1 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA
2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX
77030, USA
3 Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX
77030, USA
4 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA
5 Department of Medicine, Baylor College of Medicine, Houston, TX 77030,
USA
6 Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX
77030, USA
Author for correspondence (e-mail:
khirschi{at}bcm.tmc.edu)
Accepted 22 September 2003
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SUMMARY |
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Key words: Vascular development, Retinoic acid, Endothelial cell cycle control, Mouse
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Introduction |
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The vascular plexus that initially forms does so in the absence of blood
flow. However, extensive remodeling of the plexus into a branched circulatory
network (Risau et al., 1988b)
is dependent upon the onset of flow, suggesting that mechanical forces, as
well as soluble factors within circulation are needed for normal vessel
development. Vascular remodeling is a complex process that involves cell-cell
(gap junctions) (Hirschi et al.,
2003
; Kruger et al.,
2000
) and cell-matrix (Francis
et al., 2002
) interactions, as well as the coordination of
multiple signaling pathways. Signaling pathways implicated in this process
include: TGFß and TGFßRI and II, which regulate endothelial cell
matrix production and maturation (Dickson
et al., 1995
; Larsson et al.,
2001
); ephrin B and Ephb receptors, which modulate cell-cell
interactions and patterning (Gerety and
Anderson, 2002
; Oike et al.,
2002
); and angiopoietin 1 and the Tie2 receptor
(Sato et al., 1995
;
Suri et al., 1996
), as well as
PDGFß and PDGFRß (Lindahl et
al., 1997
; Soriano,
1994
), which mediate endothelial-induced mural cell (pericytes or
smooth muscle cells) recruitment. Other pathways, including retinoid
signaling, have been implicated in cardiovascular development; however, a
cellular role for retinoids in mammalian blood vessel assembly has not been
defined.
Retinoids are derivatives of Vitamin A (retinol) that exert a wide variety
of profound effects on vertebrate development
(DeLuca, 1991;
Zile, 2001
). Vitamin A is an
essential nutrient, required for crucial biological functions in quantities
that far exceed what can be metabolically generated. Thus, survival of
mammalian and avian species is dependent upon acquisition of adequate dietary
vitamin A, which must be enzymatically converted to active retinoic acid (RA).
The terminal step in RA synthesis is carried out by members of the class I
aldehyde dehydrogenase (ALDH) family
(Duester, 2000
;
McCaffery and Drager, 2000
;
Ulven et al., 2000
). Among
these, retinaldehyde dehydrogenase 2 (Raldh2; Aldh1a2 -
Mouse Genome Informatics) has high substrate specificity for retinaldehyde
(Zhao et al., 1996
). Embryos
lacking this enzyme are RA deficient and die in utero at E10.5
(Niederreither et al.,
1999
).
The RA signal is transduced through two families of ligand dependent
transcriptional regulators, RA receptors (RARs) and retinoid X receptors
(RXR), which bind as heterodimers to DNA motifs (RA response elements, RAREs)
and thus regulate the transcriptional activity of target genes (reviewed by
Mangelsdorf et al., 1994; Chambon,
1996). Nutritional vitamin A deficiency (VAD) in the rat
(Wilson and Warkany, 1949
), as
well as RAR and RXR knockouts in the mouse, yield many defects, including
abnormal ventricular trabeculation, defective outflow tract septation and
aortic arch malformations (Mendelsohn et
al., 1994
; Sucov et al.,
1994
, Kastner et al.,
1994
; Kastner et al.,
1997
). Quail embryos that develop under full VAD display more
severe heart defects and lack omphalolmesenteric vessel formation
(Heine et al., 1985
).
