Pulmonary vascular dysfunction secondary to pulmonary arterial hypertension: insights gained through retrograde perfusion

Abstract

Here, we tested the hypothesis that severe pulmonary arterial hypertension impairs retrograde perfusion. To test this hypothesis, pulmonary arterial hypertension was induced in Fischer rats using a single injection of Sugen 5416 followed by 3 wk of exposure to 10% hypoxia and then 2 wk of normoxia. This Sugen 5416 and hypoxia regimen caused severe pulmonary arterial hypertension, with a Fulton index of 0.73 ± 0.07, reductions in both the pulmonary arterial acceleration time and pulmonary arterial acceleration to pulmonary arterial ejection times ratio, and extensive medial hypertrophy and occlusive neointimal lesions. Whereas the normotensive circulation accommodated large increases in forward and retrograde flow, the hypertensive circulation did not. During forward flow, pulmonary artery and double occlusion pressures rose sharply at low perfusion rates, resulting in hydrostatic edema. Pulmonary arterial hypertensive lungs possessed an absolute intolerance to retrograde perfusion, and they rapidly developed edema. Retrograde perfusion was not rescued by maximal vasodilation. Retrograde perfusion was preserved in lungs from animals treated with Sugen 5416 and hypoxia for 1 and 3 wk, in lungs from animals with a milder form of hypoxic hypertension, and in normotensive lungs subjected to high outflow pressures. Thus impaired retrograde perfusion coincides with development of severe pulmonary arterial hypertension, with advanced structural defects in the microcirculation.

INTRODUCTION

Pulmonary arterial hypertension represents a diverse family of diseases that are characterized by medial hypertrophy and hyperplasia of arteries and arterioles, adventitial thickening, inflammatory cell infiltration, occlusive neointimal lesions of variable severity, arterial dropout, or rarefaction and right ventricular hypertrophy that progresses to failure (4, 12, 31). Patients with pulmonary arterial hypertension receive vasodilator therapy, but the pulmonary arterial pressure (P_pa) in these patients is only modestly reduced. The relative insensitivity to vasodilator therapy suggests that a fixed obstruction to blood flow contributes importantly to the elevation in pulmonary vascular resistance (28). However, the extent to which matrix remodeling within the vessel wall (19, 21, 23, 30, 34, 45), occlusive neointimal lesions (2, 7, 8, 36, 40, 42), and/or rarefaction (6, 29, 46) contributes to a fixed obstruction has not been fully resolved.

Occlusive neointimal lesions are thought to arise from overgrowth of apoptosis-resistant endothelial cells that encroach on the lumen (36). In the so-called cancer paradigm of vascular remodeling (27), vascular injury initiates uncontrolled endothelial growth, with a “loss of the law of the monolayer.” Although endothelial cell injury seems to initiate this vascular response, the mature occlusive lesion contains diverse cell types (38, 44). The trigger for such an endothelial injury is unknown but seems to be dependent on hemodynamic stress. For example, pulmonary artery banding prevents these lesions from forming and even reverses some lesions that have already formed (1, 43). However, the idea that a hyperproliferative cell phenotype leads to vascular occlusion has been contrasted with evidence that rarefaction is seen with advancing disease (6, 46). The concept of rarefaction implicates cellular degeneration, rather than hyperproliferation, as a central feature of vascular remodeling. Occlusive neointimal lesions and rarefaction could both impair blood flow and contribute to increased vascular resistance (6, 29).

Most of the increase in pulmonary vascular resistance in pulmonary arterial hypertension is attributed to the arterial segment. However, the relative contributions of vasoconstriction, vascular remodeling, and rarefaction to increased resistance have been questioned (29). There remains some uncertainty as to whether increased venous resistance could also impact blood flow distribution in pulmonary arterial hypertension (29). Here, we tested whether retrograde perfusion could be used to assess the impact of vascular remodeling in pulmonary arterial hypertension on blood flow. Fischer rats were used in this study, as in them Sugen 5416 followed by 3 wk of hypoxia and a return to normoxia induces severe pulmonary arterial hypertension that progresses to death; pulmonary vascular remodeling is progressive and severe (3, 14, 47). By 5 wk in the Sugen 5416, hypoxia, and normoxia model, the hypertensive pulmonary circulation does not tolerate retrograde perfusion.

MATERIALS AND METHODS

Animals.

