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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 184-192
The Vascular Endothelial-Cadherin Promoter Directs
Endothelial-Specific Expression in Transgenic Mice
By
S. Gory,
M. Vernet,
M. Laurent,
E. Dejana,
J. Dalmon, and
P. Huber
From the Commissariat à l'Energie Atomique (CEA),
Laboratoire de Transgenèse et Différenciation Cellulaire,
Département de Biologie Moléculaire et Structurale,
Grenoble, France; and the Istituto di Ricerche Farmacologiche Mario
Negri, Milan, Italy.
 |
ABSTRACT |
Vascular endothelial-cadherin (VE-cadherin) is a calcium-dependent
adhesive molecule, exclusively and constitutively expressed in
endothelial cells. Analysis of the VE-cadherin promoter fused to a reporter gene in bovine aortic endothelial cells showed three major functional regions. The proximal region alone ( 139, +24) promoted nonspecific transcription; the addition of the (289, 140) and (2226, 1190) domains abolished transcription in
fibroblasts while expression in endothelial cells remained unchanged,
suggesting that fragments (2226, +24) and longer contain the full
endogenous promoter activity. To study the transcriptional specificity
of the promoter region in vivo, we generated transgenic mice carrying the chimeric construct containing the (2486, +24) region. The promoter directed reporter expression in all examined organs of adult
transgenic mice. During embryonic development, transgene expression was
detected at the early steps of vasculogenesis. Later, the expression
persisted during development of the vascular system and was restricted
to the endothelial layer of the vessels. Together, these data provide
evidence for specific regulatory regions within the VE-cadherin
promoter. Furthermore, the identification of DNA sequences restricting
gene expression to the endothelium has many potential applications for
the development of animal models of cardiovascular or angiogenic
diseases or for the delivery of therapeutic molecules.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT of the vascular system is
one of the earliest and most critical steps during vertebrate
embryogenesis. In this process, morphogenic events, which lead to the
formation of the mature vascular endothelium, are closely associated
with successive differentiation stages. The acquisition of the
endothelial phenotype by mesodermal precursors has been essentially
documented by the expression of stable or transient
markers.1 Currently, our understanding of the molecular
cues that confer the endothelial phenotype is extremely limited. A
useful starting point for gaining insights into the mechanisms that
underlie endothelial differentiation is to characterize the regulatory
regions of genes that are specifically expressed in this cell type.
An important objective of endothelial promoter study is to define
molecular tools capable of directing endothelial-specific expression of
proteins of interest. Such promoters can be useful in the development
of transgenic animals in order to produce, for instance, animal models
of human vascular diseases, endothelial-specific expression of
dominant-negative mutants, or conditional gene targeting.
At present, the number of known endothelial specific (or at least
restricted) proteins is small. These include, but are not limited to,
von Willebrand factor,2 platelet/endothelial cell adhesion
molecule-1 (CD31),3 preproendothelin-1,4
tie-1,5 and P-selectin.6 However, these
molecules are not exclusively expressed in the endothelium. Other
markers such as E-selectin,7 vascular endothelial
growth factor receptors 18,9 and 2,10 and a
tyrosine-kinase receptor called tie-25 are specifically
expressed in endothelial cells, but essentially after cell activation
with inflammatory cytokines (for E-selectin) or during vascular
proliferation (for the vascular endothelial growth factor receptors and
tie-2).
In this report, we characterized the promoter activity of the
VE-cadherin (CD144) gene. This molecule, which belongs to the cadherin
family of adhesive receptors,11 is specifically localized at the interendothelial junctions, where it plays a crucial role in
vascular assembly.12 This protein is exclusively and
constitutively expressed by the endothelium of all types of
vessels.13,14 Moreover, VE-cadherin is expressed by
endothelial precursors as early as embryonic day 7.5 (E7.5) in the
mouse embryo.14 The gene of this molecule thus represents a
good candidate to study endothelial-specific transcriptional
mechanisms. Therefore, we were prompted to investigate the role of the
5 -flanking region of mouse VE-cadherin gene in promoter
activity. In previous work, we have identified and cloned the murine
gene coding for VE-cadherin as well as 10 kb of upstream
sequence.15 This gene contains 12 exons spanning more than
36 kb; the first exon is entirely untranslated and starts at one
transcriptional site. In this report, we used a 5-deletion strategy
and transient transfection assay to delineate cis-acting DNA
fragments in VE-cadherin promoter. Furthermore, we could define
a promoter fragment sufficient to confer endothelial specific
expression of a reporter gene in transgenic mice.