Mice deficient for Raldh2 exhibit a similar phenotype as quail VAD
embryos. Raldh2 is first expressed in the posterior mesoderm of the
mouse embryo during gastrulation (Niederreither et al., 1997), and is
expressed in the visceral endoderm at E7.5-8.5 (B.L.B., L.L., Pascal Dolle and
K.K.H., unpublished) at the time of endodermal-mediated vascular induction in
adjacent mesoderm. Importantly, retinoic acid receptors are expressed in
endothelial cells in the adjacent mesoderm (B.L.B., L.L., Pascal Dolle and
K.K.H., unpublished). Lack of Raldh2 expression in the yolk sac is
correlated with disrupted formation of extra-embryonic vessels in
Raldh2-/- mutants
(Niederreither et al., 1999).
However, Raldh2-/- embryos also exhibit defects in heart
looping morphogenesis, and severely hypoplastic atria and sinus venosus
development (Niederreither et al.,
2001
). Vascular defects can be the indirect result of cardiac
malformations; therefore, we aimed to determine whether RA directly regulates
vascular cell behavior and blood vessel formation using
Raldh2-/- mice.
Histological examination revealed vascular malformations in RA-deficient yolk sacs and embryos prior to the onset of cardiac function and blood circulation. Cellular and molecular analyses indicated that RA was not required for endothelial cell differentiation, but was required for endothelial cell maturation and the control of cell cycle progression. Raldh2-/- mutants exhibited suppressed p21 and p27 induction, enhanced endothelial cell proliferation, and disrupted vascular plexus remodeling and patterning. Continuous RA supplementation of maternal diet restored cell cycle control and rescued the observed vascular defects in Raldh2-/- embryos and yolk sacs. Hence, RA plays a crucial role in mammalian vascular development, and is required throughout development to control endothelial cell growth and vascular remodeling. Such insight may be useful for the modulation of neovascularization during the progression of prevalent pathologies in which uncontrolled endothelial cell growth is a primary defect, or for the control of vessel assembly ex vivo in tissue engineering applications.
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Materials and methods |
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Histology and Immunohistochemistry
Mouse embryos and yolk sacs (E8.25-9.5) were fixed in 4% paraformaldehyde
for 2-3 hours; embedded in 3% agarose and then paraffin wax; sectioned, or
quick-frozen in liquid nitrogen; embedded in OCT compound; and sectioned.
Hematoxylin and Eosin staining of 5-10 µm paraffin wax sections of
embryonic and yolk sac tissue was performed according to standard methods.
Immunohistochemistry of 7-10 µm frozen sections was performed, as described
(Noveroske et al., 2002),
using the following primary antibodies (diluted in 4% NGS/3% BSA/PBS blocker),
incubated for 2-4 hours at room temperature: rat anti-VE-cadherin (Pharmingen,
San Diego, CA), 1:150 dilution; mouse anti-SM-
-actin (DAKO,
Carpenteria, CA), 1:1000 dilution; mouse anti-p21, 1:200 (NeoMarkers,
Freemont, CA); and mouse anti-p27, 1:200 (NeoMarkers). Species-specific
biotinylated secondary antibodies were diluted 1:250 and incubated for 1 hour
at room temperature. Vectastain Elite ABC kit (Vector Laboratories,
Burlingame, CA) with its corresponding peroxidase substrate Vector VIP were
used, according to the manufacturer's instructions, to reveal antibody-antigen
complexes.
Embryos and yolk sacs at various stages of development (E8.25-12.5) were
prepared for whole-mount immunohistochemistry, as described
(Noveroske et al., 2002).
Fixed tissues were rehydrated, permeabilized and incubated overnight at
4°C with one of the following primary antibodies (diluted in PBS
containing 0.2% nonfat dry milk and 0.5% Triton X-100): mouse
anti-SM-
-actin (DAKO), 1:1000; mouse anti-CD31, 1:100 (PE-CAM-1,
Pharmingen); mouse anti-p21, 1:200 (NeoMarkers); and mouse anti-p27, 1:200
(NeoMarkers). Species-specific secondary IgG-POD antibodies (Boehringer
Mannheim, Indianapolis, IN) were used at 1:250 in conjunction with the
Vectashield Elite ABC kit (Vector Laboratories) and peroxidase substrate DAB
(Sigma, St Louis, MO) to reveal antigen-antibody complexes.