All experimental procedures were performed in accordance with current provisions of the U.S. Animal Welfare Act and were approved by the Institutional Animal Care and Use Committee of the University of South Alabama. F344 rats were bred at the University of South Alabama as previously described (3, 14, 47).

Rat pulmonary arterial hypertension model.

Pulmonary arterial hypertension was induced in 7- to 8-wk-old male Fischer rats, ranging in weight from 150 to 200 g, by a single subcutaneous injection of Sugen 5416 (20 mg/kg; Semaxanib; MedKoo Biosciences, Chapel Hill, NC) on day 1, followed by exposure to 3 wk of normobaric hypoxia (10% O₂) and then reexposure to normoxia (21% O₂) for an additional 2–3 wk. In addition, to evaluate the pulmonary vascular function at the early stages of pulmonary arterial hypertension, (i.e., before formation of neointimal lesions), an additional cohort of rats was subjected to Sugen 5416 and hypoxia for 1 or 3 wk only, without reexposure to normoxia.

Rat hypoxic pulmonary hypertension model.

Hypoxic pulmonary hypertension was induced by subjecting age-matched rats to 10% oxygen for 3 wk. These animals were studied immediately following this 3-wk exposure.

Echocardiography.

Doppler flow imaging was performed using a Vevo 770 imaging system with a RMV-716 scanhead centered at 17.5 MHz (Visual-Sonics, Toronto, ON). Anesthesia was induced in pulmonary arterial hypertension animals using 3% isoflurane in oxygen. Once an anesthetic plane was achieved, animals were positioned supine on a heated platform. Anesthesia was maintained using 1–1.5% isoflurane delivered through a nose cone while animals breathed spontaneously. Heart rate and respiration were monitored throughout. Indexes of right ventricular function, including pulmonary arterial acceleration time (PAAT), pulmonary arterial ejection time (PAET), and the PAAT/PAET ratio, were evaluated from the pulsed-wave Doppler measurements. Averaged recordings represent the results from five independent experiments.

Isolated lung and assessment of lung weight and hemodynamic parameters.

Animals were anesthetized using Nembutal (65 mg/kg body wt). Once a surgical plane was achieved, as defined by the absence of a withdrawal reflex following toe and tail pinch, animals were intubated and ventilated, a sternotomy was performed, and pulmonary artery and left ventricle/atrium catheters were placed. Heart and lungs were removed en bloc and suspended in a humidified chamber, where mechanical ventilation and flow were established. Rat lungs were perfused with buffer (in mmol/l: 119.0 NaCl, 4.7 KCl, 1.17 MgSO₄, 1.18 KH₂PO₄, 23 NaHCO₃, and 5.5 glucose) containing 4% bovine serum albumin and physiological (2.2 mmol/l) CaCl₂, plus 6% autologous whole blood. Lungs were perfused at 8 ml/min in either forward or retrograde orientation for 15 min to obtain an isogravimetric status, and then flow was increased by 8 ml/min every 5 min. Pulmonary artery and venous pressures and lung weight were measured continuously, and double occlusion pressure was measured at the end of each 5-min interval.

Switching between forward and reverse flow.

Inline with the inflow and outflow tubing, stopcocks and a by-pass circuit were arranged to immediately reverse flow direction from the forward to reverse orientation. This switch permitted maintaining flow sequence of the perfusion pump, bubble trap, heat exchange, and inflow reservoir and outflow reservoir without stopping the perfusion pump, thereby avoiding intermittent flow.

Retrograde lung perfusion.

In normotensive and hypoxic rats, once forward perfusion was established, flow was reversed without disconnecting the circuit, by directing perfusate through two cross connections between the pulmonary artery and left atrial lines. Lungs were perfused with increasing flow starting from 8 ml/min, and lung weight and hemodynamic parameters were continuously assessed as described above. In addition, in another set of experiments, lungs from normotensive rats were retroperfused at a fixed flow rate of 8 ml/min. In this case, P_pa was raised to 40 cmH₂O by elevating the perfusate reservoir by 40 cm, to evaluate whether the high outflow pressure impairs retrograde perfusion. In pulmonary arterial hypertensive rats, lungs were first subjected to vasodilation in the forward-direction flow. This was achieved by adding 10 µM fasudil, a Rho kinase inhibitor (LC Laboratories) (25, 26), and 50 µM SKF-96365, a calcium channel blocker (Sigma) (26), in the circulating perfusate for 20 min, and after maximal pulmonary arterial dilation was achieved, flow was switched to retrograde perfusion.

Statistical analysis.

