1 Department of Neonatology, CHRU de Lille, France; and 2 Department of Pediatrics, Pediatric Heart Lung Center, University of Colorado School of Medicine, Denver, Colorado 80218-1088
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ABSTRACT |
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We hypothesized that altered vasoreactivity in perinatal pulmonary hypertension (PH) is characterized by abnormal responses to hemodynamic stress, including the loss of flow-induced vasodilation and an augmented myogenic response. Therefore, we studied the acute hemodynamic effects of brief compression of the ductus arteriosus (DA) in control fetal lambs and in lambs during exposure to chronic PH. In both groups, acute DA compression decreased pulmonary vascular resistance (PVR) by 20% at baseline (day 0). After 2 days of hypertension, acute DA compression paradoxically increased PVR by 50% in PH lambs, whereas PVR decreased by 25% in controls. During the 8-day study period, PVR increased during acute DA compression in PH lambs, whereas acute DA compression continued to cause vasodilation in controls. Brief treatment with the nitric oxide (NO) synthase inhibitor nitro-L-arginine (L-NA) increased basal PVR in control but not PH lambs, suggesting decreased NO production in PH lambs. Chronic hypertension increased the myogenic response after L-NA in PH lambs, whereas the myogenic response remained unchanged in controls. The myogenic response was inhibited by nifedipine in PH lambs, suggesting that the myogenic response is dependent upon the influx of extracellular calcium. We conclude that chronic PH impairs flow-induced vasodilation and increases the myogenic response in fetal lung. We speculate that decreased NO signaling and an augmented myogenic response contributes to abnormal vasoreactivity in PH.
myogenic response; nitric oxide; pulmonary circulation; persistent pulmonary hypertension of the newborn; smooth muscle; calcium channels
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INTRODUCTION |
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AT BIRTH, the pulmonary circulation undergoes marked vasodilation, which allows blood flow to increase nearly 10-fold and enables the lung to assume its postnatal role in gas exchange (13, 33). Several stimuli, including drainage and absorption of fetal lung liquid, rhythmic distension of the lung, increased PO2, and altered production of several vasoactive products, including nitric oxide (NO), are known to contribute to pulmonary vasodilation at birth (4, 17, 40). The effects of several birth-related stimuli, including increased blood flow or shear stress, increased PO2, and ventilation, are partly dependent upon activation of NO synthase (NOS; see Refs. 4, 10, 28, 39). The increase in NO production accounts for nearly 50% of the abrupt fall in pulmonary vascular resistance (PVR) at birth in fetal lambs (4, 10).
In addition to its role as a vasodilator at birth, NO production also modulates fetal pulmonary vascular tone in response to diverse stimuli, including acute changes in hemodynamic forces (1, 10). In fetal lambs, acute compression of the ductus arteriosus (DA) abruptly increases blood flow and pressure and causes acute pulmonary vasodilation through the shear stress-induced release of NO (1, 10). However, despite maintenance of constant pressure, pulmonary blood flow progressively falls and PVR increases over time (1). Thus pulmonary blood flow in utero is normally maintained within narrow limits despite increases in vascular perfusion pressure resulting from active vasoconstriction. This pressure- or stretch-induced vasoconstriction, known as the myogenic response, was first described by Bayliss (8) and has been characterized in several systemic vascular beds (12, 30). However, whether high myogenic tone and active myogenic responses play critical roles in vasoregulation of the normal fetal pulmonary circulation or contribute to abnormal vascular function in chronic hypertension are unclear. Recently, inhibition of NOS activity has been shown to not only block shear stress-induced pulmonary vasodilation but also cause a striking and paradoxical vasoconstrictor response (36). These findings suggest that NO not only plays a critical role in mediating vasodilation in response to hemodynamic stimuli at birth but that NO signaling also opposes high myogenic tone in the pulmonary circulation in utero (36). Furthermore, it has been suggested that myogenic tone contributes to high PVR and the inability of several stimuli to cause sustained pulmonary vasodilation in the normal fetus (1, 2, 36, 37).
Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that is associated with diverse neonatal cardiopulmonary diseases, ranging from asphyxia, sepsis, meconium aspiration, respiratory distress syndrome, and others, or can be idiopathic (26). Physiologically, PPHN is characterized by the failure of the pulmonary circulation to dilate at birth. This leads to sustained elevation of PVR, causing extrapulmonary right-to-left shunting of blood across the DA and foramen ovule and severe hypoxemia (24, 26). PPHN contributes significantly to high morbidity and mortality in hypoxemic newborns, but its pathogenesis and pathophysiology are poorly understood.
