Main

The fetal pulmonary circulation is characterized by high PVR and low blood flow. Despite high PAP, the lung is perfused with less than 10% of the combined ventricular output during late gestation(1). Because of high PVR in the fetus, most of the right ventricular output crosses the DA into the descending aorta, thereby increasing umbilical-placental flow and gas exchange. Mechanisms that maintain high PVR in utero are incompletely understood, but may include low fetal Pao2(1,2), lack of a gas-liquid interface(3), and production of vasoconstrictor mediators such as leukotrienes(4,5) and endothelin 1(6). In addition to its high basal PVR, the fetal pulmonary circulation is characterized by its ability for time-dependent vasoregulation. Although many stimuli, such as increased Pao2, shear stress, and several pharmacologic agents, briefly increase fetal pulmonary blood flow, vasodilation is often transient as flow decreases toward baseline despite prolonged exposure to these dilator stimuli(7–9). This pattern suggests that the fetal pulmonary circulation is capable of autoregulation, an ability to oppose vasodilation with time. The effects of low Pao2, the decreased ability to sustain production of endogenous vasodilators, or the enhanced production of vasoconstrictors have been proposed as possible mechanisms to explain this phenomena. The potential contribution of the myogenic response to high PVR and to time-dependent vaso-regulation of the pulmonary circulation in the perinatal lung has not been studied in vivo.

The myogenic response is defined as the ability of vascular smooth muscle to constrict during an increase in intravascular pressure and to dilate on lowering intravascular pressure(10). The myogenic response plays a key role in autoregulation of blood flow, and has been largely documented in vitro with studies of systemic vessels and isolated perfused organs(11–14). Whether or not the pulmonary circulation is capable of a myogenic response is uncertain, as limited data are available. In isolated pulmonary arteries from adult cats, Kulik et al.(5) found that stretching resistive arteries (<1000 µm diameter) resulted in active force generation. Belik(16) studied the myogenic response in large-capacitance pulmonary arteries of adult and newborn guinea pigs. Although stretch-induced force generation was observed in both newborn and adult animals, the magnitude of the response was higher in the newborns(16). Other in vitro studies have also suggested that myogenic responses are present in lung vessels(17,18). Thus, evidence exists from in vitro studies that the pulmonary circulation can exhibit a myogenic response and that the myogenic response may be most active during the perinatal period.

We therefore hypothesize that the myogenic response may contribute significantly to high PVR and the regulation of pulmonary blood flow during the fetal life. To test this hypothesis, we studied the hemodynamic response during partial compression of the DA in chronically prepared late-gestation fetal lambs. Partial DA compression acutely increases mean PAP and pulmonary artery blood flow(7). PVR initially drops during the first 30 min of DA compression, then steadily increases toward baseline values after 1 h(7). The decrease in PVR during the first phase is considered as a consequences of a mechanical increase in blood flow and shear stress. However, partial compression of the DA may simultaneously activate two opposing responses: 1) blood flow-induced vasodilation through increased shear stress; and 2) pressure-induced vasoconstriction, such as a myogenic response, that opposes the vasodilator response. Myogenic response may be masked by shear stress-induced vasodilation, and inhibition of shear stress-induced vasodilation may reveal the underlying myogenic response. Previous studies demonstrated that shear stress-induced vasodilation is mainly mediated by NO release inasmuch as NOS blockade abolishes the vasodilator response during DA compression(19,20).

Therefore, we studied the early response during acute partial DA compression with and without inhibition of NOS. We report that acute DA compression reveals a myogenic response that is unmasked by inhibition of NO production in the intact fetal lung circulation.

METHODS

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. Nineteen mixed-breed (Columbia-Rambouillet) pregnant ewes between 124 and 128 d gestation (term = 147 d) were fasted for 48 h before surgery. Ewes were sedated with i.v. 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 into 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 into the LPA (22 gauge), main pulmonary artery (20 gauge), and left atrium (20 gauge) by direct puncture through pursestring sutures and secured, as previously described(21). An inflatable vascular occluder (In Vivo Metric, Healdsburg, CA) was placed loosely around the DA after gentle dissection of adherent connective tissue with cotton-tip 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 added to the amniotic cavity before closure of the hysterotomy. The flow transducer, catheters, and occluder were exteriorized through an s.c. 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 of heparinized saline (20 U/mL). Catheter positions were verified at autopsy. Studies were performed after a minimum recovery time of 48 h.

