governed by impaired endothelium-dependant relaxation
(21)
; in the present study, long-term endothelial
dysfunction is considered to be the result of a cascade of molecular
events leading to cellular dedifferentiation.
We thus investigated the interplay of key transcription factors in
cultures of endothelial cells, which were isolated from the aorta of
rats to obtain a better understanding of the process of cellular
dedifferentiation at the molecular level. To obtain functional
parameters, gene regulation and protein activity of individual
cytochrome P450 isoforms were investigated by measuring mRNA expression
profiles of all major rat P450 isoforms and enzyme activity for the
CYP1A1 monooxygenase substrate ethoxyresorufin. Expression of the
transcription factors c/EBP and GATA2 and that of the Oct-1 were
investigated to elucidate their specific role during cellular
dedifferentiation. Finally, gene expression of endothelial nitric oxide
synthetase (eNOS) was used as a functional marker of endothelial cells;
the modulating effects of Aroclor 1254, a well-known inducer of CYP
monooxygenases (22)
, were also studied in cultures of
endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague Dawley and Lewis rats weighing 220 ± 8 g were obtained from Charles River (Sulzfeld, Germany). Food and water were given ad libitum.
Anesthesia
Rats were anesthetized with Ketamin (anesthetic) and Rompun
(muscle relaxant) with 0.1 ml of Ketamin per 100 g body weight and
0.05 ml of Rompun per 100 g body weight. In addition, 2000
international units of heparin were given intraperitoneally prior to
surgery.
The thorax was opened by surgical procedures, and the aorta thoracalis and abdominalis were carefully prepared and excised. After washing with phosphate-buffered saline (PBS) at 37°C, 4–6 ml preheated M199 medium (Biochrom, Berlin, Germany) was pumped through the blood vessel. Thereafter, one end of the aorta was closed and 500 µl of digestive enzyme solution (Trypsin/EDTA10x, Life Technologies, Inc., Eggenstein, Germany) was pumped into the blood vessel. Then the aorta was closed completely and put into a petri dish filled with M199 medium. After an incubation period of 20 min, the aorta was opened, the enzyme solution was trapped in a small beaker, the aorta was rinsed with 5 ml DMEM (Biochrom) with 20% fetal calf serum (FCS; Biochrom), and the combined perfusate was centrifuged at 300 g and 4°C for 10 min. The supernatant was discarded and the cell pellet was suspended in 1 ml DMEM with 20% FCS, prednisolone 9.6 µg/ml, glucagon 0.014 µg/ml (Novo, Mainz, Germany), insulin 0.16 µg/ml (Hoechst, Frankfurt, Germany), penicillin 200 U/ml, streptomycin 200 µg/ml (Biochrom), heparin 15 U/ml, nonessential amino acids 10 µl/ml, and bovine endothelial cell growth factor 30 µg/ml (Roche, Mannheim, Germany). The cell suspension was placed in a 24-well plate coated with 1% gelatin. Cells were placed in an incubator at 37°C in a 5% CO2 atmosphere and used after the third passage for further experiments.
Isolation and cultivation of hepatocytes
For isolation of hepatocytes, a modified method described by
Seglen (23)
was used.
In brief, after midline incision, the portal vein was cannulated and the liver was first perfused in situ with 100 ml calcium-free Krebs Ringer buffer (KRB) for 10 min, then with 100 ml KRB and EDTA (1 mmol/l). The liver was then perfused for 8–10 min with KRB supplemented with collagenase type IV (Worthington, Freehold, N.J.) and 0.5 mM calcium chloride (Sigma, Deisenhofen, Germany). After perfusion, the liver capsule was gently removed and the dissolved liver tissue was filtrated through a nylon mesh (pore size, 100 µm) and washed twice with the washing buffer (1000 ml Hanks balanced salt solution (PAA, Cölbe, Germany) supplemented with 2.4 g HEPES (Sigma) and 2 g bovine serum albumin (BSA; Sigma). The cell pellet was resuspended in William’s E medium (Biochrom) supplemented with 5% FCS (Biochrom), 9.6 µg/ml prednisolone, 0.014 µg/ml glucagon (Novo), 0.16 U/ml insulin (Hoechst), 200 U/ml penicillin, and 200 U/ml streptomycin (Life Technologies, Inc.).