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MATERIALS AND METHODS |
Cloning procedures.
A 3.5-kb Sac I fragment isolated from clone  1,15
containing the promoter region, the first exon, and the beginning of
the first intron of mouse VE-cadherin gene, was subcloned into
pBluescript II (Stratagene, La Jolla, CA) and sequenced. (Sequence from
position 2486 to +24 is available in the EMBL databank under
accession no. Y10887). Constructs used for transient expression and
transgenic animals were produced in a multistep process. As no unique
restriction site was present within the first exon for subcloning into
the chloramphenicol acetyl transferase (CAT) reporter plasmid,
pBLCAT3,16 a polymerase chain reaction (PCR) strategy was
developed to create an artificial Xho I site downstream of
position +24. A PCR fragment was generated using clone 1 as matrix
and two primers, 5-CCCGGAAAGATCTGCTCTCT-3 and
5-CTCCACTCGAGTCTGTCCAGGGCCGAGC-3, containing the sequence centered
around the BglII site at position 1190 and the sequence from
position +6 to +24 of first exon followed by an Xho I site, respectively. This fragment was cut by BglII and Xho I
and inserted into the corresponding sites of pBLCAT3. This construct,
called 1190CAT, was verified by sequencing and constitutes the
starting material for the other CAT constructs. 773CAT, 675CAT,
289CAT, and 139CAT were obtained by insertion of Acc
I/Xho I, BamHI/Xho I,
HindIII/Xho I and Pst I/Xho I
fragments, respectively, into their cognate sites in pBLCAT3.
Constructs 515CAT and 187CAT were generated by ligation of
PvuII/Xho I and Apa I (blunted with T4 DNA
polymerase)/Xho I fragments into Sal I site, filled-in with Klenow enzyme, and Xho I site of pBLCAT3. 900CAT was
produced by inserting a filled-in Taq I fragment into the filled-in
Xho I site of pBLCAT3. For 5800CAT and 2226CAT, an
EcoRI (filled-in)/BglII and an Nhe I
(filled-in)/BglII fragments from clone 1 were ligated to
sites Sal I, filled-in, and BglII of 1190CAT.
Finally, construct 2486CAT was obtained by insertion of Spe
I/BglII fragment into Xba I and Bgl I sites of
1190CAT. The pBLCAT216 or pRSVCAT17 plasmids
containing the CAT gene driven by the herpes simplex virus
thymidine kinase (HSVTK) promoter or the long terminal repeats of the
Rous sarcoma virus, respectively, were used as positive control.
Cell culture.
Bovine aortic endothelial cells (BAEC) were prepared as described
previously18 and maintained in Dulbecco's modified
Eagle's medium supplemented with 15% fetal calf serum (Seromed,
Berlin, Germany), 100 U penicillin/mL, and 100 µg streptomycin/mL
BAEC from passages 3 to 12 were used for transfection experiments. NIH-3T3, HeLa, HepG2, HEL, and Lin 175 cell lines were obtained from
American Type Culture Collection (Rockville, MD) and
cultured in conditions identical to those for BAEC except that we used 10% fetal calf serum.
Transfections, CAT assay, and luciferase assay.
BAEC, NIH-3T3, HeLa, and HepG2 cells were transfected by the calcium
phosphate precipitation method as previously described.19 HEL and Lin 175 cells were transfected by electroporation with a gene
pulser (Bio-Rad, Hercules, CA) set at 400 V and 960 µF, in a total
volume of 800 µL. All cell lines were transfected with 3.5 pmol of
appropriate CAT construct, which is the equivalent of 10 µg
of pBLCAT3, and 5 µg of luciferase reporter plasmid, pGL3-control
(Promega, Madison, WI), to correct for variability in transfection
efficiency. Cell extracts were prepared 48 hours later and luciferase
activity was determined on an aliquot with a luciferase assay system
(Promega, Madison, WI) and a luminometer (LKB, Bromma, Sweden). The CAT
assays were performed as described20 with the equivalent of
1,200, 3,000, 600, 3,000, 150, and 200 arbitrary light units of
extracts from BAEC, NIH-3T3, HEL, HepG2, HeLa, and Lin 175, respectively, to be in the linear range of the assay. Data were
expressed as the percentage of acetylated chloramphenicol relative to
total chloramphenicol.
