Articles in PresS. Am J Physiol Regul Integr Comp Physiol (October 13, 2005). doi:10.1152/ajpregu.00629.2005
1
Revised 10/1/05
Postnatal Constriction, ATP Depletion, and Cell Death in the Mature and Immature
Ductus Arteriosus
Max Levin, M.D., Ph.D.2, Don McCurnin, M.D.3, Steven R. Seidner, M.D.3, Bradley Yoder, M.D.4,
Nahid Waleh, Ph.D.5, Seth Goldbarg, M.D.1, Christine Roman, B.A.1, Bao Mei Liu, B.A.1, Jan
Borén, M.D., Ph.D.2 and Ronald I. Clyman, M.D.1
1
Cardiovascular Research Institute and Department of Pediatrics, University of California San
Francisco, San Francisco, CA 94143; 2Wallenberg Laboratory for Cardiovascular Research,
Göteborg University, SE-413 45 Göteborg, Sweden, Departments of 3Pediatrics and 4Pathology,
University of Texas Health Science Center, San Antonio, Texas 78229; 5SRI International, Menlo
Park, CA 94025
Running Head: ATP Depletion and Open Ductus Arteriosus
Corresponding author: Ronald I. Clyman, M.D.
Box 0544, HSW 1408,University of California, San Francisco
513 Parnassus Ave, San Francisco, CA 94143-0544
phone: (415) 476-4462, FAX: (415) 502-2993
[email protected]
Total Word Count: 4730
Abstract Word Count: 239
Copyright © 2005 by the American Physiological Society.
2
Revised 10/1/05
Abstract
After birth, constriction of the full term ductus arteriosus induces oxygen, glucose and ATP
depletion, cell death and anatomic remodeling of the ductus wall. The immature ductus
frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In
addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen
or indomethacin, with increasing postnatal age. We examined the effects of premature delivery
and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth,
surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death (TUNEL-staining)
increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the
immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP
depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction;
while TUNEL-staining was logarithmically related to the degree of ductus constriction.
Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler
flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP
concentrations were significantly altered even when the immature ductus remained open after
birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses
in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature
newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its
ability to constrict after birth. This may explain its decreasing contractile response to oxygen,
indomethacin and other contractile agents with increasing postnatal age.
Key Words: baboon, hypoxia, glucose, glycogen, HIF1 alpha
3
Revised 10/1/05
Introduction
After birth the full term ductus arteriosus closes in two consecutive phases, a constriction
phase lasting several hours followed by an anatomic remodeling phase. The remodeling phase,
which permanently closes the ductus, involves massive death of smooth muscle cells in the
ductus muscle media and loss of ductus responsiveness to vasoactive stimuli (2, 3, 11, 18). In the
late gestation fetus, the ductus muscle media is supplied with oxygen and nutrients by diffusion
from its lumen and vasa vasorum (9). Postnatal constriction of the full term ductus obliterates its
lumen, obstructs flow through its vasa vasorum, and increases wall thickness producing a
marked increase in diffusion distances for oxygen and nutrients (11). Typically, the thickness of
the avascular ductus wall increases from 0.48 mm (as a fetus) to 1.09 mm during the first hours
after delivery (2, 11). This marked increase in diffusion distance produces severe oxygen,
glucose, glycogen and ATP depletion in the ductus muscle media (12). In vitro studies have
shown that cell death and anatomic remodeling in the full-term ductus correlate best with ATP
depletion; this is most marked when both tissue oxygen and glucose reserves are severely
depleted (8, 12).