Physiologic 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. The absolute values of flows were determined from the mean 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(22). Main pulmonary artery, aortic, left atrial, and amniotic catheters were connected to blood pressure transducers (TSD 104, Biopac, Santa Barbara, CA). The pressure and flow signals were continuously recorded and processed on a computer (PowerMac 7100/80 AV) 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 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 PAP and LAP divided by mean LPA blood flow. Blood samples from the main pulmonary artery catheter were used for blood gas analysis and oxygen saturation measurements (OSM 3 hemoximeter and ABL 520, Radiometer, Copenhagen).

Experimental design

Three different experimental protocols were included in this study: 1) the acute hemodynamic response to partial DA compression; 2) the effects of L-NA on the hemodynamic response to partial DA compression; and 3) the relationship between pulmonary artery pressure and pulmonary blood flow after L-NA infusion.

Protocol 1. Acute hemodynamic response to partial DA compression (n = 12 animals). To study the effects of partial DA compression, we partially inflated the DA occluder for 30 min. The degree of inflation was set to increase mean PAP by 15 mm Hg from its baseline value. Mean PAP was kept constant throughout the compression period by readjusting the degree of inflation of the occluder as needed. After 30 min of DA compression, the occluder was deflated. Mean PAP, LAP, mean AoP, amniotic pressure, and LPA blood flow were recorded at 10-min intervals, starting 30 min before and for 30 min after DA compression. During the compression period, data were recorded at 30 s, and 1, 2, 3, 4, 5, 10, 20, and 30 min. The duration of each experiment was 90 min. Arterial blood gas tensions and pH were measured at 0, 60, and 90 min.

Protocol 2. Effects of L-NA on the hemodynamic response to partial DA compression (n = 13 animals). To investigate the effects of L-NA-rationale was to unmask the effects of NO production-on the hemodynamic response to partial DA compression, we partially inflated the DA occluder after L-NA infusion. L-NA (30 mg during 10 min) was infused into the LPA 30 min before the DA compression. This dose was selected from past studies that have demonstrated effective blockade of NOS activity during acetycholine- and flow-induced vasodilation(19). The DA occluder was partially inflated for 10 min. Preliminary experiments have shown that after 10 min of DA compression, pulmonary blood flow and PVR remained constant. As in protocol 1, the degree of inflation was set to increase mean PAP by 15 mm Hg from values obtained immediately before the DA compression study period. Mean PAP was kept constant throughout the compression period. After 10 min of DA compression, the occluder was deflated. Hemodynamic parameters were recorded from 5 min before 1 and 5 min after the occlusion. During the compression period, data were recorded at 0, 5, 6, 7, and 10 min. The duration of each experiment was 20 min. Arterial blood gas tensions and pH were measured at 0 and 15 min of the experiment.

Protocol 3. Relationship between pulmonary artery pressure and pulmonary blood flow after L-NA infusion (n = 6 animals). To study the relationship between pulmonary pressure and blood flow after inhibition of NOS, successive DA compressions were performed after L-NA infusion (30 mg) at four different levels of mean PAP. L-NA was infused into the LPA 30 min before the DA compression for 10 min. Thirty minutes after beginning L-NA infusion, the DA occluder was partially inflated for 10 min to increase PAP by 10, 20, 30, or 40% from its baseline value. Mean PAP was kept constant throughout the compression period. After 10 min of DA compression, the occluder was deflated. Hemodynamic measurements were recorded before and after 10 min of DA compression. Three similar tests were successively performed at 30-min intervals at different values of mean PAP. The order of the four tests at different levels of PAP was random.

Drug preparation. L-NA solution was freshly prepared on the day of study. L-NA (Sigma Chemical Co, St. Louis, MO) at the dose of 30 mg was first dissolved in 1 M HCl, followed by dilution with normal saline (1 mL). Small volumes of NaOH (1 M) were slowly added to titrate the pH to 7.40. The drug was infused into the LPA at a rate of 6 mL/h.

