© 1996 American Heart Association, Inc.
Articles |
Targeting of Transgene Expression to the Vascular Endothelium of Mice by Homologous Recombination at the Thrombomodulin Locus
From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass.
Correspondence to Dr Robert D. Rosenberg, Department of Biology, Bldg 68-480, Massachusetts Institute of Technology, 400 Main St, Cambridge, MA 02139.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and Methods Results Discussion References |
|---|
Abstract We describe a straightforward gene-targeting technique to achieve uniform, stable, and genetically invariant expression of a transgene in the vascular endothelium of mice. To demonstrate the feasibility of this approach, the reporter gene bacterial ß-galactosidase was inserted via homologous recombination into the intronless thrombomodulin locus of murine embryonic stem cells. In this fashion, the lacZ gene is placed under the regulatory control of the endogenous thrombomodulin promoter. The expression of the transgene in adult mice recapitulated the widespread, stable, and high-level expression of the thrombomodulin gene in vascular endothelium. These data indicate that targeting of cDNAs into the thrombomodulin locus serves as a viable strategy to express transgenes in endothelial cells. Analysis of reporter gene expression revealed a heterogeneous pattern of thrombomodulin gene activity in the endothelium of the aorta and its tributaries. We also show that embryonic stem cells with a targeted thrombomodulin locus contribute in a mosaic fashion to the vascular endothelium of chimeric mice. This method for generating animals with a functionally heterogeneous cardiovascular system should provide an experimental technique for studying how localized genetic abnormalities in endothelial cell function lead to the development of vascular diseases.
Key Words: embryonic stem cells • lacZ blood vessels • chimeras • endothelial heterogeneity
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Genetic techniques that facilitate the expression of transgenes in vascular endothelial cells of experimental animals provide a powerful experimental approach for studying the role of the endothelium in the pathogenesis of cardiovascular disorders. Recombinant DNA has been introduced into the vasculature of animals by several different methods of gene transfer. For example, denuded blood vessels have been resurfaced with genetically engineered endothelial cells,1 2 and recombinant DNA has been transferred in situ using DNA-liposome complexes or viral expression vectors.3 4 5 6 7 However, these approaches usually suffer from low transfection efficiencies, transduction of cell types other than endothelial cells, and transitory patterns of gene transcription.8 9
Uniform, sustained, and high-level expression of gene products within the endothelium may be achieved either by transgenic approaches using endothelial cell–specific promoters or by gene targeting into endothelial cell–specific loci. Until recently, the transgenic approach was impossible because of our limited knowledge of the mechanisms underlying endothelial cell–specific gene regulation. In a recent report, transgenic mice were generated with a DNA construct containing the 5' upstream region and first exon of the von Willebrand factor gene coupled to the bacterial lacZ gene.10 In these animals, reporter gene activity was detected only in endothelial cells of the embryonic yolk sac and adult brain. Similar experiments carried out with the 5' upstream region of the Tie2 gene revealed expression of lacZ only in a restricted population of embryonic endothelial cells.11 Using the preproendothelin-1 promoter, expression of transgenes was achieved in the vascular wall of transgenic mice.12 Again, the foreign gene product was expressed in a heterogeneous fashion in endothelium and smooth muscle cells and was conspicuously absent from the microvascular bed of the lung and spleen. Although encouraging, these communications highlight the need for further investigations to pinpoint regulatory domains of endothelial cell–specific genes capable of driving widespread expression of transgenes in this cell type.
In the present report, we demonstrate that gene targeting results in the stable expression of heterologous gene products in vascular endothelium. We have focused our attention on the intronless thrombomodulin (TM) gene encoding a thrombin receptor present on the surface of endothelial cells throughout the vascular tree.13 14 15 16 17 Although the structures of the murine and human TM genes have been well documented,18 19 20 the cis-acting elements responsible for endothelial cell–specific expression have not yet been identified. To circumvent the need for well-characterized endothelial cell–specific promoter sequences, we inserted the bacterial lacZ gene via homologous recombination into the TM locus of murine embryonic stem (ES) cells in such a way that expression of the transgene is regulated by the endogenous TM promoter. We show that in adult mice carrying one targeted allele, the reporter gene activity mirrors the widespread expression of TM in the cardiovascular system. These data suggest that homologous recombination at the TM locus may serve as a viable strategy to achieve systemic long-term expression of transgenes in endothelial cells in a reproducible and predictable manner.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Gene Targeting and Generation of Transgenic Animals
The isolation of a genomic TM clone from 129/Sv mice has been described previously.21 A targeting vector (see Fig 1
) was
assembled in the plasmid PNT22 from a 2.4-kb genomic TM
promoter fragment, defined by an Apa I site at the 5' end,
and an Sph I site spanning the translation initiation codon
of the TM mRNA at the 3' end. This fragment was ligated into an
Sph I site introduced into the first codon of the
ß-galactosidase gene of the plasmid pSDKlacZ/SV40pA (gift from Dr
J. Rossant, Mt. Sinai Research Center, Toronto, Ontario),
containing a simian virus 40 polyadenylation signal. The TM
promoter/ß-galactosidase fusion fragment was inserted into a
unique Xho I site of plasmid PNT. The reporter gene was
followed by a selectable neomycin resistance gene
(pgkNeopA) in the sense orientation. A 3.6-kb
fragment spanning the TM mRNA region from the Sph I site at
the translational start site to an Xba I site within the 3'
untranslated mRNA region was inserted between the neo gene and the
thymidine kinase gene (pgkTKpA) of plasmid PNT.