In contrast with the full term ductus, constriction of the immature ductus frequently fails
to cause luminal obliteration and only moderately increases diffusion distances for oxygen and
nutrients within the muscle media (2, 11). The typical increase in avascular wall thickness (from
0.47 mm in the preterm fetus to 0.67 mm in the preterm newborn) is only one-third of that seen
at term (2, 11). As a result, the postnatal diffusion distances in the preterm ductus are much less
than they are at full term. This is associated with less cell death and less remodeling and leads to
persistent ductus luminal blood flow after birth (1, 2, 14). In vitro studies suggests that
preservation of ATP, glucose, and glycogen stores may explain why the immature ductus
4
Revised 10/1/05
develops less cell death (8, 12). However, the actual degree of ATP, glucose, and glycogen
depletion within the immature newborn ductus in vivo is currently not known.
In addition to the absence of anatomic remodeling, the immature newborn ductus
progressively loses its ability to constrict, both in vitro (1) and in vivo (7, 15), when exposed to
oxygen or indomethacin during the first days after birth. The explanation for the impaired
contractility of the persistently patent immature newborn ductus is currently unknown.
In this study we examined ductus from full term and immature fetal and newborn
baboons and lambs to determine how constriction of the ductus arteriosus affects its
concentrations of oxygen, glucose, glycogen, and ATP in vivo; and, to determine how these
changes relate to the appearance of cell death in the vessel’s wall and to the ability of the ductus
to constrict after birth.
Methods
Baboons
All studies were approved by the committees on animal research at the University of
California, San Francisco and the Southwest Foundation for Biomedical Research.
We used fetal and newborn baboons (Papio sp., full term = 185 d gestation) to investigate
the effects of ductus constriction on hypoxia (HIF1alpha and VEGF mRNA expression), energy
status (ATP, glucose, glycogen depletion), and cell death (TUNEL staining) (see below). Animal
care, surgery, and necropsy were performed as previously described (2, 16). Immature (125±2 d
gestation; 69% of full term) and mature (175 d gestation; 97% of full term) fetal baboons were
delivered by cesarean section and euthanized before breathing. Mature full term newborn
baboons were euthanized between 1 and 2 days after spontaneous full term delivery. Immature
newborn baboons were delivered by cesarean section at 125 d gestation, intubated with a 2-mm
5
Revised 10/1/05
endotracheal tube, and cared for in the primate intensive care nursery for the first 6 days after
delivery. Intensive Care Nursery management was performed as previously described (2, 16).
Ventilator management was designed to maintain the arterial blood gases in the following
ranges: PaO2 = 50-80 torr, PaCO2 = 35-55 torr, and pH =7.25-7.45. The immature newborn
baboons were euthanized on day 6 after delivery. Immediately before necropsy, the preterm
baboons still required mechanical ventilation with FiO2 =0.38±0.20, mean airway pressure =
9±3 cm H2O, and rate = 33±9 breaths/min.
At necropsy, the ductus was dissected in 4° C PBS solution and frozen in liquid nitrogen
(for HIF1alpha and VEGF mRNA quantification) or embedded in Tissuetek (American Master
Tech, Merced, CA) (for TUNEL staining and ATP, glucose, and glycogen analysis) and frozen
in liquid nitrogen. The dissection procedure required approximately 25 minutes before the tissue
was frozen. The ATP, glucose, and glycogen levels within the ductus are likely to change during
this period. However, the dissection protocol was identical for all animals so valid comparisons
between groups could still be made.
Doppler Examination
Our primary goal was to examine the effects of ductus constriction on hypoxia, energy
status, and cell death in the immature ductus by comparing immature fetal ductus with immature
newborn ductus that either remained partially open after birth or that closed after birth. To
assess ductus constriction, a complete echocardiographic exam including assessment of ductal
patency was performed daily using an 8-mHz transducer interfaced with a Biosound AU3
(Genoa, Italy) echocardiographic system as previously described (20). The full term newborn
baboon ductus constricts rapidly after birth and is closed by Doppler exam within 12 hours after
delivery. In contrast to the full term ductus, the immature ductus frequently remains open after
6
Revised 10/1/05
birth. Therefore, based on the Doppler exam, the ductus could be divided into five groups;
mature fetus, mature newborn-closed, immature fetus, immature newborn-open, and immature
newborn-closed. All of the immature ductus that were open at necropsy (on day 6) had been
open on the 5 preceding daily Doppler exams.