Quantitative data are presented as means ± SE. Group sizes for all figures ranged from n = 3–9. Group means were compared using one- or two-way ANOVA with a Bonferroni post hoc test as appropriate. P < 0.05 was considered statistically significant.

RESULTS

The normotensive pulmonary circulation accommodates increased blood flow in the forward and retrograde directions.

Figure 1 illustrates that an increase in perfusion is accompanied by a linear increase in P_pa (R² = 0.88; P < 0.05), within the range of perfusion rates tested. At the maximal increase in perfusion (96 ml/min), P_pa had risen by approximately threefold compared with the baseline values (Fig. 1A). Despite the increase in P_pa, pulmonary venous pressure (P_pv), and double occlusion pressure (P_do) increased only modestly (Fig. 1B). Lung weights were unchanged within the perfusion ranges of 8–88 ml/min. As perfusion increased above 88 ml/min, lung weight became less stable, displaying some variability. Edema occurred when P_do increased above 30 cmH₂O, as predicted by the classic study of Guyton and Lindsey (16). Thus normotensive isolated perfused lungs accommodate large increases in forward perfusion without a dramatic increase in either P_do or P_pv, and they remain isogravimetric.

Fig. 1.

The normotensive pulmonary circulation accommodates large increases in flow. Pulmonary arterial pressure (P_pa), pulmonary venous pressure (P_pv), double occlusion pressure (P_do), and lung weight were measured in response to increased perfusion rates. A: baseline P_pa increased ~4-fold in response to a >10-fold increase in the perfusion rate, whereas P_pv remained below 10 cmH₂O. B: this large increase in flow increased P_do ~4-fold, whereas lung weight was only increased at the highest flow rate. *P < 0.05, significantly different from baseline values; n = 7 animals.

We next tested whether the normotensive circulation accommodates retrograde perfusion, using the previously described experimental protocol. In this experimental setup, P_pv represents the driving pressure for blood flow, and so it is represented in Fig. 2A, left. P_pv increased linearly to a peak pressure of ~35 cmH₂O (R² = 0.96; P < 0.05), similar to the response observed in forward perfusion experiments, whereas P_do and P_pa (outflow pressure) were slightly increased at higher flows (Fig. 2B). Lung weight was stable up to a flow rate of 64 ml/min. Normotensive isolated perfused lungs therefore accommodate large increases in perfusion, in both forward and retrograde orientations, and they do not develop hydrostatic edema.

Fig. 2.

The normotensive pulmonary circulation accommodates large increases in retrograde perfusion. P_pa, P_pv, P_do, and lung weight were measured in response to increased perfusion rates. A: both baseline P_pv and P_pa increased ~5-fold in response to a nearly 10-fold increase in the perfusion rate. B: increased perfusion rates led to a 4-fold increase of P_do, whereas lung weight was increased only at the highest flow rate. Hatched lines represent the flow-pressure response in forward perfusion experiments. *P < 0.05, significantly different from baseline values; n = 6 animals.

Pulmonary arterial hypertension impairs blood flow through the pulmonary circulation.

Development of pulmonary arterial hypertension was confirmed in vivo using echocardiography. Figure 3A illustrates representative pulsed Doppler results obtained from five separate hypertensive animals, showing reductions in PAAT and PAAT/PAET (18, 20, 41). The Sugen 5416-, hypoxia-, and normoxia-exposed animals had a Fulton index of 0.73 ± 0.07, characteristic of severe pulmonary arterial hypertension. We noted that despite relatively little heterogeneity in right ventricular hypertrophy measured by the Fulton index, two populations of responders emerged in the forward flow-pressure response (Fig. 3B). Low responders had baseline P_pa near 50 cmH₂O, whereas high responders displayed baseline P_pa closer to 100 cmH₂O. Low and high responder P_pa increased linearly with advancing perfusion rates, although the slope of this response was greater in high than in low responders (high responder R² = 0.85 vs. low responder R² = 0.89, P < 0.05). The flow-pressure curves were left shifted in both low and high responders when compared with normotensive controls (P < 0.05), indicating the hypertensive circulation is noncompliant and cannot accommodate high perfusion rates. Despite this high P_pa, P_pv remained low over the entire range of perfusions, indicating the principal site of vascular resistance was in the arterial segment.

Fig. 3.

