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J Thorac Cardiovasc Surg 2004;127:1009-1017
© 2004 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Laboratoire de Chirurgie Expérimentale, Hôpital Marie Lannelongue, Université Paris Sud, Le Plessis Robinson, France
b Department of Pathology, McGill University, Montreal, Quebec, Canada
Read at the Eighty-third Annual Meeting of The American Association for Thoracic Surgery, Boston, Mass, May 4-7, 2003.
Received for publication April 30, 2003; revisions received June 17, 2003; accepted for publication July 22, 2003.
* Address for reprints: Elie Fadel, MD, Hôpital Marie Lannelongue, 133 Avenue de la Résistance, 92250, Le Plessis Robinson, France
fadel{at}ccml.com
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Abstract |
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METHODS: The ligated left pulmonary artery was reimplanted into the pulmonary arterial trunk. Pulmonary artery blood flow and pressure were measured 2 days and 5 weeks after reperfusion. Pulmonary artery smooth muscle thickness, endothelium-dependent relaxation, and left lung endothelial nitric oxide synthase activity and expression were assessed 5 weeks after ligation (n = 10) and 5 weeks after reperfusion (n = 10), and compared with a sham group (n = 10). Patency of the anastomoses and systemic blood supply to the lung were assessed by pulmonary angiography and nonselective thoracic aortography, respectively.
RESULTS: Angiography showed that pulmonary artery anastomoses were patent in all animals. Five weeks after reperfusion, left pulmonary blood flows were similar to those in the sham animals, and systemic blood supply to the left lung decreased. Left pulmonary vascular resistance decreased by 50% at 5 weeks after reperfusion compared with 2 days after reperfusion (P = .0009). Medial muscle thickness of the left pulmonary artery greater than 600 µm increased 5 weeks after ligation and regressed to sham values 5 weeks after reperfusion (P = .001). Endothelium-dependent relaxation was only partially restored 5 weeks after reperfusion, whereas left lung endothelial nitric oxide synthase expressions and activities returned to sham values.
CONCLUSIONS: This study shows that postobstructive pulmonary vasculopathy induced by ligation of the pulmonary artery for 5 weeks regresses after reperfusion, accounting for the progressive improvement in hemodynamics after thromboendarterectomy.
The alterations described distal to the obstruction in chronic pulmonary thromboembolic hypertension have been reproduced in animal models by unilateral ligation of 1 pulmonary artery (PA). This has been termed "postobstructive pulmonary vasculopathy" and includes the development of precapillary bronchial-to-pulmonary vascular anastomoses, PA remodeling, and abnormal PA vasoreactivity with dysfunction of the pulmonary endothelium.5,6 We hypothesized that the progressive decrease in PVR that follows the initial dramatic improvement after pulmonary thromboendarterectomy is related to regression of the postobstructive pulmonary vasculopathic abnormalities. To test this hypothesis, we assessed pulmonary hemodynamics, PA vasoreactivity, endothelial nitric oxide synthase (eNOS) function and expression, and morphologic changes in the PA bed of the left lung 5 weeks after ligation of the left PA and 5 weeks after in situ revascularization.
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Materials and methods |
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Study groups
The piglets were randomly allocated to 3 groups (n = 10 in each). The first group included animals that were studied 5 weeks after ligation of the left PA (ligated group). The second group included animals that were studied 5 weeks after ligation of the left PA followed by reanastomosis after 5 weeks (revascularized group). The third group included animals that were studied 5 weeks after dissection of the left PA without ligation (sham group).
Surgical procedures
Left PA ligation was performed as previously described.7 Briefly, anesthesia was induced with intramuscular ketamine (100 mg/kg) and maintained with intravenous pentobarbital (10 mg/kg bolus, followed by a continuous infusion of 0.1 mg · kg · min). The animals were paralyzed with pancuronium (0.3 mg/kg). After endotracheal intubation, intermittent positive-pressure ventilation was provided (MMS RET 107 ventilator, MMS, Pau, France) at a tidal volume of 15 mL/kg, a respiratory rate of 18 cycles/min, and an FIO2 of 0.5. Body temperature was kept constant at 37°C. A midline sternotomy was performed under sterile conditions, the pericardium was opened, and the intrapericardial left PA was dissected and ligated using nonabsorbable suture. The chest was closed, and the animals were allowed to recover.
