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(Circulation Research. 1996;79:773-783.)
© 1996 American Heart Association, Inc.

Articles

cAMP-Mediated Vascular Protection in an Orthotopic Rat Lung Transplant Model

Insights Into the Mechanism of Action of Prostaglandin E₁ to Improve Lung Preservation

Yoshifumi Naka, Dilip K. Roy, Hui Liao, Nepal C. Chowdhury, Robert E. Michler, Mehmet C. Oz, David J. Pinsky

the Departments of Physiology (Y.N., H.L.), Surgery (D.K.R., N.C.C., R.E.M., M.C.O.), and Medicine (D.J.P.), Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to David J. Pinsky, MD, Columbia University, College of Physicians and Surgeons, Department of Medicine, PH 10-Stem, 630 W 168th St, New York, NY 10032. E-mail djp5@columbia.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prostaglandin E₁ (PGE₁) is often added to the donor pulmonary flush solution to enhance clinical lung preservation for transplantation. Although PGE₁ is thought to act as a pulmonary vasodilator during the harvest period, the precise mechanism(s) of action whereby PGE₁ enhances lung preservation is unknown. Because cAMP levels decline in endothelial and vascular smooth muscle cells exposed to hypoxia, we hypothesized that a PGE₁-mediated increase in cAMP levels within the preserved lungs might improve pulmonary vascular homeostasis following lung transplantation. Rat lungs demonstrated a time-dependent decline in cAMP levels during hypothermic storage, with cAMP levels significantly increased by PGE₁ supplementation (2-fold by 6 hours, P<.0005). To test whether augmenting cAMP levels may enhance lung preservation, experiments were performed using an orthotopic rat left lung transplant model. Compared with controls, supplementing the preservation solution with the membrane-permeable cAMP analogue dibutyryl-cAMP resulted in dose-dependent preservation enhancement, marked by reduced pulmonary vascular resistance (6.0-fold, P<.01), improved arterial oxygenation (3.0-fold, P<.01), reduced graft neutrophil infiltration (1.5-fold, P<.05), and improved recipient survival (7.0-fold, P<.005). Similar preservation enhancement was observed with another cAMP analogue (8-bromo-cAMP) or the phosphodiesterase inhibitor indolidan. Stimulating the cAMP second messenger system by PGE₁ supplementation resulted in marked hemodynamic benefits and improved recipient survival, in parallel with reduced graft neutrophil infiltration, vascular permeability, and platelet deposition. These beneficial effects of PGE₁ were abrogated by simultaneous administration of the cAMP-dependent protein kinase antagonist Rp-cAMPS. Although an arterial vasodilator (minoxidil) resulted in significant pulmonary vasodilation during harvest, it lacked other nonvasodilating effects of PGE₁ and resulted in poor preservation. These data show that harvest vasodilation by itself is insufficient to enhance lung preservation and that PGE₁ enhances lung preservation by stimulating the cAMP-dependent protein kinase and promoting nonvasodilatory mechanisms of pulmonary protection.

Key Words: cAMP • prostaglandin E₁ • lung transplantation • protein kinase A • blood vessels

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detaching a richly vascularized organ such as the lungs from its native blood supply during transport from donor to recipient causes an obligate period of ischemia, which often results in vascular dysfunction within the newly reperfused graft. After prolonged preservation and reperfusion, neutrophils and platelets are recruited,¹ and reactive oxygen species are generated in abundance, resulting in the loss of the endogenous pulmonary vasodilator NO as well as endogenous cGMP,² an important effector arm of the NO pathway.³ Because cGMP has similar vascular effects as cAMP,^{4 5} we hypothesized that a loss of endogenous cAMP might contribute to preservation-related mechanisms of graft vascular dysfunction. This hypothesis also follows from in vitro/ex vivo experiments demonstrating that exposure of endothelial cells to hypoxia/ischemia or to oxidants results in decreased cAMP levels and changes in endothelial properties that are abrogated by cAMP analogue supplementation or PDE inhibition.^{4 6 7 8 9 10} cAMP levels similarly fall in vascular smooth muscle cells exposed to hypoxia because of increased PDE activity.¹¹

These observations led us to investigate whether cAMP levels decline in preserved lungs and whether augmenting the cAMP second messenger pathway might restore normal vascular properties after lung transplantation. To test these hypotheses, experiments were performed using a recently described model of lung transplantation¹² in which graft hemodynamics and recipient survival depend entirely on the transplanted lung. To help establish the relevance of the cAMP pathway to clinical lung transplantation, we investigated the mechanism whereby prostaglandin supplementation improves pulmonary preservation, a mechanism that is currently unknown but has been suggested to be due to prostaglandin's pulmonary vasodilating actions during the harvest period.^{13 14 15 16 17} For these experiments, we tested whether PGE₁ may be acting on the graft vasculature by raising endogenous cAMP levels and stimulating the cAMP-dependent protein kinase.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials Used to Prepare Preservation Solutions
For all experiments, the base preservation solution consisted of modified EC solution obtained from Baxter Healthcare (mEq/L: Na⁺ 10, K⁺ 115, Cl^- 15, HPO₄^2- 85, H₂PO₄^- 15, and HCO₃^- 10), modified by adding 10 mL of 10% magnesium sulfate and 50 mL of 50% glucose solution. For certain experiments, EC solution was supplemented with one or more of the following: the membrane-permeable nonhydrolyzable cAMP analogue db-cAMP¹⁸ (0 to 2 mmol/L as indicated, Aldrich), 8-Br-cAMP (0.1 mmol/L, Sigma Chemical Co), LY195115 (indolidan, a type III phosphodiesterase inhibitor,¹⁹ 10 µmol/L, Lilly Research Laboratories), PGE₁ (Prostine VR Pediatrics, 10 µg/mL, Upjohn), a combination of PGE₁ (10 µg/mL) and Rp-cAMPS (a selective inhibitor of the cAMP-dependent protein kinase,²⁰ 0.1 mmol/L, BioLog Life Science Institute), sodium butyrate (butyrate, 2 mmol/L, Sigma), 8-Br-Ad (0.1 mmol/L, Sigma), or minoxidil (a direct-acting vasodilator,²¹ 0.02 µg/mL, Sigma). For experiments in which PGE₁ was incorporated into the preservation solution, a 20 µg/kg intravenous bolus of PGE₁ was administered to donors immediately before lung harvest in addition to adding 10 µg/mL to the preservation/flush solution (this simulates the use of PGE₁ in clinical lung transplantation at our center). To address the effects of bolus intravenous PGE₁ alone on lung preservation, separate experiments were performed in which PGE₁ was given as a bolus intravenous injection (20 µg/kg) but not incorporated into the preservation/flush solution.

