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First published online 25 May 2006
doi: 10.1242/dev.02421
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INSERM, Unit 36, F-75005, Paris, France and Collège de France, F-75005, Paris, France.
* Author for correspondence (e-mail: luc.pardanaud{at}college-de-france.fr)
Accepted 2 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Circulating endothelial cells/Progenitors, Embryo, Quail-chick parabiosis, Mobilization, Bone marrow
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of the vascular plexus is an essential event during
ontogeny and mutations affecting genes involved in the emergence, the
differentiation and the maturation of the endothelial system lead to the rapid
death of embryos. The primary vascular tree develops by two processes,
vasculogenesis and angiogenesis (Risau and
Lemmon, 1988
), that relate to the intrinsic or the extrinsic
origin of endothelial cells (ECs). In the yolk sac, vasculogenesis leads to
the differentiation of the hemangioblast
(Murray, 1932), a stem cell
that gives rise to both ECs and hematopoietic cells (HCs), whereas in the
embryo, it leads to the emergence of isolated angioblasts
(Sabin, 1920). Angiogenesis
involves the development of an endothelial network from preexisting vessels
that sprout towards unvascularized regions. Following the development of the
primary vascular tree, the plexus is remodeled into arteries and veins, and
terminal differentiation is accomplished by the formation of the vascular wall
(Carmeliet, 2003).
In the adult, once the definitive vascular network is established, ECs
remain essentially quiescent with neovascularization only occurring during
physiological or pathological events. For a long time, adult
neovascularization was thought to be exclusively achieved by angiogenesis.
However, grafting experiments had suggested that circulating ECs (CECs) could
participate in neovascularization processes
(Stump et al., 1963;
Kennedy and Weissman, 1971).
CECs have since been isolated and characterized in the adult
(Asahara et al., 1997;
Shi et al., 1998;
Takahashi et al., 1999;
Gehling et al., 2000;
Lin et al., 2000;
Peichev et al., 2000). In
vitro, these cells differentiate into ECs; in vivo, they home to sites of
neovascularization, including tumoral regions or ischemic territories
(Asahara et al., 1999;
Cogle and Scott, 2004;
Urbich and Dimmeler, 2004a;
Urbich and Dimmeler, 2004b).
Furthermore, in addition to CECs, adult neovascularization seems to involve
circulating endothelial progenitor cells (EPCs)
(Urbich and Dimmeler, 2004a;
Urbich and Dimmeler, 2004b).
EPCs have important potential therapeutic applications, as their
administration could stimulate blood vessel growth in conditions of
hypo-vascularization (hind limb ischemia, myocardial infarction, stroke, wound
healing). Genetic manipulation of EPCs could also allow the inhibition of
blood vessel growth in conditions of hyper-vascularization (diabetic
retinopathy and tumorigenesis).
EPCs have been isolated from mouse embryos and were shown to form tubes in
vitro and in vivo (Vajkoczy et al.,
2003; Cherqui et al.,
2006). However, it remains unknown when and where these cells form
during embryonic development. In the quail-chick chimera system
(Le Douarin, 1969) and using
the QH1 monoclonal antibody, which is specific for HCs and ECs of the quail
species, as a marker (Pardanaud et al.,
1987), a study showed that the allantois, an avian appendage,
produces both ECs and HCs. When a quail allantois was grafted in the coelomic
cavity of a chick host, QH1+ECs and HCs colonized chick territories
and sometimes reached the host bone marrow. Owing to the distance between the
region of graft, the coelom, and the site of colonization, the bone marrow,
interstitial migration of QH1+ECs was unlikely. It was postulated
that this colonization occurred through the bloodstream
(Caprioli et al., 1998).
Although these experiments suggested that the allantois could be a site of
emergence of CECs/EPCs, the presence of these cells in the circulation and the
extent of their participation to the developing vasculature remained to be
determined.