Data analysis. The results are presented as means ± SE. The data were analyzed using repeated-measures and factorial ANOVA (StatView 4.1 for Macintosh, Abacus Concepts, Berkeley, CA). A p value < 0.05 was considered statistically significant.

RESULTS

Protocol 1. Hemodynamic response to partial DA compression. Partial compression of the DA rapidly increased mean PAP by 33 ± 3% (from 45 ± 1 to 60 ± 1 mm Hg) and LPA blood flow by 117 ± 16% (from 90 ± 6 to 195 ± 15 mL/min) at 30 min (p < 0.05). During the first minute of DA compression, PVR transiently increased by 9 ± 3% (p = 0.06) (Figs 1 and 2) and then progressively decreased below baseline values by 39 ± 5% (from 0.51 ± 0.03 to 0.30 ± 0.02 mm Hg·mL-1·min-1; p < 0.05) during sustained DA compression (Fig. 1). During acute DA compression, the heart rate increased from 154 ± 10 to 174 ± 6 beats/min (p < 0.05) (Table 1). Mean AoP did not change during DA compression (Table 1). Mean LAP before compression was 2 ± 1 mm Hg and did not change during the study period. PaO2 decreased slightly after 30 min of DA compression [17.2 ± 0.6 (30 min) versus 18.7 ± 0.8 mm Hg (baseline); p < 0.05]. The other blood gas parameters (arterial PCO2 = 51 ± 0.7 mm Hg; pH = 7.38 ± 0.01) did not significantly change during or after the compression period (Table 1).

Figure 1
figure 1

Hemodynamic response during partial DA compression. DA compression increased mean PAP and LPA blood flow (Flow). PVR increased during the first minute of DA compression and then progressively decreased. Values are expressed as means ± SEM. * p < 0.05 compared with baseline values.

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Figure 2
figure 2

Effect of partial DA compression on PVR expressed as a percentage change from baseline values. PVR increased during the first minute of DA compression and then decreased to baseline values at 2-4 min. Values are expressed as means ± SEM.

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Table 1 Arterial blood gas tension, pH, oxygen, saturation, and hemodnamic measurements before, during, and after 30 min of DA compression
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Protocol 2. Effects of L-NA on the hemodynamic response to partial DA compression. L-NA infusion increased mean PAP from 46 ± 2 to 55 ± 4 mm Hg (p < 0.05), mean AOP from 43 ± 2 to 52 ± 4 mm Hg (p < 0.05), and PVR from 0.46 ± 0.03 to 0.59 ± 0.05 mm Hg·mL-1·min-1 (p < 0.05). Partial DA compression rapidly increased mean PAP by 33 ± 4% (from 55 ± 4 to 73 ± 2 mm Hg) (Figs. 3 and 4). LPA blood flow increased during the first minute of DA compression and then progressively decreased to baseline values at 5 and 10 min of compression (Figs. 3 and 4). PVR increased steadily by 28 ± 4% (from 0.6 ± 0.02 to 0.77 ± 0.05 mm Hg·mL-1·min-1). After deflation of the DA occluder, PVR dropped rapidly to baseline values (Figs. 3 and 4). Mean AoP, heart rate, LAP, pH, and blood gas parameters did not change during the study period (Table 2).

Figure 3
figure 3

Effects of L-NA on the hemodynamic response to partial DA compression. L-NA was infused into the LPA 30 min before DA compression. DA compression increased mean PAP and LPA Flow. After a brief increase in LPA Flow, flow steadily decreased and was not different from the baseline value at 5 and 10 min of DA compression. PVR progressively increased during DA compression. Values are expressed as means ± SEM. * p < 0.05 compared with baseline values.

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Figure 4
figure 4

Time course of mean PAP, LPA Flow, and PVR in a fetal lamb pretreated with L-NA (gestational age, 132 d) during partial DA compression. Partial DA compression increased PAP and LPA Flow. However, after an initial increase, the LPA Flow steadily decreased toward a plateau value. PVR increased progressively during the first 3 min after the beginning of DA compression. The myogenic response was completed in less than 5 min.

