JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2010, 61, 5, 619-628
www.jpp.krakow.pl
I. LINDGREN1, B. ZOER2, J. ALTIMIRAS1, E. VILLAMOR2
REACTIVITY OF CHICKEN CHORIOALLANTOIC ARTERIES,
AVIAN HOMOLOGUE OF HUMAN FETOPLACENTAL ARTERIES
IFM Biology, Division of Zoology, Linkoping University, Sweden; 2Department of Pediatrics, GROW School
for Oncology and Developmental Biology, Maastricht University Medical Center (MUMC+), the Netherlands
1
The reactivity of human fetoplacental arteries is regulated by humoral and local factors of maternal and fetal origin. The
chorioallantoic (CA) arteries of bird embryos are homologous to fetoplacental arteries and fulfill the same gas-exchange
purpose without maternal influences, but their reactivity has not been studied in detail. In the present study we hypothesized
that CA arteries would respond to vasoactive factors similarly to fetoplacental arteries and the response would change during
development between maximal vascular CA expansion (15 of the 21 days incubation period) and prior to hatching.
Therefore, we analyzed the reactivity of third order arteries (~200 µm) from the CA membrane of 15 and 19 day chicken
embryos. CA arteries contracted in response to K+, the thromboxane A2 mimetic U46619, endothelin-1, acetylcholine and
acute hypoxia, but showed no reaction to α-adrenergic stimulation (phenylephrine). The nitric oxide donor sodium
nitroprusside, the adenylyl cyclase agonist forskolin, and the β-adrenergic agonist isoproterenol relaxed CA arteries precontracted with K+ or U46619. The contraction evoked by acetylcholine and the relaxations evoked by sodium nitroprusside
and isoproterenol decreased with incubation age. In conclusion, CA arteries share many characteristics with human
fetoplacental arteries, such as pronounced relaxation to β-adrenergic stimuli and hypoxic vasoconstriction. Our study will be
the foundation for future studies to explain disparate and common responses of the CA and fetoplacental vasculature.
K e y w o r d s : β-adrenergic agonist, chicken embryo, chorioallantoic membrane, hypoxic vasoconstriction, thromboxane A2,
vasoreactivity
INTRODUCTION
The early view of the human placenta as a passive organ in
which blood flow depends only on the arteriovenous pressure
difference has been modified by recent evidence that the
regulation of vasomotor tone in the vessels of the fetoplacental
circulation is important to maintain an adequate blood supply
to the fetus (1, 2). As fetoplacental blood vessels lack
autonomic innervation, control of vascular tone is mainly
influenced by humoral and locally released agents as well as by
physical factors such as flow or oxygen tension (3).
Accordingly, constriction and relaxation of fetoplacental
arteries and veins have been demonstrated in response to a
number of agonists and physical stimuli (4-6). Moreover, the
fetoplacental vasculature also shows a flow matching
mechanism similar to hypoxic pulmonary vasoconstriction
(HPV). This mechanism, termed hypoxic fetoplacental
vasoconstriction (7-9), would divert blood flow to the placental
areas with better maternal perfusion as HPV diverts blood to
the better ventilated areas of the lung (9).
The avian homologue of the mammalian placenta is the
chorioallantoic membrane (CAM), the principal respiratory
organ of the chick embryo during the last two thirds of
development (10). At day 6 of incubation (of a total of 21 days),
the CAM progressively assumes the gas-exchange function of
the area vasculosa, the previous respiratory organ (11). By days
14-15 the CAM capillary volume reaches a maximum and
around the end of day 19, the CAM degenerates when lung
ventilation is initiated during internal pipping (11). Due to the
high rates of vascular proliferation that accompanies its
development, the CAM is probably the most widely used in vivo
assay for studying angiogenesis (12). However, although this
vascular proliferation occurs while the new forming vessels are
simultaneously acquiring the capacity to regulate CAM vascular
tone, there is no information on the chorioallantoic (CA)
vascular reactivity.
The knowledge about the mechanisms which govern CA
arterial tone can lead to a better comprehension of the vascular
biology of its human analogue, the fetoplacental arteries. In the
present study we hypothesized that CA vessels are endowed with
vascular regulatory mechanisms similar to those found in human
fetoplacental arteries and that CAM vascular responsiveness
would be largest at the time when CA vessels reach the largest
degree of development. To test our hypothesis, we analyzed the
in vitro responsiveness of CA arteries to vasoactive agonists with
known activity in the human fetoplacental arteries at two
developmental stages: maximal CA vascular expansion (15d) and
close to the time of internal pipping and onset of lung respiration
(19d). The response of CAM arteries to hypoxia and the presence
or absence of periarterial innervation was also investigated.