Reanastomosis of the chronically ligated left PA was performed as described by Alley and colleagues.8 Briefly, a left posterolateral thoracotomy was performed through the fourth intercostal space. To avoid damaging the new bronchial collaterals, particularly prominent over the pericardium and near the pulmonary hilum, dissection was restricted to the first 2 cm of the left PA beyond the point of ligation and to the adjacent PA trunk. After systemic heparinization, the origin of the ligated left PA was excised, the left artery was totally clamped, and the PA trunk was partially clamped. The left PA was reimplanted end-to-side into the PA trunk using a 7-0 polypropylene running suture. Chest tube drainage was established, and the thoracotomy was closed. The animal was allowed to recover, and the chest tube was removed 24 hours later. To avoid postoperative occlusion of the anastomosis, the animals were anticoagulated with fractionated heparin until harvesting of the lungs.
Hemodynamic measurements and harvesting of the lungs
The pigs were anesthetized as previously described. A midline sternotomy was performed. Left and right PA blood flows were measured using a flow probe (Transonic System Inc, Ithaca, NY); right and left PA pressures (PAPs) were measured by direct puncture 1 cm downstream from the anastomotic site. The PVR of the right and left lungs was calculated as the mean PAP of each lung divided by the respective PA flows. Pulmonary hemodynamics were measured 2 days and 5 weeks after revascularization.
Nonselective thoracic aortography was performed 5 weeks after ligation of the left PA and 5 weeks after in situ revascularization to examine the bronchial-to-pulmonary anastomoses. After heparinization, the animals were exsanguinated, and the lungs were rapidly removed from the chest. Patency of the anastomosis was assessed by visual inspection and pulmonary angiography.
Light microscopy and morphometry
A total of 16 animals were used for this part of the study (8 animals in each of the ligated and the revascularized groups). At the time of euthanasia, the right and left lower lobes were fixed by instillation of 4% paraformaldehyde in phosphate-buffered saline through the bronchi at a pressure of 25 cm H2O until distended. The bronchi were ligated, and the lungs were left to fix for 12 to 24 hours. The lungs were then cut into 1 cm-thick slices, and 10 to 15 random sections were taken from the midsagittal slice. These were processed using routine histologic techniques and embedded in paraffin; from the blocks, 5 µm-thick sections were cut and stained with hematoxylin-eosin or Verhoeff elastic stain. The microscopic slides were grouped by lobe, randomized, and coded, and the sections were examined without knowledge of the experimental group.
A calibrated ocular micrometer on a Leitz optical microscope (Wetzlar, Germany) was used for the morphometric measurements, as previously described.5,9 The slides were systematically scanned for the measurements on the PAs, which were assessed and categorized on the basis of their associations with airways. In each lobe, we intended to find 25 arteries accompanying respiratory bronchioles, 25 arteries accompanying bronchioles, and 15 arteries accompanying bronchi. Each artery was also classified as muscular (>75% of circumference with media), partially muscular (25%-75% media), or nonmuscular (<25% media), and divided into size categories of less than 200 µm, 201 to 400 µm, and greater than 600 µm. We measured the outer diameter of these arteries at the external border of the media and the medial thickness (MT) of all muscular PAs with circular cross-sections or a long-to-short ratio less than 3:1. We studied 1636 PAs. From these measured data, we first compared the MT between groups according to the class of arterial diameter. Second, we assessed the peripheral muscularization in these PAs by plotting the percentage of each type of vessel in each category of airway and the relationship between arterial diameter and type.