The pulmonary arterial hypertensive circulation does not accommodate high perfusion rates. A: pulsed Doppler echocardiography revealed decreased pulmonary arterial acceleration time (PAAT) and PAAT/pulmonary arterial ejection time (PAET), characteristic of severe experimental pulmonary arterial hypertension (18, 20, 41). B: P_pa, P_pv, P_do, and lung weight were measured in response to increased perfusion rates. Two populations of responders were identified based upon their flow-pressure response, including high and low responders. High responders achieved P_pa >250 cmH₂O, an effect that plateaued with 40 ml/min perfusion. Low responders achieved P_pa >200 cmH₂O, an effect that plateaued between 56 and 64 ml/min perfusion. Despite this exaggerated P_pa response to flow, P_pv remained within the normal range. C: P_do was elevated at baseline perfusion rates in both high and low responders, although the P_do was significantly greater in the high responder group. P_do exceeded 30 cmH₂O by 16 ml/min in high responders and by 48 ml/min in low responders. As P_do exceeded 30 cmH₂O in both groups lung weight increased, characteristic of hydrostatic edema. Hatched lines represent the flow-pressure response in forward perfusion experiments in normotensive lungs. *P < 0.05, significantly different from baseline values; n = 4 higher responder and 6 low responder animals.

P_do was elevated in both low and high responders, when compared with the normotensive P_do (Fig. 3C). As in the normotensive pulmonary circulation, P_do exceeding 30–35 cmH₂O was paralleled by an abrupt increase in lung weight, characteristic of hydrostatic pulmonary edema. Thus the noncompliant hypertensive pulmonary circulation develops hydrostatic edema with relatively modest increases in forward perfusion.

Pulmonary arterial hypertensive lungs do not support retrograde perfusion, even after maximal vasodilation.

Following heart and lung isolation, lungs were perfused in the forward direction for 5 min at 8 ml/min to establish baseline hemodynamic values, and then switched to retrograde flow (Fig. 4). During the retrograde flow maneuver, perfusate failed to return through the circulation to the reservoir (see Fig. 5, A and B, and Supplemental Movie S1; Supplemental Material for this article is available online at the Journal website). Within minutes of initiating retrograde perfusion, the lungs became grossly edematous. Continued perfusion at 8 ml/min, in the absence of lung expansion during ventilation, led to rapid dripping of perfusate off the lung surface due to fluid clearance through open lymphatics and/or through the bronchial circulation. Shortly thereafter, copious amounts of frothy edema were cleared through the trachea. We quantified the amount of fluid that returned to the reservoir through the circulation, that could be collected dripping off the lung, and that could be collected dripping from the trachea (Fig. 5B). The vast majority of perfusate was collected from the airways. We then acutely reversed flow again, so that flow occurred in the forward direction. Even though the lung was fluid filled and was not expanding during the ventilatory cycle, flow recirculated in the forward direction; the entire cardiac output could be collected upon its return through the circulation to the reservoir (data not shown). Therefore, lungs with pulmonary arterial hypertension can accommodate forward flow, but they cannot tolerate retrograde flow, even at low cardiac outputs.

Fig. 4.

Perfusion was instantaneously switched from forward to reverse flow. Stopcocks were arranged to immediately reverse flow from the forward to reverse direction through a bypass circuit, without stopping the perfusion pump, thus avoiding intermittent flow. Right: humidified lung chamber was been removed to more clearly show the lung in relation to the perfusion apparatus.

Fig. 5.

The pulmonary arterial hypertensive circulation does not tolerate retrograde perfusion. A: after a 5-min equilibration period, retrograde perfusion was established at 8 ml/min. P_pv was elevated during retrograde perfusion, whereas P_pa remained <10 cmH₂O. B: virtually none of the perfusate returned to the reservoir through the circulation. Most of the perfusate was collected from the airways and the lymphatics or bronchial circulation. C: to ensure capillary recruitment, lungs were perfused at 8 ml/min and then flow was sequentially increased to 24 ml/min before establishing retrograde perfusion. Although P_pv was increased by the higher flow rate, P_pa remained low. D: flow did not return to the reservoir through the circulation, and again, most of the cardiac output was collected from the airways and the lymphatics or bronchial circulation. Note the increase in the y-axis scale in D vs. B.

We wondered whether low flow rates contributed to inadequate vascular recruitment. To address this issue, forward flow was initiated at 8 ml/min and increased sequentially to 24 ml/min. Retrograde perfusion was then initiated at 24 ml/min. However, even with higher flow rates, the hypertensive lungs did not accommodate retroperfusion (Fig. 5, C and D). Thus impaired retrograde perfusion cannot be simply explained by poor microvascular recruitment.