Hepatocytes were counted in a hemocytometer in the presence of 0.04%
Trypan blue solution. Two million hepatocytes per dish were cultured
between two layers of collagen in a modification of the method of Dunn
et al. (24)
and used for further experiments 48 h
postisolation.
Modulation of gene expression
Modulation of gene expression was done with Aroclor 1254 (in
DMSO), a complex chemical mixture (>80) of individual PCB isoforms and
congeners at a concentration of 10 µM for 24 h. Appropriate
controls were treated with DMSO, but not with Aroclor 1254.
Flow cytometry
The fluorescence-activated flow cytometry assay is based on a
CD31 rat fluorescein isothiocyanate (FITC) -conjugated antibody and
mouse IgG-FITC as a negative control (Serotec, Kidlington, U.K.).
Initially, cells from a confluent layer were isolated with accutase
(PAA) and washed with PBS containing 1% BSA. The resultant suspension
was centrifuged at 300 g and 4°C for 5 min. The
supernatant was discarded and the cell pellet was incubated with a CD31
antibody (Serotec) at 4°C in the dark for 30 min. After incubation
with the CD31 antibody, cells were washed twice in 1 ml PBS containing
1% FCS, resuspended, and recentrifuged at 300 g and
4°C for 5 min. Again, the supernatant was discarded and cell pellets
were resuspended in the latter buffer. Prior to the FACSCAN analysis,
cell pellets were resuspended in 500 µl PBS containing 1% BSA, and
10 µg propidiumiodide was added. Analysis were carried out on a
FACSalibur flow cytometer (Becton & Dickinson, Heidelberg, Germany)
with an Argon laser at a 488 nm excitation wave length, the green FITC
fluorescence at 530 nm and the red fluorescence of propidiumiodide at
585 nm.
EROD assay
This assay was done essentially as described by Grant et al.
(25)
. Control cells and cells treated with Aroclor 1254
were incubated with 2 µM of ethoxy-resorufin-O-deethylase (EROD) and
aliquots were removed from the cultures at 0, 1, 2, 3, and 7 h.
Appropriate aliquots were taken from the samples, treated with ammonium
acetate (200 mM, pH 4.5) and 100 U/ml of ß-glucuronidase, and
incubated at 37°C overnight. Five hundred microliters of glycine
buffer (pH 10.3) was added to the sample containing ß-glucuronidase
and subsequent fluorometric analysis was carried out on a
spectro-fluorophotometer (RF-1501, Schimadzu). The other aliquot was
treated with ammonium acetate buffer (250 µl) and glycine buffer (500
µl), but without ß-glucuronidase. The fluorometric analysis was
done at an excitation wave length of 530 nm and an emission wave length
at 585 nm. Calibration of the system was done with appropriate
standards at a concentration range of up to 100 nM.
RNA and cDNA
RNA was isolated from endothelial cells using the SV total RNA
Isolation System (Promega, Mannheim, Germany) according to the
manufacturer’s recommendation. Quality of isolated RNA was checked
using a 1.0% agarose gel. Four micrograms total RNA from each sample
was used for reverse transcription. RNA and random primer (Roche) were
preheated for 10 min at 70°C. Then 1x RT-AMV-buffer, dNTPs (Roche,
1.0 nM), 40 U RNase inhibitor (Stratagene, Amsterdam, Netherlands), 20
U AMV-RT (Promega) were added and diethyl pyrocarbonate-treated water
was given to a final volume of 20 µl. Reverse transcription was
carried out for 60 min at 42°C and stopped by heating to 95°C for 5
min. The resulting cDNA was frozen at -20°C until further
experimentation.