Quantitative PCR
We used the expression of HIF1alpha and VEGF mRNA as surrogate markers for the
degree of tissue hypoxia since their expression correlates with the degree of hypoxia in the
ductus and other tissues (17, 20). Total RNA was isolated from the frozen ductus of immature
fetuses (n=8), immature newborns whose ductus closed prior to necropsy (n=6), immature
newborns with a persistently open ductus (n=10), mature fetuses (n=8) and full term 1 to 2 day
old newborns (n=8), as previously described (20). TaqMan probes were designed using the
Primer Express program (20). An ABI PRISM 7700 Sequence detection system was used to
determine the number of PCR cycles required for product detection [cycle threshold (CT value)].
Reactions were carried out in triplicate. The fewer the number of starting copies of a gene’s
mRNA, the higher the CT value required for product detection. All reactions were repeated on at
least three separate days. Data were analyzed using the Sequence Detector version 1.6.3
program.
We used the baboon housekeeping gene MDH as an internal control to normalize the
degree of HIF1alpha and VEGF expression since its CT value is constant throughout gestation
and after birth (20). We used the method of relative gene expression, where CT (MDH-gene)
represents the difference in cycle threshold between MDH and the gene of interest (HIF1alpha or
VEGF). Each unit of CT (MDH-gene) represents a 2-fold increase in mRNA.
7
Revised 10/1/05
TUNEL staining and Enzyme-Linked Bioluminescence Imaging
We determined cell death and energy status in the following frozen, embedded ductus:
mature fetus (n=3), mature newborn-closed (n=2), immature fetus (n=6), immature newbornopen (n=6), and immature newborn-closed (n=4).
We used the TUNEL technique (Apoptag Peroxidase detection system, Intergen,
Purchase, NY) to detect cells in the early stages of DNA fragmentation and cell death as we have
described previously (12). We determined the number of TUNEL-positive nuclei, as a percent of
muscle media nuclei, by examining more than 500 nuclei in the middle two-quartiles of the
ductus wall. Histologic measurements were made as close as possible to the point of maximal
ductus narrowing which was determined by measuring the luminal area from serial (6 µm) crosssections of the frozen ductus (see below).
We used bioluminescence imaging to visualize and quantify the distribution of
metabolites within the ductus wall. This method has been described in detail elsewhere (12). A
brief methodological description is given below. 15 µm cryosections were mounted on poly-llysine slides, immediately fixed on a heating plate (95°C) for 10 minutes, and then stored at -20
°C until analysis. To calibrate the bioluminescence signal, standards were made by dissolving
different concentrations of ATP, glucose, or glycogen in phosphate buffered saline with 8% low
molecular weight gelatin. The solution was frozen and 15 µm cryosections were made and
treated in the exact same manner as the tissue sections.
To link the substrates of interest to the production of photons, different enzyme solutions
containing luciferase were used. At the time of analysis, the desired enzyme solution was applied
to the cryosection. The emitted photons were registered by a photon counting camera (C2400-47,
Hamamatsu Photonics, Japan) mounted on the microscope (Axiovert 135M, Carl Zeiss,
8
Revised 10/1/05
Germany). The light intensity in different parts of the digital image reflects the local
concentration of the studied metabolite (ATP, glucose, or glycogen). Standard curves were
prepared from the mean bioluminescence intensity of the standard preparations. A darkfield
image of the same section was also obtained to outline histological structures in the
corresponding bioluminescence image. From every ductus, 4 consecutive sections were analyzed
for each of the three metabolites. Each section was used for the analysis of one metabolite and all
measurements were performed at room temperature (23±1°C).