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Table 2 Arterial blood gas tension, pH, oxygen saturation, and hemodynamic measurements before and 10 min after partial DA compression after treatment with L-NA infusion
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Protocol 3. Relationship between pulmonary artery pressure and pulmonary blood flow after L-NA infusion. LPA blood flow measured at 10 min of partial DA compression did not change with serial increase in mean PAP (Fig. 5; upper panel). PVR increased linearly with the increase in PAP (p < 0.01) (Fig 5; lower panel).

Figure 5
figure 5

Relationship between PAP and LPA Flow (top) and PVR (bottom) after L-NA infusion. L-NA was infused into the LPA 30 min before DA compression. The DA occluder was partially inflated for 10 min to increase PAP by 10, 20, 30, or 40% above baseline values. LPA Flow and PVR were measured after 10 min of DA compression. Values are expressed as means ± SEM. * p < 0.05.

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DISCUSSION

In this study, we tested the hypothesis that the myogenic response contributes to the regulation of pulmonary blood flow during fetal life. We studied the hemodynamic response to partial acute compression of the DA with and without inhibition of shear stress-induced vasodilation by NOS blockade. Without NOS inhibition, PVR dropped progressively during the DA compression period. In contrast, DA compression after NOS inhibition caused PVR to progressively increase by 28 ± 4% from baseline. Furthermore, plateau values of PVR increased linearly with the increase in PAP. These results support the hypothesis that perinatal lung circulation can exhibit a potent myogenic response.

This study provides new insights about the mechanisms underlying regulation of pulmonary blood flow during fetal life. Partial compression of the DA acutely increases mean PAP and LPA blood flow. The increase in shear stress resulting from DA compression causes a decrease in PVR. However, this decline in PVR is preceeded by a brief increase in PVR. Previous studies demonstrated that NOS inhibition abolishes the vasodilator response to increased blood flow(19,20). After inhibition of shear stress-induced vasodilation, pulmonary blood flow did not change and PVR increased with an increase in PAP. Thus, our findings demonstrate that an active pulmonary vasoconstriction occurs in response to increased intravascular pressure after NOS inhibition. This phenomenon defines a myogenic response(10). To our knowledge, this is the first direct in vivo evidence that a myogenic response exists in the perinatal lung. These results are consistent with the hypothesis that an acute increase in PAP can generate two opposing responses: 1) pulmonary vasodilation mediated by an increase in blood flow owing to shear stress; and 2) pulmonary vasoconstriction, induced by an increase in PAP, that is stretch stress. With intact endothelial function, most of the myogenic response is masked by shear stress-induced vasodilation. However, the transient increase in PVR observed just after the DA compression may be explained by a delayed shear stress response compared with myogenic response. Furthermore, inhibition of shear stress-induced vasodilation by L-NA infusion revealed a potent myogenic response that reached its maximal value in less than 5 min.

Several studies of systemic vessels, including mesenteric, renal, and cerebral arteries, demonstrated the contribution of arteriolar myogenic response to the local autoregulation of blood flow(11,23,24). This mechanism seems also to play a role in the control of basal vascular tone and vascular resistance(25). We have demonstrated that the fetal pulmonary circulation is capable of autoregulation that becomes apparent when endothelium-dependent vasorelaxation is inhibited. Whereas myogenic responses were mostly described in systemic vessels(11–14), some previous studies also demonstrated the existence of a myogenic response in pulmonary circulation. A myogenic response was found in small resistive arteries (1000 µm diameter) of adult cats(15,17,18) and in large-capacitance pulmonary arteries of adult and newborn guinea pigs(16) and ovine fetuses(26). In newborn guinea pigs, the myogenic response is potent as the force generated for a stretch more than 120% was 100% greater than that after high K+ stimulation(16). The stretch-induced myogenic response of large pulmonary vessels was also explored in normal and pulmonary hypertensive ovine fetuses: the myogenic response was significantly greater in the fetus with pulmonary hypertension(26). Belik(16,26) hypothesized that decreased compliance of large proximal arteries resulting from a myogenic response could increase the pressure transmission of the pulse wave to the smaller resistance arteries and induce a myogenic response at this level. According to this assumption, the degree of vasoconstriction of the resistive vessels may depend, at least in part, on the myogenic response of large-capacitance arteries. Our findings provide further evidence that a potent myogenic response can be elicited in vivo in the perinatal lung. In vitro studies on isolated systemic arterioles have shown that the vasoconstriction begins within 5 to 10 s and is completed in 180 s after the imposed increase in intraluminal pressure(27). This time course of the myogenic response is consistent with our results (Fig. 4).