Lung Harvest, Preservation, and Transplantation
Inbred male Lewis rats (250 to 300 g) were used for all experiments according to a protocol approved by the Institutional Animal Care and Use Committee at Columbia University, in accordance with AAALAC guidelines. Although for lung transplant experiments, donor rats were not instrumented for hemodynamic measurements (to limit excessive donor manipulation), representative PVRs were measured in a dedicated group of animals both at baseline after anesthesia and again 30 minutes after ligation of the right PA. Lung transplant experiments were performed in the following manner: Donor rats were given 500 U heparin intravenously, and the PA was flushed with a 30-mL volume of 4°C preservation solution at a constant pressure of 20 mm Hg. When lungs are preserved in this manner, most of the infused flush solution comes out of a LA vent created in the lung donor as well as out of the pulmonary veins after transection. Using this technique, measurement of residual fluid within the lungs (with a soluble radiotracer) demonstrated that 0.4±0.2% of the infused flush solution remained as a residual within the lungs after flushing and lung harvest. The time required to deliver the 30-mL volume of preservation solution at constant infusion pressure (20 mm Hg) was recorded as an index of PVR during harvest.¹ We chose to infuse a 30-mL volume of preservation solution at 20 mm Hg because this is how lung preservation solutions are delivered in clinical practice in human lung transplantation¹⁴ and because this volume resulted in excellent blanching of the lungs, providing visual confirmation of adequate delivery.

The left lung was then harvested, a cuff was placed on each vascular stump, a cylinder was inserted into the bronchus, and the lung was submerged for 6 hours in 4°C preservation solution that was identical to the PA flush solution. Gender/strain/size-matched rats were anesthetized, intubated, and ventilated with 100% O₂ using a rodent ventilator (Harvard Apparatus). Orthotopic left lung transplantation¹² was performed through a left thoracotomy using a rapid cuff technique for all anastomoses, with warm ischemic times maintained below 5 minutes. The hilar cross clamp was released, reestablishing blood flow and ventilation to the transplanted lung. A snare was then passed around the right PA, and Millar catheters (2F, Millar Instruments) were introduced into the main PA and the LA. A Doppler flow probe (Transonics) was placed around the main PA.

Measurement of Lung Graft Function
On-line hemodynamic monitoring was accomplished using MacLab and a Macintosh IIci computer. Measured hemodynamic parameters included LA and PA pressures (mm Hg) and PA flow (mL/min). Arterial oxygen tension (PO₂, mm Hg) was measured during inspiration of 100% O₂ using a model ABL-2 gas analyzer (Radiometer). PVRs were calculated as follows: (mean PA pressure-LA pressure)/mean PA flow, expressed as mm Hg·mL^-1·min^-1. After baseline measurements, the native right PA was ligated, and serial measurements were taken every 5 minutes until the time of euthanasia at 30 minutes (or until recipient death). To determine whether longer survival was possible in lungs preserved with supplemental db-cAMP (2 mmol/L), for certain experiments (ie, to gauge the effects of db-cAMP in a subacute lung transplant model) the observation period following ligation of the native PA was extended for up to 8 hours.

Radioimmunoassay for cAMP
For certain experiments, lung cAMP content was measured by radioimmunoassay. Although cAMP content was not measured in lungs scheduled for transplantation, parallel (dedicated) experiments were performed to gauge the effects of preservation on lung cAMP content. These lungs were homogenized before or after preservation to determine cAMP content. Experiments were performed in this manner so that tissue samples of lungs destined for transplantation need not be obtained, which would have complicated surgery and hemostasis after transplantation in our model. Lungs were harvested as described above with either EC alone or EC supplemented with indolidan (10 µmol/L) or PGE₁ (10 µg/mL) and then snap-frozen in liquid nitrogen either immediately or after 6 hours of immersion in preservation solution of the same composition as that used for harvest. Tissue was homogenized for 60 seconds at 4°C in Tris-buffered saline with 0.3 mmol/L isobutylmethylxanthine (Sigma), followed by the addition of ice-cold trichloroacetic acid (6%) to further lyse cells and precipitate proteins. The trichloroacetic acid–soluble supernatant was removed from the well, extracted three times with water-saturated ether, and dried, and the pellet was resuspended in sodium acetate buffer (pH 6.2). A radioimmunoassay was then performed for cAMP according to the manufacturer's instructions (New England Nuclear), as we have reported previously.¹¹ Protein content was determined by the method of Lowry et al²² after solubilizing trichloroacetic acid–precipitated protein with sodium hydroxide (1N). Results are reported as pmol cAMP/mg protein and expressed as the mean±SEM of duplicate determinations.