Luminal Areas
In the ductus arteriosus the point of maximal narrowing (smallest luminal area) occurs
over a very short distance in the middle of the constricted vessel. Since multiple sections were
needed to make all of the metabolic and cell death measurements on a single ductus, not all of
the sections could be obtained from the region of maximal narrowing. Therefore, we determined
the luminal areas of each of the frozen sections of ductus used for ATP, glucose, glycogen, and
TUNEL analysis. This made it possible to compare measurements of ductus constriction at the
same level as the analyses.
Sheep Ductus Rings: in vitro
In order to examine how changes in ATP concentrations in the immature ductus might
affect its ability to contract, we performed measurements of isometric tension in immature sheep
ductus (at the same point in gestation (69% of full term) as the immature baboon ductus studied
above) using an organ culture - isometric tension measuring system. We utilized the immature
sheep ductus as a surrogate for the baboon ductus since the conditions for its incubation, stretch,
contractility measurements, and oxygen and glucose concentrations had been previously
established in our laboratory (8, 10). Mixed western breed lamb fetuses (104±3 d, term = 150 d
9
Revised 10/1/05
gestation) were delivered by Cesarean section and anesthetized with ketamine HCl (30 mg/kg iv)
before rapid exsanguination. The ductus was divided into two 2 mm thick rings; each ring was
mounted in a separate 20 ml organ culture bath and incubated in Krebs-bicarbonate solution
(38˚C) equilibrated with a gas mixture containing 5% CO2. The bath solution was exchanged at
a rate of 10 ml/hr (8, 10). The rings were stretched to a length that produced a maximal isometric
contractile response to increases in oxygen tension (4.0±0.1 mm) (4). The oxygen concentration
in the baths was initially 5% while the rings were being mounted. The oxygen concentration was
then switched to 80% O2 and the rings were incubated for 24 hour in either 11.1 mM [2mg/ml]
glucose or 0.56 mM [0.1 mg/ml] glucose. Isometric tensions were recorded at the start of the
experiment (5 minutes after mounting the ring) and after the 24 hours incubation in the high
oxygen environment. After the incubation the rings were embedded in Tissuetek (Miles, Inc) and
frozen in liquid nitrogen for bioluminescence imaging of ATP and TUNEL staining.
Statistics
Statistical analysis was performed by the appropriate student's t-test and by correlation and
regression analysis. When more than one comparison was made, Bonferroni’s correction was
used. Nonparametric data were compared with a Mann-Whitney test. Results are presented as
means ±SD.
10
Revised 10/1/05
Results
In the full term baboon ductus, the expression of our two surrogate markers of hypoxia
(HIF1alpha and VEGF) increased (Figure 1) and the average concentrations of ATP, glucose and
glycogen decreased (Figures 2,3) following postnatal closure. At the same time there was an
increase in the number of TUNEL-positive cells in the ductus muscle media (Figure 2D). The
concentrations of ATP, glucose and glycogen were related to the degree of ductus constriction
(area of ductus lumen versus ATP: r=0.83, p<0.08; versus glucose: r=0.96, p<0.05; versus
glycogen: r=-0.96, p<0.05, n=5). We previously found that cell death (TUNEL-positive cells) in
the full term lamb ductus increased exponentially as the ductus constricted (16). In the full term
baboon ductus there also was a significant relationship between the degree of ductus constriction
and the logarithmic transformation of the number of TUNEL-positive cells (area of ductus lumen
versus TUNEL log10: r=-0.92, p<0.05, n=5).
Similarly, in the immature baboon ductus, HIF1alpha/VEGF expression increased and
ATP concentrations decreased following delivery (Figures 1, 2A). Although the changes in
HIF1alpha/VEGF expression and ATP concentrations were significantly related to the degree of
ductus constriction (Figures 1, 2A)(area of ductus lumen versus ATP: r=0.77, p<0.01, n=16),
both HIF1alpha/VEGF expression and ATP concentrations were still significantly altered after
birth even when the immature ductus remained open and continued to have persistent luminal
blood flow (Figures 1, 2A).