Transgenic mice.
The DNA containing the promoter fragment of 2486CAT, the CAT
gene, and the small t intron and polyadenylation signal from SV40 was
liberated from plasmidic sequences by digestion with Sal I and
Sac I. Insert isolated from agarose gels and further purified
on Elutip-d columns (Schleicher & Schuell, Ecquevilly, France) was
microinjected using established procedures21 into fertilized eggs resulting from mating between B6D2F1/JIco hybrids. The
animals were screened by Southern blot with two different probes, one
containing sequences of the CAT gene alone, the other containing
sequences from the CAT gene and VE-cadherin promoter. Transgene
copy number was estimated by comparative analysis with endogenous
promoter on a phosphorimager system (Molecular Dynamics, Sunnyvale,
CA). CAT assays of F1 animal tissues were performed with 2.5 µg of
protein extract in 1 hour as previously described.20
RNA extraction and Northern blot analysis.
Total RNA were extracted from 2-month-old mouse tissues by the rapid
total RNA isolation kit (5 Prime-3 Prime, Boulder, CO). Electrophoresis
was performed by the formaldehyde method,22 using 20 µg
of RNA. Northern blot was successively hybridized with probes for CAT
(415 bp EcoRI-Sca I fragment),16
VE-cadherin (1,740 bp EcoRI-Sph I
fragment),14 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (full-length cDNA)23 transcripts.
In situ hybridization.
The techniques used were essentially as described.14 Frozen
sections (10 µm) were hybridized to 35S-labeled RNA
probes generated by in vitro transcription. Two probes were generated
for hybridization with CAT transcripts either from a 256-bp Xho
I-EcoRI fragment or from a 415-bp EcoRI-Sca I
fragment.16 Nontransgenic tissues were used as negative
control and did not show specific hybridization. For
VE-cadherin transcripts, a specific probe derived from the
first 1,740 bp of mouse VE-cadherin cDNA was used as previously
described.14
Immunohistological staining.
Embryos were fixed for 2 hours in 4% paraformaldehyde, incubated for 1 hour in 15% sucrose followed by 16 hours in 30% sucrose, and embedded
in OCT (Miles Scientific, Elkart, IN) before freezing. Sections (10-µm thick) were postfixed for 30 minutes with 4%
paraformaldehyde, rinsed for 5 minutes in phosphate-buffered saline
(PBS), and incubated for 10 minutes with 1% bovine serum albumin, for
16 hours at 4°C with primary antibodies, and for 1 hour at 22°C
with secondary antibodies, successively. Chicken antibody to CAT was
purchased from Promega (Madison, WI) and used at 1:300
dilution. Rabbit anti-mouse VE-cadherin antiserum, raised against a
specific region of VE-cadherin C-terminus,14 was diluted
1:300. As secondary antibodies, a fluorescein isothiocyanate rabbit
anti-chicken IgY antibody (Promega), and a tetramethyl rhodamine
isothiocyanate conjugated goat anti-rabbit IgG antibody (Jackson
Laboratories, West Grove, PA) were used at 1:100 dilution.
| |
RESULTS |
In vitro expression of VE-cadherin promoter.
To test whether the 5-flanking region of VE-cadherin gene had
promoter activity, fragments of this region were inserted upstream from
a promoterless CAT gene in the plasmid pBLCAT3. Eleven constructs of
different sizes, from positions 5800 to 139 bp at the 5-end to
position +24 bp at the 3-end, were analyzed by transient transfection assay, both in BAEC, which express VE-cadherin (data not shown), and in
the fibroblastic cell line NIH-3T3, which does not.14 All
of the constructs expressed the reporter gene in BAEC as indicated by
CAT enzymatic assay of cellular extracts
(Fig 1A). Constructs 139CAT, 187CAT,
and 289CAT promoted similar high levels of CAT activity. A
substantial decrease in CAT activity was observed following
transfection with longer constructs from 515CAT to 5800CAT. These
data suggest that at least one cis sequence responsible for promoter
activity in BAEC is present between positions 139 and +24 and that a
negative regulatory element is located between 515 and 290.
