Pulmonary arterial reactivity after transplantation

Pulmonary arterial reactivity after transplantation Differential effects of denervation and rejection Little is known regarding changes in reactivity of the vasculature of transplanted solid organs. Experiments were designed to differentiate the effects of denervation and rejection on the function of the endothelium and smooth muscle of pulmonary arteries in transplanted lungs of the dog. Single lungs were transplanted as autografts to study the effects of denervation or as allografts to study the additional effects of rejection. Immunosuppression was stopped in animals receiving allografts 5 days after operation, and rejection was allowed to proceed for an average of 3 days. Animals receiving autografts were studied after the same time period. There were no differences in concentrations of circulating leukocytes, platelets, or lymphocytes between the two groups. Circulating concentrations of angiotensin-converting enzyme were significantly reduced during rejection; concentrations of endothelin were unchanged. Rings of pulmonary arteries with and without endothelium were suspended for the measurement of isometric force in organ chambers. Contractions to angiotensin I and endothelin were less in rejecting than in autotransplanted arteries, whereas those to 5-hydroxytryptamine were enhanced. Contractions to norepinephrine were comparable in both autograft and rejecting aUograft arteries. Relaxations to isoproterenol were greater in the autograft than in the rejecting autografted arteries; the opposite was observed for relaxations to histamine. Endothelium-dependent relaxations to adenosine diphosphate and bradykinin but not to calcium ionophore A23187 were reduced with rejection;.relaxations to nitric oxide were unchanged. These results suggest that transplantation per se affects vascular reactivity. However, there are selective dysfunctions of receptor-operated mechanisms in arteries that are associated with rejection and that are distinct from denervation. Further, serum concentrations of angiotensin-converting enzyme may be an indicator of rejection of transplanted lungs. (J THORAC CARDIOVASC SURG 1992;103:751-62)

Folke N. Nilsson, MD, PhD (by invitation),* Christopher G. A. McGregor, MB, FRCS, and Virginia M. Miller, PhD (by invitation), Rochester, Minn.

Lng transplantation in its various forms is a successful therapy for certain patients with end-stage pulmonary disease.!" The reimplantation response and pulmonary From the Division of Thoracic and Cardiovascular Surgery, Department of Physiology and Biophysics, Mayo Clinic, Mayo Foundation, Rochester, Minn. Supported by the Mayo Clinic and Foundation. Dr. Nilsson was a visiting scientist supported by grants from the Swedish Medical Research Council, The Sweden-America Foundation, and Sahlgrenska Hospital, Gothenburg, Sweden. Read at the Seventy-first Annual Meeting of The American Association for Thoracic Surgery, Washington, D.C., May 6-8, 1991. Address for reprints: Virginia M. Miller, PhD, Department of Physiology, Mayo Clinic and Foundation, 200 First Street, SW, 901 Guggenheim Bldg., Rochester, MN 55905. *Present address: Department of Thoracic and Cardiovascular Surgery, Sahlgrenska Hospital, Gothenburg, Sweden.

12/6/34911

rejection are two common problems encountered after lung transplantation. The reimplantation response is manifest by impaired gas exchange, pulmonary opacification shown on chest roentgenograms, increased pulmonary vascular resistance, and an increase in lung water." Pulmonary rejection begins as a perivascular process with lymphocytic infiltration that progresses with more advanced rejection to the interstitium, alveoli, and bronchi' The pulmonary vasculature therefore is pivotal to both these processes. The endothelium modulates vascular tone by releasing vasoactive factors/' Alterations in the function of the pulmonary endothelium after transplantation may result from denervation of the transplanted organ or from processesassociated with rejection. Furthermore, because of the strategic position of the lungs within the circulation and the large surface area provided by the pulmonary 751

7 5 2 Nilsson, McGregor, Miller

endothelium, change in the release of vasoactive factors from the pulmonary endothelium may also have consequences at sites distant from the rejecting organ. The aim ofthis study, therefore, was to evaluate the function of the endothelium and smooth muscle in canine intralobar pulmonary arteries after denervation by single lung autotransplantation and during acute rejection after single lung allotransplantation.