We have developed a direct experimental approach to identify CECs/EPCs in developing embryos and to examine their participation to the developing vasculature. Using quail-chick parabiosis, we demonstrate that CECs/EPCs are present in the embryo and can be mobilized during angiogenic processes induced by grafting of organ rudiments on the chorioallantoic membrane (CAM) or by wounding. We also show that: (1) the emergence of these CECs/EPCs occurs early in ontogeny prior to the formation of the allantois, and is thus not restricted to this territory; (2) their mobilization is not dependent on the presence of the bone marrow; and (3) CEC/EPC mobilization does not occur during all angiogenic processes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
tissues were digested with trypsin (Gibco BRL) for 10 minutes at 37°C and
washed in phosphate-buffered saline (PBS)/10% newborn calf serum (NCS,
Invitrogen) (Fig. 1A). The
suspensions were centrifuged and resuspended in 40% chick embryonic extract,
40% PBS and 20% of penicillin-streptomycin (PS, Gibco BRL). The cell number
varied between 3x103 (six embryos for splanchnopleural
mesoderm) to 104 cells/µl (11 embryos for paraxial
mesoderm).
Embryonic blood cell harvest
Blood was harvested from quail hearts at E3 (n=33), E4
(n=22) and E5 (n=22). E15 quail blood was isolated from a
vitelline artery (n=8). The blood was transferred to a tube
containing PBS, 10% NCS and heparin (Sigma) at 4°C. To separate white and
red cells, lympholite M (Cedarlane Laboratories) was mixed V/V with the cell
suspension. After centrifugation, a cloud of white cells, present at the
interface between the two solutions, was harvested, washed in PBS/NCS,
centrifuged and resuspended in PBS/NCS (5x103 to
7x104 cells/µl).
Intracardiac injections
E2 chick (Gallus gallus, JA57) embryos were used (14-23 somite
stage). An artificial dark field was made using Indian ink injection under the
embryo. The ectoderm and the pericardium were carefully removed with fine
tweezers and 1-2 µl of cell suspension were manually injected into the
heart (Fig. 1A). The embryos or
organs were dissected between 1 and 14 days later.
Parabiosis
E2 chick and quail embryos (13-23 somite stage) were isolated with their
yolk sac and placed side by side on semisolid medium (50% agar, 20% PBS, 20%
chick yolk, 10% PS) in a 35 mm Petri dish
(Fig. 1E). The dishes were
incubated 48 hours at 37°C.
Classical parabioses were performed according to Wong and Ordahl
(Wong and Ordahl, 1996). The
surviving parabioses (244/2045=12%) were sacrificed between E7, when the two
CAMs first contacted one another, and E15. Between E7 and E11, blood smears
were prepared from chick embryos by sectioning an extra-embryonic vessel.
Smears were fixed in 4% paraformaldehyde (PAF, Sigma).
Grafts of cells and organ rudiments
Endothelin 1-producing CHO cells
(Parnot et al., 1997) or
control cells were cultured in DMEM/Ham's F12, 7.5% fetal calf serum, 1% PS.
Cells were isolated with trypsin and placed overnight in hanging drops to
obtain dense suspensions (75x104 cells/drop), which were
deposited on the chick CAM from E9 parabioses. A silicon ring delimited the
grafted region. After 1-5 days, the grafted region was fixed in PAF.
E3 quail limb, gut and lung buds, isolated as previously described
(Pardanaud et al., 1989), were
placed on the chick CAM of E6-9 parabioses and incubated for 1 to 9 days.
A part of chick liver was retrieved from two E15 parabioses and grafted on the CAM of E8 chick hosts in close contact with E3 chick limb or visceral buds. The developed associations and a part of the chick host liver were isolated 6 days later.
Wounding of the wing and CAM
On E13 parabioses, the chick CAM was sectioned and the right wing was
exposed on the CAM. Using a microscalpel, a deep longitudinal incision
(±5 mm) was made at the level of the ulna. The parabioses were
sacrificed after 6 to 48 hours. The two chick wings (wounded and
contralateral) were isolated, the feather buds were shortened, and some chick
hearts and livers were fixed.
On E8 parabioses, the wound (±1 mm) was made at the level of the digit region. The two wings were fixed 24 hours after the injury.
To wound CAMs, a silicone ring was placed on the chick CAM of E10 parabioses. Using a microscalpel, six to eight venules or arterioles were cut. After 24 hours, the CAM regions were fixed in PAF, Bouin's fluid or in 1.5% ascetic acid in cold absolute ethanol.