Myeloperoxidase Assay
Thirty minutes after ligation of the native right PA or at the time of recipient death, transplanted lungs were removed, rinsed briskly in physiological saline, and snap-frozen in liquid nitrogen until the time of myeloperoxidase assay. Tissue was homogenized in phosphate buffer (50 mmol/L, pH 5.5, 5 mL/g of tissue) containing hexadecyltrimethyl ammonium bromide (0.5%, Sigma). The assay was performed, as previously described,^{1 2 23} by thawing the sample, centrifuging at 40 000g for 15 minutes, and decanting the supernatant, which was assayed for myeloperoxidase activity using a standard chromogenic spectrophotometric technique in which test sample (0.03 mL) was added to phosphate buffer (0.97 mL) containing O-dianisidine dihydrochloride (Sigma) and hydrogen peroxide (0.0005%). Change in absorbance at 460 nm was measured over 1 minute (increase in optical density was linear over this time interval).

Measurement of Graft Wet-to-Dry Ratio
To measure edema formation in the transplanted lungs, the ratios between graft wet and dry weights were measured after 4-hour preservation and transplantation. The 4-hour preservation interval was chosen on the basis of previously published data¹² demonstrating that using a 4-hour preservation duration with EC solution should permit sufficient recipient survival to the 30-minute end point to measure edema formation. In this manner, edema could be measured at identical time points after reperfusion between control and experimental groups. Thirty minutes after ligation of the native right PA, lungs were removed with the bronchus ligated and then weighed. After preservation at 80°C for 48 hours, lungs were reweighed, and graft wet-to-dry weight ratios were then calculated.

Measurement of Graft Platelet Accumulation
Graft platelet accumulation was determined using ¹¹¹In-labeled platelets, prepared as described previously.^{1 24} Blood (5.0 mL) was taken from a gender/strain-matched donor rat and heparinized (2500 U). Platelets were isolated by differential centrifugation at 300g for 5 minutes to obtain platelet-rich plasma, which was then washed three times at 2000g for 15 minutes in 10 mL of ACD-A (mmol/L: citric acid 38, sodium citrate 75, and glucose 135). The pellet was suspended in 5 mL of ACD-A and centrifuged at 100g for 5 minutes to remove contaminating red blood cells, and the supernatant was collected. ¹¹¹In-oxyquinoline (70 µL of 1 mCi/mL, Amersham Mediphysics) was added with gentle shaking for 30 minutes at room temperature. The radiolabeled platelets were washed three times in ACD-A and resuspended in PBS, and platelet number was adjusted to 5x10⁷/mL. After completion of the vascular and bronchial anastomoses, 1.0 mL of ¹¹¹In-labeled platelet suspension was injected intravenously into the recipient. One minute after platelet infusion (immediately before reperfusion), 0.5 mL of blood was taken from the LA to determine blood radioactivity, to ascertain blood platelet concentrations, and to normalize for variations in blood loss during surgery. Five minutes after reperfusion, the native right PA was ligated, the graft was removed 10 minutes thereafter, and ¹¹¹In-platelet deposition was quantified by gamma counting. Platelet accumulation was expressed as the ratio of graft radioactivity to blood radioactivity normalized to dry weight.

Statistics
Data were evaluated using the Mann-Whitney U test or Fisher's exact test. Values are expressed as mean±SEM, with differences considered statistically significant at a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro exposure of endothelial⁶ and vascular smooth muscle cells¹¹ to a period of hypoxia causes a decline in intracellular cAMP content, leading us to investigate whether subjecting a richly vascularized organ such as the lungs to clinically relevant ischemia (organ preservation) may similarly reduce tissue cAMP levels. To investigate the effects of preservation on lung cAMP content, lungs from male Lewis rats were harvested/preserved in standard fashion using EC preservation solution, the solution most commonly used for clinical lung transplantation.¹⁴ Over the course of the preservation period, there was a significant decline in cAMP levels (Fig 1, control). When the PDE inhibitor indolidan was added to the preservation solution, lung tissue cAMP content increased 30% after 6 hours of lung preservation compared with control lungs preserved in its absence.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of hypothermic preservation on lung cAMP content. Lungs from male Lewis rats were harvested, preserved in EC preservation solution alone (control, n=12) or EC with supplemental PGE1 (20 µg/kg IV followed by 10 µg/mL in flush/preservation solution, n=12) for the indicated durations, and snap-frozen until assay. Lung cAMP content was measured by radioimmunoassay.

These data led us to implement a strategy to buttress cAMP levels in preserved lungs as a means of improving lung preservation for transplantation. For these experiments, control preservation conditions consisted of lungs preserved for 6 hours at 4°C in EC solution in the absence of cAMP supplementation. Control grafts failed rapidly during the immediate postreperfusion period, demonstrating elevated PVR, decreased PA flow, reduced arterial oxygenation, and rapid recipient demise (Figs 2A, 2C, 3, and 4 [leftmost bar]). In sharp contrast, supplementation of EC solution with db-cAMP, the membrane-permeable cAMP analogue, resulted in marked stabilization of all of these hemodynamic and functional parameters (Figs 2B, 2D, and 3): PVRs were low, PA flow was maintained after an initial slight decline caused by ligation of the native (right) PA, and graft function (arterial oxygenation) was preserved. These beneficial effects of cAMP analogue supplementation were dose dependent and translated into a significant increase in recipient survival (Fig 4).