Thermocycler reverse transcription-polymerase chain reaction
(RT-PCR)
For PCR amplification of cDNA, a 25 µl reaction mixture was
prepared containing 10x polymerase reaction buffer, 3 mM
MgCl2, 0.4 nM dNTPs(Roche), 400 nM concentration
of the 3' and 5' specific primer (synthesized by Life Technologies,
Inc.), 1 U Taq-polymerase (Roche), and 1 µl of cDNA.
PCR reactions were carried out in a thermal cycler (T3, Biometra,
Göttengin, Germany) using the following melting, annealing, and
extension cycling conditions: denaturation for 30 s at 94°C,
annealing for 60 s at 57°C, and extension for 60 s at
72°C (29 cycles) for CYPs, ß-actin, and GAPDH. PCR conditions for
GATA-2 were denaturation for 60 s at 94°C, annealing for 60 s at 62°C, and extension for 180 s at 72°C for 31 cycles. PCR
conditions for c/EBP, Oct-1, and eNOS were denaturation for 45 s at 94°C, annealing for 45 s at 58°C, and extension for
60 s at 72°C for 30 cycles. Primer sequences are shown in
Table 1
. PCR products were separated using a 1.8% agarose gel, stained with
ethidium bromide, and photographed on a transilluminator. cDNA products
were also sequenced by Seqlab (Göttingen, Germany).
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Real-time semiquantitative RT-PCR
cDNA was diluted 1:10 with nuclease-free water and 1 µl of the
diluted cDNA was added to the Lightcycler-Mastermix (0.5 µM of
specific primer, 3 mM MgCl2, and 2 µl
Master-SYBR-Green, Roche). This reaction mixture was filled up with
water to a final volume of 20 µl. PCR reactions were carried out in a
real-time PCR cycler (Lightcycler, Roche).
After an initial denaturation step at 95°C for 30 s, the PCR reaction was initiated with an annealing temperature of 57–62°C for 6 s, followed by an extension phase at 72°C for 11–33 s and a denaturation cycle at 95°C for 1 s. The PCR reaction was completed after a total of 35–45 cycles; at the end of each extension phase, fluorescence was observed and used for quantitative purposes.
A melting point analysis was carried out by heating the amplicon from 65°C to 95°C and a characteristic melting point curve was obtained for each product.
In addition a serial dilution experiment was carried out (n=3) with cDNA produced from endothelial total RNA using a 1:10 dilution regiment to estimate the mRNA expression level of the specific genes. Control samples contained water instead of cDNA; note that oligonucleotide dimers were produced in the PCR mixture, but could easily be distinguished by the melting point analysis.
Statistical analysis
The Wilcoxon signed rank test was used and differences were
considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows typical PECAM-1-positive endothelial cells, e.g., a
regular, ordered, and densely packed confluent cell layer with
condensed nuclei is observed. In contrast, PECAM-1-negative cells are
disorganized, with large intercellular spaces and less prominent nuclei
(see Fig. 1B
). Both cultures are the third passage of
isolated aortic endothelial cells.
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Flow cytometry
Endothelial cells were assayed for PECAM-1 expression by
fluorescence-activated flow cytometry. Mouse IgG coupled to FITC was
used to estimate the level of background fluorescence as shown in
Fig. 2A
.
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In one experiment, over 95% of endothelial cells were positive for
PECAM-1 (n=5); a typical result for PECAM-1-positive
cultures is shown in Fig. 2C
. Less than 0.5% of all
PECAM-1-negative cell cultures (n=8) expressed this
particular antigen (see Fig. 2B
). The viability of
PECAM-1-positive and -negative cells was studied using the
propidiumiodide stain; on average, >95% of endothelial cells were
viable irrespective of the presence or absence of PECAM-1 expression.