VEGF-induced angiogenesis on CAM
Sterile filter papers (1 cm2, Whatmann 3MM) were dipped in a
solution of cortisone acetate (Sigma) in absolute ethanol (3 mg/ml) to reduce
inflammation (Brooks et al.,
1999). After air-drying, they were placed on the CAM of chick
embryos from E10 parabioses. Recombinant human VEGF165 (R&D
Systems and ABCys) or PBS was applied on the filters (25 µl=2 µg). The
parabioses were sacrificed 4 days later; the filter papers together with the
CAM areas were removed and rinsed twice in PBS. Under the stereomicroscope,
the number of vessel branches was counted, then the filters were removed and
CAMs were processed for immunohistochemistry.
BrdU incorporation
One parabiosis on which a limb bud had been grafted on the chick CAM for 9
days, intravenously received 40 µl of BrdU (1000x, Roche
Diagnostics). After 4 hours the graft and the chick host heart were fixed in
PAF. Sections were stained with a biotinylated mouse anti-BrdU antibody (1/10
in PBS) (Pharmingen) and diaminobenzidine (Sigma). A double staining was
performed with QH1 followed by an alkaline phosphatase-conjugated secondary
antibody.
Immunohistochemistry
Unless indicated otherwise, all tissues were fixed in Bouin's fluid,
dehydrated, embedded in paraffin and sectioned (5-7.5 µm). Sections were
deparaffinized then rehydrated in PBS. QH1 (undiluted hybridoma supernatant)
staining was visualized using peroxydase-(BioRad), alkaline
phosphatase-(Clinisciences), Texas Red-(Southern Biotechnologies), Alexa 488-
or Alexa 555-conjugated secondary antibodies (Invitrogen), as previously
described (Pardanaud et al.,
1996). QH1-GRL2, QH1-LEP100, QH1-LEA double staining was performed
on 1.5% ascetic acid-absolute ethanol-fixed sections. QH1-Sambucus
nigra lectin staining was performed on PAF-fixed sections.
GRL2 (Thomas et al., 1993)
is an avian-specific monoclonal antibody recognizing HCs. GRL2 (1/100 in PBS,
overnight at 4°C) was revealed by an Alexa 488 goat anti mouse IgG1.
LEP100 monoclonal antibody stains macrophages
(Lippincott-Schwartz and Fambrough,
1986). LEP100 (1/5 in PBS, overnight at 4°C) was revealed by
an Alexa 555 goat anti mouse IgG.
LEA agglutinin (Lycopersicon esculentum, Sigma) labels macrophages and
avian venous endothelium (Navarro et al.,
2003). After rehydration, sections were pretreated with 0.025%
trypsin at 37°C for 10 minutes. Biotinylated LEA (20 µg/ml in PBS-0.1%
triton, overnight at 4°C) was revealed using Cy3 streptavidin
(Amersham).
Biotinylated Sambucus nigra lectin
(Hagedorn et al., 2005) (1/400
in PBLEC buffer-PBS pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1
mM MnCl2, 1% triton-, overnight at 4°C) recognizes avian ECs
and was revealed using Cy3 streptavidin.
In toto QH1-Sambucus nigra lectin double staining was performed on chick CAM rings. After overnight incubation in PBS-3% NCS, 0.5% triton at 4°C, the rings were washed in PBS (3x30 minutes), PBLEC (30 minutes) and biotinylated Sambucus nigra lectin (1/100 in PBLEC) was applied overnight at 4°C. After PBS washes, CAM rings were incubated overnight (4°C) in QH1, then stained with Cy3 streptavidin and Alexa 488 goat anti mouse IgM (1/100 in PBS 0.25% triton) overnight at 4°C. Rings were mounted in Mowiol (Fluka).
The anti 
-smooth muscle actin antibody (SMA, Sigma) was
diluted 1/200 in PBS, applied 2 hours at room temperature and revealed using
an Alexa 488 goat anti-mouse IgG2a.
TUNEL staining was carried out on chick PAF-fixed CAM sections, using an in situ cell death detection kit with fluorescein (Roche Diagnostics), according to the manufacturer's instructions.