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of db-cAMP on PA flow (A and B) and arterial oxygenation (PO2, C and D). All lung transplants were performed after 6-hour 4°C preservation, after which baseline measurements (pre) were performed. Immediately thereafter, the native (right) PA was ligated at time 0, and serial measurements were performed at the indicated time points. A and C, Control (EC solution alone). B and D, db-cAMP (EC solution was supplemented with the membrane-permeable nonhydrolyzable cAMP analogue db-cAMP [2 mmol/L]). Data for individual animals are shown (n=7 for both groups).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effects of db-cAMP (cAMP, 2 mmol/L) or the PDE inhibitor indolidan (PDEI, 10 µmol/L) on posttransplant hemodynamics and graft function. All lung transplants were performed after 6 hours of hypothermic preservation. Measurements were recorded at the final time at which the recipient was alive or 30 minutes after ligation of the native right PA. A, PVR. B, PA flow. C, Arterial oxygenation (PO2). Data for individual animals are shown () as well as group means (, with ±SEM) (n=7 for all groups).

View larger version (15K): [in this window] [in a new window]   Figure 4. Dose-dependent effect of db-cAMP on recipient survival. Experiments were performed as described in Fig 3. Dose response of db-cAMP showed maximal ability to enhance lung preservation at 2 mmol/L (n=7 for 0 mmol/L, n=4 for 0.002 mmol/L, n=3 for 0.02 mmol/L, n=7 for 0.2 mmol/L, and n=7 for 2 mmol/L). Recipient survival was determined at the 30-minute time point following ligation of the native (right) PA. Experiments were performed as described in Fig 3.

To demonstrate that these beneficial effects were not limited to db-cAMP but were the result of stimulation of the cAMP second messenger pathway, several alternative experimental strategies were used. Since butyrate is cleaved from db-cAMP upon entering the cell,¹⁸ butyrate alone was tested and found to have no effect on preservation compared with the control condition (Fig 5). Use of another cAMP analogue, 8-Br-cAMP, showed beneficial effects similar to those of db-cAMP, whereas the noncyclic purine analogue 8-Br-Ad was without effect (Fig 5). Because vascular smooth muscle cells exposed to hypoxia demonstrate an increase in type III PDE,¹¹ which hydrolyzes endogenous cAMP, a specific type III PDE inhibitor (indolidan)¹⁹ was added to EC preservation solution. Indolidan significantly reduced PVR, augmented PA flow, improved arterial oxygenation, and improved recipient survival compared with the control condition (Figs 3 and 5).

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of cAMP analogues (db-cAMP and 8-Br-cAMP) or the PDE inhibitor indolidan on recipient survival. Lungs were preserved in EC solution alone (control, n=7), or EC solution supplemented with db-cAMP (2 mmol/L, n=7), 8-Br-cAMP (0.1 mmol/L, n=6), indolidan (PDEI, 10 mol/L, n=7), or the side chains 8-Br-Ad (0.1 mmol/L, n=3) or butyrate (2 mmol/L, n=3). Recipient survival was determined at the 30-minute time point following ligation of the native (right) PA. Experiments were performed as described in Fig 3.

To convert our lung preservation/transplantation model to a subacute model, we performed additional experiments in which we extended the observation period in lungs that had been preserved in the presence of db-cAMP (2 mmol/L). These experiments demonstrated that the recipient rat survived for a mean of 5 hours (range, 2 hours 13 minutes to 7 hours 25 minutes) after ligation of the native (right) PA after lung transplantation. This survival in recipients of db-cAMP–treated lungs was significantly greater than that of control recipients transplanted with untreated lungs, 90% of which were dead by 30 minutes. These subacute survival data, in which only early survival was improved by cAMP supplementation, led us to recognize the stringency of lung transplant conditions in this model. To objectively establish the stringency of this model, and recognizing that the left lung in rats is relatively much smaller than the right lung, the following experiments were performed: Baseline PVRs were measured in nontransplanted control rats, after which the right PA was ligated (as was done in the lung transplant experiments), and PVRs were again measured after 30 minutes. These data demonstrate a mean PVR of 0.3±0.06 mm Hg·mL^-1·min^-1 in nontransplanted control rats in the absence of right PA ligation, which increased to 0.9±0.1 mm Hg·mL^-1·min^-1 (a 3-fold increase) by 30 minutes after right PA ligation. When PVR data were obtained after lung transplantation/right PA ligation in control rats (from Fig 3A), it is apparent that there is a 7-fold increase in PVR caused by the preservation/transplantation process itself. In contrast, similar comparisons reveal that PVRs are increased to a much smaller degree (1.2-fold and 2.1-fold) after lung transplantation when the lungs are preserved in the presence of db-cAMP or indolidan, respectively.

Because there is abundant experimental evidence to suggest that neutrophil recruitment following reperfusion is a major cause of pulmonary reperfusion injury^{25 26} and that stimulation of the ß-adrenergic/cAMP pathway has important antineutrophil effects,²⁷ we next investigated whether stimulating this pathway would attenuate postreperfusion graft leukostasis. Using a chromogenic assay of the neutrophil-specific enzyme myeloperoxidase to quantify tissue neutrophil deposition,²³ these studies showed that stimulation of the cAMP pathway with either a cAMP analogue (db-cAMP) or PDE inhibitor (indolidan) decreased graft neutrophil infiltration (Fig 6).

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of db-cAMP (cAMP, 2 mmol/L) or indolidan (PDEI, 10 µmol/L) on graft neutrophil accumulation. Myeloperoxidase activity (MPO), indicating change in absorbance at 460 nm measured over 1 minute (Abs460nm/min), was used to quantify neutrophil deposition. Data for individual animals are shown () as well as group means (, with ±SEM) (n=7 for all groups).