When endothelial cells were isolated, cultivated, and passaged from Lewis rats >80% were PECAM-1 positive (n=8), whereas the success rate with Sprague Dawley rats was on average only 25–30% (n=9). This indicates a potential species difference for the successful isolation and subsequent cultivation of endothelial cells.
EROD activities as a marker substrate for cytochrome P450
monooxygenase
Ethoxyresorufin was incubated with endothelial and hepatocyte cell
cultures (positive control) as described in Materials and Methods. As
shown in Fig. 3A
, PECAM-1-positive endothelial cells metabolized
ethoxyresorufin in a time-dependent fashion and responded to treatment
with Aroclor 1254, a well-known inducer of this particular enzyme
system. In strong contrast, PECAM-1-negative cells metabolized
ethoxyresorufin only at minimal rates (see Fig. 3B
), the
difference in activity being ~50-fold between both cultures. An
approximate threefold higher rate of EROD activity could be determined
if hepatocyte cultures were compared with PECAM-1-positive endothelial
cell cultures 1 h after incubation (see Fig. 3C
).
However, if incubations with hepatocyte cultures were continued for up
to 7 h, a significant decline in the amount of EROD metabolites
could be observed. When these samples were subjected to digestion with
ß-glucuronidase, a large amount of metabolites was recovered,
indicating hepatocytes to be capable of catalyzing the glucuronidation
of EROD oxidative products (see Fig. 3D
). This was not seen
with endothelial cell cultures; indeed, endothelial cells may not be
capable of catalyzing this particular type of reaction. Based on the
results shown in Fig. 3D
and within 1 h of incubation,
~50% of the overall metabolic activity can be accounted for by the
glucuronidase activity in hepatocytes. After 2 h of incubation,
roughly 80% accounted for the glucuronide metabolites, which increased
to 90% and 95%, respectively, after 3 and 7 h. This shows that
PECAM-1-positive or -negative endothelial cells are incapable of
carrying out glucuronidation of EROD metabolites.
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Gene expression studies
Figure 4
depicts the gene expression profiles for CYP1A1, CYP1A2, CYP2B1/2,
CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, and the housekeeping genes
ß-actin and GAPDH. A comparison of PECAM-1-positive and
PECAM-1-negative cells provides clear evidence for CYP1A1 to be
strongly expressed in PECAM-1-positive cells and hepatocyte cultures;
in PECAM-1-negative cells, however, only a minor band can be detected,
and this finding correlates with the EROD activity described above.
CYP1A2 mRNA transcripts were detected in neither PECAM-1-positive or
-negative endothelial cell cultures. In contrast, CYP2B1 was expressed
in PECAM-1-positive cells, but again no mRNA transcripts could be
detected for PECAM-1-negative cells with the PCR assay. CYP2C11 mRNA
transcripts were detected in PECAM-1-positive cells, but
PECAM-1-negative cells produced only a faint band in bromide-stained
agarose gels. Expression of CYP2E1 mRNA was stronger in
PECAM-1-negative than -positive cells. Neither CYP3A nor CYP4A1 gene
transcripts could be detected in PECAM-1-positive or PECAM-1-negative
cells, but the housekeeping genes ß-actin and GAPDH were uniformly
expressed throughout all cultures.
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We investigated hepatocyte cultures as positive controls for CYP
monooxygenase gene expression, and there is clear-cut evidence for
CYP1A1 to be strongly expressed in controls and Aroclor 1254-treated
cultures. ICYP1A2 mRNA transcripts were also detected. When compared to
PECAM-1-positive cells, CYP2B1 mRNA was stronger expressed in
hepatocyte cultures; in the case of CYP2C11, expression levels were
equal for hepatocytes and PECAM-1-positive endothelial cells. The
expression of CYP2E1 did not change in hepatocyte cultures upon
treatment with Aroclor 1254, and mRNA transcripts for PECAM-1-negative
endothelial cells and for hepatocyte cultures are similar. As shown in
Fig. 4
, CYP3A1 and CYP4A1 are expressed at the mRNA level in rat
hepatocyte cultures, and we can demonstrate that treatment with Aroclor
1254 enhanced the gene expression of the latter two genes ~threefold
(see below). The housekeeping genes ß-actin and GAPDH are similar and
are uniformly expressed in controls and Aroclor 1254-treated cell
cultures.