Material and methods Experimental design. Mongrel dogs of either sex (21 ± 1.8 kg) were used. Donors and recipients were matched for size and weight. Intralobar pulmonary arteries (2 to 5 mm in diameter) from the lower lobe were studied in three groups: lungs from unoperated dogs (control animals), denervated lungs from dogs receiving autotransplants, and acutely rejecting lungs from dogs receiving allotransplants (rejecting lungs). Animal care was in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Anesthesia and analgesia. After premedication with fentanyl (0.03 rug/kg) and atropine (0.5 mg/kg intramuscularly), general anesthesia was induced with ketamine (10 to 12 rug/kg intravenously). Animals were intubated and anesthesia was maintained with a continuous infusion of ketamine (1 gm), etomidate (40 mg), and fentanyl (20 mg) diluted in 250 ml 5% glucose. Postoperatively, butorphanol tartrate (IO mg/dog, intramuscularly) was administered every 4 to 6 hours for the first 24 hours. Donor operation. Median sternotomy, thymectomy, and anterior pericardiectomy were performed. The azygos vein was ligated and the venae cavae, aorta, and trachea were encircled with umbilical tape. Heparin (3 rug/kg) and methylprednisolone acetate (I 50 mg) were given intravenously. Additionally, an infusion of the prostaglandin E, synthetic analog (500 /J-g/250 glucose solution 5%; Upjohn Company, Kalamazoo, Mich.) was begun at the completion of the dissection and continued until the systemic pressure fell by approximately 30%. The lungs were flush-perfused with Euro-Collins solution (60 ml/kg; 4 0 C) through a 5F-gauge catheter placed in the main pulmonary artery. Pulmonary arterial pressure during perfusion was not allowed to rise above the donor's previously measured mean pulmonary arterial pressure. Topical cooling of the lungs with cold (4 0 C) saline was done during pulmonary perfusion. Ventilation was continued (with room air), and, at the end ofthe perfusion, the trachea was clamped at end-inspiration. The heart-lung block was excised and the left lung dissected from the block in the inflated state for transplantation. Left lung allotransplantation. Recipient animals underwent a left lateral thoracotomy through the fifth intercostal space. Extrapericardial left pneumonectomy was performed. The donor lung was wrapped in sponges soaked in cold (4 0 C) saline, and the lung surface was continuously irrigated with cold saline during performance of the anastomoses. The left atrial and pulmonary arterial anastomoses were performed with continuous 4-0 and 5-0 Prolene sutures (Ethicon, Inc., Somerville, N.J.), respectively. The donor lung was separately intubated with a