Semi-quantitative reverse transcriptase polymerase chain reaction
(sqRT-PCR)
Yolk sacs from E8.5 embryos were dissected and pooled according to
genotype, then RNA was extracted using TRIzol Reagent (Invitrogen) and
purified as specified by the manufacturer. RNA (1 µg) was reverse
transcribed with random primers and Superscript Reverse Transcriptase II
(Invitrogen) at 42°C for 50 minutes and 72°C for 15 minutes.
One-twentieth of the RT reaction was PCR amplified using Taq polymerase
(Invitrogen) and specific primers as follows: Flk1 (forward,
5'-gccaatgaaggggaactgaagac-3'; reverse,
5'-tctgactgctggtgatgctgtc-3'), VE-cad (forward,
5'-ttgcccagccctagcaacctaaag-3'; reverse,
5'-accaccgccctcctcatcgtaagt-3'), Tie2 (forward,
5'-atggactctttagccggctta-3'; reverse
5'ccttatagcctgtcctcgaa-3'), Ang1 (forward,
5'-cagtggctgcaaaaacttga-3'; reverse,
5'-tctgcacagtctcgaaatgg-3'), Cx40 (forward,
5'-atgggtgactggagcttccc-3'; reverse,
5'-cacaaagatgatctgcagtaccc-3'), HPRT (forward,
5'-gctggtgaaaaggacctct-3'; reverse,
5'-cacaggactagaacacctgc-3'). Reactions for each primer set were
run at their optimized annealing temperature, within their determined linear
range and with no RT controls. HPRT was used as an internal control for
semi-quantitative comparison. Each RT-PCR experiment was performed with 4-5
separate RNA collections.
Quantification of cell proliferation and apoptosis in situ
Proliferation
Embryos and yolk sacs (E8.25-9.5) were fixed for 2-3 hours in 4%
paraformaldehyde in PBS at 4°C. Endogenous peroxidase activity was
quenched via incubation with 3% H2O2 in PBS for 30
minutes, followed by washing in PBS containing 0.2% BSA and 0.1% triton.
Tissues were incubated with anti-phosphohistone 3 antibodies (1:200; Upstate,
Lake Placid, NY) overnight at 4°C, then with peroxidase-linked anti-rabbit
secondary antibodies (1:250); antigen-antibody complexes were revealed using
DAB substrate. Nuclei within tissues were revealed via mounting slides with
Vectashield containing DAPI (Vector). The numbers of phosphohistone 3-positive
and DAPI-stained nuclei in each of three high-power fields was counted in five
yolk sac samples from wild-type and Raldh2-/- embryos;
data represent the mean±s.d. for each group. Immunostaining with
anti-phosphohistone 3 antibodies was similarly performed on frozen sections of
wild-type and Raldh2-/- yolk sac tissues; sections were
co-stained with anti-VE-cadherin and DAPI, as described above.
Apoptosis
Detection of apoptotic cells was performed on 10 µm frozen sections
using the DeadEnd colorimetric TUNEL assay system (Promega, Madison, WI).
Briefly, slides were fixed in 4% paraformaldehyde for 15 minutes, washed in
PBS and permeabilized with 20 µg/ml Proteinase K solution for 10 minutes.
Sections were then washed in PBS, repeat fixed in 4% paraformaldehyde, washed
in PBS and equilibrated for 10 minutes in equilibration buffer (200 mM
potassium cacodylate, 25 mM Tris-HCl, pH 6.6, 0.2 mM DTT, 2.5 mM cobalt
chloride, 0.25 mg/ml BSA). In situ 3'OH ends of DNA fragments were
labeled with biotinylated nucleotide mix (250 µM biotinylated nucleotides,
10 mM Tris-HCl, pH 7.6, 1 mM EDTA) by terminal deoxynucleotidyl transferase
(25 u) for 1 hour at 37°C. Sections were then sequentially washed in
2xSSC and PBS, and blocked with 0.3% H2O2 for 3
minutes. Biotinylated DNA was detected by incubating with Streptavidin HRP
(1:500 in PBS) for 30 minutes and addition of DAB substrate.
