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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-03-0955.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Molecular Medicine, Beth Israel
Deaconess Medical Center, Boston, MA.
An important limitation of standard transgenic assays is that
multiple copies of the transgene are inserted randomly into the mouse
genome, resulting in line-to-line variation in expression. One way to
control for these variables is to target a single copy of the transgene
to a defined locus of the mouse genome by homologous recombination. In
the present study, we have used such an approach to target the
promoters of 2 different genes, namely von Willebrand factor (VWF) and
Flt-1, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus.
Consistent with previous findings in standard transgenic animals, we
report that the VWF promoter contains information for expression in a
subset of endothelial cells in the heart, skeletal muscle, and brain.
In contrast, the Flt-1 promoter directs expression in all vascular beds
except for the liver. The Flt-1 transgene was active in the endothelium
of tumor xenografts, whereas the VWF promoter was not. Under in vitro
conditions, conditioned medium from tumor cells resulted in a
significant up-regulation of Flt-1 mRNA and promoter activity, but no
change in VWF levels. Taken together, these results suggest that (1)
Hprt locus targeting is a valuable tool for studying vascular
bed-specific gene regulation, (2) the VWF and Flt-1 promoters are
regulated by distinct transcriptional mechanisms in the intact
endothelium, and (3) tumor angiogenesis results in the differential
activation of endothelial cell-specific promoters.
(Blood. 2002;100:4019-4025) At one time, the endothelial lining was considered
to be an inert layer, serving to separate flowing blood from underlying tissue. Over ensuing years, it became increasingly appreciated that the
endothelium is a highly active organ involved in regulating the
trafficking of cells and nutrients, vasomotor tone, and
hemostasis.1 What is important to recognize is that the
various functions of the endothelium are differentially regulated in
time and space, giving rise to endothelial cell heterogeneity and
vascular diversity.
Based on our previous work, we proposed a model of endothelial cell
gene regulation that emphasizes the critical role of vascular bed-specific signaling pathways.2 Initial support for
this model was derived from studies of the von Willebrand factor (VWF) promoter. In multiple independent lines of standard transgenic mice, a
small 734-bp fragment of the human promoter was shown to contain
information for expression in a subset of endothelial cells within
blood vessels of the brain,3 while the inclusion of the
additional 5' flanking sequence as well as the first intron of the gene
directed more widespread expression in the microvasculature of the
heart and skeletal muscle.4 These results suggested that
the VWF gene was regulated in a modular fashion. In other words,
overall expression was mediated by the sum of distinct signaling
pathways each communicating with different regions of the promoter.
These findings were prototypic of a more generalized phenomenon within
the endothelium. For example, we reported that a 1600-bp fragment of
the human eNOS promoter contains information for expression in large
and small vessels of the brain, heart, and skeletal muscle as well as
in the aorta.5 In contrast, a 1200-bp region of the Egr-1
promoter directed expression in the endothelium of the heart and brain
of the adult mouse, but not in other vascular beds.6
Promoters from the Tie-1,7 Tie-2,8 vascular
endothelial-cadherin,9 and human
preproendothelin10 genes were similarly shown to direct expression in limited subsets of endothelial cells under in vivo conditions.
An important limitation of the standard transgenic mouse assay is that
multiple copies of the transgene are inserted randomly into the mouse
genome, often resulting in significant line-to-line variation in
expression. To control for these variables, we recently employed
homologous recombination to insert a single copy of a transgene into a
defined locus of the mouse genome.11,12 In these latter
studies, a transgenic cassette containing either the 1600-bp eNOS
promoter or the Tie-2 promoter-enhancer coupled to the LacZ
cDNA was targeted to the hypoxanthine phosphoribosyltransferase (Hprt)
locus by homologous recombination. In each case, the promoter retained
tissue specificity when integrated into this site. Moreover, the level
and pattern of transgene expression was independent of orientation,
relative to that of the endogenous Hprt locus.11 Taken
together, these results argued against an overriding effect of the
surrounding Hprt locus on transgene expression and suggested that
the targeting strategy could be employed for rapid throughput screening
of endothelial cell-specific promoters.