We next tested whether vasodilation would improve retrograde perfusion. Lungs from pulmonary arterial hypertensive rats were initially perfused in the forward orientation to establish baseline hemodynamics, and then the circulation was maximally dilated using the combination of the Rho kinase inhibitor fasudil (10 µM) and the calcium channel blocker SKF-96365 (50 µM). Fasudil and SKF-96365 treatment reduced P_pa by 31% (Fig. 6A); the lowest pressure obtained in these lungs was 28.5 cmH₂O. After 20 min of forward perfusion with fasudil and SKF-96365, retrograde perfusion was initiated (Fig. 6B). Once again, perfusate return to the reservoir was impaired. We noted that the fulminant edema fluid collected from the trachea possessed blood, suggesting the increase in hydrostatic pressure injured fragile, or dysfunctional, endothelium in the hypertensive circulation (Fig. 6C). Most of the perfusate was collected from the trachea whereas a minor amount of perfusate was obtained from the combination of lymphatic or bronchial drainage and circulatory return (Fig. 6D). Thus vasodilation is not sufficient to rescue retrograde perfusion in pulmonary arterial hypertension.

Fig. 6.

Maximal P_pa dilation does not enable retrograde perfusion in pulmonary arterial hypertensive lungs. After a 5-min equilibration period, fasudil (10 µM) and SKF-96365 (50 µM) were recirculated for 20 min, and then retrograde perfusion was established. A: maximal vasodilation reduced forward flow P_pa by ~31%. B: P_pv during retrograde perfusion was ~10% lower than the corresponding P_pa during forward perfusion. P_pa during retrograde perfusion was <10 cmH₂O, respectively. C: immediately after retrograde perfusion was established, lung weight increased and edema fluid containing blood was collected from the airways and the lung surface. D: the majority of perfusate was collected as edema fluid that was being cleared through the airways. Minor fractions of inflow perfusate were collected from the lung cleared through lymphatic channels or the bronchial circulation and from the pulmonary circulation, respectively. *P < 0.05, significant difference.

The hypoxia-induced pulmonary hypertensive circulation supports retrograde perfusion.

We examined whether the circulation in animals with hypoxia-induced hypertension would support retrograde perfusion. These animals develop elevated P_pa with medial and adventitial remodeling that causes right ventricular hypertrophy, but they do not develop occlusive neointimal lesions. The Fulton index in these animals was 0.5 ± 0.03 (vs. a normal range of 0.25–0.30), characteristic of pulmonary hypertension. However, the severity of this remodeling was less pronounced than it was in the Sugen 5416-, hypoxia-, and normoxia-exposed animals.

Hypoxic hypertensive lungs were isolated and forward perfused for 5 min to establish baseline hemodynamic status, and then retrograde perfusion was initiated. With retrograde perfusion the P_pv (inflow pressure) approached 10 cmH₂O at a perfusion rate of 8 ml/min (Fig. 7A) and increased linearly with advancing flow rates. P_do and P_pa increased modestly. The lung remained isogravimetric until the flow rate reached 80 ml/min, consistent with evidence that chronic hypoxia remodels the microcirculation and strengthens the capillary endothelial barrier (Fig. 7B) (32, 37). Throughout the experimental protocol, perfusate was successfully recirculated, with no obstruction to retrograde perfusion. Thus hypoxic hypertension does not prevent retrograde perfusion.

Fig. 7.

Lungs with hypoxic hypertension accommodate retrograde perfusion despite high outflow pressures. P_pa, P_pv, P_do, and lung weight were measured in response to increased perfusion rates. A: P_pv pressure at baseline perfusion rates was ~10 cmH₂O and increased to 45 cmH₂O at 80 ml/min, while P_pa increased from 4 to 23 cmH₂O over the full range of perfusion rates. B: P_do remained below 30 cmH₂O throughout the experiment, and the lung remained isogravimetric until flow rate was 80 ml/min. Hatched lines represent the flow-pressure response in forward perfusion experiments in normotensive lungs. *P < 0.05, significantly different from baseline values; n = 5 animals.

The normotensive circulation allows for retrograde perfusion, even when outflow pressure is significantly elevated.