Retinoic acid rescue of Raldh2-/- mutants
To determine to what extent vascular defects observed in
Raldh2-/- embryos and yolk sacs could be rescued, and to
identify the crucial period(s) during vascular development when RA is needed,
maternal diet was supplemented with RA during defined treatment periods that
correlated with specific stages of vessel assembly (E7.5-8.5; E7.5-9.5;
E7.5-12.5). To prepare RA-supplemented diet, all trans RA (Sigma) from a 5
mg/ml ethanol stock suspension was diluted in 50 ml of water and mixed with 50
g of powdered chow to a final concentration of 100 µg/g food, which was
partially covered with aluminum foil and renewed each day for up to 5 days.
E12.5 embryos were analyzed histologically.
Endothelial cell proliferation assay
Endothelial cells were isolated from adult bovine aortas, as described
(Gimbrone, 1976), and grown in
Dulbeco's Modified Essential Medium (DMEM) containing 10% calf serum (CS),
penicillin, streptomycin and glutamine. For proliferation assays, endothelial
cells were trypsinized, counted using a Coulter Counter and plated in 24-well
culture dishes at 10,000 cells per well in 0.5 ml DMEM/2% CS. Cells were
incubated at 37°C for up to 3 days in the presence of 0-1 µM all-trans
retinoic acid. At each time point, triplicate samples were trypsinized and
counted. Data represent the mean±s.d. of total endothelial cell number
in each experimental group from at least three experiments.
Cell cycle analysis
Endothelial cells were plated and treated with RA as described above for
cell proliferation assay; however, after trypsinization, cells were fixed in
100% methanol, then incubated with 10 µg/ml propidium iodide (Calbiochem,
SanDiego, CA). The cell samples were then filtered through 35 µm nylon mesh
and DNA analyzed within an hour on a flow cytometer (Beckon Dickinson
FACScan). FACS analysis figures are shown for one representative experiment;
data represent averages from at least three experiments.
Western blot analysis
Total protein was isolated from endothelial cells that were cultured in the
presence of 0-1 µM retinoic acid for up to 3 days, as previously described
(Hirschi et al., 1996), and
electrophoresed in 7 to 15% SDS-polyacrylamide gels (10 µg protein per
lane). After transfer to a 0.2 µm PVDF membrane (Millipore, Bedford, MA),
the membranes were blocked in 5% nonfat dry milk (in PBS with 0.1% Tween20)
for 1 hour, then incubated for 1 hour with primary antibodies. Antibodies
included mouse monoclonal anti-p21 (Lab Vision, Fremont, CA), diluted 1:500 in
blocking solution; mouse anti-p27 (1:750; BD Transduction, SanDiego, CA);
anti-RB (1:1:500; Lab Vision); anti-cyclin D1 (1:1000; Rockland,
Gilbertsville, PA); anti-cyclin D2 (1:1000; Lab Vision); anti-Cdk2 (1:1000;
Rockland); anti-Cdk4 (1:750; Rockland); and anti-cytoskeletal actin (1:1000;
Santa Cruz). After primary antibody incubation, membranes were washed three
times in 0.1% Tween20 in PBS for 20 minutes, then exposed to species-specific
secondary antibodies conjugated to horseradish peroxidase (1:10,000; Amersham
Life Sciences, Arlington Heights, IL). Antigen-antibody complexes were
revealed using an enhanced chemiluminescence system (ECL-Plus kit; Amersham);
autoradigraphs were quantified using a digital imaging system (Alpha Innotech,
CA).