Because NO contributes significantly to normal regulation of vascular tone in the perinatal pulmonary circulation, it has been suggested that PPHN may be partly due to decreased NO production (4, 34, 41). To better understand the etiology and pathophysiology of PPHN, our laboratory and others have developed an experimental model of chronic intrauterine pulmonary hypertension caused by partial compression or ligation of the DA in the late-gestation ovine fetus (6, 31). In this model, chronic DA compression causes sustained intrauterine pulmonary hypertension without increased pulmonary blood flow or hypoxemia (6). After delivery, PVR remains elevated with extrapulmonary right-to-left shunting and hypoxemia, despite mechanical ventilation with hyperoxia (6). Past studies have also demonstrated blunted endothelium-dependent vasodilation and downregulation of endothelial NOS expression (29, 34, 41). Mechanisms by which impaired NO signaling alters pulmonary vasoreactivity and contributes to the failure of PVR to fall at birth are incompletely understood.
Therefore, we hypothesize that, in addition to impaired NO production, altered pulmonary vasoreactivity in PPHN is characterized by an unmasked or increased myogenic response in the pulmonary circulation. To test this hypothesis, we performed serial studies of the acute hemodynamic effects of brief DA compression in chronically prepared fetal lambs with chronic pulmonary hypertension and control animals. We report that chronic pulmonary hypertension in utero rapidly impairs flow-induced vasodilation, blocks basal NO production, and augments a potent myogenic response. In addition, we found that treatment with nifedipine abolished the myogenic response in lambs with severe pulmonary hypertension, suggesting that the myogenic response is dependent on activation of voltage-gated calcium channels.
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METHODS |
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Animal Preparation
All animal procedures and protocols used in this study were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Fourteen mixed-breed (Columbia-Rambouillet) pregnant ewes between 124 and 128 days gestation (term = 147 days) were fasted for 48 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (total dose: 2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes were kept sedated but breathed spontaneously throughout the surgery. Under sterile conditions, the fetal lamb's left forelimb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with lidocaine (2 ml, 1% solution). Polyvinyl catheters (20 gauge) were advanced in the ascending aorta and the superior vena cava after insertion in the axillary artery and vein. A left thoracotomy exposed the heart and great vessels. Catheters were inserted in the left pulmonary artery (LPA), main pulmonary artery, and left atrium by direct puncture through purse string sutures, as previously described (3). An inflatable vascular occluder (In Vivo Metric, Healdsburg, CA) was placed loosely around the DA after gentle dissection of adherent connective tissue with cotton-tipped swabs. An ultrasonic flow transducer (Transonic Systems, Ithaca, NY) was placed around the LPA to measure blood flow. The uteroplacental circulation was kept intact, and the fetus was gently replaced in the uterus. An additional catheter was placed in the amniotic cavity to measure pressure. Ampicillin (500 mg) was infused in the amniotic cavity before closure of the hysterotomy. The flow transducer cable, catheters, and vascular occluder were exteriorized through a subcutaneous tunnel to an external flank pouch. The ewes recovered rapidly from surgery, generally standing in their pens within 6 h. Food and water were provided ad libitum. Catheters were maintained by daily infusions of 2 ml heparinized saline (20 U/ml). Catheter positions were verified at autopsy. Studies were performed after a minimum recovery time of 48 h.Physiological Measurements
The flow transducer cable was connected to an internally calibrated flowmeter (Transonic Systems) for continuous measurements of LPA blood flow. The output filter of the flowmeter was set at 100 Hz. Absolute values of flows were determined from means of phasic blood flow signals from at least 30 cardiac cycles with zero blood flow defined as the flow value immediately before the onset of systole. Main pulmonary artery, aortic, left atrial, and amniotic catheters were connected to blood pressure transducers (TSD 104; Biopac System, Santa Barbara, CA). The pressure and flow signals were continuously recorded and processed on a computer using an analog-to-digital converter system (Biopac). The data were sampled at a rate of 200 samples/s. Pressures were referenced to the amniotic cavity pressure. Calibration of the pressure transducers was performed daily with a mercury column manometer. Heart rate was determined from the phasic pulmonary blood flow signal. PVR in the left lung was calculated as the difference between mean pulmonary artery and left atrial pressures divided by mean left pulmonary blood flow. Blood samples from the main pulmonary artery catheter were used for blood gas analysis and oxygen saturation measurements.Experimental Design