Past studies suggest that "reflex" pulmonary vasoconstriction caused by distension of the main pulmonary artery may be an additional mechanism that contributes to the physiologic findings reported here(28–32). Juratsch and colleagues(28–31) proposed that distension of the main pulmonary artery causes an immediate increase in PAP as measured distal to the inflated balloon, and that this mechanism exists in human newborns with PPHN. In these studies, stretch-induced stress of the main pulmonary artery was hypothesized to cause distal pulmonary vasoconstriction through a reflex mechanism, in which sensing of stretch in the main pulmonary artery leads to neural transmission of a vasoconstrictor signal to distal vessels. However, this neural mechanism has not been consistently observed in several studies, and it has been suggested that this response may be caused by mechanical effects and not neural reflex activity(28,32). In addition, the identification of a stretch-induced myogenic response in isolated pulmonary arteries in vitro(15–17) suggests that a neural reflex vasospasm mechanism caused by main pulmonary artery distension is unlikely to be sufficient to completely explain these observations. However, our observations cannot exclude the possible contribution of a neural mechanism in these whole animal studies.

There are two potential limitations in our study. First, factors other than NO produced by the vascular endothelium, such as endothelium-derived hyperpolarization factor and prostacyclin(7,33–35), also mediate relaxation of vascular smooth muscle cells after an increase in shear stress. Hence, inhibition of NOS may not completely abolish blood flow-induced vasodilation. However, previous investigations support the hypothesis that NO is the major effector of flow-mediated vasodilation. Although meclofenamate partially blunts the rise in fetal pulmonary blood flow during partial compression of the DA(7), L-NA nearly abolishes this response(19). Furthermore, aspirin or indomethacin do not inhibit the effects of flow-stimulated endothelial cells on vascular reactivity(36). Finally, prostacyclin causes vasodilation partly through NO release(37). However, although NO release appears to play the greater role, we cannot rule out that prostacyclin or endothelium-derived hyperpolarization factor release by the shear stress-induced vasodilation could have partly counteracted the increase in PVR after DA compression. In these conditions, the myogenic response could have been underestimated. Second, although the relative increase in mean PAP expressed as a percentage of change was similar in control and L-NA-treated animals during partial DA compression, the absolute maximal PAP was higher after L-NA infusion because basal PAP was higher. The hypothesis that the acute rise in PAP during DA compression may have impaired ventricular function cannot be completely ruled out. However, the fact that the right ventricle was able to generate and sustain the rise in PAP does not support this hypothesis. Moreover, PVR was calculated as the ratio of the driving pulmonary artery pressure (PAP - LAP) to the resulting LPA blood flow. Thus, as long as the right ventricle is able to sustain PAP, acute changes in PVR do not appear to be dependent on ventricular function.

In summary, our findings provide evidence that a potent myogenic response exists in the perinatal lung. This stretch-induced vasoconstriction can be revealed after inhibition of the vasodilator response to shear stress. In these conditions, pulmonary blood flow does not increase with PAP, i.e. pulmonary blood flow is controlled by autoregulation. Numerous pathophysiologic situations were found to impair endothelium-dependent vasorelaxation. During fetal life, NOS activity can be modulated by PaO2, and low fetal PaO2 appears to suppress endogenous NO activity(38,39). In a model of experimental PPHN, acetylcholine-induced fetal pulmonary vasodilation is lost early(40). Thus, as severe hypoxemia or PPHN may impair endothelium-dependent vasoreactivity, it is likely that in these pathophysiologic settings, shear stress-induced vasodilation may also be reduced or abolished. Therefore, we speculate that the myogenic response may become the predominant regulatory mechanism of vascular tone and contribute significantly to high PVR and altered vasoreactivity. Whether the inability to achieve the normal decline in PVR at birth in infants with PPHN is explained by a persistent autoregulatory mechanism in the pulmonary circulation remains an open question.