To mimic the high arterial resistance in pulmonary arterial hypertension, normotensive lungs were isolated and forward perfused at 8 ml/min to establish baseline hemodynamic parameters, flow was reversed, and then the outflow tract (pulmonary artery reservoir) was elevated above the heart to elicit an outflow pressure of 40 cmH₂O. This change in pressure was sensed throughout the circulation, as P_pv (inflow pressure) and P_pa (outflow pressure) were 40 cmH₂O (Fig. 8A). Within minutes of elevating the P_pa, the lung became grossly edematous. Unlike the fluid collected from the airways of the Sugen 5416-, hypoxia-, and normoxia-treated animals, however, this fluid did not contain blood (Fig. 8B). Thus elevated vascular pressure causes a hydrostatic edema resulting in loss of large volumes of the returning perfusate into the tissues and airways (Fig. 8C), but this increase in pressure does not prevent retrograde perfusion.

Fig. 8.

Normotensive lungs accommodate retrograde perfusion despite high outflow pressures. A: after retrograde perfusion was established, the outflow (pulmonary artery) reservoir was increased by 40 cmH₂O, resulting in an increase in both P_pa and P_pv. B: lungs rapidly became edematous, and clear edema fluid was collected from the airways. C: approximately half of the perfusate was collected from the airways and ~40% of the perfusate returned to the reservoir.

The 1- and 3-wk hypertensive lungs allow for retrograde perfusion.

We determined when the circulation becomes intolerant to retrograde perfusion in the Sugen 5416, hypoxia, and normoxia model. Pulmonary arterial hypertension progressively worsened over 1 (Sugen 5416 and 1 wk of hypoxia), 3 (Sugen 5416 and 3 wk of hypoxia), and 5 wk (Sugen 5416, 3 wk of hypoxia, and 2 wk of normoxia) of treatment. There was no difference in the Fulton index between 3- and 5-wk animals (P = NS) (Fig. 9A), although retrograde perfusion was tolerated at the 3-wk time point (Fig. 9B). Retrograde flow at the 1-wk time point was tolerated over a wide range of flows, but at the 3-wk time point, retrograde flow was only tolerated through 16 ml/min. Pulmonary arterial and venous resistances increased significantly between the 3- and 5-wk time points (Fig. 9, C and D, and Table 1). Occlusive lesions were not seen at 1 wk, were infrequent at 3 wk, and were common by 5 wk (Fig. 9, E–G).

Fig. 9.

Retrograde perfusion is observed 1 and 3 wk after Sugen 5416 and hypoxia treatment. A: the Fulton index demonstrates progressive right ventricular hypertrophy in response to Sugen 5416 and hypoxia exposure, which reaches a maximum 3 and 5 wk after treatment. RV, right ventricular; LV, left ventricular; S, septum. B: the pulmonary circulation accommodated retrograde flows 1 and 3 wk posttreatment. C and D: pulmonary arterial and venous resistances were calculated from (P_pa − P_do)/CO and (P_do − P_pv)/CO, respectively, where cardiac output (CO) is represented by a perfusion rate of 8 ml/min. The differences between arterial and venous resistances are plotted to represent the segment-specific contributions to pulmonary vascular resistance (PVR). *P < 0.05, significantly different from baseline values; n = 5–10 per group. C: the arterial (inflow) to venous (outflow) (a-v) resistance difference increased significantly by the 5-wk time point in forward flow experiments, illustrating the principal site of resistance is upstream of the capillary bed. D: the venous (inflow) to arterial (outflow) (v-a) resistance difference is negative during retrograde perfusion, illustrating a significant site of resistance downstream from the capillary bed. ND, not determined. E–G: representative histology illustrates progressive vascular lesion severity in 1 (E)-, 3 (F)-, and 5-wk (G) treatments. Arrowheads illustrate sites of vascular occlusion, and arrows indicate medial hypertrophy of arteries.

Table 1.

Pulmonary vascular resistance was significantly increased in both the arterial and venous segments of hypertensive lungs

	Forward Flow		Retrograde Flow
	Inflow (artery)	Outflow (vein)	Inflow (vein)	Outflow (artery)
Normotension	0.41 ± 0.05†	0.26 ± 0.06	0.10 ± 0.03†	0.31 ± 0.05
Pulmonary arterial hypertension
1 wk	0.92 ± 0.26†	0.26 ± 0.05	0.35 ± 0.11*	0.53 ± 0.06
3 wk	2.27 ± 0.21*†	0.37 ± 0.07	1.15 ± 0.40*	1.62 ± 0.19
5 wk	6.59 ± 0.88*†	1.65 ± 0.43*	ND	ND

Values are means ± SE with pulmonary vascular resistance (PVR) in cmH₂O·ml⁻¹·min⁻¹. PVRs are shown at baseline flow rates (e.g., 8 ml/min) during forward and retrograde flow. Arterial and venous resistances are increased in animals with pulmonary arterial hypertension when compared with normotensive controls. The outflow resistances are higher than the inflow resistances during retrograde perfusion, indicating high resistance downstream from the capillary bed. Note that animals treated for 5 wk could not be perfused in the retrograde orientation; therefore, the resistances from these animals were not determined (ND).