620
The experimental work was performed at the Department of
Pediatrics, GROW School for Oncology and Developmental
Biology, Maastricht University Medical Center (MUMC+), the
Netherlands.
MATERIAL AND METHODS
Egg incubation and vessel isolation
All experiments were performed in accordance with the
Dutch law of animal experimentation and were approved by the
local Ethical Committee. Fertilized eggs from a pure broiler sire
strain were obtained from Breeding Research and Technology
Centre van Hendrix Genetics B.V. (Boxmeer, the Netherlands).
Eggs were incubated in 37.8°C, 45% relative humidity and
turned 90° every hour (Model 25HS, Masalles Comercial).
CAM arteries were sampled at 15 and 19 days of incubation
(hatching at 21 days). After cutting the roots of the
extraembryonic vessels at the point of exiting the abdominal
cavity, the egg shell lined with the CAM was rinsed with room
tempered Krebs-Ringer Bicarbonate (KRB) buffer and, with the
aid of a dissection microscope, tertiary branches of the CA artery
(in situ external diameter ~200 µm) were carefully isolated and
cut into ~2 mm long rings. Care was taken to keep the
endothelium intact. Only one CA artery ring per egg was used.
Recording of arterial reactivity
Chorioallantoic artery rings were mounted between an
isometric force transducer (Kistler Morce DSC 6) and a micro
displacement device in a myograph (model 610 M, Danish
Myotechnology) using stainless steel wires (diameter 40 µm).
The myograph organ baths were filled with KRB (38°C and
bubbled with 95% O2: 5% CO2). After a 30 min equilibration, the
vessels were distended to a resting tension corresponding to a
transmural pressure of 1.33 or 2.66 kPa, the mean arterial
pressure at 15d and 19d respectively (13). The average final
diameter of the vessels at the above transmural pressures did not
differ significantly between the age groups (490±28.5 and
590±45.5 µm for 15d and 19d, respectively). After 30 min of
stabilization under resting tension, a control contraction was
elicited by raising the K+ concentration of the buffer (to 62.5
mM) in exchange for Na+. The preparations were washed three
times and allowed to recover before a new stimulation.
Contractile agonists were evaluated under resting tone.
Cumulative concentration-response curves to K+ (4.75-125
mM), the thromboxane A2 mimetic U46619 (1 nM-1 µM),
endothelin-1 (ET-1; 1 nM-100 nM) and the α1-adrenoceptor
agonist phenylephrine (Phe; 10 nM-100 mM) were constructed
by increasing the concentration of the drug. In a group of
experiments, the responses to K+ and U46619 were analyzed in
the presence of the nitric oxide (NO) synthase (NOS) inhibitor
Nω -nitro-L-arginine methyl ester (L-NAME, 0.1 mM).
Relaxant agonists were evaluated during contraction induced
by 62.5 mM K+ or 1 µM U46619. Time controls showed that
KCl and U46619 developed a steady-state developed tension
over the course of the experiment. Concentration-response
curves to acetylcholine (ACh; 10 nM-100 mM), bradykinin
(10 nM- 0.1 mM), the NO donor sodium nitroprusside (SNP; 10
nM-100 mM), the β-adrenergic agonist isoproterenol (Iso; 1 nM
-1 mM) and the adenylate cyclase activator forskolin (Forsk;
1 nM-10 mM) were carried out. In 19 day embryos,
concentration-response curves to acetylcholine were also
performed after blocking cyclooxygenase 1 and 2 with
indomethacin (Indo; 10 µM).
To study the effects of acute hypoxia, the organ chambers
were wrapped in cling film to stabilize oxygen partial pressures,
which was checked by a blood gas analyzer (ABL 510
Radiometer). Hypoxia was induced by switching the gas mixture
to the organ bath from 95% O2/5% CO2 (yielding a pO2 of
74.7±1 kPa) to 95% N2/5%CO2 (yielding a pO2 of 2.48±0.1 kPa).