|
The murine ES cell line D323 (obtained from Dr R.O. Hynes, Massachusetts Institute of Technology, Cambridge, Mass) was routinely propagated as undifferentiated stem cells on a feeder layer of mitotically inactivated primary mouse embryonic fibroblasts in DMEM containing 4.5 g/L glucose, 10 mmol/L HEPES, 0.1 mmol/L ß-mercaptoethanol, 2 mmol/L glutamine, 0.1 mmol/L MEM/nonessential amino acids, 15% heat-inactivated fetal calf serum (Intergen), and 103 U/mL recombinant murine leukemia inhibitory factor (ESGRO, GIBCO BRL). For transfection with the DNA targeting construct, a single cell suspension of ES cells was prepared by trypsinization, and the cells were washed twice in HEPES-buffered saline (mmol/L: HEPES 25, NaCl 134, KCl 5, and Na2HPO4 0.7, pH 7.1), suspended at 2x107 cells per milliliter in the same buffer containing 20 µg/mL of Not I–linearized DNA, and electroporated at 600 V/cm and 500 µF with a gene-pulser (Bio-Rad). The cells were replated on neomycin-resistant feeder layers, and stably transfected cells were selected in medium containing 200 µg/mL of (active) G418 (GIBCO BRL) and 2 µmol/L Gancyclovir (gift of Syntex, Palo Alto, Calif). Drug-resistant colonies were isolated after 7 to 9 days and individually expanded. ES cell clones with a targeted mutation were identified by Southern blot hybridization analysis of genomic DNA. lacZ-targeted ES cells were injected into C57Bl/6 host blastocysts and transferred to the uterus of pseudopregnant females. Chimeric animals were bred to C57Bl/6 females to achieve germline transmission of the mutated allele.
Measurement of TM mRNA and TM Protein Levels
The steady-state
level of TM mRNA was estimated by RNase
protection experiments performed on total RNA isolated from the tissues
of wild-type and heterozygous TMlacZ–targeted mice essentially as
described previously.21 Receptor levels in various organs
were determined with a double-monoclonal antibody assay using the
TM-specific antibodies 34A and 201B as outlined
elsewhere.21 24
Analysis of ß-Galactosidase Gene Expression and
Immunohistochemistry
For detection of reporter gene expression, the
animals
were perfused with PIPES-buffered paraformaldehyde (0.1
mol/L PIPES [pH 6.9], 2 mmol/L MgCl2, 2%
paraformaldehyde, and 1.25 mmol/L EGTA). Subsequently,
the organs were removed and incubated in perfusion buffer for 3 hours.
Tissue fragments were rinsed twice in PBS, equilibrated in PBS/30%
(wt/vol) sucrose, embedded in OCT compound, and frozen on dry ice.