After at least 48 h of recovery from surgery, animals were randomly assigned to either the control (n = 7) or the chronic pulmonary hypertension (n = 7) group. The duration of the study was 8 days. Chronic pulmonary hypertension was induced by inflating the vascular occluder by slowly infusing a small volume of saline in the cuff around the DA, causing a partial compression of the DA (1, 24). The degree of inflation was set to increase mean pulmonary artery pressure (PAP) by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the study period by readjusting the degree of inflation of the occluder during the first 2 h of DA compression and then by maintaining a constant inflation pressure within the occluder. Briefly, a 10-ml saline syringe was connected to the occluder. The piston was pressed continuously by a spring, the length of which was set to obtain the target PAP. Mean PAP was measured at least two times daily to readjust the pressure in the occluder, if necessary. In the chronic pulmonary hypertension group, DA compression before each acute study was released by completely deflating the occluder 20 min before the beginning of each experiment. In control animals, the vascular occluder was not chronically inflated, but serial measurements of hemodynamic and arterial blood gas tensions were recorded (as described below).Four different protocols were included in this study. These include 1) serial measurements of the acute hemodynamic response to partial DA compression in pulmonary hypertension and control fetal lambs (protocol 1); 2) serial determinations of the hemodynamic effects of nitro-L-arginine (L-NA), an NOS inhibitor, on basal pulmonary vascular tone (protocol 2); 3) serial assessments of the acute hemodynamic response to partial DA compression after L-NA treatment (for assessment of the myogenic response; protocol 3); and 4) studies of the acute hemodynamic response to partial DA compression after treatment with nifedipine (a voltage-operated calcium channel blocker; protocol 4). Comparisons were made between serial measurements from the control and the chronic pulmonary hypertension (PPHN) study groups.
Protocol 1: Serial measurements of the acute hemodynamic response to partial DA compression in pulmonary hypertension and control fetal lambs. To study serial changes in the hemodynamic response during acute partial compression of the DA, we partially inflated the DA occluder for 10 min after recording baseline measurements. The degree of inflation was set to increase mean PAP by 15 mmHg from baseline values. Mean PAP was kept constant throughout the compression period by readjusting the degree of inflation of the occluder, as needed. After 10 min of DA compression, the occluder was released rapidly (deflated). Mean PAP, left atrial pressure, mean aortic pressure (AoP), amniotic pressure, and left pulmonary blood flow were recorded at 5-min intervals starting 20 min before compression (baseline) and for 10 min after the DA compression period. The duration of each experiment was 40 min.
Protocol 2: Serial determinations of the hemodynamic effects of NOS inhibition on basal pulmonary vascular tone. To determine serial changes in the acute effects of NOS inhibition on basal hemodynamic variables, L-NA (30 mg over 10 min) was infused in the LPA after the completion of protocol 1 (i.e., 40 min after the beginning of the experiment). This dose was selected from published studies that have demonstrated that this dose of L-NA inhibits ACh, oxygen, and flow-induced pulmonary vasodilation (4, 10, 36, 39). Hemodynamic parameters were recorded at 5-min intervals, starting from the beginning of L-NA infusion and for 30 min. The duration of each experiment was 30 min.
Protocol 3: Serial assessment of the acute hemodynamic response to partial DA compression after L-NA treatment. To investigate the effects of L-NA on the hemodynamic response to acute partial DA compression, we partially inflated the DA occluder at the end of the second part of the experiment. The DA occluder was partially inflated for 10 min, and the degree of inflation was set to increase PAP by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the compression period. After 10 min of DA compression, the occluder was deflated. Hemodynamic parameters were recorded at 5-min intervals for 10 min during and 10 min after the compression period. The duration of each experiment was 20 min.
Protocol 4: Studies of the acute hemodynamic response to partial DA compression after nifedipine treatment. To investigate the effects of voltage-operated calcium channel blockade on the hemodynamic response to acute partial DA compression, we partially inflated the DA occluder after treatment with nifedipine, a voltage-operated calcium channel blocker. Nifedipine (0.8 mg) was infused for 10 min in the LPA. Preliminary studies showed that this dose does not affect basal pulmonary vascular tone in the fetus. After completion of drug infusion, the DA occluder was partially inflated for 10 min. As described above, the degree of inflation was set to increase mean PAP by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the compression period. After 10 min, the occluder was deflated. Hemodynamic parameters were measured at 5-min intervals during and after the compression period.