In situ hybridization
In situ hybridization was performed on chick CAM sections, using avian
EC-specific c-ets1 or VEGF-R2 antisense riboprobes
(Vandenbunder et al., 1989;
Eichmann et al., 1993). The
protocol, previously described (Eichmann
et al., 2000), was followed by QH1 immunostaining revealed by a
Texas Red-conjugated anti mouse IgM.
Quantification of QH1+ cells
Observation and counting were performed with Leica or Olympus microscopes.
Confocal images were acquired using a Leica TCS SP2 confocal microscope. For
each harvested tissue and organ, serial sections were prepared. The number of
QH1+ cells in chick tissues was counted manually (x25
objective, final magnification x110) on a total of 12,000 sections. To
calculate the percentage of QH1+ECs integrated in vessels or in the
interstitium, one section was randomly chosen from 24 different samples and
the number of QH1+ECs integrated in vessels versus all
QH1+ECs was determined. The same procedure was applied on 12
different samples to calculate this percentage during mobilization
processes.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cell suspensions were injected into E2 chick host hearts
(n=10, Fig. 1A).
Histological analysis was carried out using the QH1 monoclonal antibody, which
recognizes quail ECs and HCs but does not label quail mural cells
(Pardanaud et al., 1987;
Etchevers et al., 2001).
Serial sections prepared from injected embryos at E3 showed that
QH1+EC integrated everywhere in the embryo. Owing to the
intracardiac injection, a majority of QH1+ECs directly reached the
carotids and were found in the perineural mesenchyme surrounding the
diencephalon and the mesencephalon (Fig.
1B,C). They also participated to the vascularization of the heart,
mesonephros, liver (data not shown) and limb buds, where they were detected up
to 14 days after injection (Fig.
1D).
Emergence of CECs/EPCs
To determine if CECs/EPCs circulated in the embryo, we developed an ex ovo
parabiosis model (Fig. 1E). E2
quail and chick blastoderms were isolated together with their developing yolk
sac and placed on a semi-solid medium in a petri dish. Sectioning of the
marginal sinus of each embryo and suturing of the edges facilitated
establishment of vascular connections. After 48 hours, histological analysis
(n=6, stages 15-19HH) (Hamburger
and Hamilton, 1951) showed that the embryos had established
vascular anastomoses, as attested by the presence of QH1+HCs and
ECs in chick territories (Fig.
1F). Circulating QH1+ECs and HCs invaded virtually all
chick tissues, including the yolk sac vessels, the perineural vascular plexus,
the heart, the aorta (Fig. 1G)
and the splanchnopleural mesoderm. CECs/EPCs were thus present in the
circulation at E2 and reached blood vessels, albeit in low numbers.
To confirm the presence of CECs/EPCs in the circulation, blood was harvested from quail embryos at different developmental stages and white cells were injected into E2 chick hearts (Fig. 1A, right panel). Histological analysis between E3 and E16 showed that blood collected from E3 (n=4), E4 (n=11), E5 (n=2) and E15 (n=4) quail embryos contained QH1+CECs/EPCs that participated to the host vascularization, in particular in the head, the visceral organs and the yolk sac (Fig. 1H). As observed with injections of angioblasts, CECs/EPCs survived at least until E16. In all three experimental models, i.e. intracardiac injections of white blood cells or EPC-containing tissues and ex-ovo parabioses, QH1+CECs/EPCs were either integrated into small vessels or present in the interstitium.
CECs/EPCs in classical parabiosis
To follow the behavior of embryonic CECs/EPCs during later stages, we
performed chick-quail parabioses. An E2 quail embryo with its yolk was
transferred into an E2 chick egg (Fig.
1I). After several days, the two CAMs established contact and
developed vascular connections between the embryos. The surviving parabioses
(12%) were sacrificed between E7 and E15
(Fig. 1I,J). To verify that
vascular connections were established, blood smears from the chick blood were
examined for the presence of QH1+ cells
(Fig. 1K). Vascular bridges had
not yet formed at E7, as attested by the absence of QH1+ cells in
the chick blood (0/13). Vascular connections began to form between E8 and E10
(39/70) and were always present after E10 (161/161).