After 10-20 min of hypoxic exposure, the gas mixture was
switched back to 95% O2/5% CO2.
Scanning electron microscopy
In order to discard a damage of the endothelium during the
myograph study, the endothelial surface of a subset of vessels
(n=8 of each age) was examined by scanning electron microscopy,
as previously described (14). After the myograph study, rings were
fixed overnight at 4°C in 2.5% glutaraldehyde in 0.13 mol/L
cacodylate buffer, pH 7.4. After being rinsed twice in buffer,
specimens were postfixed in 1% ostium tetroxide at 4°C for 1.5
hours, rinsed three times in cacodylate buffer, and dehydrated
through graded concentrations of ethanol. The specimens were
critical point-dried from 100% ethanol with liquid CO2. After the
specimens were dried, they were bisected to expose the luminal
surface, mounted on stubs, and sputter-coated with gold. The
samples were viewed and photographed at 10 kV with a scanning
electron microscope (Philips XL30, Eindhoven, the Netherlands).
Immunohistochemistry of the chorioallantoic membrane
Following fixation in 4% paraformaldehyde (60 min) whole
mount sections of the chorioallantoic membrane of 19 day old
embryos were permeabilized in phosphate-buffered saline (0.1 M,
pH 7.2) containing 0.25% Triton X-100 and incubated overnight
with a mouse anti-neurofilament antibody (4H6, dilution 1:10,
obtained from the Developmental Studies Hybridoma Bank). For
immunoperoxidase staining, the membranes were incubated with
rabbit anti-mouse IgG (1:50, DakoCytomation Z0259) for 60 min
followed with incubation in PAP mouse monoclonal (1:50,
DakoCytomation P0850) and reacted with 0.5% 3,3’
diaminobenzidine tetrahydrochloride diluted in Tris-HCl buffer
(0.1 pH 7.6) containing 0.015% H2O2. Segments of mesenteric
membranes from adult chickens were processed in identical
fashion and served as positive controls.
Data analysis
All results are presented as mean±S.E. unless otherwise
stated. Contractions are expressed as active wall tension (N m-1)
or as percentage of the contraction induced by K+ (62.5 mM) in
the same vessel. Relaxations are expressed as percentage of
depression of the contractions evoked by K+ or U46619. Potency
(expressed as pD2= -log EC50) and efficacy (expressed as
maximal effect, Emax) was determined for each artery by fitting
individual concentration-response data to a nonlinear sigmoid
regression curve and interpolating (GraphPad Prism 4; GraphPad
Software Inc.). In the case of Iso, where the drug produced a
biphasic response, only the first part of the curve (relaxation)
was analyzed. The difference between mean values from 15d
and 19d chicken embryos was assessed by Student’s t-test. The
fiduciary level of significance was set to P<0.05.
Drugs and solutions
The composition of the KRB buffer was (in mM): NaCl
118.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, KCl 4.7, CaCl 2.5
and glucose 5.5. Solutions containing different K+ concentrations
were prepared by replacing part of the NaCl with an equimolar
amount of K+. Forsk and ET-1 were obtained from Alexis
621
A
B
125
KCl
93.75
KCl
125
93.75
62.5
0.5 N/m
0.5 N/m
62.5
31.25
31.25
10 min
5 min
D
Phe
0.5 N/m
1.2
Force (N m-1)
C
-8
-7
-6
-5
0.8
0.4
-4
0
31.25
10 min
62.5
93.75
KCl (mM)
125
Fig. 1. Concentration-dependent contractile effects to K+ and phenylephrine. Contractile effects of K+ (A and B, 15 and 19 day old,
respectively) and phenylephrine (Phe, C, 19 day old) in isolated chorioallantoic arteries from chicken embryos (total incubation time
21 days). D summarizes the contractile effects of increasing concentrations of K+ (open bars 15 day olds, n=6 and solid bars 19 day
olds, n=10, respectively). Data are presented as mean±S.E.
Biochemicals and U46619 from Cayman Chemicals. All the
other drugs were obtained from Sigma-Aldrich. All drugs were
dissolved in deionised water, except Forsk and Indo, which were
initially dissolved in ethanol and U46619, which was initially
dissolved in DMSO. Total DMSO or ethanol added to the organ
bath was, at the most, 0.1% v/v and did not affect arterial tone.