Cryosections of 8 to 10 µm thickness were mounted on
surface-treated glass slides, air-dried, and postfixed briefly
with 1% formaldehyde in PBS. The sections were then rinsed in PBS and
incubated for 4 to 16 hours at 30°C in PBS containing 5 mmol/L
K4Fe(CN)6x3H2O, 5 mmol/L
K3Fe(CN)6, 2 mmol/L
MgCl2, 0.02% Nonidet P-40, 0.01% SDS, and 1 mg/mL
4-chloro-5-bromo-3-indolyl-ß-galactopyranoside (X-Gal). Stained
sections were rinsed again with PBS, counterstained with eosin,
dehydrated through graded alcohol, and mounted for photographic
documentation. Staining of whole organs and tissue fragments was
achieved by perfusing animals as described above, postfixing the organs
or tissue samples for 3 hours in perfusion buffer, and washing twice
for 2 hours each in PBS. The samples were then incubated with the
chromogenic ß-galactosidase substrate X-Gal as
described above. Immunohistochemical detection of TM in cryosections
was performed by postfixing air-dried sections for 1 minute at
4°C in acetone. Samples were rinsed briefly in PBS and then processed
for immunohistochemistry with the TM-specific monoclonal antibodies 34A
and 201B16 24 and secondary antibody/horseradish
peroxidase conjugates according to the instructions of the supplier
(Vectastain ABC, Vector Laboratories).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Insertion of the ß-Galactosidase Gene Into the TM Locus by Homologous Recombination
The organization of the intronless TM gene in D3 ES cells and the design of the targeting DNA construct are shown in Fig 1
(top). Homologous recombination between the targeting
construct and the endogenous TM gene results in the
insertion of the ß-galactosidase/neomycin genes into the start
codon of the TM gene and the transcription of a hybrid mRNA molecule,
consisting of 157 nucleotides of TM 5' untranslated mRNA
followed by the lacZ open reading frame. At the same time, the
integration of the reporter construct disrupts the TM gene by
separating the protein coding region from its promoter and creating a
loss-of-function mutation. The targeting construct was
introduced by electroporation into the D3 line of ES cells, and
individual colonies were isolated after selection in G418 and
Gancyclovir. Of a total of 581 colonies screened, eight were identified
as homologous recombinants by Southern blot hybridization
analysis of genomic ES cell DNA (Fig 1, bottom): integration of
the lacZ and neomycin genes into one genomic TM allele creates a
restriction fragment–length polymorphism of the TM locus that
is detected by hybridization of Bgl II–digested and
BamHI-digested DNA to probes external to the region
contained in the targeting vector. One clone showed an aberrant
restriction pattern indicative of a rearrangement in the 5' promoter
region and was excluded from further analysis. Using this
targeting vector, the frequency of homologous recombination events was
about two times higher than in previous experiments (1 in 80 versus 1
in 160) in which repetitive sequence elements present in the 5'
promoter region of the TM gene had been included in the targeting
construct.21 A line of TMlacZ–targeted mice was
established from ES cell–derived germ-line chimeras through
breeding with C57Bl/6 mice. TM mRNA and protein levels in several
organs of heterozygous TMlacZ–targeted mice were reduced to 52±6%
and 47±7%, respectively, compared with the organs from wild-type
littermates (n=3, average of two determinations for lung, spleen, and
kidney in each animal). They were free of overt thrombotic
complications, displayed wild-type reproductive
performance, and were phenotypically normal. Homozygous
TMlacZ–targeted mice failed to survive early postimplantation
development beyond embryonic day 9.5. The developmental defect was
similar to that observed earlier in TM-deficient mice21
and was evident as an overall growth retardation at embryonic day 8.5,
followed by a rapid resorption of mutant embryos.
ß-Galactosidase Expression in TMlacZ–Targeted
Mice
The organs of heterozygous F1 TMlacZ mice (8 to 12 weeks after
gestation) were examined for ß-galactosidase expression by
staining of cryosections or intact tissue fragments with the
chromogenic substrate X-Gal (Fig 2).
Reporter gene expression was scored positive if
simultaneously processed control sections from
nontransgenic sex-matched littermates exhibited no lacZ staining.
The endothelium of most blood vessels within the spleen
(Fig 2A), heart (Fig 2B), lung (Fig
2D), skin, body wall, bones,
retina, skeletal muscle, gastrointestinal tract, and choroid plexus
contained readily detectable levels of ß-galactosidase. In
contrast, the liver and kidney displayed marked differences in the
staining between large vessels and the microvascular bed. In the liver,
only the luminal intima of large hepatic vessels was
consistently lacZ positive. Occasional staining was seen in the
arterial intima of Glisson's triads, whereas
ß-galactosidase activity was absent in sinusoidal
endothelium and liver parenchymal cells. In the kidney,
X-Gal reaction product was detected in the larger vessels as well
as the afferent or efferent glomerular arterioles. In
contrast, only a small fraction of endothelial cells
lining the glomerular capillaries contained detectable lacZ
activity. The distribution of the X-Gal reaction product in
microvessels of the cortical brain parenchyma was markedly
heterogeneous; lacZ-staining vessels were frequently
observed in proximity to blood vessels that lacked detectable enzyme
activity (Fig 2C). This heterogeneous pattern was
reproduced by immunohistochemical localization of
endogenous TM in the brains of heterozygous TMlacZ mice
(not shown) and contrasted with the more uniform distribution of the
antigen in the brains of wild-type littermates, which express
twofold higher levels of antigen (Fig 2E and
2F).
|
An immunohistochemical analysis of TM antigen distribution was also performed on tissue sections from the liver, kidney, heart, and lung of heterozygous TMlacZ mice (not shown); in the liver and the kidney, TM antigen was detected predominantly in the endothelium of larger vessels. Staining of renal glomerular capillaries was variable and of lower intensity. Homogeneous and intense immunostaining was observed in all vessels of the myocardium, spleen, and lung. This distribution of TM antigen in the vascular beds of different organs was consistent with results from earlier immunohistochemical studies14 15 16 17 and resembled the expression pattern of the lacZ reporter gene.