Immunoprecipitation and detection of protein-protein complexes
Total protein was isolated from endothelial cells that were cultured in the
presence of 0-1 µM all-trans retinoic acid for up to three days, as
described (Hirschi et al.,
1996). Cell lysates were pre-cleared using protein A/G agarose,
and incubated with primary antibodies overnight at 4°C. The following
primary antibodies were used to immunoprecipitate proteins from cell lysates:
anti-p21, 1:1000; anti-p27, 1:750; anti-cyclin D1, 1:1000; anti-cyclin D2,
1:1000; anti-Cdk2, 1:1000; and anti-Cdk4, 1:1:750. Immunoprecipitated protein
was pelleted, rinsed, resuspended in gel loading buffer, then boiled and
pelleted again. The supernatant was subjected to western analysis, as
described above.
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Results |
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Vascular plexus formation and remodeling
Embryos and yolk sacs were dissected at E8.25-8.5 (3- to 6-somite stage),
prior to the onset of cardiac function and systemic blood circulation,
photographed, then genotyped and processed for further analyses. Upon
morphological examination, it was apparent that a vascular plexus and red
blood cells had formed in both wild-type and Raldh2-/-
yolk sac tissues (Fig. 1A-D).
Hematoxylin and Eosin staining of 5 µm paraffin sections of yolk sac tissue
suggested that although a vascular plexus formed in both wild type and
Raldh2-/- mutants (Fig.
1C,D), the vessels within Raldh2-/- plexi were
dilated (Fig. 1D).
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To determine whether the endothelial tubes formed in wild-type and
Raldh2-/- tissues acquired a vessel wall composed of mural
cells (smooth muscle cells or pericytes), yolk sac tissues isolated at E9.5
were immunostained with antibodies against SM--actin, which is among
the first proteins to be significantly upregulated in mesenchymal progenitors
in response to endothelial cell recruitment and contact
(Hungerford et al., 1996
;
Hirschi et al., 1998
). In E9.5
wild-type tissues, a high level of SM-
-actin expression was evident
around all large vessels of the yolk sac
(Fig. 1G), and associated with
a small proportion of vessels in the head region and along the dorsal aorta
within the embryo proper (not shown). By contrast, whole-mount staining of
Raldh2-/- mutants revealed that the endothelial tubes in
yolk sacs (Fig. 1H) and embryos
(not shown) were not invested with cells highly expressing SM-
-actin,
suggesting lack of endothelial-mediated mural cell recruitment and/or
differentiation.
Endothelial cell maturation
As morphological defects were apparent in Raldh2-/-
mutants prior to the onset of cardiac function and vessel wall formation, we
focused all subsequent cell and molecular analyses on mutants at E8.25-8.5. In
so doing, we were able to define the direct effects of RA on endothelial cell
behavior and early vessel formation and avoid potential confounding effects of
RA deficiency that may occur at later stages of development, such as disrupted
hemodynamic forces and systemic oxygen/nutrient distribution.
We performed whole-mount immunostaining of E8.25-8.5 embryos and yolk sacs with antibodies against PECAM1, an early marker of endothelial cells. We found that in wild-type yolk sacs, endothelial tubes were similar in diameter, evenly distributed, and exhibited some `pruning' into smaller vessel structures (Fig. 2A, arrows). By contrast, endothelial tubes within Raldh2-/- yolk sacs were dilated, with little evidence of appropriate remodeling (Fig. 2B, arrows). Within wild-type embryos, vascular plexus formation was evident within the head (Fig. 2C) where distinct margins of vascularization were notable (arrows). By contrast, the vascular plexi within the heads of Raldh2-/- embryos were diffusely distributed (Fig. 2D). Vessels within the somitic regions of Raldh2-/- mutants were also dilated and inappropriately patterned (Fig. 2F, arrows) relative to wild type (Fig. 2E).
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Control of vascular cell proliferation and apoptosis
The dilated and immature vessel structures in Raldh2-/-
yolk sacs and embryos could result from multiple endothelial cell
dysfunctions. Given the proposed role of retinoids in growth control and
programmed cell death in other systems, we examined whether vessel defects in
the Raldh2-/- mutants resulted from lack of endothelial
cell growth control or suppressed apoptosis during vascular development. To
obtain a measure of cell proliferation in vivo, we immunostained wild-type and
Raldh2-/- yolk sac and embryonic tissue (five tissues in
each group) with antibodies against phosphohistone 3, which is detectable only
in cells undergoing mitosis. Representative stained tissues are shown in
Fig. 3A,B; there was
consistently more phosphohistone 3 staining in Raldh2-/-
tissues.