In the present study, we have extended these observations by targeting
the VWF and Flt-1 promoters to the Hprt locus of mice. We show that the
VWF and Flt-1 promoters direct expression in different subsets of
endothelial cells in embryonic and adult organs as well as in tumor
xenografts. This report confirms the results of previous VWF standard
transgenic studies. Moreover, it is the first study to demonstrate
endothelial cell activity of the Flt-1 promoter in vivo. Finally, the
data support the notion that vascular bed-specific gene expression is
mediated, at least in part, by signals residing in the extracellular milieu.
Cell culture and transfections
Plasmids and targeting vectors
RNA isolation and RNase protection assays HUVECs were serum-starved in EBM-2 plus 0.5% FBS for 18 hours. The cells were then incubated with serum-starved medium alone (control) or tumor cell-conditioned medium for an additional 24 hours, at which time they were harvested for total RNA using the Trizol reagent (Invitrogen, Carlsbad, CA). Flt-1 riboprobe was synthesized from pGEM-hFlt-1 with T7 RNA polymerase, whereas VWF riboprobe was synthesized from pGEM-hVWF with SP6 RNA polymerase. RNase protection assays were performed with an RPA III kit (Ambion) according to the manufacturer's instructions. Densitometry was used to calculate the intensity of Flt-1, VWF, and glyceraldehyde phosphate dehydrogenase (GAPDH) signals.Generation and analysis of Hprt-targeted transgenic mice The Flt-1 and VWF transgenes were targeted to BK4-ES cells (a generous gift from Sarah Bronson), and homologous recombinants were used to generate Flt-1-lacZ-Hprt or VWF-lacZ-Hprt chimeric mice as previously described.11,12 Chimeric males were bred to C57BL/6 females to obtain agouti offspring. Female agouti offspring were then bred to C57BL/6 males to generate hemizygous male mice. Genotyping was performed by polymerase chain reaction (PCR) analysis or Southern blot analysis of mouse tail genomic DNA. Analysis of embryos and adult tissues was carried out as previously described.3,11 LacZ staining of tissues from Flt-1-lacZ-Hprt and VWF-lacZ-Hprt mice was carried out in parallel.Generation of tumors in mice Confluent Lewis lung carcinoma and B16-F1 cells were washed with PBS, trypsinized, and collected by brief centrifugation at room temperature. A total of 1.5 × 106 cells was suspended in 100 µL PBS, and the resulting suspension was injected subcutaneously into the right flank of adult Hprt-targeted mice. The tumors were allowed to grow for 14 days (0.25-0.5 cm3), at which time the mice were killed and tumor tissues were harvested for LacZ staining. There were 2 independent Lewis lung carcinoma and 2 independent B16-F1 xenografts analyzed in each of the Flt-1-lacZ-Hprt and VWF-lacZ-Hprt lines.
Generation of Flt-1 and VWF Hprt-targeted mice In a previous study, a fragment of the human Flt-1 promoter containing DNA sequences between 748 bp and +284 bp, relative to the
start site of transcription, was shown to direct high-level expression
in cultured endothelial cells.15 To verify whether this
region contains information for expression in vivo, we employed homologous recombination to target a single copy of the
Flt-1-lacZ transgene to the Hprt locus of mice (Figure
1). We obtained a total of 4 recombinant
ES cell clones. We used 2 of these to generate chimeric mice. The
chimeric males were then bred to C57BL/6 females to obtain germ-line
transmission. To date, the mice have been back-crossed for 4 generations. We have previously shown that the human VWF promoter
containing the 2182 bp 5' flanking region and the first exon and
intron of the gene directed expression in the microvascular endothelium
of the heart and skeletal muscle in standard transgenic
mice.4 To determine the reproducibility of these results
in the context of the Hprt locus, we generated Hprt-targeted animals
with a transgenic cassette containing the same fragment of the VWF
promoter coupled to the LacZ reporter gene
(VWF-lacZ-2) (Figure 1). We used 2 recombinant ES cell
clones to generate chimeric mice. These mice have been back-crossed to C57BL/6 for 5 generations.