After having established that cAMP supplementation or PDE inhibition could have important pulmonary protectant effects, we wondered whether PGE₁ might exert its pulmonary protectant effects by acting through the cAMP pathway.^{28 29 30} This question is of particular clinical relevance, because PGE₁ is often used in clinical lung transplantation for reasons that are largely anecdotal, based on the hypothesis that the vasodilation provided by PGE₁ during harvest facilitates rapid and uniform delivery of hypothermic preservation solution throughout the lungs.^{13 14 16 17} Initial experiments were performed to measure the effects of PGE₁ supplementation on tissue cAMP content during preservation. These studies demonstrated that PGE₁ supplementation resulted in a rapid increase in tissue cAMP content at the time of preservation, which, although it declined somewhat during 6 hours of subsequent preservation, still remained significantly greater than the cAMP content of control lungs preserved in its absence (Fig 1).

This PGE₁-induced increase in pulmonary cAMP content was paralleled by improved graft function. PGE₁ supplementation resulted in reduced PVR, increased PA flow, improved arterial oxygenation (Fig 7), and improved recipient survival (Fig 8) compared with the control condition. To address the effects of bolus intravenous PGE₁ alone on lung preservation, separate experiments were performed in which PGE₁ was given as a bolus intravenous injection (20 µg/kg) but not incorporated into the preservation/flush solution. These data demonstrated an intermediate level of survival when compared with the other groups (control lung survival, 14% [n=7]; PGE₁ by initial bolus, 50% survival [n=4]; and PGE₁ by bolus and in flush/preservation solution, 88% survival [n=8]). Hemodynamic and oxygenation data similarly fell in an intermediate range (data not shown). To explore whether these effects were due to stimulation of the cAMP-dependent protein kinase,⁴ a specific inhibitor of the cAMP-dependent protein kinase (Rp-cAMPS)²⁰ was incorporated into the flush/preservation solution in addition to PGE₁. Rp-cAMPS abrogated the hemodynamic, functional, and survival benefits of PGE₁ (Figs 7 and 8), strongly suggesting an intermediary role for cAMP-dependent protein kinase in PGE₁'s pulmonary preservative effects.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effects of PGE1 (PG) on posttransplant hemodynamics and graft function. All lung transplants were performed after 6 hours of hypothermic preservation. Measurements were recorded at the final time at which recipient was alive or at 30 minutes after ligation of the native right PA. A selective inhibitor of the cAMP-dependent protein kinase (Rp-cAMPS, 0.1 mmol/L) was used in combination with 10 µg/mL PG (PG+Rp). A, PVR. B, PA flow. C, Arterial oxygenation (PO2). Data for individual animals are shown () as well as group means (, with ±SEM) (n=7 for control and PG+Rp, n=8 for PG).

View larger version (18K): [in this window] [in a new window]   Figure 8. Effect of PGE1 (PG) supplementation and antagonism of the cAMP-dependent protein kinase on recipient survival. PG+Rp indicates Rp-cAMPS in combination with PG (n=7 for control, n=8 for PG, and n=7 for PG+Rp).

Experiments were next designed to answer questions related to potential physiological mechanisms whereby PGE₁ may be acting to enhance pulmonary preservation. To test whether PGE₁ exerts its beneficial effects via its vasodilating action during harvest, we measured the flushing time required to deliver identical volumes of preservation solution at identical flushing pressure as a reflection of PVR during harvest. Compared with the control condition, PGE₁ significantly shortened the flushing duration (Fig 9), indicating that it does indeed function as a vasodilator during harvest. Consistent with the expected mediation by cAMP-dependent protein kinase,⁴ PGE₁-induced harvest vasodilation was abolished by Rp-cAMPS. To investigate whether harvest vasodilation alone would be sufficient to enhance pulmonary preservation, a potent direct acting vasodilator (minoxidil)²¹ was added to EC preservation solution and as expected, resulted in a significant reduction in the PA flush time, indicative of strong harvest vasodilating effects. However, in contrast to PGE₁, minoxidil was associated with a high degree of graft neutrophil infiltration (Fig 10), poor graft hemodynamics (not shown), and poor recipient survival (Fig 9).

View larger version (25K): [in this window] [in a new window]   Figure 9. Effects of vasodilators added to the preservation solution on pulmonary vasodilation during harvest (solid bar) and recipient survival (hatched bar). The time required to deliver the 30-mL volume of preservation solution at constant infusion pressure (20 mm Hg) was recorded as an index of PVR during harvest. Recipient survival was determined at the 30-minute time point after ligation of the native (right) PA. PG indicates PGE1; PG+Rp, Rp-cAMPS in combination with PG; Minox, the direct-acting vasodilator minoxidil. Values are mean±SEM (n=7 for control [EC alone], n=8 for PG [10 µg/mL], n=7 for PG+Rp [PG, 10 µg/mL; Rp, 0.1 mmol/L], and n=7 for Minox [0.02 µg/mL]). *P<.005 vs control; #P<.05 vs control, PG+Rp, and Minox.

View larger version (14K): [in this window] [in a new window]   Figure 10. Effects of PGE1 (PG, 10 µg/mL), PG combined with Rp-cAMPS (PG+Rp; PG, 10 µg/mL; Rp, 0.1 mmol/L), or minoxidil (Minox, 0.02 µg/mL) on graft neutrophil accumulation. Myeloperoxidase activity (MPO), indicating change in absorbance at 460 nm measured over 1 minute (Abs460nm/min), was used to quantify neutrophil deposition (n=7 for control, PG+Rp, and Minox; n=8 for PG). Data for individual animals are shown () as well as group means (, with ±SEM).