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| Fig 1.
Functional analysis of VE-cadherin promoter by
transient transfection. 5-deleted fragments of the mouse
VE-cadherin promoter were fused to the CAT gene as indicated in
the text and transfected into BAEC (A) or NIH-3T3 cells (B). The HSVTK
promoter was used as positive control. "0" states for the
promoterless plasmid pBLCAT3. In each assay, the luciferase expression
plasmid, pGL3, was cotransfected, and CAT assays were normalized
according to the luciferase activity. The equivalent of 1,200 or 3,000 arbitrary light units of BAEC or NIH-3T3 cells extracts, respectively,
was used to be in the linear range of the CAT assay. Each data is the
average of 6 to 10 independent experiments. Standard deviations are
indicated. *P < .01; #P < .02;
 P < .05.
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In NIH-3T3 cells, construct 139CAT exhibited strong promoter
activity (Fig 1B). However, in contrast to BAEC, serial decreases in
expression were observed following transfection with 187CAT and
289CAT. Constructs 289CAT to 1190CAT had minimal activity in
these cells and constructs 2226CAT, 2486CAT, and 5800CAT had
only background expression. Together, these data suggest that a basic
and ubiquitous promoter is located between +24 and 139; in addition,
in nonendothelial cells, at least two negative regulatory elements are
present between 140 and 289 and between 1190 and 2226. Most
importantly, these data also suggest that constructs 2226CAT and
longer have endothelial-specific expression.
To substantiate the cell-specific expression capacity of these
constructs, 2486CAT was transfected in other nonendothelial cell
lines. As shown in Fig 2, 2486CAT, as
opposed to viral promoters, had no promoter activity in cells of
epithelial, hepatocytic, or erythro-megakaryocytic origin, which
confirms the data obtained in NIH-3T3 cells. The fact that the promoter
was silent in two hematopoietic cell lines is of particular interest,
as endothelial and hematopoietic lineages have several common markers
and a common origin, further suggesting the restricted expression
potential of this promoter fragment.
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| Fig 2.
Expression of the VE-cadherin 2486CAT fusion
gene in various cell lines. Construct 2486CAT and pGL3 plasmid were
cotransfected in cells of epithelial (HeLa), hepatocytic (HepG2), or
erythro-megakaryocytic origin (HEL and Lin175). The HSVTK (see Fig 1)
or the Rous sarcoma virus (RSV) promoters was used as positive control.
CAT activities were determined with the equivalent of 150, 3,000, 600, or 200 light units of extracts from HeLa, HepG2, HEL, or Lin175,
respectively. Data are the average of 3 independent experiments.
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Generation of VE-cadherin-CAT transgenic mice.
To definitely address the transcriptional specificity of the
VE-cadherin gene 5-flanking region, we developed a mouse
transgenic model with 2486CAT. This construct, depleted of plasmidic
sequences, was microinjected into fertilized eggs. Offspring were
screened for integration of the transgene by Southern blot analysis
with two probes hybridizing either with the CAT gene or the
promoter region. Among 28 live-born offspring, two transgenic founders were obtained and were called mouse 23 and mouse 28. Copy number was
estimated by comparing hybridization signal obtained for the transgene to that obtained for the endogenous promoter. Two transgenic lines were derived, line 23 and line 28, which contained 8 and 16 copies of the transgene, respectively. Analysis of the progeny of the
founder animals showed that the transgene was transmitted at a
frequency of 50%.
Transgene expression in adult tissue extracts.
To determine the expression of the transgene in adult mice, protein
extracts from various tissues of offspring of transgenic founders were
prepared and assayed for CAT enzymatic activity. For both lines, all
solid tissues examined developed a CAT activity (Table 1), whereas no activity was
detectable with tissues from normal mice despite long incubation times
(data not shown). Data were higher for line 28 than for line 23, but in
both cases, the activity profiles were similar, indicating the same
expression pattern, which appeared to be positively correlated with the
vascularization level of the different tissues. The activities were
particularly high in lung and heart. Liver, brain, spleen, kidney,
thymus, and skin expressed comparable and substantial amounts of CAT
activity. As expected, blood cells did not express the enzyme but
traces of activity could be detected in the plasma, suggesting either that some CAT molecules could enter the secretory pathway or more likely that this enzyme was liberated by lysed cells from endothelial cell turnover. In agreement with these data, 2% to 5% of CAT activity was detected in the supernatant of transfected BAEC.