Real-time semiquantitative RT-PCR
The relative gene expression of CYP1A1 is shown in Fig. 5a
, with CYP1A1 mRNA expression in control hepatocytes
being set as the 100% value. When hepatocyte cultures were treated
with Aroclor 1254 for 24 h, gene expression increases ~fourfold
and in PECAM-1-positive endothelial cells ~sixfold. If this gene
expression is compared with control PECAM-1-positive endothelial cells,
however, an approximate threefold increase is observed.
PECAM-1-negative cells did not respond to Aroclor 1254 treatment, and
overall expression of individual genes except for CYP2E1 and the
housekeeping genes was significantly suppressed when compared to the
appropriate controls.
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CYP1A2 mRNA was only detected in hepatocyte cultures, and its
expression was induced ~sixfold upon treatment with Aroclor 1254
(Fig. 5b
). CYP2B1/2 increased ~twofold in Aroclor
1254-treated hepatocyte cultures, but PECAM-1-positive endothelial
cells did not respond to this treatment; with PECAM-1-negative
endothelial cell cultures, gene expression of CYP2B1/2 was
significantly reduced or even absent, as shown in Fig. 5c
.
Gene transcripts for CYP2C11 did not increase upon treatment with
Aroclor 1254 irrespective of the cell type, e.g., hepatocyte or
PECAM-1-positive endothelial cell cultures, but was significantly
reduced in PECAM-1-negative cells (~10% of original value, see Fig. 5d
).
CYP2E1 mRNA transcripts did not change upon treatment of hepatocyte
cultures with Aroclor 1254, and a similar result was obtained when
PECAM-1-positive and -negative endothelial cells were compared (Fig. 5e
). Unlike the other CYP genes, PECAM-1-negative
endothelial cells had a strong expression of CYP2E1. As stated above,
CYP3A and CYP4A transcripts were not expressed in any of the
endothelial cells cultures, whereas in hepatocyte cultures a strong
expression of the latter genes could be observed, especially upon
treatment with Aroclor 1254, the relative increase being ~threefold
(see Figs. 5f-h
). GAPDH gene expression was identical
throughout all cell cultures and was not altered upon treatment with
Aroclor 1254, as shown in Fig. 5i
.
eNOS gene expression
The gene expression of eNOS was studied because of its unique
expression in functional endothelium, and its expression differed
dramatically when PECAM-1-positive and -negative cultures of
endothelial cells were compared. The data shown in Fig. 6
and
7d
provide strong evidence for eNOS gene expression to be
reduced to 5% of control values.
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There was also evidence for eNOS gene expression to be reduced to
~80% in Aroclor 1254-treated endothelial cell cultures, thus
indicating a process of cellular deregulation on chemical treatment
(see Fig. 7d
).
Gene expression of nuclear transcription factors
The nuclear transcription factors GATA-2 and c/EBP were
chosen for their known role in gene regulation of PECAM-1. GATA-2
expression was reduced to ~10% of control values in PECAM-1-negative
cells, as shown in Fig. 7a
. A similar finding is obtained
when c/EBP gene expression is studied in PECAM-1-positive and
-negative endothelial cell cultures; indeed, the gene expression of
c/EBP was reduced to ~10% of control values in cell cultures
lacking PECAM-1 expression (see Fig. 7b
). Treatment of
PECAM-1-positive endothelial cell cultures with Aroclor 1254 resulted
in a significant increase of transcription factor mRNA expression
(c/EBP ~6-fold, GATA-2 ~11-fold) when compared with controls,
but endothelial cells not expressing PECAM-1 did not respond to this
treatment (Fig. 7a, b
).