In addition to its effects to inhibit neutrophil adhesion to endothelial cells, the cAMP pathway contributes to other important endothelium-based mechanisms of vascular homeostasis, including endothelial anticoagulant properties and barrier function.^{5 6 31} To investigate whether stimulation of the cAMP-dependent protein kinase by PGE₁ may augment these additional nonvasodilatory protective mechanisms, we measured the ability of PGE₁ to inhibit graft platelet deposition and edema formation, as well as the ability of Rp-cAMPS to abrogate these beneficial effects. Supplementation of the cAMP pathway with PGE₁ not only enhanced graft function and reduced graft neutrophil infiltration (Figs 7 and 10) but decreased graft platelet deposition and reduced pulmonary edema (Fig 11). PGE₁'s effects to inhibit neutrophil and platelet accumulation and reduce edema formation were completely abrogated by antagonism of cAMP-dependent protein kinase (Figs 10 and 11). These data indicate that harvest vasodilation alone is insufficient to enhance pulmonary preservation and suggest that the beneficial effects of PGE₁ were not exclusively due to its actions as a vasodilator.

View larger version (16K): [in this window] [in a new window]   Figure 11. Effects of PGE1 (PG, 10 µg/mL) and PG combined with Rp-cAMPS (PG+Rp; PG, 10 µg/mL; Rp, 0.1 mmol/L) on formation of pulmonary edema and graft platelet accumulation. A, Wet-to-dry ratio of graft was used to quantify pulmonary edema (n=8 for control, n=9 for PG, and n=4 for PG+Rp). B, 111In-labeled platelet deposition, expressed as the ratio of graft radioactivity to blood radioactivity, was used to measure graft platelet deposition (n=6 for control, n=6 for PG, and n=4 for PG+Rp). Data for individual animals are shown () as well as group means (, with ±SEM).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent experimental evidence points to a pivotal role of the vasculature within a preserved and transplanted organ in the ultimate fate of the graft during the early reperfusion period.^{1 2 11 32} In critical transplantation scenarios, such as transplantation of the heart or lungs, early graft failure is tantamount to recipient death. The richly vascularized lungs appear to be particularly vulnerable to ischemic insult, with elevated PVR and poor gas exchange being the unwelcome harbingers of recipient demise.¹⁴ Current clinical strategies to improve pulmonary graft vascular function are limited and controversial. Current optimal clinical pulmonary preservation consists of lung harvest with hypothermic modified EC solution, a high potassium–based electrolyte solution. Profound vasoconstriction during the harvest period has been variously reported to be due to a combination of hypothermia, denervation of the harvested lungs, and the high potassium solution used for the PA flush.³³ Because it has been suggested that vasodilators enhance pulmonary preservation by lowering PVR during harvest, resulting in more rapid and effective distribution of hypothermic preservation solution,¹⁴ PGE₁ is often given to the donor and included in the preservation solution at the time of harvest.^{13 15 16 17} The benefit of PGE₁ has remained controversial, however, and the mechanism of action has remained largely speculative, which perhaps accounts for its continued sporadic clinical use.

The present study is the first to show that PGE₁ is not only beneficial in the rat orthotopic lung transplant model but that its mechanism of benefit is related to nonvasodilator properties, such as its ability to reduce neutrophil and platelet accumulation in the transplanted lungs and maintain endothelial barrier function. Our data with the rat lung transplant model are consistent with previous data showing that prostaglandins may prevent neutrophil adhesion to injured endothelium,³⁴ platelet aggregation,^{28 35} and vascular leakiness.³⁶ In addition, our data show that the potent vasodilator minoxidil, which lacks these other important vascular effects,²¹ is a poor pulmonary preservative. These data are consistent with previous reports of poor pulmonary protective activity of another direct-acting vasodilator, hydralazine.^{1 37}

These studies demonstrate that PGE₁ acts to increase cAMP levels in preserved lungs and that the beneficial vascular effects of PGE₁ on PVR, arterial oxygenation, endothelial barrier function, graft neutrophil and platelet accumulation, and recipient survival are mediated by the cAMP-dependent protein kinase. In addition to PGE₁ supplementation, other means of stimulating the cAMP pathway appear to be strikingly effective at enhancing pulmonary preservation, including use of cAMP analogues or the type III (cAMP-specific) PDE inhibitor indolidan.¹⁹ These beneficial actions are not limited to vasodilation but extend to diminishing leukostasis and improving gas exchange as well. These data are consistent with previous data showing that ß-adrenergic receptor agonists and cAMP analogues have a number of important vascular properties in addition to pulmonary vasodilation; they preserve endothelial barrier function under hypoxic/ischemic conditions,^{4 6 7 8 9 10 38} prevent neutrophil adhesion to endothelium,²⁷ and even help to maintain the normal anticoagulant phenotype of the vascular wall³⁹ (including antiplatelet effects^{28 40 41} ). Although the 33% reduction in cAMP levels in control lungs during the 6-hour preservation period may appear modest, increasing these levels with either a PDE inhibitor or PGE₁ was quite effective in improving graft function. The apparent discrepancy between modest alterations in whole-lung homogenate cAMP levels and graft vascular function may be explained by recognizing that cAMP and endogenous PDEs are found in multiple cell types, such as endothelial cells, vascular smooth muscle cells, and airway cells, and may be compartmentalized within cells. Therefore, analysis of cAMP levels in extracts of crude tissue homogenates provides only a rough measure of the potential activity of the cAMP second messenger pathway.