RESULTS
Contractile responses
Isolated CA artery rings of 15d and 19d embryos responded
with tonic contraction to K+ up to a concentration of 93.75 mM
(Fig. 1A and 1B). The response did not differ between ages (Fig.
1D). The effect of all other contractile agents was subsequently
expressed as % of the contraction induced by K+ (62.5 mM) in
the same vessel. Phe did not evoke any significant change in the
tone of the chorioallantoic arteries (Fig. 1C).
ET-1 (Figs. 2A and 2B) and U46619 (Figs. 2C and 2D)
induced a concentration-dependent contraction with a threshold
concentration of 10 nM. Dose-response curves to ET-1 and
U46619 could not be adequately fitted because maximum effect
was not reached at the highest concentration of the drug used
(Figs. 2E and 2F, respectively). When the contractile effect of
equimolar (1 µM) concentrations of U46619 and ET-1 were
compared, a higher contractile efficacy was observed for
U46619. We observed that vasomotion (example shown in Fig.
2A and 2C) was present in all 15d vessels stimulated with ET-1
and U46619, but absent in all vessels from 19d embryos. The
same phenomenon was observed in 100% of 15d arteries and
88% of 19d arteries contracted with U46619 (>0.1 µM). No
significant differences between age groups were observed for
the magnitude of ET-1- or U46619-induced contraction (Figs.
2E and 2F, respectively). The NOS inhibitor L-NAME (0.1
mM) induced a slight contraction in the 19d CA arteries
(7.23±0.9% of the contraction induced by 62.5 mM K+, n=6). In
contrast L-NAME-induced contraction was not observed in the
15d vessels. The presence of L-NAME did not significantly
affect the contractile response to KCl or U46619 at any age
(results not shown).
Response to ACh and bradykinin
In CA arteries contracted with K+ or U46619, ACh induced an
additional concentration-dependent increase in wall tension (Figs.
3A, 3B and 4A). In order to discard that the lack of ACh-induced
relaxation was due to an inadvertent damage of the endothelium
during the experiment, we tested endothelial integrity in a subset
of vessels (n=8 of each age) by scanning electron microscopy. We
observed that during the experimental procedure the endothelium
of the CA arteries was not removed (Fig. 5). The maximal
contraction induced by ACh was significantly larger in 15d than in
19d (Fig. 4A). ACh-induced contraction was not affected by the
presence of Indo (results not shown). ACh applied under resting
tone (i.e. without pre-contraction) had a negligible effect on wall
tension and bradykinin did not affect the tone induced by K+ or
U46619 in 15 and 19d CA arteries (results not shown).
Relaxation responses
SNP relaxed K+-contracted vessels in a concentrationdependent manner (Fig. 3C, 3D and 4B) without reaching a
plateau at the highest concentration tested (0.1 mM). Starting
from concentrations higher than 10 µM, the relaxant effect of
SNP was significantly higher in 15d than in 19d arteries.
622
A
B
ET-1
ET-1
-9
0.5 N/m
0.5 N/m
-7
-8
-7
-8
-9
10 min
10 min
C
D
U46619
U46619
-6
0.5 N/m
-7
0.5 N/m
-6
-8
-9
-7
-9 -8
10 min
E
10 min
400
F
% of KCl contraction
% of KCl contraction
400
200
0
-10
-9
-8
-7
Log [U46619] (M)
-6
-5
200
0
-10
-9
-8
Log [ET-1] (M)
-7
-6
Fig. 2. Concentration-dependent contractile effects to endothelin-1 and U46619. Representative traces of concentration-dependent
contractile effects to increasing concentrations of endothelin-1 (ET-1, A and B, 15 and 19 day old, respectively) and U46619 (C and
D, 15 and 19 day old, respectively) in isolated chorioallantoic arteries from chicken embryos (total incubation time 21 days).
Vasomotion (cyclical variations in vasomotor tone) was observed in all 15 day vessels stimulated with ET-1 and U46619 (A, C). A
summary of the contractile effects of increasing concentrations of ET-1 and U46619 expressed as percent of KCl precontraction is
shown in E and F (open symbols 15 day old, n= 4 and 6, ET-1 and U46619, respectively. Solid symbols 19 day old, n=6 and 9, ET-1
and U46619, respectively). Data points are presented as mean±S.E.