Northern blot analysis of transgenic tissue RNA further established
transgene expression in adult organs and showed comparable RNA profile
between CAT and VE-cadherin (Fig
3).
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| Fig 3.
Northern blot analysis of CAT, VE-cadherin, and GAPDH
mRNA in adult transgenic tissues. RNA from wild-type lung (1) and
transgenic heart (2), lung (3), brain (4), liver (5), kidney (6), and
thymus (7) were hybridized to CAT (A), VE-cadherin (B), and GAPDH (C)
probes. Similar expression profiles could be observed between CAT and
VE-cadherin in transgenic tissues.
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In situ and immunohistologic detection of CAT gene expression in
transgenic mice.
To analyze the expression of the reporter gene at the cellular level,
in situ hybridization and immunohistologic stainings were performed on
frozen tissue sections. Transgenic animals used for this study were
obtained by mating transgenic males with wild-type females. The in situ
experiments were conducted using two different probes derived from the
CAT gene or a probe derived from mouse VE-cadherin cDNA (see
Materials and Methods). Both CAT probes were found to be highly
specific for CAT gene product and yielded identical results. No
specific signal was observed when nontransgenic tissues or embryos were
used (see Fig 6C). Both transgenic lines were investigated in this
study and showed the same distribution pattern for CAT
expression.
The endothelial-specific expression of CAT gene was first
determined at the protein level by colabeling of tissue sections with
CAT and VE-cadherin antibodies; a representative example shows
identical staining pattern for both proteins
(Fig 4A and B). Furthermore, in situ
hybridizations performed on adjacent sections, using either the
CAT or the VE-cadherin probes, indicated expression of
both transcripts in the same vascular structures (Fig 4C through F) and
confirmed the endothelial-specific expression of the transgene. Because
of the high background level produced by the anti-CAT polyclonal
antibody, subsequent analyses were performed by in situ hybridization.
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| Fig 4.
Comparison of transgene and VE-cadherin
expression. Costaining of a E13.5 embryo section with anti-CAT (A) and
anti-VE-cadherin (B) antibodies showed an identical expression
pattern, as illustrated here for the oral area. In situ hybridizations,
performed with either the CAT (C,E) or the VE-cadherin
(D,F) probe on adjacent sections (10 µm), confirmed specific
endothelial expression of CAT at the transcript level. (C,D)
Oral area; (E,F) hindlimb; scale bars represent 100 µm.
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Primary and progressive expression of CAT gene occurred in the vascular
system of the developing embryo, perfectly matching the expression of
the endogenous VE-cadherin gene.14 At E7.5, CAT expression was only observed in primitive blood islands
consisting of hemangioblastic precursors and in allantois, at the sites
of early vasculogenesis (Fig
5)1,24; no signal could be detected in the embryo proper
or in the wild-type maternal decidua. As the embryo developed further,
CAT signal became more intense and colocalized with capillaries
and larger vessels of embryonic and extraembryonic tissues.
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| Fig 5.
Transgene expression at E7.5. In situ hybridization of a
transversal section with CAT probe, shown as bright-field (A)
or dark-field image (B). The transgenic embryo is located in the
wild-type maternal decidua. CAT transcripts were detected in
the mesodermal aggregates of the yolk sac from which blood islands are
derived (arrow), and in the inner part of allantois (arrowhead). Scale
bar represents 100 µm; al, allantois; am, amnion; d, decidua; n,
neuroectoderm; ys, yolk sac.