The Journalot Thoracic and Cardiovascular Surgery

sterile endotracheal tube and inflated with 100% oxygen on a single occasion to reduce shunting before the bronchial anastomosis was performed with interrupted 4-0 Prolene sutures. The omentum, which was freed and tunneled up behind the sternum through a midline abdominal incision before the thoracotomy, was wrapped around the bronchial anastomosis. From removal to established blood flow, the ischemic time of the transplanted lung was 60 ± 10 minutes. Left lung autotransplantation. Autotransplantation was performed after a left thoracotomy through the fifth intercostal space. The left pulmonary artery, left main-stem bronchus, and pulmonary veins with a cuff of left atrium were exposed and clamped. The left atrial cuff and bronchus were divided and the pulmonary vesselsflushed with cold (4 0 C) modified Euro-Collins solution (30 ml/kg), The pulmonary artery was then divided and the three anastomoses performed as described for allotransplantation. The ischemic time was 65 ± 13 minutes. Immunosuppression. The dogs receiving allotransplants were immunosuppressed with intravenous cyclosporine (1 to 3 mg/kg/day to maintain plasma concentration of 200 to 250 ng/rnl) and azathioprine (2 mg/kg/day), starting with the first dose immediately before induction of anesthesia. Methylprednisoloneacetate (I 25 mg intravenously) was given every 8 hours for 24 hours, with the first dose at the time of reperfusion. It was then reduced for 2 days with doses of 2 rug/kg and 1 rug/kg, respectively. After 5 days, when animals had no radiographic signs of pulmonary infiltration, cyclosporine and azathioprine were discontinued. Antibiotic treatment for all animals consisted of nafcillin sodium (1 gm) every 8 hours for the first 24 hours, followed by gentamicin (60 mg) and kefcillin (I gm) daily (all given intravenously) until the day they were killed. Investigations. Blood samples were taken for complete blood counts, electrolytes, creatinine, angiotensin-converting enzyme (ACE), renin, and endothelin preoperatively and postoperatively at day 4 and at the time the animals were killed. ACE levels were measured spectrophotometrically. Renin and endothelin levelswere measured by radioimmunoassay. At the time of killing, bacterial and fungal cultures were taken from tracheal secretions and blood. Blood concentration of cyclosporine was monitored twice a week. Chest radiographs were obtained immediately postoperatively and after 5 days. When rejection was suspected from clinical signs, radiographic confirmation was obtained. Rejection occurred an average of 3.1 (range 2 to 5) days after discontinuation of immunosuppression, at which point the dogs were killed. The dogs receiving autotransplants similarly were killed after 8 days. Animals were anesthetized with pentobarbital sodium (30 mg/kg, intravenously) and exsanguinated. The heart-lung block was excised, and the intralobar arteries from the lower lobes were dissected and placed in modified Krebs-Ringer bicarbonate solution (NaCl 118.3, KCI4.7, CaCh 2.5, MgS04 1.2, KH zP04 1.2, NaHC0 3 25.0, calcium disodium edetate 0.26, and glucose 11.1 mmol/ L, control solution) for studies in organ chambers. The other lung lobes were infused with 10% buffered formalin for histologic assessment. Organ chamber experiments. After cleaning of connective tissue, the pulmonary arteries were cut into rings of 4 to 5 mm length. In randomly selected rings, the endothelium was removed deliberately by gently rubbing the luminal surface with the tip of a pair of watchmakers' forceps. The rings were suspended between a fixed point and a force transducer (Gould UC-2; Viggo-Spectramed Inc., Critical Care Division,Oxnard,

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Pulmonary arterial reactivity after transplantation

April 1992

753

Table I. Mixed venous concentration of leukocytes, lymphocytes, and platelets in dogs with lung transplants Autotransp/anted (n = 5)

Rejecting (n = /2) allotransp/anted Preoperative Leukocytes (x109/L) Lymphocytes (%) Platelets (x I09 IL)

Eight days postoperative

Preoperative

Eight days postoperative

13.1 ± 5.5* 74 ± 2 230 ± 23

6.7 ± 0.9 78 ± 8 253 ± 28

14.5 ± 6.3* 85 ± 5 238 ± 47

9.7 ± 3.7 68 ± 13 243 ± 25

Data are shown as mean ± standard error of the mean. ·Significant difference from preoperative levels by Student's

I

test for paired observations, p

< 0.05.

Table II. Serum concentration of hormones from mixed venous blood sample in dogs with lung transplants Rejecting allotransp/ant

ACE (U/L) Renin (ng/rnl/hr) Endothelin (pgl ml)

Autotransp/ant

Preoperative

Eight days postoperative

Preoperative

Eight days postoperative

I\.4 ± 1.7 (6) 6.5 ± \.9 (8) 7.3 ± 0.7 (9)

4.3 ± 0.4* (5) 15.7 ± 3.3* (8) 16.6 ± 9.3 (8)

9.5 ± \.5 (6) 2.4 ± 0.9 (5) 8.1 ± 0.6 (4)

8.4 ± \.5 (6) 15.2 ± 3.2* (5) 11.1 ± 4.2 (4)