Iso and Forsk also relaxed CA arteries (Figs. 3E-3F and 3G3H, respectively). Whereas Forsk fully reverted the tone evoked
by K+ or U46619 (Figs. 4D and 4F), Iso only induced a partial
relaxation (Figs. 4C and 4E) and high concentrations (>1 µM)
triggered vasoconstriction (Fig. 3E and 3F). Forsk-induced
relaxation was not significantly affected by age or the precontractile agent. In contrast, Iso showed a higher relaxant
efficacy in the 15d vessels pre-contracted with U46619 than in
the 15d vessels pre-contracted with K+. These pre-contractionrelated differences in Iso-induced relaxation were not observed
in the 19d vessels. No significant differences to the relaxant
potency and efficacy of Iso between ages were found (pD2
7.57±0.07 vs. 7.25±0.08 for 15d vs. 19d K+ pre-contracted
arteries, respectively and 8.07±0.24 vs. 7.97±0.19 for 15d vs.
19d U46619 pre-contracted arteries, respectively). However, as
shown in Fig. 4C and Fig. 4E, some particular concentrations of
Iso evoked lower relaxation in 19d when compared with 15d.
Response to hypoxia
In non-precontracted CA rings, hypoxia evoked a tonic
contraction that was relatively stable after ~5 min in both 15d
and 19d vessels (Fig. 6A, top and bottom, respectively). The
hypoxia-induced contraction was completely reversed upon
reoxygenation. No age-related differences were observed in the
response of CA arteries to hypoxia (Fig. 6B).
CAM immunohistochemistry
Positive specific staining of neurofilaments is shown in the
mesenteric plexus in Fig. 7A and has also been observed in other
embryonic vessels such as in the aortic arch (Altimiras,
unpublished results).The absence of innervation of the CAM
was confirmed by the absence of specific staining against
chicken neurofilaments, as shown in Fig. 7B.
623
A
-4
-5
B
ACh -9
-6
KCl
KCl
10 min
10 min
C
-4
-5
-6
-7
0.5 N/m
0.5 N/m
ACh -8
-8 -7
D
SNP -8
SNP
-8
-7
-6
-7
-5
-4
-6
KCl
-4
0.5 N/m
0.5 N/m
-5
KCl
10 min
10 min
E
Iso
-9
F
-8
-5
-7
-7
0.5 N/m
0.5 N/m
-6
KCl
Forsk
-9
-5
-6
KCl
10 min
G
-4
Iso -9 -8
10 min
Forsk
H
-8
-9
-8
-7
-7
-6
KCl
-5
10 min
0.5 N/m
0.5 N/m
-6
KCl
-5
10 min
Fig. 3. Representative traces of putative relaxant agonists. Concentration-response curves to putative relaxant agonists during contraction
with 62.5 mM K+. The figure shows the effects of acetylcholine (ACh, A and B), the NO-donor sodium nitroprusside (SNP, C and D), the
β-adrenergic agonist isoproterenol (Iso, E and F) and the adenylate cyclase activator forskolin (Forsk, G and H) in isolated chorioallantoic
arteries from 15 (A, C, E and G) and 19 (B, D, F and H) day old chicken embryos (total incubation time 21 days).
DISCUSSION
Gas exchange organs like the placenta or the postnatal lung
possess high flow, low resistance vascular beds in which O2
tension plays a critical regulatory role (9, 15). Our results show
important similarities between the vascular reactivity of chicken
CA arteries and reactivity reported for human fetoplacental
arteries. These include lack of responsiveness to α-adrenergic
receptor stimulation, marked relaxation to β-adrenergic
stimulation, no relaxation to known endothelium-dependent
vasodilators (i.e. ACh and bradykinin), and presence of hypoxic
vasoconstriction. In addition, when arteries from the CAM at its
“functional peak” (15d) were compared with those close to the
end of its function (19d), we observed selective changes in ACh-
624
A
B
-125
% of precontraction
-100
0
25
-75
*
*
-50
*
*
*
50
*
75
*
-25
0
125
-10
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
log[ACh], M
C
D
*
% of precontraction
25
*
-3
-6
-5
-4
-6
-5
-4
0
50
75
75
100
100
125
125
-10
-9
-8
-7
-6
-10
-5
-9
-8
-7
log[Forsk], M
log[Iso], M
F
0
25
% of precontraction
-4
25
*
50
-5
log[SNP], M
0
E
*
*
100
0
25
50
50
*
75
75
100
100
125
125
-10
-9
-8
-7
-6
-5
log[Iso], M
-10
-9
-8
-7
log[Forsk], M
Fig. 4. Concentration-dependent effects of putative relaxant agonists. Effects of increasing concentrations of acetylcholine (ACh, A),
the NO-donor sodium nitroprusside (SNP, B), the β-adrenergic agonist isoproterenol (Iso, C, E) and the adenylate cyclase activator
forskolin (Forsk, D, F) in isolated chorioallantoic arteries from 15 (open symbols) and 19 (solid symbols) day old chicken embryos
(total incubation time 21 days). The vessels were pre-contracted with 62.5 mM K+ (A-D) or 1 µM U46619 (E, F). Relaxation is
expressed as percentage of K+ (A-D) or U46619 (E and F) pre-contraction. n=6 to 9 in all experiments, except for E, in which 15d n=4.