QH1+CECs/EPCs were present in various tissues, irrespective of their embryonic origin: head, limbs, bone marrow, feather buds, dermis, muscles, heart, mesonephros, metanephros, spleen, liver, thymus, lung, gallbladder, gut, pancreas, yolk sac and CAM (Fig. 1L,M and data not shown).
Most QH1+CECs/EPCs were found in the interstitium. In addition
to their distinctive elongated morphology, these ECs were characterized by
their expression of three EC-specific markers: Sambucus nigra lectin
(Fig. 2A-C), c-ets-1
(not shown) and VEGFR-2 (Fig.
2D,E). Elongated QH1+ECs did not express macrophage
markers LEP (Fig. 2F-H) or LEA
(Fig. 2I-L) while many round
QH1+HCs were LEP+ and LEA+
(Fig. 2F-L). Elongated
QH1+ECs were also negative for the GRL2 antigen, which labels
thrombocytes, myeloid and erythroid progenitors
(Thomas et al., 1993)
(Fig. 2M-O). By contrast, some
round QH1+HCs expressed GRL2
(Fig. 2M-O). Taken together,
this labeling showed that ECs could be clearly distinguished from HCs both by
morphology and marker expression. As expected, quail HCs of the myeloid and
macrophage lineage are present in chick tissues after parabiosis
(Fig. 2P). QH1+
Kupffer-like HCs were observed in the liver and granulocytes or osteoclasts in
the bone marrow (Fig. 2Q).
QH1/SMA double staining showed that QH1+HCs or CECs/EPCs
were not preferentially associated with the vessel wall
(Fig. 2R-T).
Counting of the number of QH1+CECs/EPCs integrated in the interstitium of chick tissues showed that their number never exceeded 50 cells/mm3 in hearts, livers or wings retrieved from E14-15 parabioses (n=22), while this number decreased to 3-6 cells/mm3 in E8 parabioses (whole embryos, n=2; wings, n=9). Interestingly, in the brain, this number was less than 1 cell/mm3 (E9, n=3; E14, n=3).
In all chick tissues, a small but significant fraction of QH1+CECs/EPCs integrated into vessels, including capillaries, veins and more rarely lymphatics (33/571=6%, Fig. 3A-F), but never reached major vessels or formed whole tubes. Confocal microscopy of in toto QH1/Sambucus nigra lectin double staining on chick CAM showed that QH1+/Sambucus+ ECs could be integrated into the superficial chick vascular plexus as isolated cells (Fig. 3F) and, in few cases, as small groups of cells (not shown). Under the superficial layer, QH1+/Sambucus+ ECs often connected together and formed vascular cords (Fig. 3G-I). These structures did not form a lumen as we never observed consecutive sections of QH1+ endothelia. At the limit of these cords, vascular tips protruded and could contact the endothelial plexus (Fig. 3G,H) or chick QH1-/Sambucus+ cords (Fig. 3I). Sometimes, these cords were found to bridge the chick vasculature (Fig. 3I).
To determine if CECs/EPCs conserved migratory potential after tissue integration, a secondary grafting experiment step was performed using two E15 parabioses. A piece of the chick liver from a parabiosis was retrieved and grafted on an E8 chick host CAM in close contact with either E3 chick limb or visceral buds. In the four cases examined 6 days later, each grafted `parabiotic' liver contained one QH1+EC (Fig. 3J). In two cases, one QH1+EC migrated to an associated limb (Fig. 3K) and one to a grafted visceral bud (not shown). Furthermore, QH1+ECs reached sinusoids in two host livers (Fig. 3L). Taken together, all the experiments presented here confirmed the existence of a small reservoir of CECs/EPCs in the embryo.
Mobilization of CECs/EPCs
Wounds on the wing
The kinetics of CECs/EPCs mobilization were studied during the wound
healing process. An incision in the ulna was performed on a wing of chick
embryos from E13 parabioses (Fig.
4A) and the embryos were sacrificed 6-48 hours later. Macroscopic
observation showed that the healing process took place rapidly as the wound
depth was reduced within 6 hours (Fig.