Each study was performed after at least 48 h of recovery. The first day of study was designated as "day 0." Study protocols were performed at day 0 (before initiating chronic DA compression in the pulmonary hypertension group), at 48 h (days 2, 4, 6, and 8 for protocol 1), or at 96 h (days 4 and 8 for protocols 2 and 3) intervals. Protocol 4 was performed in control and hypertensive lambs on day 9. At each study, arterial blood gas tensions and pH were measured during the baseline period, at the end of the first DA compression period (30 min), after L-NA infusion at 70 min, and at the end of the second DA compression period (80 min). One animal in each group was excluded from the study at days 6 and 8 because of technical problems with the DA or fetal demise.Drug Preparation
L-NA and nifedipine solutions were freshly prepared just before the study. L-NA (Sigma Chemical, St. Louis, MO) at the dose of 30 mg was dissolved in 1 M HCl and diluted in normal saline (1 ml final volume). NaOH (1 M) was added in small increments to titrate the pH of the solution to 7.40. Nifedipine (0.8 mg; Sigma Chemical) was dissolved in 0.1 ml of 100% ethanol and diluted in normal saline (1 ml final volume). Each drug (L-NA and nifedipine) was infused at a rate of 0.1 ml/min.Data Analysis
The results are presented as means ± SE. Data were analyzed by using repeated-measures and factorial ANOVA (Stat View 4.1 for Macintosh; Abacus Concepts, Berkeley, CA). Post hoc analysis was performed with Newman-Keuls testing. A P value <0.05 was considered statistically significant. ![]() |
RESULTS |
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Protocol 1: Serial Determinations of the Acute Hemodynamic Response to Partial DA Compression in Hypertensive and Control Animals
Day 0 studies.
The acute hemodynamic response to partial compression of the DA was
similar in the control and pulmonary hypertension groups when studied
on day 0 (before initiating chronic DA compression in the
hypertension group). In the control group, brief compression of the DA
rapidly increased mean PAP by 35% (P < 0.05 vs.
baseline) and LPA blood flow by 82% (P < 0.05) at 10 min (Fig. 1). In the pulmonary
hypertension group, mean PAP increased 32%, and LPA blood flow
increased by 74% (P < 0.05 for each parameter; Fig. 1). During acute DA compression, PVR decreased 20 ± 5% below
baseline values in both groups (P < 0.05; Figs. 1 and
2). Heart rate increased from 149 ± 13 (baseline) to 169 ± 9 beats/min and from 154 ± 11 (baseline) to 176 ± 14 beats/min in the control and pulmonary hypertension groups, respectively (P < 0.05 for
baseline vs. peak values within each group). Mean left atrial pressure
was 2 ± 1 at baseline and did not change during the study period.
Values for mean AoP, arterial blood gas, and pH parameters were not
different between study groups at baseline (Table
1) and did not change significantly
during the DA compression period.
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Day 2 studies.
After 2 days of hypertension, release of the DA occluder decreased mean
PAP from 62 ± 4 to 54 ± 5 mmHg in the hypertensive group
(Fig. 3). Reinflation of the DA occluder
rapidly increased mean PAP by 33 ± 4% in the control group and
by 31 ± 5% in the pulmonary hypertension group
[P = not significant (NS) between groups; Fig. 3].
However, although LPA blood flow increased nearly twofold in the
control group (P < 0.05), there was no change in LPA
blood flow in the chronic pulmonary hypertension group. PVR increased
in the chronic pulmonary hypertension group but not in the control
group (Fig. 3). During acute DA compression, heart rate increased in
both groups (from 145 ± 11 to 167 ± 12 beats/min in the
control group and from 151 ± 11 to 178 ± 14 beats/min in the chronic pulmonary hypertension group; P < 0.05, treatment vs. baseline within each group). Left atrial pressure, mean
AoP, and arterial blood gas parameters did not change during the study period.
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Day 4 studies.
After 4 days of DA compression, release of the DA occluder reduced mean
PAP from 64 ± 2 to 55 ± 4 mmHg (Fig.
4). After recovery from chronic DA
compression, acute inflation of the DA occluder increased mean PAP by
41 ± 4 and by 36 ± 5% in the control and pulmonary
hypertension groups, respectively. Although LPA blood flow increased by
more than twofold in the control group (104 ± 14%;
P < 0.05 vs. baseline), there was no change in LPA
blood flow during acute DA compression in the pulmonary hypertension group (Fig. 4). During the DA compression period, PVR decreased from
0.57 ± 0.03 to 0.41 ± 0.05 mmHg · ml1 · min
1 in the
control group (P < 0.05) but increased from 0.62 ± 0.03 to 0.99 ± 0.1 mmHg · ml
1 · min
1 in the
chronic pulmonary hypertension group (P < 0.05; Figs. 2 and 4). During acute DA compression, heart rate increased in both
groups, but there was no difference between study groups. Mean left
atrial pressure, mean AoP, and arterial blood gas parameters did not
change during or after the compression period and were not different
between groups.