^*P < 0.05, significantly different from normotensive control values;

^†P < 0.05, significantly different from the corresponding outflow values; n = 5–10 per group.

Fig. 9—Continued.

DISCUSSION

Our studies support the idea that blood can be perfused equally well through the normotensive pulmonary circulation in both forward and retrograde orientations (9, 10). The normotensive pulmonary circulation accommodates significant increases in perfusion with relatively little increase in pressure, and it does so without developing hydrostatic edema. In contrast, in the Sugen 5416, hypoxia, and normoxia model, the noncompliant pulmonary arterial hypertensive circulation accommodates significantly less forward flow, is susceptible to hydrostatic edema, and possesses an absolute intolerance to retrograde perfusion. Such intolerance to retrograde perfusion is not observed in the early stages of vascular remodeling but becomes evident as the disease progresses. Mechanisms accounting for this observation remain poorly understood.

We documented the development of severe pulmonary arterial hypertension in our studies (3, 14, 47). Echocardiography showed hypertrophy of the right ventricle with apparent dilation and reduced performance, and the Fulton index in these animals exceeded 0.7. Increasing perfusion in the forward orientation in pulmonary arterial hypertensive lungs caused an abrupt elevation in P_pa that led to abnormally high P_do and hydrostatic edema. Despite uniformly hypertrophied right ventricles, we noted an unexplained heterogeneity in the pulmonary vascular pressure responses to increased perfusion in the lungs of these animals. P_pa increased to 250 cmH₂O in some animals. The high pressures documented here would not be tolerated in vivo; rather, they reflect extreme ranges of the noncompliant circulation in an experimental protocol not limited by the failing heart. Pulmonary arterioles represented the predominant site of resistance in all animals; however, venous resistance was also elevated. Increased P_do was closely paralleled by a susceptibility to pulmonary edema with hemorrhage at low cardiac outputs, consistent with hydrostatic causes of pulmonary edema (16). The vascular leak site responsible for pulmonary edema in this experimental setting is unknown. Arteries, capillaries, and veins may all be susceptible (24, 35). In addition, fluid could access intrapulmonary bronchopulmonary anastomoses that may expand in pulmonary arterial hypertension and be cleared through lymphatic channels (15, 33). These findings are compatible with an underlying endothelial permeability defect in the Sugen 5416, hypoxia, and normoxia experimental model, as previously documented (14, 47).

The increased vascular resistances reported here are similar to the human condition, where the principal elevation in vascular resistance is found in the arterial segment in pulmonary arterial hypertension (11). Kafi et al. (22) analyzed the P_pa decay curve after balloon occlusion, and reported elevated “capillary” pressures in patients with group 1 pulmonary arterial hypertension. They recommended this value reflected increased microvascular pressure in resistance arteries and arterioles, in vessels ≈100 µm in internal diameter, based on comparison of data using balloon occlusion with those using micropuncture techniques (17). However, Rol et al. (29) proposed that the venous segment contributes to increased vascular resistance and, furthermore, that neither vasoconstriction of, nor structural changes in, arterial resistance vessels are sufficient to explain increased total pulmonary vascular resistance in pulmonary arterial hypertension. Pulmonary venous resistance was increased in our studies, especially at the 5-wk pulmonary arterial hypertension time point. Factors responsible for increased pulmonary vascular resistance, and for the distribution of vascular resistances, in the hypertensive circulation are still in question.

In our studies, flow was first initiated in the forward direction, and then without suspending flow and/or pressure across the vascular circuit, retrograde perfusion was initiated. This maneuver led to an abrupt cessation of return perfusion through the hypertensive arteries, suggesting the outflow (e.g., arterial) resistance was too high. We questioned whether excessive vasoconstriction contributed to this impairment to retrograde perfusion. Fasudil and SKF96365 were used to elicit maximal vasodilation (25, 26). These vasodilators reduced P_pa ≈30%. Vasodilation improved retrograde perfusion, but it did not rescue the return flow. In addition, retrograde perfusion was tolerated in hypoxic hypertensive animals even though they display increased vascular tone. It therefore seems that excessive vasoconstriction contributes modestly to the high outflow resistance that impairs retrograde perfusion in the pulmonary arterial hypertensive animals.