Data points are presented as mean±S.E.; * P<0.05 15 day vs. 19 day old embryos.
induced contraction as well as SNP- and Iso-induced relaxation.
Thus, chorioallantoic arteries display similar vasoactive
mechanisms as their human homolog, fetoplacental arteries.
Developmental changes in the reactivity of chorioallantoic
arteries
Knowledge about the ontogeny of vasoactivity in human
fetoplacental vessels is limited because most research has been
conducted on term placentas. Placental trophoblasts are an
important bio-interface between the maternal and the fetal
compartments because they produce a spectrum of hormones
and vasoactive compounds including thromboxane A2, ET-1,
prostacyclin, and NO (16-18), but it is unknown if this local
release changes during pregnancy. A developmental increase in
K+-induced contraction (19) and a decrease in angiotensininduced contraction (20) of human fetoplacental arteries have
been reported and they could serve to protect the uteroplacental
625
A
B
Fig. 5. Scanning electron micrographs of chorioallantoic artery endothelium. Representative examples of scanning electronic
micrographs of chorioallantoic artery rings from 15d (A) and 19d (B) chicken embryos. Previous to the scanning electron microscopy,
the vessels were mounted in a myograph, where it showed contraction in response to ACh. The micrographs show a continuous
endothelial surface without damage, ensuring that the endothelium was not mechanically removed during the experimental procedure.
The scaling bars correspond to 10 µm.
A
0.1 N/m
HYPOXIA
5min
0.1 N/m
HYPOXIA
5 min
B
0.16
Area s-1
0.12
0.08
0.04
0
15
19
Age (days)
Fig. 6. Responses of chorioallantoic arteries to hypoxia.
Hypoxia was induced by switching the organ bath gas mixture
from 95% O2: 5% CO2 to 95% N2: 5% CO2. The duration of the
hypoxic treatment is represented by the shaded boxes above each
representative trace. Isolated chorioallantoic arteries from 15
and 19 day old chicken embryos displayed a tonic hypoxic
vasoconstriction (A, top trace 15 days and bottom trace 19 days).
The average hypoxic contraction of chorioallantoic arteries was
expressed as the area under the contraction/seconds of hypoxia
and was not significantly different between 15 day (open bar,
n=6) and 19 day (closed bar, n=11) embryos (B). The results in
B are presented as mean±S.E.
vasculature from the alterations in maternal vasoactive factors
that accompany normal pregnancy (20).
Because the chick embryo lacks maternal influences such a
protective mechanism is not needed. Nevertherless,
chorioallantoic arteries of 15d chick embryos responded to K+evoked depolarization and to a variety of agonists indicating the
presence of electro- and pharmaco-mechanical coupling. In
contrast to the less mature systemic arteries (21), CA arteries can
regulate flow in the CAM and the mechanisms are already
functional at the time when it reaches a maximal capillary
volume (22). As the chick gets closer to hatching, the increments
in pulmonary blood flow during internal pipping are coupled to
a decrease in chorioallantoic blood flow (23). Thus, the
maturation of CA vasoactivity could serve to balance the
prevailing vasodilatory tone during development, which could
explain the significant drop in SNP- and Iso-induced relaxation.
In vitro investigations of the contractile properties of intact
vascular segments are performed using two principally different
methods. Vessels can be mounted either as isometric
preparations (‘wire-myograph’), when force development is
measured at a certain diameter, or cannulated as isobaric
preparations where the vessel is exposed to a transmural pressure
and allowed to change diameter (‘pressure-myograph’) (24-26).