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At E13.5, the transgene was strongly expressed in the vasculature of
most organs, including lung, heart, gut, kidney, and limb buds
(Fig 6). In comparison, expression in the
liver was weaker, but still readily detectable. The head oral and neck
areas also expressed high levels of CAT transcripts. The
perineural vascular plexus and the choroid plexus were highly positive
with CAT probes. Conversely, no signal could be detected within
capillaries of the brain and spinal chord. This expression profile
suggests a discrimination between the blood-brain barrier, which is
already established at this stage, and the "permeable"
endothelium of the brain.25,26 Higher magnification clearly
shows silver grains over cells lining the luminal side of the aorta
(Fig 7A) and the trabeculae of the
endocardium (Fig 7B); capillaries within the myocardium also showed
intense labeling. In the intestine, CAT transcripts were
localized in the capillaries at the periphery of the epithelial barrier
(Fig 7C). Lung (Fig 7D) contained a dense labeling in the
microcirculation surrounding the bronchi. As shown Fig 7E, the
transgene was expressed in the capillary network of the liver and the
hepatic venous plexus. VE-cadherin RNA stainings, performed in
parallel, showed expression pattern comparable to that of CAT RNA (Fig
7A through E).
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| Fig 6.
Transgene expression at E13.5. Parasagittal sections of
E13.5 transgenic (A, bright field and B, dark field) or wild-type (C,
dark field) animals, hybridized with CAT antisense probe. With
the exception of the brain, specific signals were detected in the
vasculature of all the embryo. Probe did not show any reactivity in
nontransgenic mice. Scale bar represents 15 µm; b, brain; c, choroid
plexus; d, duodenum; h, heart; k, kidney; li, liver; lm, limb; lu,
lung; u, umbilic.
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| Fig 7.
Endothelial expression of the CAT gene in various
tissues of E13.5 embryos. Embryo sections were hybridized with
CAT (A-E) or VE-cadherin (A-E) antisense transcripts.
Comparison of gene expression in aorta (A-A), heart (B-B), intestine
(C-C), lung (D-D), and liver (E-E). Scale bars represent 50 µm
(A-A) or 100 µm (B-E, B-E); b, bronchi; c, capillary; e,
endocardium; my, myocardium; ve, ventricle; vp, venous plexus.
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In 2-month-old mice, in situ analysis of sections from various tissues
confirmed previous data obtained with E13.5 embryos (Fig 8 and not shown). In particular,
functionality of organs like lung or liver, which really takes place
after birth, did not affect CAT expression pattern (Fig 8A and B). As
previously discussed for E13.5 embryos, expression was weaker in
intrahepatic vessels in comparison to other visceral derivatives;
however, this expression was similar among the different vessel types, namely, sinusoidal endothelium, centrilobular veins (Fig 8A), and
portal vein (data not shown).
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| Fig 8.
Endothelial expression of the transgene in adult organs.
In the lung (A), silver grains are located over endothelial cells of
capillaries (arrows) interspersed between pneumocytes. CAT
expression in the sinusoids and centrilobular vein of the liver (B).
Scale bar is 45 µm; a, alveolae; c, centrilobular vein; s,
sinusoids.
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Taken together, these data show that the expression of the transgene is
(1) endothelial-specific, (2) universal along the vascular tree, with
the exception of the brain capillaries, and (3) constitutive during
development from the early steps of vasculogenesis.
| |
DISCUSSION |
This report demonstrates that approximately 2,500 bp of the mouse
VE-cadherin gene promoter region is sufficient to direct appropriate transcription in vivo in the endothelium of adult mice and
during vascular development. Overall, the transgene expression pattern
is similar to that of endogenous VE-cadherin.
In situ hybridization indicates that all types of vessels expressed the
transgene, except brain capillaries, where highly specialized junctions
are established at mid-gestation.25 Endogenous VE-cadherin expression was also shown to be downregulated in
the blood-brain barrier, but to a lesser extent as transcripts were still detectable in brains of E17.5 embryos.14 This may
represent a higher sensitivity of the promoter fragment to this
organotypic regulation than the endogenous promoter. Interestingly, in
vivo studies performed with the von Willebrand factor promoter
identified a small DNA sequence (487/+246) restricting reporter gene
expression to endothelial cells of adult brain,27 whereas a
larger fragment (containing 2,182 bp of 5-flanking sequence, the first
exon and the first intron) targeted expression within endothelial cells in the brain, heart, and skeletal muscle.28 These limited
patterns of expression strongly suggest the existence of regional
differences in endothelial regulatory factors, as well as
subtype-specific cis-acting DNA domains. In this respect, the
(2486/+24) VE-cadherin and the (487/+246) von
Willebrand factor fragments are functionally complementary to
target endothelial expression in adult mice. In the liver,
differentiation of sinusoids from capillaries, which occurs between E17
and E19 in the mouse embryo,29 did not affect CAT
expression. This result is in agreement with a previous study reported
by Scoazec et al,30 who demonstrated that VE-cadherin is a
homogenous marker of continuous and sinusoidal endothelia in the liver.