Isolated PA ring studies
Isolated PA ring studies were performed as previously described.6 Briefly, at the end of each experiment, intra-PA segments were dissected out and placed in warm Krebs-Henseleit buffer composed of NaCl, 118.3 mmol/L; KCL, 4.7 mmol/L; CaCl2, 1.5 mmol/L; NaHCO3, 25 mmol/L; MgSO4, 1.1 mmol/L; KH2PO4, 1.2 mmol/L; and glucose, 5.6 mmol/L. Isolated PAs were cleaned and cut into rings 3 to 4 mm in length (1-2 mm outer diameter); 3 to 4 rings were obtained from each animal. The rings were then mounted on stainless steel hooks, suspended in 10-mL tissue baths, and connected to force-displacement transducers (LB-5, Showa-sokki, Tokyo, Japan) to measure force change using a chart recorder (LR 4210, Yokogawa, Tokyo, Japan). The baths were filled with 10 mL of Krebs-Henseleit buffer and aerated at 37°C with a mixture of 95% O2 and 5% CO2. PA rings were initially stretched to produce a preload of 1g of force and allowed to equilibrate for 60 to 90 minutes. Pilot studies involving length tension analysis of PA rings showed that this amount of preload provided an optimal resting tension, which was also similar to the optimal resting tension (1.060 ± 0.040g) reported by Liu and colleagues10 in piglet PA rings. During this period, the Krebs-Henseleit buffer in the tissue baths was changed every 10 minutes. After incubation with indomethacin (10–5 mol/L) for 60 minutes, a concentration-response curve to the thromboxane analog U46619 (10–9 mol/L-10–6 mol/L) was constructed. The rings were then washed, and the developed force was allowed to return to baseline. Next, the rings were precontracted with U46619 to generate approximately 1g of developed force. Once a stable contraction was obtained, cumulative doses of acetylcholine (10–9-10–4 mol/L), endothelium-dependent vasodilator calcium ionophore A23187 (10–10 mol/L-3.10–7 mol/L), or endothelium-independent vasodilator sodium nitroprusside (10–9 mol/L-10–4 mol/L) were added to the bath, and changes in endothelium-dependent relaxation were assessed. These rings were washed again and allowed to equilibrate to baseline levels.
In addition to the change in force, responses were assessed on the basis of determination of the concentration that produced 50% of the maximal response (EC50) extrapolated from a plot of log concentration versus percentage of maximal response. The contractile responses to U46619 were expressed in absolute values (milligrams), and the maximum relaxation to acetylcholine, A23187, and sodium nitroprusside was expressed as the percentage of the U46619-induced precontraction (0% indicated no relaxation and 100% indicated relaxation equal in magnitude to the amount of precontraction).
Drugs
A23187 (calcium ionophore), sodium nitroprusside, acetylcholine hydrochloride, and indomethacin were purchased from Sigma Chemical Company (St Louis, Mo), and U46619 was provided by Upjohn (Kalamazoo, Mich).
Measurement of eNOS activity
Calcium-dependent and -independent NOS activities were determined in left lung homogenates from the 3 groups as previously described.6 The lungs were frozen in liquid nitrogen immediately after removal. Tissue was homogenized on ice, using an Ultraturax blender (Kinematica, Lucerne, Switzerland), in 4 volumes of buffer containing 50 mmol/L Tris-HCL (pH 7.4), 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1 mmol/L ethyleneglycoltetraacetic acid (EGTA), 0.1% 2-mercaptoethanol, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, and 1 mmol/L phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 100,000g for 1 hour at 5°C. To remove soluble proteins, the pellet was resuspended in homogenization buffer containing 1 mol/L KCl and allowed to stand on ice for 5 minutes before centrifugation at 100,000g for 30 minutes at 5°C. The supernatant fraction was discarded, and the pellet (membrane fraction) was resuspended in homogenization buffer containing the detergent CHAPS (20 mmol/L), 1 mol/L KCl, and glycerol (10% vol/vol) and then allowed to stand on ice for 30 minutes before centrifugation at 100,000g for 30 minutes at 5°C. Activity of eNOS in the supernatant (membrane fraction) was determined by measuring the calcium-dependent conversion of [3H]L-arginine to [3H]L-citrulline