The average concentrations of glucose and glycogen decreased in the immature ductus
after birth (Figures 2B, 2C). Although the presence of glucose depletion was significantly related
to the degree of ductus constriction (Figures 2B), the presence of glycogen depletion appeared to
be independent of the absolute degree of constriction (Figure 2C).
11
Revised 10/1/05
In the immature ductus, there was also a significant increase in the number of TUNELpositive cells (p<0.05) following delivery (Figure 2D). Although the degree of TUNEL-positive
staining was significantly related to the degree of ductus constriction (TUNEL log10 versus area
of ductus lumen: r=-0.69, p<0.01, n=16), extensive cell death (>15% TUNEL-positive cells)
only occurred when the ductus was closed and there was no Doppler flow through its lumen
(Figure 2D).
In the immature ductus, the degree of TUNEL staining was significantly related to the
degree of ATP and glucose depletion (Figure 4A, 4B). ATP concentrations were directly related
to tissue glucose concentrations (r=0.52, p<0.05, n=16). Glycogen concentrations did not appear
to be related to either the concentrations of ATP (data not shown) or the degree of TUNEL
staining in the immature ductus (Figure 4C).
We used ductus arteriosus from preterm lamb fetuses to determine if the decrease in ATP
concentrations in the immature open ductus could affect ductus contractility. We examined the
contractile behavior of immature sheep ductus in vitro exposed to 80% O2 and two different
concentrations of glucose. At low glucose concentrations, ATP concentrations were not
maintained and fell to 31% of the starting (0 hr) fetal ductus levels (Figure 5). This is similar to
the drop in ATP concentration observed in the immature newborn baboon ductus that remains
open after birth in vivo (Figure 2). There was no difference between the two experimental groups
in the incidence of cell death (TUNEL staining) during the in vitro incubation (24 hours). Ductus
with normal concentrations of glucose and ATP contracted when exposed to 80% O2 for 24
hours; in contrast, ductus with low glucose and low ATP failed to contract when exposed to 80%
O2 (Figure 5).
12
Revised 10/1/05
Discussion
We found that after delivery, constriction of the full term newborn baboon ductus
arteriosus is associated with oxygen, glucose, glycogen and ATP depletion and subsequent cell
death in the ductus muscle media (Figures 1, 2). These findings confirm similar observations
made in the full term lamb ductus arteriosus (12). Prior in vitro studies have suggested that
ductus muscle media cell death is due to ATP depletion and energy failure rather than to classical
apoptotic mechanisms (8, 12). Cell death is most marked when both tissue oxygen and glucose
reserves are severely depleted (8, 12).
The preterm ductus fails to develop the same degree of cell death after birth as the full
term ductus (2, 16). However, prior in vitro studies have shown that, if the preterm ductus
develops the same degree of tissue hypoxia and glucose depletion as the full term ductus, it is
capable of developing a similar degree of severe ATP depletion and cell death (8, 12). The
present studies are the first to demonstrate that constriction of the preterm ductus in vivo is
associated with glucose and ATP depletion (Figures 2, 3), in addition to oxygen depletion
(Figure 1), in the ductus wall. In the preterm ductus, cell death (TUNEL staining) in vivo is
significantly associated with ATP and glucose depletion just as it is in the full term ductus
(Figure 4).
In the full term newborn ductus arteriosus, severe oxygen, glucose, glycogen and ATP
depletion, cell death and tissue remodeling occur even before there is complete loss of luminal
blood flow (11) (12); this is due to the marked increase in diffusion distances for oxygen and
nutrients that occur after birth (11) (12). The present study demonstrates that the preterm ductus
must completely obliterate its luminal blood flow before it develops the same degree of in vivo
13
Revised 10/1/05
hypoxia, glucose depletion and cell death as found in the full-term newborn ductus (Figures 1,
2).