CAT transcripts were detected as early as E7.5 in blood islands
and allantois of mice embryos, thus marking the early stages of
vasculogenesis. Thereafter, the CAT gene was expressed in the vasculature of visceral, as well as parietal derivatives (ie, head,
limbs, and kidney), which are vascularized by vasculogenic (the
assembly of vessels from newly differentiated endothelial cells) and
angiogenic (the sprouting of capillaries from preexisting vessels)
mechanisms, respectively.1,31 Previous
studies32 suggest the existence of different regulatory
domains for directing expression during angiogenesis and
vasculogenesis. If this hypothesis is correct, our data suggest that
VE-cadherin promoter contains both elements.
The 5-deletion analysis, performed in BAEC, showed three important
domains involved in the transcriptional regulation of the
VE-cadherin gene: a proximal domain, located between positions 139 and +24, promoting transcription in a cell line-independent fashion and two negative domains between 289 and 140 bp and between 2226 and 1191 bp that abolish transcriptional rate in NIH-3T3 (Fig 9). Functional analysis of DNA
sequences of the (139/+24) region, as well as DNA-protein
interaction studies, indicated several binding sites for transcription
factors, such as Sp1, Sp3, and Ets factors.33 A fine
functional dissection of the two other regions is underway to determine
the transcription factors and the DNA elements that control
VE-cadherin expression and, more generally, to gain further
insights in endothelial transcriptional mechanisms. Such a regulation
with positively acting proximal domains and cell-type specific
silencing domains is a unique feature among studied endothelial
promoters, but is reminiscent of some promoters conferring
tissue-specific expression in other cell-types, such as
 IIb integrin,34
the immunoglobulin kappa gene,35 or the
 1-crystallin gene.36 However, it is
conceivable that these silencing domains bind multiple positive and
negative factors acting in a interdependent fashion.
A recent report indicates that a combination of the tie-2
promoter with an intron fragment allows the expression of the
LacZ reporter gene in all endothelial cells of transgenic mice,
throughout embryogenesis and adulthood.37 LacZ
staining of adult vessels is surprising, as tie-2 expression is
downregulated in quiescent endothelium.5 As evoked by the
authors, this may be due to the loss of negative regulatory elements or
it may represent  -galactosidase accumulation rather than active
transcription. In this report, histologic investigations were
preferentially performed by in situ hybridization to visualize
transcripts rather than protein accumulation. Moreover, as CAT
mRNA is notoriously unstable in eukaryotic cells, the hybridization
signal better reflects the transcriptional activity of the promoter.
VE-cadherin promoter may be widely used to target gene
expression in the endothelium of transgenic mice in order to study various pathophysiologic conditions. Animal models where genes able to
change the antithrombotic or hemostatic properties of endothelial
cells, to decrease the neointima formation, or to limit angiogenic
growth can be developed using the VE-cadherin promoter alone or
in concert with cis-responsive elements that could modulate
transcriptional activity.
Another potential application of the VE-cadherin promoter is
gene therapy. An important problem with gene therapy is the specificity of gene transfer, and the possible deleterious side effects that might
result from the expression of a therapeutic gene candidate in
nonrelevant cells. The possibility of modulating endothelial cell
responses by directing the expression of a desired gene within these
cells via the VE-cadherin promoter is an attractive therapeutic goal.
| |
ACKNOWLEDGMENT |
We thank D. Vittet for helpful discussion. We are grateful to F. Aubouy
for artwork, to T. Kortulewski and F. Breviario for excellent technical
assistance, and to Y. Senis for careful reading of the manuscript.
| |
FOOTNOTES |
Submitted March 9, 1998;
accepted August 26, 1998.
Supported by the Commissariat à l'Energie Atomique, France.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to P. Huber, PhD, CEA-Grenoble,
DBMS-TDC, 17, rue des Martyrs, 38054 Grenoble, France.
| |
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Abstract
Introduction