One of the limitations to the present study is that although cAMP supplementation causes marked early benefits (at 30 minutes), even initially well-preserved lungs failed after 5 hours. It is possible that technical factors may have contributed to the limited long-term survival, including the need for repeated anesthesia during the observation period, inflammatory mediator release from the necrotic right lung (only the right PA, not the right PV, was ligated), and difficulties in achieving complete hemostasis for a prolonged observation period. Perhaps most important, we believe that the limited longer-term survival is based on the extreme stringency of the lung transplant model used for the present studies. Although this particular model has in previous studies enabled us to gain insights into early-acting vascular mechanisms of lung preservation, additional studies performed in the present work demonstrate objectively that conditions in this model are extreme. In this rat model, a small single-lobed lung (the left) is preserved and transplanted; this left lung in rats is relatively much smaller than the right lung, which is a large four-lobed structure. Nevertheless, this model was adopted because the left lung has a single common pulmonary vein that can be reconstructed rapidly using a single-cuff technique, enabling us to limit warm ischemic times during the implantation procedure. After preservation, the left lung is transplanted with microsurgical technique, after which the native PA is ligated. This necessitates that the entire cardiac output be directed toward the newly transplanted lung and that the animal survive completely as a result of the function of this postischemic lung. In addition to its relevance to double lung transplantation in humans, the obvious benefit of using such a model is that it enables one to establish the adequacy of lung preservation without the confounding influence of a normal lung on both hemodynamics and gas exchange. Although in the present subacute studies we were unable to demonstrate longer-term benefit from cAMP stimulation, these data nevertheless provide useful information concerning the relative merits of cAMP stimulation on early graft function, recipient survival, and indices of graft vascular function, such as permeability, platelet accumulation, and leukosequestration. In addition, it permits comparison with a number of previously published reports from our own laboratory. It is tempting to speculate that had the lung transplant model been less stringent, then longer-term benefits may have been observed with PGE₁/cAMP stimulation.

On the basis of our data, it appears that the proximal mechanism of death in recipients of poorly preserved lungs stems from flow limitations to the pulmonary graft rather than a primary failure of gas exchange. Characteristically, elevated PVR precedes right ventricular failure, which is marked by right ventricular dilation by visual inspection. Arterial oxygenation, even in failing grafts/dying animals, does not decline to levels that are incompatible with life. We observed a similar phenomenon in an orthotopic baboon lung transplant model, in which we transplanted the lung after prolonged preservation.⁴² In those experiments, even in the presence of elevated PVR, pulmonary venous sampling revealed elevated PO₂. These observations lead us to hypothesize that arterial oxygenation is maintained to a modest degree by inhalation of 100% oxygen and prolonged transit times through the vasoconstricted pulmonary vasculature. In the present experiments, even though arterial oxygenation by itself was not sufficiently low to cause recipient demise, arterial oxygenation was significantly lower in recipients of failing compared with successfully preserved grafts. This indicates that arterial oxygenation can serve as an indicator of the effectiveness of preservation.

Data from the present study, along with other published data, provide a framework from which to view the mechanism of action of cAMP analogues and PGE₁ to improve lung preservation. The relationship between cAMP levels and endothelial monolayer permeability has been demonstrated in several reports. In the work of Ogawa et al,⁶ hypoxic exposure of endothelial cells (used as a paradigm for ischemia) was shown to cause time- and dose-dependent increases in monolayer permeability due to retraction of lateral cell margins under hypoxic conditions.⁵ Increases in monolayer permeability correlated inversely with cAMP levels, which were shown to decline secondary to reduced adenylate cyclase activity. In this hypoxic model, barrier function was restored by applying cAMP analogues or by increasing endogenous cAMP by inhibiting endothelial G_i activity, thereby increasing activity of adenylate cyclase. In our more recent study, in which we exposed endothelial monolayers to TNF,³⁹ we observed that TNF exposure caused a time- and dose-dependent increase in permeability coefficients of endothelial monolayers, also inversely related to intracellular cAMP levels. Under conditions of TNF exposure, barrier function could be restored to normal levels by membrane-permeable cAMP analogues. The work of Suttorp et al⁷ suggests that the increased hydraulic conductivity of endothelial cell monolayers exposed to hydrogen peroxide (caused by a reduction of cAMP content) could be reversed by PDE inhibition (especially in the presence of PGE₁) or by stimulating the cAMP-dependent protein kinase (PKA),⁷ suggesting an integral role of PKA in the maintenance of endothelial barrier function.

Looking further into the molecular mechanisms by which cAMP/PKA modulates endothelial monolayer permeability, cAMP analogue supplementation appears to act to increase PKA activity, by increasing the phosphorylation of myosin light chain kinase, which in turn inhibits its activity.^{43 44 45} Because in its phosphorylated state the 20-kD myosin light chain mediates the centripetal retraction of the lateral margins of endothelial cells,⁴⁶ agents that inhibit myosin light chain kinase activity (such as PKA agonists) preserve myosin light chains in their unphosphorylated state,⁴⁵ thereby maintaining endothelial monolayer barrier function.

In addition to the data described in the present study, there is a significant body of work that corroborates these findings with cultured endothelial cells to decreased pulmonary capillary permeability in the lungs. In several reports involving an isolated blood-perfused rabbit lung model, the increase in capillary filtration coefficients caused by ischemia and reperfusion was reversed by stimulating the cAMP second messenger pathway.^{8 9 38} In a sheep model, ß-adrenergic stimulation prevented thrombin-induced increases in pulmonary transvascular fluid and protein exchange.³⁸ Data from the present experiments, in which PGE₁ supplementation elevates cAMP levels and reduces posttransplant pulmonary edema formation (these antiedema effects of PGE₁ are blocked by inhibiting the cAMP-dependent protein kinase), suggest that similar mechanisms may be responsible for the protective effects of PGE₁ against edema formation in the setting of lung transplantation.