One theoretical disadvantage of comparing transgene expression across different lines of Hprt-targeted mice is the possible effect of mixed genetic background on promoter activity. In the studies below, we have taken 3 measures to control for this possibility. First, we have analyzed mice that are genetically identical across lines, namely F1 agoutis (50% E129: 50% C57). The major drawback of this strategy is that all targeted mice at the F1 stage are by definition heterozygous females. Therefore, the X-linked transgene is inactivated in one-half of all cells. However, in both the VWF-lacZ-Hprt and Flt-1-lacZ-Hprt lines, the pattern of transgene expression was identical in F1 female agoutis compared with male hemizygous mice from subsequent generations. Second, we have routinely analyzed and compared transgene expression in large numbers of mice (n > 8) from the same generation. In any given line, the expression pattern was consistently identical between littermates and between litters of the same generation. Finally, we have back-crossed the VWF-lacZ-Hprt and Flt-1-lacZ-Hprt lines to wild-type C57 mice for at least 4 generations, with no observable differences in either the pattern or level of transgene expression. Comparison of Flt-1 and VWF promoter activity in embryos Heterozygous females were mated with 6- to 8-week-old Flt-1-lacZ-Hprt and VWF-lacZ-Hprt hemizygous males. Embryos were removed and processed for LacZ staining at day E10.5. Whole mounts of Flt-1-lacZ-Hprt embryos revealed uniform staining in the endothelium of superficial vessels, the dorsal aorta, intersomitic vessels, caudal veins, as well as blood vessels of the yolk sac (Figure 2A-B). In whole mount studies of the VWF-lacZ-Hprt embryos, -galactosidase activity was limited to the telencephalon and head
region, and to a lesser extent the umbilical vessels (Figure 2C-D). In
contrast, the endogenous VWF gene is more widely expressed in the
embryonic vasculature (Coffin et al16 and data not shown).
These findings indicate that while DNA sequences between 748 bp and
+284 bp of the human Flt-1 gene are sufficient for directing widespread vascular expression in the embryo, additional sequences in the VWF
promoter are required to confer authentic expression at this stage of development.
Comparison of Flt-1 and VWF promoter activity in adult mice To determine the activity of the Flt-1 and VWF transgenes in the postnatal period, we carried out LacZ stains of whole mounts and cryosections from adult organs. In whole mounts of Flt-1-lacZ-Hprt mice, reporter gene activity was detected in large vessels of the brain (Figure 3A,C), within the substance of the heart (Figure 3E,G), in blood vessels of the thigh muscle (Figure 3I), diaphragm (Figure 3K), and chest wall (Figure 3M), and in the lung (Figure 3O). Whole mounts of the kidney and spleen, although limited by high background staining, showed a clear increase in LacZ staining in the Flt-1 mice (data not shown). In whole mounts of VWF-lacZ-Hprt organs,-galactosidase
activity was evident in both small and large vessels of the brain
(Figure 3B,D), microvessels (and occasional veins) of the heart (Figure
3F,H), blood vessels of the thigh muscle (Figure 3J), diaphragm (Figure
3L), and chest wall (Figure 3N). In contrast to the
Flt-1-lacZ-Hprt mice, there was no detectable expression in
whole mounts of the lung, spleen, or kidney (Figure 3P, and data not
shown).
LacZ staining of tissue sections revealed significant
differences between the Flt-1-lacZ-Hprt and
VWF-lacZ-Hprt lines. In the brain of
Flt-1-lacZ-Hprt mice, reporter gene activity was detected in
the endothelial lining and smooth muscle cell layer of occasional
arteries, whereas the VWF transgene was expressed in the endothelium of
a subset of both large and small blood vessels (data not shown). In the
heart, Flt-1-lacZ activity was present in the endothelium
and smooth muscle cells of occasional large arteries as well as in
cardiomyocytes (Figure 4A). In contrast, expression of the VWF transgene was limited to the endothelium of
myocardial capillaries and occasional veins (Figure 4B,H shows LacZ-positive capillaries). Both the Flt-1 and VWF promoters
directed expression in the microvascular endothelium of skeletal muscle and the thymus (data not shown). In sections of the kidney,
Comparison of Flt-1 and VWF promoter activity in tumor xenografts The results of recent studies suggest that the expression of the endogenous VWF gene and the VWF promoter in the microvascular bed of the heart is mediated, at least in part, by a platelet-derived growth factor (PDGF)-dependent signaling pathway.17 We were interested in determining whether Flt-1 activity is similarly modulated by the extracellular milieu. Previous studies have demonstrated that the Flt-1 gene is up-regulated in the endothelium of tumor blood vessels.18 Therefore, we employed a tumor xenograft model to study the relationship between tumor angiogenesis and Flt-1 promoter activity. In these experiments, either Lewis lung carcinoma or B16-F1 melanoma cells were injected subcutaneously into Flt-1-lacZ-Hprt and VWF-lacZ-Hprt mice. The resulting tumors were harvested 14 days later and processed side-by-side for LacZ staining. Lewis lung carcinoma and B16-F1 melanoma tumors harvested from Flt-1-lacZ-Hprt lines revealed uniform and strong-galactosidase activity in the majority of neo-vessels (Figure
5A-B). In contrast,
VWF-lacZ-Hprt-derived xenografts failed to stain for
LacZ, despite the presence of reporter gene activity within
the capillaries of adjacent skeletal muscle (Figure 5C-D). These
findings suggest that the Flt-1 and VWF promoters are differentially
regulated during tumor angiogenesis.