In
contrast to the almost uniform expression of ß-galactosidase
in the larger blood vessels of the major organ systems, the luminal
surface of the thoracic and abdominal aorta showed patchy staining (Fig
3). lacZ-positive cells were found in small clusters or
streaks oriented along the direction of blood flow. This
heterogeneity contrasted with the homogenous and
intense staining of the endothelium in smaller arteries
branching from the aorta (Fig 3C). We also note that the
abdominal
aorta consistently contained more strongly lacZ-positive
endothelial cells than did the thoracic aorta.
|
Reporter gene activity
was also documented in nonvascular structures,
such as brain meninges (Fig 4A), interstitium of the
testes (Fig 4B), and the skin. Some neuronal cells showed
ß-galactosidase activity above background levels, but these cells
did not exhibit immunohistochemically detectable antigen expression.
Analysis of blood smears and of rib and femoral bone marrow
failed to reveal lacZ reporter activity in platelets,
megakaryocytes, or white blood cells and their progenitors (not shown).
No readily detectable staining was observed in whole mounts of the
thoracic pleura (not shown). Expression in the dermis was easily
detected in tissue obtained by tail biopsy (Fig 4C) and was
used to
identify heterozygous carriers of the mutation on a routine basis. The
pattern of reporter gene expression described above was identical in
three consecutive generations of backcrosses onto a C57Bl/6
background.
|
Generation of Chimeric Animals With a Genetically
Heterogeneous Vascular
Endothelium
After injection into host blastocysts, ES cells contribute
predominantly to a pool of totipotent cells located within the inner
cell mass that eventually gives rise to the embryo. The individual
organs and tissues of the adult animal consist of a mosaic mixture of
cells originating to varying degrees from the wild-type host
blastocyst and the genetically distinct ES
cells.25 26 The
contribution of ES cells to the vascular endothelium
was investigated by determining the lacZ expression pattern in chimeric
animals. Five animals displaying 70% to 95% chimerism in coat color
patterns were chosen for analysis.
The
ß-galactosidase–positive ES cell–derived
endothelial cells were observed in the vascular bed of
all organs in which the reporter gene was also expressed at detectable
levels in the endothelium of TMlacZ mice (Fig 5). The relative
proportions and the distribution of
lacZ-targeted and wild-type endothelial cells
varied between different organs of a given animal. In the
myocardium, areas with a relatively high proportion of ES
cell–derived endothelial cells were frequently
found adjacent to regions virtually devoid of blue staining (Fig
5A).
The endothelium of the lung and spleen displayed a more
even distribution of lacZ-positive cells (Fig 5C and
5D). In contrast
to the high degree of chimerism in the brain meninges, the vasculature
within the brain consistently contained few ES
cell–derived endothelial cells, resulting in a
localized focal blue staining of isolated vessels (Fig 5B).
Compared
with heterozygous TMlacZ animals, the endothelium of
the aorta and iliac artery contained a reduced number, but similar
distribution, of lacZ-positive cells in all chimeras (Fig 5E
and 5F).
Mosaicism was also evident in numerous other smaller vessels, such as
in the body wall, or in skeletal muscles, where reporter expression was
apparent over restricted vessel fragments.
|
These data show that ES cells with a genetically modified TM locus have the capacity to contribute in a mosaic pattern to the vascular endothelium in chimeric mice, thus creating a genetic heterogeneity of the endothelial cell population.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
In the present communication, we describe a straightforward approach for targeting expression of transgenes to murine vascular endothelium. We have inserted the lacZ reporter gene by homologous recombination into the TM locus of murine ES cells. We then show that the activity pattern of lacZ reproduces the expression profile of the endogenous receptor.14 15 16 17 The most intense uniform staining was observed in the vascular endothelium of the heart, lung, and spleen. In the kidney, reporter gene activity was predominantly associated with larger arteries, as well as afferent and efferent glomerular arterioles. In the liver, ß-galactosidase activity was similarly localized in the endothelium of major vessels. These data substantiate earlier findings14 15 16 17 27 indicating both interorgan and intraorgan differences in the levels of TM gene product. In contrast to other reports,16 28 brain endothelial cells clearly possessed lacZ activity, and the presence of TM antigen was confirmed by immunohistochemical analysis. The previous failure to identify TM in murine brain microvascular endothelium may be explained by differences in the sensitivity of methods used to detect the receptor and/or the loss of antigen during tissue processing.15 17
Reporter gene expression in the endothelium of the aorta was remarkable for the patchy distribution of endothelial cells expressing lacZ and contrasted sharply with the homogeneous endothelial staining of its tributaries. This transition in staining intensity may be related, in part, to regulation of TM gene expression by hemodynamic forces, as recently documented under in vitro conditions.29 30 In fact, the human TM promoter contains the sequence motif GAGACC, which has been identified as a shear-stress response element in the platelet-derived growth factor B-chain promoter and in other genes.31 It is not known whether a similar cis-acting sequence element is present in the promoter of the murine TM gene. The patchy distribution of lacZ-positive cells in aortic endothelium is also reminiscent of the previously reported heterogeneous pattern of expression of endothelin and von Willebrand factor in large-vessel intima.32 Furthermore, our results indicate that the aorta may be more sensitive to the development of thrombotic lesions because of the decreased amount (compared with other vessels) of TM per given endothelial surface area. This observation might explain how spontaneous thrombosis occurs preferentially in the abdominal aorta and internal carotid artery in human homozygous carriers of the fibrinogen Naples allele.33 34
TM expression has also been documented in a number of cell types outside the vasculature, including the meninges, mesothelial cells, dermal keratinocytes, and platelets.16 17 35 36 37 38 39 In mice carrying the TMlacZ allele, the X-Gal reaction product is detectable in brain meninges and dermis but is conspicuously absent from mesothelial cells and bone marrow/peripheral blood cell lineages. The above discrepancy in the apparent tissue distribution of lacZ and the endogenous gene product may be due to true species differences, variations in the sensitivities of detection assays, tissue-specific differences in the stability of lacZ mRNA, or nonphysiological interactions between DNA elements within the ß-galactosidase gene and the TM promoter.