Retrograde perfusion elicited rapid alveolar flooding in the hypertensive circulation. We wondered whether alveolar flooding could collapse arterioles and prevent retrograde perfusion. To test this possibility, the arterial pressure was increased by raising the outflow reservoir in normotensive lungs during the retrograde maneuver. Whereas elevating the outflow pressure increased capillary pressure and caused rapid alveolar flooding, the alveolar flooding did not prevent retrograde flow. We thought that perhaps alveolar flooding plus the hypercontractile state of arterioles in the hypertensive circulation could combine to prevent retrograde flow. We therefore increased the outflow pressure in the hypertensive circulation during the retrograde perfusion, in an attempt to prevent an abrupt pressure drop from the arterioles to the arteries (data not shown). However, this maneuver did not rescue retrograde perfusion. These results argue against the idea that high alveolar pressure collapses arterioles to form the critical obstruction to flow.

Rarefaction may contribute to increased pulmonary vascular resistance, although the significance of rarefaction to disease progression has been a point of controversy (6). We considered whether arteriolar dropout could account for impaired retrograde perfusion. It seems unlikely that microvessel degeneration explains an inability to accommodate retrograde blood flow if the remaining blood vessel lumens are patent. Moreover, retrograde perfusion was largely unimpeded in hypoxic hypertensive lungs. Thus, if rarefaction contributes to impaired retrograde perfusion in severe pulmonary arterial hypertension, it is likely to be a modest effect that potentiates other primary causes.

Severe pulmonary arterial hypertension in humans, and in this animal model, is characterized by development of occlusive neointimal lesions. The relevance of neointimal occlusive lesions to pulmonary arterial resistance, and to progression of pulmonary arterial hypertension in general, continues to be a matter of debate. Tuder et al. (39) estimated that either 224,000 lesions within arterioles 200 µm in diameter, or more than 2,000,000 lesions within arterioles 40 µm in diameter, would be required to increase P_pa in the human, suggesting they are not likely to contribute to increased pulmonary vascular resistance. Similarly, Burrowes et al. (5) concluded that up to 65% of the pulmonary circulation would have to be occluded to cause an acute increase in P_pa. Vascular remodeling in group 1 pulmonary hypertension is highly heterogeneous, and it is thought that only ≈30% of the resistance vessels have reduced luminal diameters (29). However, in preliminary studies using the Sugen 5416, hypoxia, and normoxia model, three-dimensional analysis of the remodeled pulmonary hypertensive circulation revealed that sections along a contiguous vascular segment are only intermittently occluded (13). Thus the prevalence of these occlusive lesions may be more widespread than currently realized. Whole lung three-dimensional pathology will be required to fully understand the incidence, prevalence, and severity of occlusive neointimal lesions. Time course studies revealed that retrograde flow was supported until the point at which occlusive neointimal lesions became more widespread, reflecting advancing vascular dysfunction. Future studies will be required to determine whether occlusive neointimal lesions are a major contributor to impaired retrograde perfusion.

In summary, we report two novel observations in these studies. First, increased perfusion is sufficient to disrupt the endothelial cell barrier in pulmonary arterial hypertension, consistent with recent reports of a hyperpermeability defect. Even modest increases in perfusion rate lead to pulmonary edema with hemorrhage. This permeability defect is not limited to the arterial segment and likely includes both capillary and venous segments. Second, as the severity of pulmonary arterial hypertension progresses, the circulation acquires an absolute intolerance to retrograde perfusion. The mechanism(s) for such an intolerance to retrograde flow remains an active area of study. Retrograde perfusion offers novel insight into the functional status of the remodeled hypertensive pulmonary circulation.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-66299 and HL-60024 (to T. Stevens).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.S. conceived and designed research; C.Z., E.S.C., and L.B. performed experiments; C.Z., E.S.C., L.B., and T.S. analyzed data; L.B., I.F.M., and T.S. interpreted results of experiments; C.Z., E.S.C., and L.B. prepared figures; T.S. drafted manuscript; C.Z., L.B., I.F.M., and T.S. edited and revised manuscript; T.S. approved final version of manuscript.