It has been argued that certain characteristics of the vessels are
critically dependent on which method is used and that the in vivo
situation corresponds more closely to an isobaric condition than
to an isometric condition. Nevertheless, in terms of assessing the
pharmacological activity of drugs on isolated blood vessels, the
use of isometric recording procedures, which were utilized in the
present study, are largely considered to be adequate (24-26).
Acetylcholine-induced contraction of chorioallantoic arteries
ACh has been widely used in numerous vascular beds to
stimulate endothelium-dependent relaxation (27-29). In the
chicken embryo, ACh induces an endothelium-dependent and, at
least partially, NO-mediated relaxation of pulmonary and
systemic arteries (14, 30-32). In addition, ACh and other
endothelium-dependent agonists also induce the release of
endothelium-derived contracting factors (EDCFs) such as
prostanoids or reactive oxygen species (33, 34). ACh-induced
626
A
B
Fig. 7. Micrographs of neurofilament staining. Neurofilament staining with the 4H6 anti-neurofilament mouse monoclonal antibody
distinctly stained perivascular nerves in the mesenteric plexus from an adult chicken (A), but not in the CAM from the 19 day old fetus
(B). nf – nerve fibers, bv – blood vessel. The scaling bar corresponds to 100 µm.
contraction is interpreted either as a sign of endothelial
dysfunction in which EDCFs are insufficiently counteracted by
NO or other endothelium-derived relaxing factors (33, 34), but
has also been attributed to the direct action on muscarinic
receptors of vascular smooth muscle cells in the absence of
functional endothelium-dependent vasodilatation. However, in
the embryonic chicken ductus arteriosus (35) and CA arteries
(present work) we have observed ACh-induced contraction. In
the ductus arteriosus, low concentrations of ACh elicited a
relaxation, followed by a contraction at higher concentrations
(35). ACh-induced contraction was completely endotheliumdependent and involved a cyclooxygenase-1-derived TP receptor
agonist (35). In CA arteries we observed that ACh-induced
contraction was present in endothelium-intact vessels and was
not blocked by Indo, but the exact mechanism behind the AChvasoconstriction warrants further investigation. In contrast to
ACh, the NO donor SNP relaxed CAM arteries, indicating that
the guanylate cyclase pathway of relaxation is active.
Nevertheless, it should be noted that SNP showed a low relaxant
efficacy when compared with compounds acting through the
adenylate cyclase pathway (i.e. Iso and Forsk).
As ACh arises from parasympathetic activity and is rapidly
degraded in the synaptic cleft, circulating levels of ACh are low.
Since the CA vasculature lacks innervation, it is not likely to be
exposed to significant levels of ACh in vivo. Thus, while the ACh
induced contraction in CAM arteries is mechanistically interesting,
it might play less of a regulatory role from a physiological point of
view. Similarly, receptor-mediated endothelium-derived relaxation
is not a major mechanism in the human fetoplacental circulation
because fetoplacental arteries do not relax in response to
endothelium-dependent agents (such as ACh, bradykinin, or
A23187), but they relax in response to NO donors (4, 36). Instead,
it has been proposed that flow-induced NO production contributes
to the maintenance of low resistance circulation in normal
pregnancy (15, 37). This mechanism is also likely in the CAM,
because eNOS is present in CA vessels (38) and detectable
amounts of NO can be measured across the eggshell during
embryonic development (39). In the present work, we observed
that the NOS inhibitor L-NAME induced a slight contraction in the
19d CA arteries. This might be taken as an indication of a basal
production of NO. However, L-NAME did not contract the 15d CA
arteries and did not affect the contractile response to K+ or
U46619. Therefore, whether the CA arteries release NO and the
stimuli leading to its production is yet to investigate.
Bradykinin induces endothelium-dependent relaxations of
many vessels from various species (29). However, we did not
observe any relaxant effect of bradykinin in K+- or U46619contracted chicken CA arteries. Previously, we reported that
bradykinin did not relax the chicken ductus arteriosus (a vessel
that in contrast to the CA arteries shows ACh-induced
relaxation) (14). Nevertheless, it should be noted that bradykinin
shows low, if any, affinity for the ornithokinin (the “avian
bradykinin”) receptor when compared with the authentic ligand
ornithokinin ([Thr6,Leu8]bradykinin) (40).