The present investigation demonstrates that gene targeting of
exogenous cDNAs into the TM locus represents a viable strategy
for creating animal models of cardiovascular disease.
This approach allows widespread and high-level expression of
transgenes in adult endothelium that is not possible
with the currently available endothelial
cell–specific promoters. Thus, expression of procoagulant gene
products should allow us to generate murine models of the
hypercoagulable state, whereas the expression of cell-adhesion
molecules or chemokines might produce murine models with the early
inflammatory lesion of atherosclerosis. We also note
that the gene targeting technique is not prone to variability in
transgene expression because of integration site–specific effects.
This advantage may be particularly relevant to the study of polygenic
phenotypes in which the penetrance of a primary genetic defect
is modulated by other genes and should facilitate their identification.
One potential drawback of the present model is the
loss-of-function mutation created by the insertion of an
exogenous cDNA into the TM locus. Although heterozygous TM-deficient
mice with 50% of normal receptor levels do not exhibit overt
pathological abnormalities,21 the defect may become
symptomatic when the foreign gene product interferes
with the function of the hemostatic mechanism. We are currently in the
process of introducing targeted mutations into the TM gene that are
expected to increase the biological activity of the receptor to 
180%
as described for variant human TM genes.40 41
Subsequent
targeting of the wild-type TM allele in these mouse lines
should result in the generation of mice expressing levels of TM that
are within the limits of naturally occurring variability in different
mouse strains (Reference 42 and authors' unpublished data). Finally,
it is important to recognize that expression levels of a heterologous
gene targeted into the TM locus will be modulated by the same
cytokines and hormones that regulate transcription of the
endogenous gene.43 44 45
The present study also reveals that ES cells with a targeted TM locus become integrated to a significant extent into the endothelium of chimeric mice, leading to an artificial genetic heterogeneity of the vascular bed. The mosaic composition of the vascular tree in ES cell chimeras may result in microheterogeneity (as observed in aortic endothelium), in the presence of large regions of transgenic vasculature adjacent to areas with normal blood vessels (such as shown in the myocardium), in transgene expression limited to small and isolated stretches of blood vessels (as in the brain), or in a more uniform distribution of ES cell–derived endothelial cells (as found in the lung and spleen). These differences in the contribution of ES cells to the vasculature in different organs were consistently present in all chimeras and are probably related to the developmental pattern of vascularization in a given organ.
These observations suggest that targeted ES cells can be used to create animals with a functionally heterogeneous vascular system and hence permit us to devise animal models that are otherwise difficult or impossible to establish. For instance, experimental variation of the overall degree of chimerism could result in different systemic levels of secreted endothelial gene products, such as activators or inhibitors of fibrinolysis. Locally confined expression of cell surface–associated components, on the other hand, would allow us to determine whether and how localized production of procoagulants, chemokines, or cell adhesion molecules like selectins can lead to the development of vascular lesions.
Finally, we are presently exploiting the chimeric approach to bypass the embryonic lethal effect of a TM-null mutation. Preliminary results indicate that homozygous TM-deficient ES cells contribute to the vasculature of chimeric mice in a fashion similar to that described here for TMlacZ–targeted cells. The analysis of mice generated by this technique should enable us to ascertain the pathophysiological consequences of the null mutation in localized areas of the vascular system.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grant 1RO1 HL-53396-01. Drs Aird and Husain are both recipients of Clinician-Scientist Awards from the Medical Research Council of Canada. We would like to thank Stephen Kennel (Oak Ridge Laboratories) for his generous gift of TM-specific monoclonal antibodies.