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Day 6 studies.
In the chronic pulmonary hypertension group, release of the DA occluder
decreased mean PAP from 68 ± 2 to 55 ± 4 mmHg (Fig. 5). Subsequently, the DA occluder was
partially inflated to increase mean PAP by 35 ± 3 and by 36 ± 6% in the control and chronic pulmonary hypertension groups,
respectively (Fig. 5). LPA blood flow increased by 108 ± 16%
(from 85 ± 5 to 177 ± 21 ml/min), and PVR decreased by
29 ± 7% (from 0.56 ± 0.05 to 0.4 ± 0.07 mmHg · ml1 · min
1) during
acute DA compression in the control group (P < 0.05). In contrast, LPA blood flow did not change in the pulmonary
hypertension group during acute DA compression (Fig. 5). Unlike the
controls, PVR increased by 50 ± 8% during acute DA compression
in the chronic pulmonary hypertension group (Fig. 5;
bottom). Mean left atrial pressure, mean AoP, and arterial
blood gas parameters did not change during the study period and were
not different between groups.
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Day 8 studies.
In the chronic pulmonary hypertension group, acute release of the DA
decreased mean PAP from 71 ± 4 to 60 ± 6 mmHg. Partial compression of the DA rapidly increased mean PAP by 37 ± 3% in the control group (from 49 ± 2 to 67 ± 3 mmHg;
P < 0.05) and by 30 ± 6% in the chronic
pulmonary hypertension group (from 60 ± 6 to 78 ± 6 mmHg).
LPA blood flow increased by 89 ± 12% (from 109 ± 5 to
206 ± 18 ml/min) in the control group (P < 0.05)
but did not change with acute DA compression in the chronic pulmonary hypertension group. During acute DA compression, PVR decreased from
0.44 ± 0.03 to 0.32 ± 0.04 mmHg · ml1 · min
1 in the
control group (P < 0.05), but PVR increased from
0.62 ± 0.06 to 0.95 ± 0.10 mmHg · ml
1 · min
1 in the
chronic pulmonary hypertension group (Figs. 2 and
6). Mean left atrial pressure, mean AoP,
and arterial blood gas parameters did not change during the study.
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Protocol 2: Serial Determinations of the Hemodynamic Effects of NOS Inhibition on Basal Pulmonary Vascular Tone
Day 0 Studies.
At day 0, L-NA treatment increased PVR in both
study groups from 0.53 ± 0.07 (baseline) to 0.72 ± 0.06 mmHg · ml1 · min
1 and from
0.54 ± 0.05 (baseline) to 0.78 ± 0.04 mmHg · ml
1 · min
1 in the
control and pulmonary hypertension groups, respectively [P < 0.05 for baseline vs. treatment within each
group (Fig. 1) and at 40-70 min (Fig.
7)]. Mean AoP increased after
L-NA treatment by 20% in both groups (P < 0.05 vs. baseline). Values for heart rate, left atrial pressure,
arterial blood gas values, and pH did not change during this study
period and were similar between groups.
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Day 4 studies.
In the control group, L-NA infusion increased PVR from
0.52 ± 0.05 to 0.78 ± 0.07 mmHg · ml1 · min
1
(P < 0.05 vs. baseline) and decreased LPA blood flow
(P < 0.05). In contrast, L-NA treatment
did not change PVR or LPA blood flow in the chronic pulmonary
hypertension group (at 40-70 min; Fig. 4). The increase in PVR
after L-NA treatment, as expressed as percentage change
from baseline, was greater in control animals than in the chronic
pulmonary hypertension group (Fig. 7). In contrast to the pulmonary
circulation, mean AoP increased after L-NA treatment in
both groups. Heart rate, left atrial pressure, pH, and arterial blood
gas parameters remained unchanged during this study period, and these
parameters were not different between groups.
Day 8 studies.
L-NA infusion increased mean PAP from 48 ± 4 to
59 ± 5 mmHg (P < 0.05; treatment vs. baseline
values) and increased PVR from 0.45 ± 0.04 to 0.62 ± 0.03 mmHg · ml1 · min
1 in the
control group (P < 0.05; at 40-70 min; Fig. 6).
In contrast, L-NA did not change mean PAP, LPA blood flow,
or PVR in the chronic pulmonary hypertension group (Figs. 6 and 7).