Conditioned medium from tumor cells induces Flt-1, but not VWF, mRNA, and promoter activity To determine whether the differential activity of the Flt-1 and VWF transgenes in tumor endothelium could be recapitulated in tissue culture, we incubated serum-starved primary human endothelial cells with tumor cell-conditioned medium. The endothelial cells were harvested 24 hours later for total RNA and assayed for Flt-1 and VWF mRNA expression by RNase protection assays. As shown in Figure 6A, incubation of HUVECs with conditioned medium from Lewis lung carcinoma or B16-F1 melanoma cells resulted in a small but statistically significant induction of Flt-1 mRNA levels (2.21- and 1.87-fold, respectively). In contrast, VWF mRNA levels remained unchanged. In the next set of experiments, the Flt-1 or VWF promoters were coupled to the luciferase reporter gene and the resulting plasmids were transiently transfected into HUVECs. The cells were then incubated in the presence or absence of tumor cell-conditioned medium and assayed 24 hours later for luciferase activity. Addition of conditioned medium from either Lewis lung carcinoma or B16-F1 melanoma cells resulted in a significant induction of Flt-1 promoter activity (1.97- and 1.98-fold, respectively; P < .01) (Figure 6B), but no change in VWF promoter activity. To determine whether the effect of conditioned medium on Flt-1 promoter activity was specific to malignant cells and was not simply an artifact of the cell culture system, we incubated the transfected HUVECs with supernatants from primary human keratinocytes and renal epithelial cells. As shown in Figure 6B, Flt-1 activity was not increased under these conditions. Together, these results correlate with the findings in vivo and suggest that differential activation of the Flt-1 promoter in tumor vasculature is governed, at least in part, by tumor cell-derived soluble mediators.
Based on the results of standard transgenic mouse assays, we previously proposed a model of endothelial cell gene regulation which emphasized the critical role of vascular bed-specific signaling pathways that begin in the extracellular environment and end at the level of the promoter.2 However, these studies were limited by the unpredictable effect of copy number and integration site on promoter activity. In the present study, we have employed a gene targeting strategy to control for these variables. We have shown that when integrated into the same genomic locus, promoters from 2 different genes, VWF and Flt-1, contain information for expression in different subsets of endothelial cells. These data add strong support to our model of modular endothelial cell-specific gene regulation. Particularly reassuring was the observation that the Hprt-targeted VWF
transgene directed expression in the same vascular beds as previously
reported for the standard transgenic mice, namely the heart, skeletal
muscle, and brain.4 The only difference observed between
the Hprt-targeted and standard transgenic animals was the presence of
The limited distribution of Vascular endothelial growth factor (VEGF) exerts its biologic functions
through 2 endothelial cell-specific affinity tyrosine kinase
receptors, Flt-1 and KDR/Flk-1.21,22 The role of Flt-1 in
the endothelium is not well understood. Mice that are null for Flt-1
die before birth and are associated with abnormal, disorganized blood
vessels.23 However, mice that lack the tyrosine kinase domain of Flt-1 show normal development, arguing against a critical role for Flt-1 signaling in the endothelium.24 The human
Flt-1 promoter has been previously cloned and characterized under in vitro conditions. A 1-kb fragment of the 5' flanking region of the
human Flt-1 promoter was shown to direct endothelial cell-specific expression in transient transfection assays.15 Moreover,
when stably transfected into ES cells, a 2.2-kb fragment of the mouse promoter was reported to direct endothelial cell-specific expression in embryoid bodies.25 The immediate upstream promoter of
the human Flt-1 gene contains 5 Ets-binding motifs, 2 GC boxes,
and a cAMP response element (CRE).26 In a previous study,
Flt-1 expression was shown to be mediated by a cooperative interaction between the CRE at This study is the first to identify a fragment of the Flt-1 promoter
that directs expression in the intact endothelium. Mice targeted with
the Flt-1-lacZ construct expressed the transgene in
virtually every vascular bed with the notable exception of the liver.