Based on our data, another potentially important mechanism whereby stimulating the cAMP pathway improves lung preservation is the reduction of graft leukosequestration. The effects of cAMP analogues or PDE inhibitors that lead to decreased neutrophil adhesion have been studied in detail in previous studies. In 1974, Bryant and Sutcliffe⁴⁷ showed that db-cAMP and PGE₁ both inhibit granulocyte adhesion to capillary tubes ex vivo. In the work of Boxer et al,²⁷ endothelial cells were shown to release cAMP, which impaired adhesion of adjacent PMNs. In a study directed at the effects of another prostaglandin on PMN adhesion, PGI₂ (which increases intracellular cAMP in endothelial cells) was shown to inhibit the adhesion of PMNs to endothelial cells in culture as well as lungs in an isolated lung model; in fact, cAMP analogues had a dose-dependent effect to inhibit PMN adhesion in the in vitro system.⁴⁸ PGI₂ inhibits the adherence of PMNs to nylon or wool fiber, with impaired adherence being transient and correlating with rises in intracellular cAMP within the PMN.⁴⁹ Although there may be several mechanisms by which cAMP diminishes PMN adhesiveness, it appears that elevating intracellular cAMP within PMNs inhibits mobilization and surface expression of the neutrophil ß₂ integrin, CD11b/CD18, as well as shape change of the PMN.⁵⁰ Although increased CD11b/CD18 expression may not be required for stimulated neutrophil adherence to cultured endothelium,⁵¹ Derian et al⁵⁰ conjecture that qualitative changes in CD11b/CD18, or diminished expression in submaximally stimulated PMNs, may underlie the antiadhesive actions of cAMP.

There is considerable overlap between the ability of either the cAMP or the NO/cGMP pathways to affect the vasculature with respect to vasodilation, inhibition of neutrophil and platelet accumulation, and maintenance of endothelial barrier function.³ It is not surprising, therefore, that the cAMP and NO/cGMP pathways have similar beneficial effects to improve pulmonary graft function.^{1 2} In in vivo experiments, such as those performed in the present work, there is likely to be some overlap between these two cyclic nucleotide pathways; for instance, the type III (cAMP-specific) PDE, whose activity is increased in hypoxic vascular smooth muscle, is cGMP inhibitable.¹⁹ Therefore, one might expect that the loss of endogenous NO levels and reduced cGMP levels following pulmonary reperfusion might result in disinhibition of the hydrolytic activity of PDE III, further lowering cAMP levels. This may explain why indolidan (a type III PDE inhibitor¹⁹ ) was effective at enhancing lung preservation. It is unlikely, however, that the present experimental results can be explained by direct activation of the cGMP-dependent protein kinase by the cAMP analogues used in this study, because N⁶-monobutyryladenosine 3',5'-monophosphate (the active compound formed after db-cAMP enters the cell¹⁸ ) and 8-Br-cAMP are, respectively, 313-fold and 53-fold less potent than cGMP in activating the cGMP-dependent protein kinase.⁵² The ability of Rp-cAMPS (a nonhydrolyzable competitive antagonist of the cAMP-dependent protein kinase, which binds to cAMP-dependent protein kinase without causing activation²⁰ ) to abrogate the beneficial effects of PGE₁ in the present study strongly suggests that the cAMP-dependent protein kinase is important in enhancing lung preservation independent of the NO/cGMP pathway.

Taken together, these data demonstrate that cAMP analogues or agents that raise endogenous cAMP levels (such as a PDE inhibitor or PGE₁) enhance pulmonary preservation for transplantation in an orthotopic rat left lung transplant model, with an important intermediary role for the cAMP-dependent protein kinase. In contrast to a simple harvest vasodilator such as minoxidil, which is a poor pulmonary preservative, stimulation of the cAMP second messenger pathway normalizes gas exchange and pulmonary blood flow, attenuates graft neutrophil and platelet accumulation, reduces pulmonary edema, and improves recipient survival after transplantation. These observations underscore the importance of nonvasodilator protective mechanisms of cAMP stimulation and highlight the importance of maintaining vascular homeostasis within the graft for the success of lung transplantation.

	Selected Abbreviations and Acronyms

 

8-Br-Ad = 8-bromoadenosine 8-Br-cAMP = 8-bromoadenosine 3',5'-monophosphate ACD-A = acid/citrate/dextrose anticoagulant db-cAMP = N6,O2'-dibutyryladenosine 3',5'-monophosphate EC = Euro-Collins LA = left atrium (atrial) PA = pulmonary artery (arterial) PDE = phosphodiesterase PGE1, PGI2 = prostaglandin E1 and I2 PKA = protein kinase A PMN = polymorphonuclear leukocyte PVR = pulmonary vascular resistance Rp-cAMPS = Rp isomer of adenosine 3',5'-monophosphorothioate TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported in part by grants from the Cystic Fibrosis Foundation (Dr Pinsky) and the Public Health Service (HL-55397, Dr Pinsky) and a Grant-in-Aid from the American Heart Association (Dr Pinsky). Dr Oz is the recipient of a Robert E. Gross Research Scholarship and is an Irving Assistant Professor of Surgery, and Dr Pinsky is a Clinician-Scientist of the American Heart Association.

	Footnotes

 
Previously presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, as part of the Cournand and Comroe Young Investigator Competition (Circulation. 1995;92[suppl I]:I-H).

Received January 25, 1996; accepted June 11, 1996.

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