4B). QH1 staining showed that already at 6 hours, a significantly
greater number of QH1+CECs/EPCs invaded the wounded wings compared
with the number of QH1+CECs/EPCs in control chick wings
(Fig. 5A). The number of
QH1+CECs/EPCs remained high up to 48 hours after wounding,
corresponding to E15, the last day before the quail hatched
(Fig. 5A). The invading
QH1+CECs/EPCs were distributed uniformly throughout the wounded
wing, even in areas distant from the wound site. Most of the
QH1+ECs were isolated and located in the interstitium of the
dermis, the perichondrium, the muscles, the feather buds and, in a few cases,
the bone marrow (Fig. 4C).
Their integration in host endothelia remained rare and restricted to small
vessels (36/774=5%). In all cases, the mobilization of CECs/EPCs was
restricted to wounded wings, as the number of QH1+ECs invading the
contralateral wings (Fig. 4D)
or other organs, such as the heart and the liver, was not altered
(Fig. 5A). The total number of
QH1+HCs in wounded wings varied greatly between experiments but no
significant difference between control, contralateral and wounded limbs was
found (Fig. 5B). However, the
proportion of extravasated QH1+HCs appeared higher in the operated
wings than in the controls, in particular in the vicinity of the wound
(Fig. 4C,D). Thus, it seemed
that wounding stimulated mobilization of circulating QH1+
cells.
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Grafts of organ rudiments
To test whether CECs/EPCs were mobilized during angiogenesis and
vasculogenesis processes, E3 chick limb buds (n=11) or visceral
organs (n=4) were grafted on the chick CAM in E10 parabioses. After 1
to 9 days (Fig. 4I,J), while
numerous QH1- chick vessels vascularized the grafts, a great
contingent of quail QH1+CECs/EPCs migrated into the rudiments
(Fig. 4K,L). Most of these
cells were isolated and migrated interstitially. Besides QH1+ECs,
QH1+HCs also invaded the tissues. Four days after grafting, we
noted that the number of QH1+CECs/EPCs was higher in the limb buds
than in the visceral organs (Fig.
5D).
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Grafts of endothelin 1-producing cells
Suspensions of endothelin 1-producing CHO cells, known to stimulate
angiogenesis (Cruz et al.,
2001), or control cells were deposited on the chick CAM from E9
parabioses. The eggs were sacrificed after 1-5 days. Although control cells
never survived in these conditions (n=3), transfected cells gave rise
to well developed nodules at the surface and inside the CAM (n=7,
Fig. 6A,B). These nodules were
vascularized by chick vessels in which QH1+CECs/EPCs could be
integrated (Fig. 6B,C).
However, the majority of QH1+CECs/EPCs were interstitially located
(Fig. 6B,C).
VEGF-induced angiogenesis
VEGF application on chick CAM from E10 parabioses induced a sprouting
angiogenic response (Fig. 6D,E)
quantified by a higher number of vessel branches
(Fig. 5E). Histological
analysis showed significant inflammation leading to the invasion of numerous
QH1+HCs in the CAM (Fig.
6F,G). QH1+CECs/EPCs were present in the interstitium
and the capillary endothelium of the treated areas. Their number did not vary
significantly between VEGF-treated and PBS-treated CAMs
(Fig. 5F,
Fig. 6F,G). The vascular
density being different in these two groups, as determined by the increase in
vessel branching after VEGF treatment (Fig.
6F,G), we calculated the number of quail QH1+CECs/EPCs
per branch. Despite a certain variability between cases, this ratio was not
statistically different in PBS-treated and VEGF-treated CAMs
(Fig. 5G). Thus, VEGF-induced
sprouting angiogenesis on the CAM did not mobilize
QH1+CECs/EPCs.
Mobilization of CECs/EPCs occurs prior to bone marrow formation
We studied the mobilization of CECs/EPCs before the bone marrow
differentiated. In the first set of experiments, the chick wing of E8
parabioses was wounded (n=9). In the second set of experiments, a
chick limb bud was grafted on the chick CAM of E6.5 parabioses
(n=11). In both cases, the QH1 analysis was carried out at E9, a
developmental stage when the quail bone marrow had not yet differentiated.