in the reaction mixture. The enzyme extract (25 µL) was added to 200 µL of the reaction mixture containing 50 mmol/L Tris-HCl (pH 7.4), 10 µmol/L tetrahydrobiopterin, 1 mmol/L dithiothreitol, 10 µg/mL calmodulin, 4 µmol/L flavin adenine dinucleotide, 4 µmol/L flavin mononucleotide, 2 µmol/L L-arginine, 1 Kcpm/µL L-[3H] arginine, and 1 mmol/L nicotinamide adenine dinucleotide, with or without 1 mmol/L CaCl2. After 40 minutes of incubation at 37°C, the reaction was stopped with 2 mL of a solution containing 20 mmol/L Na acetate, pH 5.5, 1 mmol/L L-citrulline, 2 mmol/L EDTA, and 0.2 mmol/L EGTA. The mixture was applied to a 1-mL Dowex AG 50WX8 column (Dow Chemical Company, Roissy, France), and [3H]L-citrulline was eluted with 2 mL of distilled water. The radioactivity in the eluate was measured using liquid scintillation spectroscopy, and the concentration of protein in the enzyme extract was determined according to the method of Lowry.10a
Lung eNOS protein
Lung eNOS protein levels were determined as previously described.6 Tissue was homogenized on ice using a Ultraturax homogenizer (Kinematica) in 4 volumes of buffer containing 50 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 0.1% 2-mercaptoethanol, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 1 mmol/L phenylmethylsulfonyl fluoride, and 20 mmol/L CHAPS and allowed to stand on ice for 30 minutes before centrifugation at 3000g for 10 minutes at 5°C.
The supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins in the gel were transferred to a nitrocellulose membrane by electroblotting in a transblot Bio-Rad transfer apparatus (Bio-Rad Laboratories, Hercules, Calif) for 12 hours at 4°C. Before the transfer, the gels, Whatman filter paper, and nitrocellulose membrane were soaked in electroblotting buffer (25 mmol/L Tris-HCl; 193 mmol/L glycine; 20% methanol, pH 8.0) for 15 minutes. After transfer, the membrane was blocked using 1X TBST (0.15 mol/L NaCl; 10 mmol/L Tris-HCl, pH 8.0; 0.05% Tween-20; and 5% bovine serum albumin) for 1 hour at room temperature. The eNOS protein was detected by incubating the membrane overnight at 4°C with mouse polyclonal anti-eNOS (Interchim; Asnières, France) diluted 1:1000. The membrane was washed 3 times in 1X TBST. Specific protein was detected using a horseradish peroxidase-conjugated secondary antibody and ECL reagents (Amersham, Arlington Heights, Ill). eNOS immunoreactivity was quantified using a semi-automated image analysis device (NIH image 1.52; Bethesda, Md) that quantifies both the area and the intensity of immunoreactive bands using a ScanJet II scanner with DeskScan II (Hewlett- Packard, Palo Alto, Calif) software. Results are reported in arbitrary units.
Statistical analysis
All results are reported as means ± SEM. One-way analysis of variance was performed, followed by Fisher's test for between-group comparisons. For the morphometric data, MT measurements were determined by analysis of variance and Tukey's Honestly Significant Difference multiple comparison test; changes in peripheral muscularization were determined by the 
2 test. Correlations between eNOS activity and eNOS protein levels were performed using simple linear regression. All statistical analyses were performed using Statview IV (Abacus Concept, Berkeley, Calif), Systat version 10.2 (Systat Software Inc, Richmond, Calif), or, for the 2 test, Excel 2000 (Microsoft Corporation, Redmond, Wash).
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Results |
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Total pulmonary blood flows and right PVR were similar in the sham, ligated, and revascularized groups. Left mean PAP values were not obtained in the ligated group, and they were not significantly different between the revascularized (14.4 ± 1.2 mm Hg) and sham (12.3 ± 0.9 mm Hg) groups. In the revascularized animals, although there was no difference in left mean PAP values, the left PVR decreased from 21.7 ± 1.5 mm Hg · L · min 2 days after reperfusion to 12.5 ± 0.9 mm Hg · L · min (P = .0009) 5 weeks after reperfusion. The values of left PVR were lower in sham-operated animals (5.7 ± 0.5 mm Hg · L · min) than in animals studied 2 days (P = .0001) or 5 weeks (P = .001) after reperfusion.