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To determine which cell types within Raldh2-/- tissues exhibited increased mitotic index, cross-sections of E8.5 yolk sacs were immunostained for phosphohistone 3 and co-stained with DAPI; mitotic index was calculated as described above. We found no difference in the mitotic index of cells within the visceral endoderm layer of wild-type and Raldh2-/- yolk sacs (Fig. 3C); however, there was a significant increase in mitosis in the mesodermal layer of Raldh2-/- yolk sacs compared with wild type. Coimmunostaining for phosphohistone 3 and endothelial cell marker VE-cadherin demonstrated that the mitotic cells in the Raldh2-/- yolk sacs were predominantly vascular endothelial cells (Fig. 3E). There was no difference in the level of apoptosis among endothelial cells in the wild-type and Raldh2-/- yolk sacs (data not shown).
We examined the expression of cell cycle inhibitors via whole-mount and section immunostaining of wild-type and Raldh2-/- yolk sacs. We found significantly reduced levels of p21 (Fig. 4B) and p27 (not shown) in Raldh2-/- tissues, compared with wild type (Fig. 4A). Section staining revealed that p21 expression in the wild-type yolk sacs was predominantly in endothelial cells of blood vessels in the mesodermal layer of the yolk sac (Fig. 4C, arrows); p21 protein was essentially absent in Raldh2-/- yolk sacs (Fig. 4D).
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Endothelial cells were cultured for up to 72 hours in the presence of 0-1
µM all-trans RA, and the expression of cell cycle associated proteins was
examined. Via western blot analyses, we determined that 1 µM RA induced the
expression of p21 (Fig. 4E) and
p27 (not shown), members of the Cip/Waf family of Cdk inhibitors, within 8
hours, but had no affect on the expression of members of the Ink4 family of
Cdk inhibitors (p15, p18, p19) or p53 (data not shown). Retinoid treatment
also had no effect on the expression of cyclins A, B, D3 or E, or on the
expression of Cdc2, or Cdk2, 5 or 6 (data not shown). Surprisingly, retinoids
upregulated the expression of cyclin D1 and D2 proteins and Cdk4 (not shown),
which are associated with progression of cell cycle from G1 to S phase. The
upregulation of cyclin D proteins, in conjunction with the upregulation of p21
and cell cycle arrest of fibroblasts has been previously reported
(Sinibaldi et al., 2000), but
the biological significance is not clear.
To determine whether RA directly regulates endothelial cell proliferation, we cultured endothelial cells for 3-5 days in the presence of 0-1 µM all-trans RA or 0-1 µM synthetic agonists (BioMol) of the RXR or RAR receptor families - methoprene acid (MA; RXR-specific agonist) and TTNPB (RAR-specific agonist). RA significantly suppressed the increase in endothelial cell number seen over time in culture in untreated cells, as did the RAR agonist TTNPB (Fig. 5A); RXR agonist MA had no effect on endothelial cell number. Thus, it appears that RA regulates endothelial cell number in a process specifically mediated via RAR receptors. The effects of RA on endothelial cell number were dose-dependent from 0.1 to 5 µM (data not shown). After cell counting, control and RA-treated endothelial cells were fixed and stained with propidium iodide, then subjected to fluorescence activated cell sorting (FACS) to determine their cell cycle distribution. In response to RA, there was a significant decrease in the proportion of endothelial cells in S phase and an increase in the proportion of cells in G1 phase (Fig. 5B). There was no evidence of RA-induced endothelial cell apoptosis, which corroborates our in vivo findings.