During the wound healing process, QH1+CECs/EPCs invaded the wounded wings (Fig. 6H). Their number was significantly increased compared with the number of QH1+CECs/EPCs that colonized control or contralateral wings (Fig. 7). Compared with wounding of wings of parabioses at E13, the number of invading CECs/EPCs was reduced. However, owing to technical constraints, both the site and the size of the wound were different, so these results are difficult to compare directly. QH1+HCs were also present in these tissues (Fig. 6H) but their number varied greatly and was not significantly different between the experimental groups (not shown).
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We conclude that the differentiation of the bone marrow is not essential to mobilize CECs/EPCs.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We first verified that avian EPCs survived and differentiated in the
circulation as recently shown for murine ECs
(Yamashita et al., 2000), then
chose the quail-chick parabiosis model to experimentally demonstrate the
existence of CECs/EPCs. Previous studies using quail-chick parabioses between
E6 and E10 never detected QH1+ECs in the chick embryo
(Kurz and Christ, 1998;
Kurz et al., 2001). The
absence of QH1+ECs in those tissues was probably related to our
observation that vascular connections between quail and chick CAM were not
systematically established prior to E10 and that the participation of
QH1+CECs/EPCs to chick vasculature between E7 and E10 was very
low.
Recent studies showed that ex vivo expanded embryonic EPCs were recruited
during adult tumorigenesis (Vajkoczy et
al., 2003; Yurugi-Kobayashi et
al., 2003). When fluorescent E7.5 mouse EPCs were introduced in
the circulation of rat C6 glioma tumor-bearing nude mice, they were
specifically arrested within the tumor vasculature, extravasated into the
interstitium and sometimes incorporated into functional capillaries
(Vajkoczy et al., 2003).
Unlike our observations, mouse EPCs never participated to the host vasculature
in normal organs. This could be due to: (1) the method of analysis using
intravital fluorescence videomicroscopy, which only allowed superficial
measures while our histological analysis screened the organ vasculature and
detected rare QH1+ECs; and/or (2) the adult mouse model, in which
organogenesis was completed and the vascular system was quiescent, while our
experiments concerned growing embryos in which organogenesis and endothelial
growth were actively ongoing.
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). The low mitotic index of CECs/EPCs we obtained agreed with
this finding and might explain why the majority of migrating
QH1+ECs were alone and never formed a clone. An equivalent low
mitotic index is also observed in normal E6-7 chick aortic ECs
(Seifert et al., 1992).
Concerning the behavior of EPCs, recent studies showed that invading cells
participate to functional blood vessels
(Asahara et al., 1999;
Vajkoczy et al., 2003;
Yurugi-Kobayashi et al., 2003;
Tepper et al., 2005). In our
parabiosis experiments, QH1+CECs/EPCs could be distinguished from
QH1+ circulating HCs by their distinctive elongated morphology and
their expression of various EC, but not HC, markers. Of note, we have not
observed incorporation of CECs/EPCs or circulating HCs in the vascular wall in
any of the experiments performed. However, as QH1 does not recognize
pericytes, we cannot exclude the existence of quail-derived circulating
pericytes progenitors or transdifferentiation of circulating cells.
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)
reported that interstitially located EPCs could influence neovascularization
in a paracrine manner by releasing proangiogenic factors.
Although the number of CECs/EPCs integrated in normal developing tissues
was always low, CECs/EPCs were rapidly mobilized during wound healing after an
incision in a chick wing. The recruitment of circulating cells to sites of
wound healing was also shown in the adult
(Asahara et al., 1999;
Sivan-Loukiavona et al., 2003; Galiano et
al., 2004; Montesinos et al.,
2004; Tepper et al.,
2005). As in the embryo, adult CECs/EPCs migrated interstitially
in the wounded tissue or reached small endothelia but never formed complete
tubes (Asahara et al., 1999).
In our model, the number of mobilized CECs/EPCs was at least doubled in
wounded wings by comparison with control or contralateral limbs. In the adult,
a similar ratio was found (Montesinos et
al., 2004). Furthermore, the increase in number of
QH1+CECs/EPCs already occurred after 6 hours and remained stable
until 48 hours, suggesting a rapid mobilization during the establishment of an
initial vascular supply to the wounded ischemic tissue.