Light microscopy and morphometry
Qualitatively, the vessels, airways, and parenchyma of the contralateral right lobes of both the ligated and revascularized groups were unremarkable except for a few foci of chronic inflammation. The muscular PAs had a normally thin media sandwiched between internal and external laminae (Figure 1, A). The bronchial vessels were small and normally distributed around airways, particularly bronchi.
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The morphometric data corroborated the qualitative descriptive findings. The significant findings for the MT plotted according to arterial diameter (Figure 2) occurred in the PAs greater than 600 µm in diameter. The arteries in the ligated group showed a significantly thicker media than those in the contralateral control right lungs of both the ligated and revascularized groups (P < .05-.01); they were also thicker than those of the revascularized left lobes (P < .05), consistent with regression of the medial thickening in this group.
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Isolated left PA ring study
Maximal contraction to U46619 was higher in the sham group than in the revascularized and ligated groups (5729 ± 245 mg vs 2514 ± 422 mg, P = .001 and 5729 ± 245 mg vs 1853 ± 431 mg, P = .0001, respectively). The U46619 EC50 was similar in the 3 groups.
The relaxation response to sodium nitroprusside was not affected by PA ligation or reperfusion. Maximal relaxation in response to acetylcholine (Figure 3, A) was lower in the ligated group than in the revascularized (P = .04) and sham (P = .007) groups, and higher in the sham group than in the revascularized group (P = .05). No differences in the acetylcholine EC50 were seen between the 3 groups.
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eNOS activity
The calcium-dependent NOS activity in the left lungs of the ligated group was lower than in the sham and revascularized groups, and there were no differences between the sham and revascularized groups (Figure 4, A). Calcium-independent NOS activity of the left lung was similar in the 3 groups.
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Discussion |
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Chronic thromboembolic obstruction of the PAs in humans can be reproduced in experimental animals by chronic unilateral ligation of a PA. Our previous ex vivo studies showed that PVR distal to the site of ligation increased in dogs5 and piglets6,7 after long-term lung ischemia. To extend these findings to in vivo conditions, we developed an animal model in which the left lung was reperfused by reimplantation of the left PA end-to-side into the PA trunk after a period of chronic left PA ligation. At 2 days after reperfusion of the previously ligated left lung, we found a 4-fold elevation in the PVR compared with normal. This increase was not explained by a persistent obstruction of the left PA because the reanastomosed PAs were patent at the end of the procedure, as confirmed by angiography and subsequently by direct visual inspection; neither was this increase in PVR caused by ischemia-reperfusion injury of the left lung because it was markedly attenuated in this model of chronic lung ischemia compared with acute ischemia.7 Rather, we believe that this increase in PVR was related to the functional and structural alterations that develop distal to the obstruction of the distal vascular bed. As reported in our previous studies in piglets, dogs, and rats,5-7,11,12 postobstructive pulmonary vasculopathy is characterized by (1) a marked increase in collateral bronchial blood flow through bronchial to pulmonary precapillary anastomoses; (2) a prominent increase in pulmonary vasoreactivity associated with a decrease in eNOS activity, eNOS protein expression, and eNOS-dependent relaxation of the left PAs; and (3) structural changes that include proliferation of new bronchial vessels and an increase in PA MT. Similar functional and morphologic vascular changes have been described in the lungs of patients with chronic thromboembolic obstruction of the PAs.13 The mechanisms responsible for this pulmonary postobstructive vasculopathy remain unclear. We suspect that ischemic injury to the distal pulmonary parenchyma initiates a complex sequence of events including endothelial cell damage, local release of inflammatory mediators, and angiogenic factors that induce remodeling of the PAs, bronchial artery proliferation, and endothelial dysfunction.