The endogenous Flt-1 gene is normally expressed in the vasculature of
the liver,29,30 suggesting that additional promoter
elements are required for expression in this vascular bed. In most
organs, Previous studies have demonstrated increased expression of Flt-1 and Flk-1 in tumor endothelium.18,33-36 Moreover, in standard transgenic studies, an Flk-1 promoter containing 939 bp in combination with intronic enhancer sequences was shown to direct expression in the vascular beds of experimental melanoma, fibrosarcoma, and mammary adenocarcinoma.37 Our results suggest that like the Flk-1 transgene, the Flt-1 promoter also contains information for expression in the neovasculature of tumor xenografts. In contrast, the VWF promoter was not active in this vascular bed. Despite parallels in expression between the Flk-1 and Flt-1 transgenes in tumor endothelium, the promoter sequences from these 2 genes share few similarities.38,39 Therefore, it seems likely that the effect is mediated by different transcriptional control mechanisms. There is little knowledge about the signaling pathways that induce the
expression of Flt-1 and/or Flk-1 in tumor microvessels. In a recent
study, the addition of tumor cell-conditioned medium or VEGF to HUVECs
resulted in reduced cell surface expression of Flt-1, but increased
Flt-1 mRNA.40 These latter effects were abrogated by
preincubation with anti-VEGF antibodies.40 Indeed, other
studies have shown that VEGF induces Flt-1 expression in endothelial
cells.41 In addition, hypoxia has been shown to induce the
expression of Flt-1 in cultured endothelial cells.42 The
results of the present study support a role for one or more tumor
cell-derived soluble mediator(s) in mediating the up-regulation of
Flt-1 expression during tumor angiogenesis. Further studies will be
required to identify the nature of these signals. It is noteworthy that
tumor endothelial cells have been shown to express increased levels of
Ets-1.43,44 These observations suggest that the Ets motif
at The differences in expression pattern between single-copy transgenes
that have been targeted to a defined locus of the genome clearly point
to the existence of vascular bed-specific transcriptional networks.
Indeed, the results of the present study add to a growing list of
Hprt-targeted endothelial cell-specific transgenes that now include
VWF, Flt, eNOS,11 and 2 different fragments of the Tie-2
promoter.12 As summarized in Table
1, each of the promoters directs
expression in a unique subset of endothelial cells or blood vessel
types. We conclude that the various DNA sequences represent novel
molecular markers for endothelial cell heterogeneity. An important goal
for future studies will be to elucidate promoter elements that mediate
expression in different types of endothelial cells and to determine the
extent to which these regions are responsive to extracellular or
microenvironmental signals.
We are grateful to Sarah Bronson for providing us with reagents for Hprt locus targeting. We acknowledge Jason Guan for technical help.
Submitted March 27, 2002; accepted July 12, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-03-0955.
Supported by National Institutes of Health grants HL 60585-04, HL 63609-02, and HL 65216-03.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William C. Aird, Beth Israel Deaconess Medical Center, Molecular Medicine, RW-663, 330 Brookline Ave, Boston, MA 02215; e-mail: waird{at}caregroup.harvard.edu.
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Abstract
Introduction
-[32P] UTP-labeled 413-bp human Flt-1 antisense
riboprobe was incubated with 10 µg yeast RNA (lane 1) or total RNA
from untreated HUVECs (lane 2), HUVECs treated with conditioned medium
from Lewis lung carcinoma (LLC) cells (lane 3), or HUVECs treated with
conditioned medium from B16-F1 melanoma cells (lane 4). An