Oct-1 gene expression
Oct-1 is a known silencer for the transcriptional activation of a
wide range of physiologically important genes, including the CYP1A1
gene. We show in this study that Oct-1 gene expression is significantly
elevated (~14-fold) in PECAM-1-negative cells, but its expression is
minimal in PECAM-1-positive cells (see Fig. 6
and Fig. 7c
).
This finding correlates well with CYP1A1 mRNA expression, which was
significantly reduced in endothelial cell cultures lacking PECAM-1
expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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to play a major role in endothelial
function. We also show that highly differentiated endothelial cells are
capable of metabolizing (oxidizing) xenobiotic substances and that the
expression of cytochrome P450 monooxygenases is regulated, at least in
part, by the transcription factors GATA-2, c/EBP, and the
transcriptional silencer Oct-1.
The rationale for the selection of the above-named transcription
factors is based on their importance in regulating gene expression in
the endothelium, e.g., PECAM-1, eNOS, and CYP monooxygenases (16
, 26
, 27)
, and our experimental strategy encompassed a concomitant
assessment of transcription factors and subsequent genes regulated by
these transcription factors. In the case of GATA-2 and c/EBP, evidence
is already available for PECAM-1 gene expression to be regulated, at
least in part, by these transcription factors (16
, 17)
.
GATA-2 also regulates ICAM-2 (28)
and endothelin gene
expression (29)
, whereas Oct-1 was recently discovered as
a silencer for CYP1A1 monooxygenases (30)
and for the
blood coagulating von Willebrand factor (31)
. Figure 8
depicts known binding sites for transcription factors in the PECAM-1
and CYP1A1 gene (16
, 27)
. The differential gene expression
of transcription factors in conjunction with flow cytometry and
morphological examinations provides the rational for a potential
mechanism of cellular dedifferentiation based on gene activation and
silencing of a battery of genes by distinct transcription factors. We
also show that induction with chemical agents, such as Aroclor 1254,
result in a coordinate mRNA induction of GATA-2 and c/EBP and of CYP
monooxygenase mRNA in highly differentiated endothelial cells, which
are positive for PECAM-1 (based on flow cytometry measurements with
CD31 fluorescent-labeled antibodies) and eNOS gene expression; it was
remarkable that abolition of PECAM-1 expression was accompanied by
highly significant reductions in GATA-2, c/EBP, and eNOS gene
expressions, whereas Oct-1 expression was massively increased. We thus
propose a process of endothelial cellular dedifferentiation that is
governed by the interplay of activation of Oct-1 and by silencing of
other transcription factors and subsequent down-regulation of a battery
of target genes, some of which were investigated in this study.
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Endothelial dysfunction is of major importance in vascular disease.
From a clinical point of view, atherosclerosis is a disease
characterized by endothelial dysfunction, particularly impairment of
endothelium-dependent relaxation (32)
. The
well-known vasospasm of diseased arteries appears to be related to a
defective generation or delivery of NO (33)
. Coronary
spasm leading to myocardial infarction is extremely relevant clinically
and could arise from this problem. We can show in this study that eNOS
gene expression is almost completely abolished in dedifferentiated
endothelial cells; likewise, the expression of CYP monooxygenases and
particular of CYP1A1 is dramatically reduced as shown by our gene
expression and protein activity data. The endothelium-derived
hyperpolarizing factor (EDHF) is a cytochrome P450 1A-linked metabolite
of arachidonic acid, as suggested by Adeagbo (7)
. Adeagbo
perfused the rat mesenteric prearteriolar bed with various solutions
and showed EDHF to most likely be a P4501A1-catalyzed metabolite of
arachidonic acid. In a more recent study by Coceani et al.