CA arteries lack α-adrenergic response, but relax to βadrenergic stimulation
In this study, we demonstrated a lack of innervation in the
chicken chorioallantoic arteries (Fig. 7). Just as in the human
fetoplacental arteries, this lack of nerve input leaves the control
of vascular tone to humoral and locally released factors. In
addition to physical factors, eicosanoids, ET-1, and
catecholamines have been implicated in the physiological
regulation of placental blood flow and the human fetoplacental
vessels show a high responsiveness to these agents (4, 41, 42).
Accordingly, we observed that chicken CA arteries were highly
responsive to the thromboxane A2 mimetic U46619 and, to a
lesser extent, ET-1. When we analyzed the response to adrenergic
agents, we observed that the β-adrenoceptor agonist Iso induced
a marked relaxation, but we were unable to detect an effect of the
α-adrenoceptor agonist Phe. Similarly, human fetoplacental
vessels do not respond to Phe or norepinephrine (36, 41), but
relax in response to Iso and other β-adrenoceptor agonists (6). It
has been suggested that the β2-adrenoceptor plays one of the main
roles in the regulation of fetoplacental circulation (6).
627
CA vascular tone, hypoxic vasoconstriction and implications
for embryonic circulatory homeostasis
CA arteries respond to hypoxia with contraction, a response
shared with vessels from other gas exchange organs such as the
lungs and the placenta. It has been suggested that the
mechanisms of hypoxia sensing and signalling in human
fetoplacental arteries are similar to those in pulmonary arteries
(8, 9, 43, 44) so it is likely that similar mechanisms are
operational in CA vessels, but their characterization is beyond
the scope of the present work.
Hypoxic vasoconstriction in CA vessels would serve the
same purpose as in other gas exchangers, i.e. to match
ventilation with perfusion. During natural incubation,
convection of air around the egg is limited due to the contact
with the brood patch and the nest floor, which decreases eggshell
conductance (45) and generates an anisotropic pattern of oxygen
diffusion in the egg (46), but it is not known if less ventilated
areas are also less perfused. Changes in total CA blood flow
during generalized hypoxia have been reported, but there is little
agreement between studies. Using different flow measurement
techniques a decreased CA flow to 10% O2 has been shown (47),
but also a relative increase to 0% O2 (48) and even no changes
in CA flow (49). The disagreement stems from the conflicting
direct vasomotor response to low oxygen between embryonic
and CA blood vessels and the humoral vasomotor effect of
circulating catecholamines released during hypoxia (50). In
agreement with the current study low oxygen would cause direct
CA vasoconstriction and indirect CA vasodilation mediated by
β-adrenergic receptors, but an integrated understanding of the
circulatory changes to hypoxia await further studies.
CONCLUSIONS
In the present study we show that the chicken CA arteries
share many contraction and relaxation characteristics with the
human fetoplacental arteries. HPV (32), hypoxic systemic
vasorelaxation (51), normoxic contraction of the ductus
arteriosus (52), and hypoxic contraction of the CAM arteries
(present work) are present in the chicken, making this species an
attractive model organism for the investigation of putative
common vascular mechanisms (53). This first characterization
of the CAM artery will serve as the starting point of further
investigation to explain the disparate and common responses.
Acknowledgements: The authors would like to thank Ida
Gustafson for the help with the immunohistochemistry and
optical microscopy work and Hans Duimel, from the Department
of Electron Microscopy of the Maastricht University for his
collaboration in processing and visualizing the scanning images
of the vessels. The 4H6 monoclonal antibody developed by
Halfter was obtained from the Developmental Studies
Hybridoma Bank maintained by the University of Iowa. Funding
to J.A. from the Swedish Research Council for Environment,
Agricultural Sciences and Spatial Planning (FORMAS) and the
Wallenberg Foundation is greatly acknowledged.
Conflict of interests: None declared.
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R e c e i v e d : March 24, 2010
A c c e p t e d : September 24, 2010
Author’s address: Dr. Isa Lindgren, Department of Physics,
Chemistry and Biology (IFM), Linkopings Universitet, S-581 83
Linkoping, Sweden; Phone: +46(0)13 281332; Fax +46(0)13
281399; E-mail: [email protected]