Mean AoP increased from 44 ± 3 to 55 ± 4 mmHg
(P < 0.05) in the control group and from 49 ± 3 to 57 ± 3 mmHg (P = 0.05) in the pulmonary
hypertension group. Heart rate, left atrial pressure, pH, and arterial
blood gas parameters remained unchanged during this study period.
Protocol 3: Serial Determinations of the Acute Hemodynamic Response to Partial DA Compression After L-NA Treatment
Baseline studies (day 0). Partial DA compression after L-NA treatment rapidly increased mean PAP from 58 ± 3 to 76 ± 2 mmHg in the control group (P < 0.05) and from 58 ± 2 to 72 ± 2 mmHg in the chronic pulmonary hypertension group (P < 0.05 and P = NS for comparisons of mean PAP during compression between groups; at 70-80 min; Fig. 1). The increase in PVR during DA compression after L-NA treatment was not different between groups. After deflation of the DA occluder, PVR fell to baseline values within each group. Mean AoP, left atrial pressure, pH, and blood gas parameters did not change during the study period.
Day 4 studies.
After L-NA treatment, partial DA compression increased mean
PAP by 30 ± 3 and by 30 ± 5% in the control and pulmonary
hypertension groups, respectively (at 70-80 min; Fig. 4). LPA
blood flow did not change during DA compression after the
L-NA period in either group. PVR progressively increased in
both groups (P < 0.05, treatment vs. baseline within
each group; P = NS between study groups). The
percentage change of PVR during DA compression was not different between control and pulmonary hypertension groups (Fig.
8). Mean AoP, left atrial pressure, pH,
and arterial blood gas parameters did not change during the study in
either group.
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Day 8 studies.
Partial compression of the DA after L-NA treatment rapidly
increased mean PAP by 32 ± 5% in the control group and by
36 ± 4% in the chronic pulmonary hypertension group
(P = NS between groups). LPA blood flow did not change
during acute DA compression in either group. PVR progressively
increased from 0.62 ± 0.03 to 0.78 ± 0.03 mmHg · ml1 · min
1 in the
control group (P < 0.05; baseline vs. compression
values) and from 0.71 ± 0.07 to 1.06 ± 0.07 mmHg · ml
1 · min
1 in the
chronic pulmonary hypertension group (P < 0.05 for
baseline vs. compression values; at 70-80 min; Fig. 6). The
increase in PVR during DA compression was greater in the chronic
pulmonary hypertension group than in the control group (Fig. 8). Mean
AoP, left atrial pressure, pH, and arterial blood gas parameters did not change during this study period in either group.
Protocol 4: Acute Hemodynamic Response to Partial DA Compression After Inhibition of Voltage-Operated Calcium Channels in Control and Hypertensive Lambs
At day 9, mean PAP decreased from 71 ± 4 to 61 ± 5 mmHg after acute release of the DA occluder in the chronic pulmonary hypertension group. Nifedipine infusion did not change mean PAP, LPA blood flow, or PVR in either study group. Partial compression of the DA after nifedipine treatment rapidly increased mean PAP by 38 ± 5% in the control group (from 44 ± 3 to 61 ± 3 mmHg; P < 0.05) and by 33 ± 6% in the chronic pulmonary hypertension group (from 52 ± 6 to 69 ± 6 mmHg; P < 0.05). The percent change in PAP during acute DA compression was not different between study groups. After nifedipine treatment, DA compression increased LPA blood flow by 122 ± 17% (from 102 ± 8 to 226 ± 24 ml/min) in the control group (P < 0.05) but did not change LPA flow in the chronic pulmonary hypertension group (from 98 ± 9 to 126 ± 19 mmHg · ml
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DISCUSSION |
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In this study, we tested the hypothesis that chronic intrauterine pulmonary hypertension impairs the pulmonary vascular response to changes in hemodynamic forces, as assessed by serial studies of acute compressions of the DA. In normal fetal lambs, acute compression of the DA causes progressive pulmonary vasodilation, which is blocked by NOS inhibition, suggesting that NO mediates shear stress-induced vasodilation. After NOS inhibition or in the pulmonary hypertension lambs, LPA blood flow increases rapidly during the first few minutes but decreases rapidly toward baseline levels (36, 37). In addition, acute DA compression after NOS inhibition causes a marked pulmonary vasoconstrictor response, suggesting that NOS blockade unmasks a potent myogenic response in the normal fetal lung. In contrast to controls, exposure to chronic pulmonary hypertension impairs flow-induced vasodilation and enhances the myogenic response during acute DA compression in the absence of NOS inhibition. Overall, we found that, in the late-gestation fetus: 1) flow-induced pulmonary vasodilation was absent and pressure-induced vasoconstriction was unmasked after 2 days of pulmonary hypertension; 2) the vasoconstrictor response after NOS blockade was diminished by 4 days of pulmonary hypertension; and 3) pressure or stretch-induced vasoconstriction, i.e., the myogenic response, was enhanced after 8 days of chronic pulmonary hypertension. In addition, we have also demonstrated that nifedipine treatment abolished this myogenic response, demonstrating that influx of extracellular calcium through voltage-operated channels is critical for stretch-induced vasoconstriction in the hypertensive lung. These results support the hypothesis that chronic pulmonary hypertension abolishes flow-induced vasodilation and increases the myogenic response in the perinatal lung.