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|
Circulating RA rescues Raldh2-/- vascular
defects
It was previously determined that the effects of Raldh2 deficiency
on the embryo could be partially rescued by providing RA to the embryos via
maternal diet and blood circulation
(Niederreither et al., 2001;
Niederreither et al., 2002
).
RA administration at a dosage of 100 µg/g food (see Materials and methods)
does not interfere with RARE-lacZ reporter transgene activity and
safely avoids teratogenic effects in wild-type embryos
(Niederreither et al., 2002
).
We used this system to determine to what extent vascular defects observed in
Raldh2-/- embryos and yolk sacs could be rescued and to
identify the crucial period(s) during vascular development when RA is needed.
We supplemented maternal diet with RA during defined developmental periods
that correlated with specific stages of vessel assembly. In this manner, we
studied the impact of circulating RA on endothelial tube formation (E7.5-8.5
RA supplementation); vascular remodeling, including initial mural cell
recruitment (E7.5-9.5 RA supplementation); and vessel assembly, including
mural cell differentiation and vessel wall formation (E7.5-12.5 RA
supplementation).
Pregnant Raldh2+/- females were fed powdered chow containing 100 µg/g all-trans RA from E7.5-8.5, E7.5-9.5 or E7.5-12.5; all mice were killed at E12.5. Wild-type embryos from the experimental (Raldh2+/-) litters and E12.5 wild-type embryos from chow-fed animals were used as controls, and there was no difference in the development of these embryos, indicating that RA had no effect on the development of wild-type embryos. Almost all mutants of RA-fed mothers survived to E12.5, whereas mutant embryos of chow-fed females died at E10.5. The embryos exposed to RA from E7.5-8.5 (during endothelial tube formation) exhibited the lowest degree of rescue of vascular development (Fig. 7, column 2). The yolk sacs of these embryos exhibited formation of some large vessels that are never formed in untreated mutants (arrowheads), but all smaller caliber vessels (arrows) were significantly dilated relative to wild-type animals also exposed to RA (Fig. 7, column 1). Exposure to RA from E7.5-9.5 (encompassing early vascular remodeling) induced formation of a larger number of branched vessels (arrowheads), but dilation of all vessels (arrows) was still evident (Fig. 7, column 3). Exposure to RA throughout the entire period of vascular development (E7.5-12.5) lead to vascularization of the mutant yolk sacs equivalent to wild-type littermates (Fig. 7, column 4); large (arrowheads) and small (arrows) vessels were similar in caliber and distribution to those observed in wild type (column 1).
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Discussion |
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We found that in the absence of RA (Raldh2-/- mutants), mesodermal progenitors do differentiate into endothelial cells that form a primitive vascular plexus. However, the blood vessels that constitute the plexi of RA-deficient embryos are immature, abnormally dilated and never become remodeled into a competent circulatory network capable of sustaining embryonic development. We determined that the dilation and inappropriate patterning of blood vessels observed in the absence of RA was associated with lack of endothelial cell cycle control. Uncontrolled endothelial cell growth during early vessel formation in Raldh2-/- mutants disrupted later stages of vessel assembly, as well, including endothelial-induced mural cell recruitment and differentiation.
The fact that circulating RA, provided to the embryo through maternal diet,
restored cell cycle control and rescued the observed vascular defects in
Raldh2-/- embryos and yolk sacs indicates that endothelial
cells themselves need not produce active forms of RA during development.
However, given that active RA would not normally be obtained from maternal
circulation, which typically contains the precursor retinol, our studies
indicate that functional embryonic Raldh2 is required for normal
vascular development. Furthermore, as RA was needed during the entire period
of vascular development to suppress endothelial cell growth and restore
vascular remodeling, it appears that continuous regulation of the production
or degradation (White et al.,
1997; White et al.,
1996
) of this soluble effector is necessary for appropriate vessel
formation and function.