(33)
, a contractoral tension of the ductus arteriosos is
sustained by cytochrome P450-linked mechanisms. The authors isolated
ductus from near-term fetal lambs and guinea pigs to investigate the
effects on both muscle tone and endothelium for the formation of
1-aminobenzotriazol, a suicide substrate for cytochrome P450
monooxygenase reactions. The authors provide clear evidence that
1-aminobenzotriazol relaxation is due to suppression of a contractile
mechanism and not to activation of prostaglandin and NO-mediated
relaxing mechanism with cytochrome P450 being a key component in this
reaction cycle (34)
. Cytochrome P450
monooxygenase-regulated signaling of Ca2+ entry
in human and bovine endothelial cells was reported by Graier et al.
(4)
, who show that depletion of endothelial
Ca2+ stores activates microsomal P450
monooxygenase activity, which in turn synthesizes
5,6-epoxyeicosatrienoic acid. This arachidonic acid metabolite is in
turn a second messenger for activation of endothelial calcium entry.
The inhibition of NO-mediated responses by 7-ethoxyresorufin, a
substrate and competitive inhibitor of cytochrome P450 in rat aortic
rings, was studied by Li and Randt (6)
. Clear evidence was
obtained suggesting bioactivation of NO-donating substances (sodium
nitroprusside and glyceryl trinitrate) by CYP monooxygenases.
One of the first reports on cytochrome P4501A1 activity and its
induction in the endothelium of vertebrate heart was published by
Stegemann and co-workers (2)
. These authors show
microsomal EROD activity to be similar to those induced in the liver of
a teleost fish. With immunohistochemical methods, scup heart sections
of ~2–4 µm were stained with a monoclonal antibody;
immunohistochemical studies showed P4501A1 to be detectable only in
endothelial cells of the endocardium and coronary vasculature. Using
human umbilical vein endothelial cells (HUVECs), Farin et al.
(3)
provided clear evidence for the expression of
cytochrome P450s and microsomal epoxide hydrolase in primary cultures
of HUVECs. Using RT-PCR or Northern blot hybridization methods, a
number of cytochrome P450 isoforms could be detected at the mRNA level
(CYP2A1, CYP3A, CYP1A1, CYP1A2, but not CYP2B6). When these primary
cell cultures were treated with either Arochlor 1254 and/or
ß-naphthoflavone CYP1A1, mRNA levels were increased but not the
levels of CYP3A1 and CYP4A1 mRNA subsequent to PCB or
ß-naphthoflavone treatment (3)
. CYP1A1 and CYP2B was
also detected in primary and immortalized rat brain endothelial cells,
suggesting that cerebrovascular endothelial cells may express different
P450 isoforms when compared to arterial cells of aortic endothelium
(35).
In the present study, detailed information on the CYP gene expression of rat aortic endothelial cells was obtained and included positive identification of CYP1A1, CYP2B1/2, CYP2C11, and CYP2E1 mRNA in highly differentiated arterial endothelium; with the exception of CYP2E1, PECAM-1-negative cells basically lacked the gene expression of any of the CYP isoforms investigated. As shown above, CYP monooxygenases play an important role in vascular relaxation, and therefore the findings of the present study provide a comprehensive assessment of CYP gene expression during endothelial dysfunction.
In conclusion, the present investigation provides new insight into the molecular mechanisms of endothelial dysfunction based on the interplay of nuclear transcription factors and target gene expression. It is also evident that endothelial dysfunction is accompanied or even mediated by changes in oxidative metabolism. Endothelial dysfunction is governed not only by loss of eNOS gene expression, but also by a lack of PECAM-1 expression, a cellular adhesion molecule with great importance for vascular biology. Thus, endothelial dysfunction is governed by several events, including loss of eNOS and PECAM-1 expression. Whether the gene silencer Oct-1 is solely responsible for this process requires further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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