Wounds on the CAM surprisingly showed that CEC/EPC mobilization was effective only when veins were injured. This result could be related to a mechanical effect involving a rapid vasoconstriction on arteries following injury. This vasoconstriction limited the ischemia around wounds and the CEC/EPC mobilization did not occur. This result could also imply that arteries and veins would have different properties regarding the capacity to respond to an injury and to mobilize CECs/EPCs. This difference might be an additional way to identify the nature of invasive vessels in tumors. The quail-chick parabioses could be an interesting model to study this point by grafting tumors on chick CAM.
The significant difference between QH1+ECs invading the
grafted limb buds and the visceral organs was probably linked to the
vascularization process operating in these tissues
(Pardanaud et al., 1989;
Pudliszewski and Pardanaud,
2005). The limb territory, which is vascularized through
angiogenesis, was more permissive to the invasion of QH1+CECs/EPCs
than were the visceral buds, which are vascularized through vasculogenesis.
This invasion could represent an early vascular mobilization that permitted to
the grafted ischemic tissues to establish the first vascular connections with
the host CAM vessels and avoid necrosis.
As mentioned above, the VEGF-induced sprouting angiogenesis on the CAM did
not mobilize CECs/EPCs. Although this result seemed unexpected, VEGF did not
always mobilize adult EPCs either (De
Palma et al., 2003; Ruzinova
et al., 2003). VEGF-stimulated EPCs did not systematically
increase the formation of vessels (Young
et al., 2002) but seemed to act indirectly on angiogenesis via the
recruitment of bone marrow-derived circulating cells
(Grunewald et al., 2006;
Zentilin et al., 2006). In our
model, CECs/EPCs appeared to be principally involved during angiogenic
responses related to ischemia and not requiring sprouting, at least during the
initial response phase.
We also showed here that CECs/EPCs do not originate from the bone marrow as
they were present at stages when this organ was not yet developed.
Furthermore, the mobilization of CECs/EPCs was not dependent on this organ as
it was effective while the marrow was not differentiated. These observations
agreed with recent findings that concluded that the bone marrow was not the
source of EPCs (Beck et al.,
2003; Gothert et al.,
2004; Hillebrands et al.,
2002; Machein et al.,
2003; Voswinckel et al.,
2003) but rather appeared as a niche containing vascular wall
progenitors and HCs directly mobilized during neoangiogenesis process
(Rajantie et al., 2004;
Grunewald et al., 2006). The
bone marrow could also provide a useful environment for the multiplication of
EPCs because, in our work, their number was more important at stages when this
hematopoietic organ had developed.
The original model of E2 parabiosis demonstrated that CECs/EPCs were
present early in ontogenesis. To map precisely the origin of CECs/EPCs,
quail-chick yolk sac chimeras are currently being generated. In previous
studies using E1 yolk sac chimeras, associating a chick blastoderm with a
quail yolk sac, scarce QH1+ECs could be found in E2.5-E4 chick
embryos, mainly in the cephalic region
(Cuadros et al., 1992). These
results indicate a yolk sac origin of CECs/EPCs also recently suggested by La
Rue et al. (La Rue et al.,
2003) using a retroviral labeling strategy.
The lack of lineage-specific markers in the avian model did not permit to
distinguish whether QH1+ CECs correspond to progenitors cells or to
`mature' ECs, but owing to their great ability to reach sites of angiogenesis,
we favor the hypothesis of EPCs. It has been recently shown that mature CEC
were more sticky and remained sequestered within the microvasculature of
organs, while undifferentiated cells gained access to tumor microvasculature
(Vajkoczy et al., 2003).
In conclusion, we show that embryonic CECs/EPCs are already present early
in development and are mobilized during angiogenic processes even in the
absence of the bone marrow. The next step will be to determine the factors
that regulate mobilization and homing of these cells. It would be also
interesting to know how physiopathological conditions such as hypoxia or
diabetes modulate the emergence and the role of CECs/EPCs: a recent model of
diabetic chicken (Larger et al.,
2004) could be used in our parabiosis model.
|
ACKNOWLEDGMENTS |
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