In the immature ductus, tissue oxygen concentrations (Figure 1) appear to be more
significantly affected by postnatal constriction than are tissue glucose concentrations (Figure
2B). In vitro studies have shown that, under similar conditions of hypoxia, the immature ductus
has higher concentrations of tissue glucose than the full term ductus (12). This appears to be due
to increased glucose availability (12).
When ductus arteriosus (or other vessels) are studied in vitro, the primary substrate
utilized during glycolysis and ATP generation is glucose, rather than glycogen (12, 13). The
present in vivo findings support these in vitro findings; in the immature ductus, ATP
concentrations are directly related to tissue glucose concentrations but not to glycogen
concentrations. Similarly, cell death (TUNEL staining) is related to tissue glucose concentrations
but not to glycogen concentrations in the immature ductus. Although partial constriction of the
immature ductus was associated with glycogen depletion (Figure 2C-immature newborn-open),
tight constriction produced regions of the ductus wall where glycogen concentrations were
actually greater than those observed in the fetus (Figure 3). We have previously shown that
ductus rings, incubated in vitro under hypoxic conditions, develop both regions of glycogen
depletion as well as regions of glycogen surplus; interestingly, the regions of glycogen surplus
are more commonly seen in the immature than the mature ductus (12). We hypothesize that this
adaptive mechanism could add to the ability of the immature ductus to tolerate episodes of
hypoxia and nutrient shortage making it more resistant to developing postnatal cell death and
permanent closure.
14
Revised 10/1/05
Cell death and remodeling of the preterm ductus only occur when there has been
complete loss of luminal blood flow, with profound tissue anoxia and ATP depletion (Figure
2D)(16). The current studies show that there are significant decreases in tissue oxygen
(HIF1alpha/VEGF expression) (Figure 1) and ATP concentrations (Figure 2A) in the preterm
ductus even when it continues to have persistent luminal blood flow after birth. Smooth muscle
contractility is impaired by both hypoxia (10) and ATP depletion in the ductus (Figure 4) as well
as in other vessels(5, 6, 19). In the tightly constricted closed ductus, cell death and anatomic
remodeling continue to hold the ductus closed despite the impaired muscle contractility.
Metabolic depletion and diminished contractility still occur in the persistently patent ductus,
however, cell death and tissue remodeling fail to develop and fail to obstruct the patent lumen.
These findings have important implications for clinical care. Prior studies have shown
that indomethacin is most effective in closing the preterm PDA when it is given
“prophylactically” within 24 hours of birth(15). Its ability to produce ductus closure decreases
with increasing postnatal age. We hypothesize that, with increasing postnatal age, depletion of
oxygen and ATP in the persistently patent ductus decreases its ability to constrict when exposed
to indomethacin or other vasoconstrictors (1). Our current findings may explain why
indomethacin becomes less effective in constricting the immature ductus with increasing
postnatal age (15).
Acknowledgements
The authors thank all the personnel that support the BPD Resource Center: the animal husbandry
group led by Drs. D. Carey and M. Leland, the NICU staff (H Martin, D Correll, S Gomez, S
Ali, L Kalisky, L Nicley, R Degan, S Salazar), the Wilford Hall Medical Center neonatal fellows
15
Revised 10/1/05
who assist in the care of the animals, and the UTHSCSA pathology staff (V Winter, L Buchanan,
K Symank, Y Valdes and K Mendoza) who perform necropsies.
Grants
Supported by grants from U.S. Public Health Service (NIH grants HL46691, HL56061, HL52636
BPD Resource Center, and P51RR13986 Primate Center facility support) and by a gift from the
Gates Foundation. Seth Goldbarg is a research fellow with the Stanley J. Sarnoff Endowment for
Cardiovascular Research.
References
1.
Clyman RI, Campbell D, Heymann MA, and Mauray F. Persistent responsiveness of
the neonatal ductus arteriosus in immature lambs: a possible cause for reopening of patent ductus
arteriosus after indomethacin induced closure. Circulation 71: 141–145, 1985.