Received September 27, 1995; accepted November 30, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
1. Conte MS, Birinyi LK, Miyata T, Fallon JT, Gold HK, Whittemore AD, Mulligan RC. Efficient repopulation of denuded rabbit arteries with autologous genetically modified endothelial cells. Circulation. 1994;23:2161-2169.
2. Geary RL, Clowes AW, Lau S, Vergel S, Dale DC, Osborne WR. Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally transduced smooth muscle cells: a model for local and systemic gene therapy. Hum Gene Ther.. 1994;5:1211-1216. [Medline] [Order article via Infotrieve]
3. Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H, Kaneda Y, Ogihara T, Dzau VJ. Evidence for direct local effect of angiotensin in vascular hypertrophy: in vivo gene transfer of angiotensin converting enzyme. J Clin Invest.. 1994;94:978-984.
4. Takeshita S, Gal D, Leclerc G, Pickering JG, Riessen R, Weir L, Isner JM. Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation: in vitro and in vivo findings in a rabbit model of vascular injury. J Clin Invest.. 1994;93:652-661.
5.
von der Leyen HE, Gibbons GH, Morishita R, Lewis NP,
Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy
inhibiting neointimal vascular lesion: in vivo transfer of
endothelial cell nitric oxide synthase gene.
Proc Natl Acad Sci U S A.. 1995;92:1137-1141.
6. Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG, Epstein SE, Finkel T. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1994;91:10732-10736.
7.
Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ,
Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell
proliferation after arterial injury.
Science.. 1994;265:781-784.
8.
Muller DWM, Gordon D, San H, Yang Z, Pompili VJ, Nabel
GJ, Nabel EG. Catheter-mediated pulmonary vascular
gene transfer and expression. Circ Res.. 1994;75:1039-1049.
9.
Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene
expression after intravenous DNA delivery into adult
mice. Science.. 1993;261:209-211.
10.
Aird WC, Jahroudi N, Weiler-Guettler H, Rayburn HB,
Rosenberg RD. Human von Willebrand factor gene sequences
target expression to a subpopulation of endothelial
cells in transgenic mice. Proc Natl Acad Sci
U S A.. 1995;92:4567-4571.
11. Schlaeger TM, Qin Y, Fujiwara Y, Magram J, Sato TN. Vascular endothelial cell lineage-specific promoter in transgenic mice. Development.. 1995;121:1089-1098. [Abstract]
12. Harats D, Kurihara H, Belloni P, Oakley H, Ziober A, Ackley D, Cain G, Kurihara Y, Lawn R, Sigal E. Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter. J Clin Invest.. 1995;95:1335-1344.
13.
Esmon CT, Owen WG. Identification of an
endothelial cell cofactor for thrombin catalyzed
activation of protein C. Proc Natl Acad Sci U S A.. 1981;78:2249-2252.
14.
Maruyama I, Bell CE, Majerus PW. Thrombomodulin
is found on endothelium of arteries, veins,
capillaries, and lymphatics, and on syncytiotrophoblast of human
placenta. J Cell Biol.. 1985;101:363-371.
15. DeBault LE, Esmon LE, Olson JR, Esmon CT. Distribution of the thrombomodulin antigen in the rabbit vasculature. Lab Invest.. 1986;54:172-178. [Medline] [Order article via Infotrieve]
16. Ford VA, Wilkinson JE, Kennel SJ. Thrombomodulin distribution during murine development. Roux Arch Dev Biol.. 1993;202:364-370.
17. Boffa MC, Burke B, Haudenschild CC. Preservation of thrombomodulin antigen on vascular and extravascular surfaces. J Histochem Cytochem.. 1987;35:1267-1276. [Abstract]
18.
Jackman RW, Beeler DL, Fritze LMS, Soff G, Rosenberg
RD. Human thrombomodulin gene is intron depleted: nucleic acid
sequences of the cDNA and gene predict protein structure and suggest
sites of regulatory control. Proc Natl Acad Sci
U S A.. 1987;84:6425-6429.
19.
Shirahi T, Shiojiri S, Ito H, Yamamoto S, Kusumoto H,
Deyashiki Y, Maruyama I, Suzuki K. Gene structure of human
thrombomodulin, a cofactor for thrombin-catalyzed activation of
protein C. J Biochem.. 1988;103:281-285.
20.
Dittman WA, Kumada T, Sadler JE, Majerus PW. The
structure and function of mouse thrombomodulin: phorbol
myristate acetate stimulates degradation and synthesis of
thrombomodulin without affecting mRNA levels in hemangioma
cells. J Biol Chem.. 1988;263:15815-15822.
21.
Healy AM, Rayburn HB, Rosenberg RD, Weiler-Guettler
H. Absence of the blood-clotting regulator thrombomodulin
causes embryonic lethality in mice before development of a functional
cardiovascular system. Proc Natl Acad Sci
U S A.. 1995;92:850-854.
22. Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, van den Oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature.. 1994;368:419-425. [Medline] [Order article via Infotrieve]
23. Doetschman TC, Eistetter H, Katz H, Schmidt W, Kemler R. The in vitro development of blastocyst derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol.. 1985;87:27-45. [Medline] [Order article via Infotrieve]
24. Kennel SJ, Lankford T, Hughes B, Hotchkiss JA. Quantitation of a murine lung endothelial cell protein, P 112, with a double monoclonal antibody assay. Lab Invest.. 1988;59:692-701. [Medline] [Order article via Infotrieve]
25.
Beddington RS, Robertson EJ. An assessment of
the developmental potential of embryonic stem cells in the midgestation
mouse embryo. Development.. 1989;105:733-737.
26. Williams BO, Schmitt EM, Remington L, Bronson RT, Albert DM, Weinberg RA, Jacks T. Extensive contribution of Rb-deficient cells to adult chimeric mice with limited histopathological consequences. EMBO J.. 1994;13:4251-4259. [Medline] [Order article via Infotrieve]
27.
Ford VA, Stringer C, Kennel SJ. Thrombomodulin
is preferentially expressed in Balb/c lung microvessels.
J Biol Chem.. 1992;267:5446-5450.
28.
Dittman WA, Majerus PW. Structure and function
of thrombomodulin: a natural anticoagulant. Blood.. 1990;75:329-336.
29.
Malek AM, Jackman R, Rosenberg RD, Izumo S.
Endothelial expression of thrombomodulin is reversibly
regulated by fluid shear stress. Circ Res. 1994;74:852-860.
30. Takada Y, Shinkai F, Kondo S, Yamamoto S, Tsuboi H, Korenaga R, Ando J. Fluid shear stress increases the expression of thrombomodulin by cultured human endothelial cells. Biochem Biophys Res Commun.. 1994;205:1345-1352. [Medline] [Order article via Infotrieve]
31.
Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey
CF, Gimbrone MA. Platelet-derived growth factor B chain
promoter contains a cis-acting fluid
shear-stress-responsive element. Proc Natl Acad
Sci U S A.. 1993;90:4591-4595.
32. Tomlinson A, Van Vlijmen H, Loesch A, Burnstock G. An immunohistochemical study of endothelial cell heterogeneity in the rat: observations in `en face' Haeutchen preparations. Cell Tissue Res.. 1991;263:173-181. [Medline] [Order article via Infotrieve]
33. Koopman J, Haverkate F, Lord ST, Grimbergen J, Mannucci PM. Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia: homozygous substitution of Bß 68 Ala–Thr. J Clin Invest.. 1992;90:238-244.
34. Lord ST. Fibrinogen. In: High KA, Roberts HRR, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: Marcel Dekker; 1991:51-74.
35.
Suzuki K, Nishioka J, Hayashi T, Kosaka J.
Functionally active thrombomodulin is present in human
platelets. J Biochem.. 1988;104:628-632.
36. Imada S, Yamaguchi H, Nagumo N, Katayanagi S, Iwasaki H, Imada M. Identification of fetomodulin, a surface marker protein of fetal development as thrombomodulin by gene cloning and functional assays. Dev Biol.. 1990;140:113-122. [Medline] [Order article via Infotrieve]
37.
McCachren SS, Diggs J, Weinberg JB, Dittman WA.
Thrombomodulin expression by human blood monocytes and by human
synovial tissue lining macrophages. Blood.. 1991;78:3128-3123.
38. Raife TJ, Lager DJ, Madison KC, Piette WW, Howard EJ, Sturm MT, Chen Y, Lentz SR. Thrombomodulin expression by human keratinocytes: induction of cofactor activity during epidermal differentiation. J Clin Invest.. 1994;93:1846-1851.
39. Mizutani H, Hayashi T, Nouchi N, Ohyanagi S, Hashimoto K, Shimizu M, Suzuki K. Functional and immunoreactive thrombomodulin expressed by keratinocytes. J Invest Dermatol.. 1994;103:825-828. [Medline] [Order article via Infotrieve]
40.
Clark JH, Light DR, Blasko E, Parkinson JF, Nagashima
M, McLean K, Vilander L, Andrews WH, Morser J, Glaser CB. The
short loop between epidermal growth factor-like domains 4 and 5 is
critical for human thrombomodulin function. J
Biol Chem.. 1993;268:6309-6315.
41.
Nagashima M, Lundh E, Leonard JC, Morser J, Parkinson
JF. Alanine-scanning mutagenesis of the epidermal growth
factor-like domains of human thrombomodulin identifies critical
residues for its cofactor activity. J Biol
Chem.. 1993;268:2888-2892.