These findings are the first demonstration that an increased myogenic response contributes to abnormal vasoreactivity in chronic pulmonary hypertension in vivo. Past experimental studies of chronic pulmonary hypertension in newborn and adult animals have demonstrated several potential mechanisms of abnormal vascular tone (5). Such mechanisms include impaired production of endogenous vasodilators, including NO and prostacyclin, increased production of vasoconstrictors such as endothelin (ET)-1, or abnormal smooth muscle cell responsiveness to vasoactive stimuli (5, 20, 21, 29, 34, 35, 41). Previous work in this model of perinatal pulmonary hypertension has also demonstrated decreased soluble guanylate cyclase expression and activity (35), increased cGMP-specific (or, type 5) phosphodiesterase activity (15), and increased ET-1-mediated vasoconstriction (20, 21). Thus past studies suggest that chronic pulmonary hypertension in utero alters the balance of endogenous vasoactive substances in favor of increased tone (5). This present study suggests that an enhanced myogenic response also contributes to abnormal vasoreactivity in chronic pulmonary hypertension. In addition to the role of NO, the altered response to acute hemodynamic stress may be partly due to decreased production or activity of other dilators, including prostacyclin or an endothelium-derived hyperpolarizing factor, or increased production of vasoconstrictor products, such as ET-1, thromboxane, or leukotrienes. However, recent studies suggest that cyclooxygenase products, such as prostacyclin or thromboxane, are unlikely to play a significant role in modulation of the myogenic response (43). Recent studies have suggested an important role for cytochrome P-450 metabolites of arachidonic acid, including 20-hydroxyeicosatetraenoic acid, in pressure-induced vasoconstriction of cerebral and renal arteries in vitro (14, 23, 38). This mechanism has not yet been studied in the fetal lung circulation. In addition to the loss of the braking effect of NO production on the myogenic response in the normal fetus, chronic hypertension also augments the myogenic response, thereby contributing to abnormal vasoreactivity due to enhanced stretch-induced vasoconstriction. The effects of prolonged hypertension on the acute response to hemodynamic forces are partly due to decreased endogenous NOS activity (29, 34, 41), which unmasks the underlying myogenic response (36, 37). However, the presence of an enhanced myogenic response in this model suggests an additional mechanism that underlies abnormal vasoreactivity in perinatal pulmonary hypertension.
The myogenic response is defined as the ability of vascular smooth muscle to constrict during an increase in intravascular pressure and to dilate upon lowering intravascular pressure (8, 12, 30). The myogenic response plays a key role in autoregulation of blood flow to the brain, kidney, and other systemic vascular beds, but little is known about the presence and physiological roles of myogenic responses in the pulmonary circulation. Past studies have described stretch-induced vasoconstriction in the pulmonary circulation, but these findings were based exclusively on in vitro studies of isolated arteries (9, 25). More recently, studies in chronically prepared fetal lambs have demonstrated that a potent myogenic response exists in the pulmonary circulation in vivo (36). In the normal fetus, acute DA compression rapidly increases PAP and pulmonary artery blood flow, and PVR rapidly decreases (1). However, despite maintaining PAP constant over time, PVR steadily increases above baseline values after 1 h. These time-dependent changes in PVR during acute DA compression likely represent the effects of the following two opposing stimuli: 1) flow-induced vasodilation because of an abrupt increase in shear stress and 2) pressure- or stretch-induced vasoconstriction resulting from activation of a strong myogenic response that opposes the vasodilator response. Shear stress-induced pulmonary vasodilation is primarily mediated by NO release (10). DA compression after NOS inhibition causes PVR to increase in the normal fetal lamb, suggesting that NO synthesis blockade unmasks the myogenic response (36).