2.
Clyman RI, Chan CY, Mauray F, Chen YQ, Cox W, Seidner SR, Lord EM, Weiss
H, Wale N, Evan SM, and Koch CJ. Permanent anatomic closure of the ductus arteriosus in
newborn baboons: the roles of postnatal constriction, hypoxia, and gestation. Pediatric Research
45: 19–29, 1999.
3.
Clyman RI, Mauray F, Roman C, Heymann MA, and Payne B. Factors determining
the loss of ductus arteriosus responsiveness to prostaglandin E2. Circulation 68: 433–436, 1983.
4.
Clyman RI, Mauray F, Wong L, Heymann MA, and Rudolph AM. The
developmental response of the ductus arteriosus to oxygen. Biol Neonate 34: 177–181, 1978.
5.
Coburn RF, Moreland S, Moreland RS, and Baron CB. Rate-limiting energydependent steps controlling oxidative metabolism-contraction coupling in rabbit aorta. J Physiol
448: 473-492, 1992.
6.
Dillon PF. Influence of cellular energy metabolism on contractions of porcine carotid
artery smooth muscle. J Vasc Res 37: 532-539, 2000.
7.
Firth J and Pickering D. Timing of indomethacin therapy in persistent ductus. Lancet
II(8186): 144, 1980.
8.
Goldbarg S, Quinn T, Waleh N, Roman C, Liu BM, Mauray F, and Clyman RI.
Effects of hypoxia, hypoglycemia, and muscle shortening on cell death in the sheep ductus
arteriosus. Pediatric Res 54: 204-211, 2003.
9.
Goldbarg SH, Takahashi Y, Cruz C, Kajino H, Roman C, Liu BM, Chen YQ,
Mauray F, and Clyman RI. In utero indomethacin alters O2 delivery to the fetal ductus
arteriosus: implications for postnatal patency. Am J Physiol Regul Integr Comp Physiol 282:
R184-190., 2002.
16
Revised 10/1/05
10.
Kajino H, Chen YQ, Seidner SR, Waleh N, Mauray F, Roman C, Chemtob S, Koch
CJ, and Clyman RI. Factors that increase the contractile tone of the Ductus Arteriosus also
regulate its anatomic remodeling. Am J Physiology 281: R291-R301, 2001.
11.
Kajino H, Goldbarg S, Roman C, Liu BM, Mauray F, Chen YQ, Takahashi Y, Koch
CJ, and Clyman RI. Vasa vasorum hypoperfusion is responsible for medial hypoxia and
anatomic remodeling in the newborn lamb ductus arteriosus. Pediatric Research 51: 228-235.,
2002.
12.
Levin M, Goldbarg S, Lindqvist A, Sward K, Roman C, Liu BM, Hulten LM, Boren
J, and Clyman RI. ATP depletion and cell death in the neonatal lamb ductus arteriosus.
Pediatric Research 57: 801-805, 2005.
13.
Lynch RM and Paul RJ. Compartmentation of glycolytic and glycogenolytic
metabolism in vascular smooth muscle. Science 222: 1344-1346, 1983.
14.
Narayanan M, Cooper B, Weiss H, and Clyman RI. Prophylactic indomethacin:
Factors determining permanent ductus arteriosus closure. J Pediatr 136: 330-337, 2000.
15.
Schmidt B, Davis P, Moddemann D, Ohlsson A, Roberts RS, Saigal S, Solimano A,
Vincer M, and Wright LL. Long-term effects of indomethacin prophylaxis in extremely-lowbirth- weight infants. N Engl J Med 344: 1966-1972., 2001.
16.
Seidner SR, Chen Y-Q, Oprysko PR, Mauray F, Tse MM, Lin E, Koch C, and
Clyman RI. Combined prostaglandin and nitric oxide inhibition produces anatomic remodeling
and closure of the ductus arteriosus in the premature newborn baboon. Pediatric Research 50:
365-373, 2001.