42. Ford VA, Kennel SJ. An intracisternal A-particle DNA sequence is closely linked to the thrombomodulin gene in some strains of laboratory mice. DNA Cell Biol.. 1993;12:311-318. [Medline] [Order article via Infotrieve]
43.
Conway EM, Rosenberg RD. Tumor necrosis factor
suppresses transcription of the thrombomodulin gene in
endothelial cells. Mol Cell Biol.. 1988;8:5588-5592.
44.
Weiler-Guettler H, Yu K, Soff G, Gudas LJ, Rosenberg
RD. Thrombomodulin gene regulation by cAMP and retinoic acid in
F9 embryonal carcinoma cells. Proc Natl Acad Sci
U S A.. 1992;89:2155-2159.
45.
Yu K, Morioka H, Fritze LMS, Beeler DL, Jackman JW,
Rosenberg RD. Transcriptional regulation of the thrombomodulin
gene. J Biol Chem.. 1992;267:23237-23247.
This article has been cited by other articles:
 |
|
 |
|
  B. Bolinger, P. Krebs, Y. Tian, D. Engeler, E. Scandella, S. Miller, D. C. Palmer, N. P. Restifo, P.-A. Clavien, and B. Ludewig Immunologic ignorance of vascular endothelial cells expressing minor histocompatibility antigen Blood, May 1, 2008; 111(9): 4588 - 4595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Vanhooke, J. M. Prahl, C. Kimmel-Jehan, M. Mendelsohn, E. W. Danielson, K. D. Healy, and H. F. DeLuca CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1{alpha}-hydroxylase promoter activity in the skin PNAS, January 3, 2006; 103(1): 75 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. Melo, M. Gnecchi, A. S. Pachori, D. Kong, K. Wang, X. Liu, R. E. Pratt, and V. J. Dzau Endothelium-Targeted Gene and Cell-Based Therapies for Cardiovascular Disease Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1761 - 1774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Cooley Murine Model of Neointimal Formation and Stenosis in Vein Grafts Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1180 - 1185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-M. You, I. N. Mungrue, W. Kalair, T. Afroze, B. Ravi, A. M. Sadi, R. Gros, and M. Husain Conditional Expression of a Dominant-Negative c-Myb in Vascular Smooth Muscle Cells Inhibits Arterial Remodeling After Injury Circ. Res., February 21, 2003; 92(3): 314 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Weiler, V. Lindner, B. Kerlin, B. H. Isermann, S. B. Hendrickson, B. C. Cooley, D. A. Meh, M. W. Mosesson, N. W. Shworak, M. J. Post, et al. Characterization of a Mouse Model for Thrombomodulin Deficiency Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1531 - 1537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Edelberg, P. D. Christie, and R. D. Rosenberg Regulation of Vascular Bed-Specific Prothrombotic Potential Circ. Res., July 20, 2001; 89(2): 117 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Isermann, S. Hendrickson, K Hutley, M Wing, and H Weiler Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block Development, January 3, 2001; 128(6): 827 - 838. [Abstract] [PDF]
|
|
|
|
|
|
F. M. Faraci and C. D. Sigmund Vascular Biology in Genetically Altered Mice : Smaller Vessels, Bigger Insight Circ. Res., December 3, 1999; 85(12): 1214 - 1225. [Full Text] [PDF]
|
|
|
|
|
|
J. Guan, P. V. Guillot, and W. C. Aird Characterization of the Mouse von Willebrand Factor Promoter Blood, November 15, 1999; 94(10): 3405 - 3412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Kullo, R. D. Simari, and R. S. Schwartz Vascular Gene Transfer : From Bench to Bedside Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 196 - 207. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Carmeliet, L. Moons, and D. Collen Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis Cardiovasc Res, July 1, 1998; 39(1): 8 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Cowan, D. Tsang, C. M. Pedic, L. R. Abbott, T. A. Shinkel, A. J.F. d'Apice, and M. J. Pearse The Human ICAM-2 Promoter is Endothelial Cell-specific in Vitro and in Vivo and Contains Critical Sp1 and GATA Binding Sites J. Biol. Chem., May 8, 1998; 273(19): 11737 - 11744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Husain, K. Bein, L. Jiang, S. L. Alper, M. Simons, and R. D. Rosenberg c-Myb–Dependent Cell Cycle Progression and Ca2+ Storage in Cultured Vascular Smooth Muscle Cells Circ. Res., May 19, 1997; 80(5): 617 - 626. [Abstract] [Full Text]
|
|
|
|
|
|
E. M. Conway, S. Pollefeyt, D. Collen, and M. Steiner-Mosonyi The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis Blood, January 15, 1997; 89(2): 652 - 661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H Weiler-Guettler, W. Aird, H Rayburn, M Husain, and R. Rosenberg Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos Development, July 1, 1996; 122(7): 2271 - 2281. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||