The myogenic response may play important physiological roles in the maintenance of high PVR and time-dependent autoregulation of pulmonary blood flow in the normal fetus. Several physiological and pharmacological stimuli cause fetal pulmonary vasodilation, but vasodilation is often transient despite continued exposure to the dilator stimulus (5). For example, increases in fetal arterial PO2 cause a two- to threefold increase in blood flow after 1 h, but, despite maintaining high arterial PO2, blood flow falls progressively over time (7). Similar patterns have also been observed during prolonged treatment with pharmacological vasodilators, including ACh, bradykinin, tolazoline, and dilator prostaglandins (5). Mechanisms that oppose pulmonary vasodilation in the fetus are uncertain, but we speculate that this time-dependent process represents an autoregulatory mechanism caused by stretch-induced activation of a strong myogenic response. Although treatment with a selective ETA receptor blockers improves blood flow during prolonged DA compression, the myogenic response cannot simply be explained by increased ET-1-mediated vasoconstriction (2). Interestingly, endothelium-independent agonists, such as 8-bromo-cGMP, atrial natriuretic peptide, and inhaled NO, cause more sustained fetal pulmonary vasodilation during prolonged treatment than endothelium-dependent agonists (22). These findings have led to the speculation that the time-dependent loss of vasodilation may represent an inability of the fetal lung to sustain NO production in response to endothelium-dependent stimuli (5).
Thus the fetal pulmonary circulation is characterized by the presence of a strong myogenic response that is increased by chronic pulmonary hypertension. Mechanisms by which hypertension augments the myogenic response in the developing lung are uncertain but are likely to primarily be the result of altered calcium handling in vascular smooth muscle cells, secondary to changes in potassium ion channel activity, intracellular signaling, and autocrine or paracrine factors (11, 14, 16, 18, 23, 27, 38). In this study, inhibition of voltage-operated calcium channels inhibited the myogenic response, as has been observed previously for the myogenic response in systemic vessels (19, 32, 42). Dihyhdropyridines abolish pressure- or stretch-induced increases in intracellular calcium concentration in smooth muscle from isolated rat mesenteric arteries (42). In addition, activation of voltage-operated calcium channels enhances the myogenic response in skeletal muscle arteries (19). Finally, voltage dependence of the L-type channel predicts that the 20- to 35-mV depolarization resulting from stretch of vascular smooth muscle increases the open probability of voltage-gated calcium channels by 10- to 15-fold (32). It is possible, however, that nifedipine may have blocked the myogenic response in hypertensive lambs by an alternate mechanism, but their effects in this model are most likely the result of inhibition of calcium channels.
Our results further support the hypothesis that the myogenic response is mediated by activation of voltage-operated calcium channels and that calcium influx is likely a key downstream event in the signaling pathway. These data do not rule out an "upstream" role of other types of channels that could regulate voltage-operated calcium channels through depolarization. Vasoconstriction in response to increased intravascular pressure is mediated by changes in the activation states of potassium and calcium ion channels, resulting in depolarization of vascular smooth muscle cells and an influx of calcium. Activation of phosopholipases C and A2 and protein kinase C and increased production of lipid mediators are associated with the development of myogenic tone (11, 14, 16, 18, 23, 27).
In conclusion, we report that chronic hypertension dramatically alters the response of the fetal pulmonary circulation to abrupt changes in hemodynamic forces, as characterized by the loss of flow-induced vasodilation and enhancement of a potent myogenic response. These observations are likely due in part to marked impairment of endogenous NOS activity, as reflected by the loss of L-NA-mediated vasoconstriction and the finding that NOS inhibition blocks flow-induced vasodilation. In addition, hypertension likely directly alters the vascular smooth muscle cell's response to stretch stress or pressure-induced vasoconstriction, causing further augmentation of the myogenic response resulting from increased activity of voltage-operated calcium channels. We speculate that chronic hypertension decreases NO production, inhibiting the normal vasodilator response to birth-related stimuli and causing paradoxical vasoconstriction to acute hemodynamic stress. In addition, hypertension alters smooth muscle function, causing an enhanced myogenic response. We further speculate that interventions that specifically target mechanisms underlying the myogenic response may improve the treatment of patients with severe pulmonary hypertension.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the March of Dimes Research Foundation (to S. H. Abman) and National Heart, Lung, and Blood Institute Specialized Center of Research Program Grants HL-46481 (to S. H. Abman) and RO1 HL-68702 (to S. H. Abman).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. H. Abman, Pediatric Heart Lung Center, B-395, Dept. of Pediatrics, The Children's Hospital, 1056 E. 19th Ave., Denver, CO 80218-1088 (E-mail: steven.abman{at}uchsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 July 2001; accepted in final form 27 August 2001.
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