17.
Semenza GL. O2-regulated gene expression: transcriptional control of cardiorespiratory
physiology by HIF-1. J Appl Physiol 96: 1173-1177; discussion 1170-1172, 2004.
18.
Slomp J, Gittenberger-de Groot AC, Glukhova MA, Conny van Munsteren J,
Kockx MM, Schwartz SM, and Koteliansky VE. Differentiation, dedifferentiation, and
apoptosis of smooth muscle cells during the development of the human ductus arteriosus.
Arterioscler Thromb Vasc Biol 17: 1003-1009., 1997.
19.
Taggart MJ and Wray S. Hypoxia and smooth muscle function: key regulatory events
during metabolic stress. J Physiol 509 (Pt 2): 315-325, 1998.
20.
Waleh N, Seidner S, McCurnin D, Yoder B, Liu BM, Roman C, Mauray F, and
Clyman RI. The Role of Monocyte-Derived Cells and Inflammation in Baboon Ductus
Arteriosus Remodeling. Pediatric Research 57:254-62, 2005.
17
Revised 10/1/05
Figure legends
Figure 1. Real Time PCR measurements of HIF1alpha and VEGF in the immature and mature
ductus. Ten nanograms of cDNA from individual ductus were placed in separate wells and
analyzed by Taqman Real Time PCR. CT(MDH-gene) represents the difference in cycle
threshold (CT) between the expression of housekeeping gene MDH and the individual gene of
interest. Each unit of CT(MDH-gene) represents a 2-fold increase in a gene’s mRNA. Ductus
were grouped according to the Doppler exams: mature fetus, mature newborn-closed, immature
fetus, immature newborn-open, and immature newborn-closed. *p<0.05; **p<0.01.
Figure 2. TUNEL-positive staining and average ATP, glucose and glycogen concentrations in the
immature and mature ductus. Each metabolite’s concentration (µmol/gram wet weight) was
determined throughout the entire ductus wall. We determined the number of TUNEL-positive
nuclei, as a percent of muscle media nuclei, by examining more than 500 nuclei in the middle
two-quartiles of the ductus wall. See legend to Figure 1. *p<0.05; **p<0.01.
Figure 3. ATP, glucose, and glycogen concentrations in fetal and neonatal ductus from immature
and mature baboons. Bioluminescence imaging was used to map the distribution of different
metabolites within 15 µm cryosections of fetal and newborn ductus.
Figure 4. Relationship between cell death (TUNEL-staining) and average ATP, glucose and
glycogen concentrations in the immature and mature ductus. For the purposes of statistical
analysis the percent of TUNEL-positive nuclei was logarithmically transformed to the base 10.
18
Revised 10/1/05
Figure 5. The effects of low glucose and ATP concentrations on isometric tension in rings of
preterm fetal lamb ductus arteriosus. Two rings per ductus were obtained from 7 preterm fetal
lambs. The rings were incubated for 24 hours in bath solutions containing 80% oxygen and either
0.56 or 11.1 mM glucose. The rings were stretched to the same initial lengths (0.56 mM glucose:
length=4.1±0.6 mm; 11.1 mM glucose: length=3.9±0.6) and starting (0 hours) tensions. TUNEL
measurements: See Legend for Figure 2. After the 24 hours incubation, there were no differences
in TUNEL-positive staining (0.56 mM glucose: TUNEL-positive=2.7±2.0%; 11.1 mM glucose:
TUNEL-positive=0.4±0.5%, p=NS). ATP concentration at 0 hours (fresh frozen rings) of
preterm fetal ductus arteriousus: 0.34±0.09
mol/g w.w.. *p<0.05.
19
Figures
Figure 1
Figure 2
Revised 10/1/05
20
Figure 3
Figure 4
Revised 10/1/05
21
Figure 5
Revised 10/1/05