It was unknown until now whether the alterations characteristic of postobstructive pulmonary vasculopathy are reversible after reperfusion of the chronically ischemic lung. Our animal model of in vivo reperfusion offers a unique opportunity to test this hypothesis. Indeed, we observed a marked regression of both the functional and morphologic abnormalities of the left lung after 5 weeks of reperfusion, specifically, the bronchial-to-pulmonary anastomoses and the normal PA remodeling. In addition, the activity and expression of the left pulmonary eNOS also improved in parallel toward normal values; in contrast, recovery of pulmonary vasoreactivity was incomplete in the reperfused lung. The sum total of these findings might account for the reduction in left pulmonary PVR to values intermediate between those of ischemic and normal lungs.
Our study has important implications. Progressive long-term improvements in PVR have been observed in patients with chronic thromboembolic pulmonary hypertension after reestablishment of the pulmonary circulation in the previously obstructed vascular bed. Because our model adequately reproduces the morphologic and functional changes in vasculature seen in humans in the postobstructive pulmonary vascular bed, we believe that long-term improvement in PVR after pulmonary thromboendarterectomy similarly mirrors the regression of this vasculopathy. Moreover, in patients with the most severe forms of postembolic pulmonary hypertension, thromboendarterectomy may fail to lower PVR because of an extensive postobstructive pulmonary vasculopathy, thereby increasing the risk of postoperative morbidity and mortality.14 In these patients, a recent study indicates that chronic prostacyclin administration before thromboendarterectomy decreases postoperative PVR.15 This treatment may be effective not only by dilating the pulmonary vasculature but also by inhibiting vascular growth and remodeling.
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Discussion |
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Dr Fadel. In fact, our aim was to study the postobstructive vascular bed and not the remaining perfused PA bed. We observed a hypertrophied systemic blood supply to the lung, and one of us, Dr Rene Michel, has already shown that the PVR was increased in the postobstructive bed. We believe that our animal model reproduces the pathologic changes observed in human postembolic pulmonary vasculopathy. Local release of proinflammatory mediators and angiogenic factors is likely because we observed an increased production of vascular endothelial growth factor in the acute ischemic lung (unpublished data; Fadel E, MD, 2000).
Dr Frank L. Hanley (Stanford, Calif). Dr Wechsler's comments notwithstanding, these observations may have more application to many congenital problems in which we see iatrogenic or non-iatrogenic occluded PAs that may need reconstruction after long periods of complete occlusion.
In regard to the vascular resistance decrease, there could be 2 factors involved. When these arteries are occluded for long periods of time, the conduit component of the artery becomes hypoplastic because there's no flow in it. If you reperfuse the artery acutely with reanastomosis, those arteries don't immediately achieve a normal diameter, so there is going to be significant resistance along the conduit portion before you get to the microvasculature. Do you know whether your decrease in resistance is a result of gradual reestablishment of normal diameter of the conduit component or whether there are real changes in the small arterioles that lead to the decrease?
Dr Fadel. This is a very interesting question. We agree that the large PAs beyond the ligation site are hypoplastic, but we didn't find any difference in diameter of the small PAs between the control, ligated, or reperfused groups. The distal PA bed seems somewhat preserved and reperfused by the bronchial circulation. However, PVR mainly depends on the distal vascular bed. So, we believe that the decrease of PVR, after the decrease observed postoperatively, is related to changes that occurred in the microvascular system.
Dr Randall B. Griepp (New York, NY). Did any of these animals have reperfusion pulmonary edema? And if they did, can you say anything about how much media, how much musculature in the media, was left in those animals that did have edema?
Dr Fadel. In this study, the lung was studied 5 weeks after reperfusion, so we did not find any pulmonary edema in the reperfused lung. In previous studies, we found a pulmonary edema when the lung was studied immediately after reperfusion of the ligated PA. We also found the same edema when the lung was reperfused using an endarterectomy through the media of a previously obstructed PA by an adherent thrombus. We don't believe that the endarterectomy of the proximal PA bed could be responsible for the pulmonary edema. Indeed, it seems a real reperfusion edema.
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