Our in vivo and in vitro data collectively suggest that RA signaling
suppresses endothelial cell replication via the upregulation of Cdk inhibitors
of the Cip/Waf family. Consistent with our findings, previous studies
demonstrated that the expression of p21 and p27 is transcriptionally
(Liu et al., 1996;
Sasaki et al., 2000
) and
post-transcriptionally (Dimberg et al.,
2002
) regulated by RA. We found that in endothelial cells in
vitro, the inhibitors p21 and p27 competitively bind to Cdk4 and cyclin D
proteins and, thus, prevent their interactions, which are needed for
Cdk4-dependent progression from G1 to S phase of cell cycle
(Ekholm and Reed, 2000
). This
mechanism of RA control of proliferation has been previously demonstrated for
lymphocytes (Naderi and Blomhoff,
1999
) and myeloid cells
(Dimberg et al., 2002
), as well
as tumor cells (Hsu et al.,
2000
; Suzui et al.,
2002
; Zhang et al.,
2001
).
Although the timing of RA-induced expression of p21 and p27 in endothelial
cells within 8 hours in vitro is consistent with transcriptional or
post-transcriptional regulation, it is possible that RA signaling in vivo
functions in conjunction with other signaling pathways to modulate endothelial
cell maturation and growth control. For example, TGFß signaling is
associated with the regulation of endothelial cell extracellular matrix
production (Goumans et al.,
1999), which may contribute to growth control. In addition to
upregulation of p21 at 8 hours, we also observed an increase at 72 hours;
therefore, it is possible that RA induces other signals such as the TGFß,
which contribute to endothelial cell cycle control via matrix production and
cell-matrix mediated interactions. A signaling hierarchy among these factors,
and their potential contribution to endothelial cell cycle control in vivo,
are currently under investigation. However, the fact that maternally derived
RA is sufficient to rescue the endothelial cell proliferation defects in
Raldh2-/- mutants indicates that if other signaling
pathways are involved in the control of endothelial cell cycle progression,
they are downstream of RA signaling.
Endothelial cell proliferation has also been proposed to be passively
controlled via the VEGFA receptor Flt1. Similar to
Raldh2-/- mutants, Flt1-deficient embryos exhibit
abnormally high levels of endothelial cell replication, associated with
disrupted vascular remodeling and vessel assembly
(Fong et al., 1995). Flt1 is
proposed to modulate endothelial cell proliferation by limiting the local
availability of VEGFA and, thereby, preventing the stimulation of pathways
that trigger replication (Kearney et al.,
2002
). Consistent with this idea, we found that Flt1 mRNA
expression was downregulated in the Raldh2-/- embryos;
however, VEGFA levels were concomitantly reduced (B.L.B., L.L., Pascal Dolle
and K.K.H., unpublished). Therefore, it is unlikely that decreased Flt1
receptor availability in Raldh2-/- mutants would lead to
enhanced endothelial cell replication induced by excessive VEGFA
signaling.
Given that circulating RA suppresses endothelial cell cycle progression
during vessel assembly, it may be possible to use RA (provided intravenously
or orally) to treat pathologies in which uncontrolled neovascularization is a
primary defect, such as in angiogenesis-dependent tumorigenesis or
retinopathy. In support of this idea, it was previously suggested that
suppression of endothelial cell proliferation contributes to RA-induced growth
inhibition of squamous cell carcinoma in vivo
(Lingen et al., 1996).
In summary, we have demonstrated that RA plays a crucial role in the control of mammalian vascular development. The primary role of RA appears to be the suppression of endothelial cell growth during vascular remodeling and patterning; no other soluble factor, to date, has been shown to control this process in vivo. Requirement of a circulating factor to inhibit endothelial cell growth may explain why the establishment of fully formed, quiescent blood vessels is coincident with and dependent upon the onset of blood circulation. Further elucidating the mechanism(s) by which RA signaling controls endothelial cell cycle control and vascular remodeling during normal development should provide the necessary insights into the control of aberrant neovascularization associated with prevalent pathologies.
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ACKNOWLEDGMENTS |
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Footnotes |
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The Niederreither and Hirschi laboratories contributed equally to this
work
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