Data are shown as means ± standard error of the mean. (n), number of animals. ·Significant difference from preoperative levels by one-way analysis of variance; p

Calif.) for the measurement of isometric force by two stainless steel wires inserted into the lumen of the ring. The rings were then placed in organ chambers filled with 25 ml of the control solution at 37° C and bubbled with 95% oxygen-5% carbon dioxide. Rings with and without endothelium were studied in parallel. They were equilibrated at a passive tension of less than 1 gm for 30 minutes. After this time, each ring was stretched progressively to the optimal point on its length-tension curve as determined by the tension developed to potassium chloride (20 mmol/L) at each level of stretch. Maximal contraction to potassium chloride (60 mmol/L) was then measured. After another equilibration period of 30 minutes, cumulative concentration-response curves were obtained to norepinephrine 00-9 to 10-5 mol/L), phenylephrine (10-9 to 10-5 mol/L), 5-hydroxytryptamine (5-HT) (10-9 to 10-5 mOI£L), angiotensin I 00-9 to 10-6 mol/L), or endothelin (10-1 to 10-7 mol/L), Responsesto angiotensin I were determined in the absence and presence of the ACE inhibitor captopril (10-5 mol/L) (E. R. Squibb & Sons, Inc., Princeton, N.J.). To study the relaxations of the vessels, the rings were contracted with a submaximal concentration (10-6 to 3 X 10-6 mol/L) of phenylephrine. Cumulative concentration-response curves to isoproterenol 00-9 to 10-6 mol/L), adenosine diphosphate (10-9 to 10-4 moll L), histamine (10-9 to 10-4 mol/L), or bradykinin (10-10 to 10-6 mol/L) were obtained. Relaxations to the calcium ionophore A23187 (Sigma Chemical Co., St. Louis, Mo.) (10-9 to 10-6 mol/L) were obtained only in rings with endothelium; relaxations to nitric oxide (3 X 10-9 to 10-5 mol/L) were obtained in rings without endothelium. Responses to bradykinin were obtained in the absence and presence of indomethacin (10-5 mol/L) and captopril (10-5 mol/L). Not more than four concentration-response curves were obtained in each ring. Chemical and drugs. The following drugs were used: adenosine diphosphate, angiotensin I, bradykinin, calcium ionophore A23187 (Sigma), captopril (Squibb), endothelin-l (Peninsula Laboratories Inc., Behnont, Calif.), histamine (Sigma),

< 0.05.

5-HT creatine sulfate (Sigma), indomethacin (Sigma), dl-isoproterenol (Sigma), I-norepinephrine bitartrate (Sigma), and phenylephrine (Sigma). Unless specified, drugs were prepared daily in distilled water and kept on ice. Indomethacin was dissolved in a solution of Na2C03 (final bath concentration, 2 X 10-5 mol/L). A23187 was dissolved in dimethyl sulfoxide (Sigma; final bath concentration, 8.2 X 10-3 mol/L). Neither the Na2C03 nor the dimethyl sulfoxide in the concentration used affected the responses of the tissues. Nitric oxide from a cylinder (Union Carbide, Danbury, Conn.) was used to fill a glass gas bulb fitted with a silicone injection septum. A volume of gas was removed with a glass syringe and injected into another glass gas bulb that had been filled with 100 ml of distilled water (bubbled with helium for approximately 3 hours) to give stock solutions of nitric oxide (4 X 10-5 mol/L, 4 X 10-4 mol/L, and 4 X 10-3 mol/L),? Concentrations of the drugs are reported as the final molar concentration in the organ chamber. Calculations and statistical analysis. The results are expressed as mean ± standard error of the mean. In all experiments n equals the number of rings, each taken from different animals. Where appropriate, the effectiveconcentration causing 50% of the maximal responses (ED50) was calculated for individual concentration-response curves and the mean of these values reported as the negative logarithm of the molar concentration. Since rings with and without endothelium of the same blood vesselwere studied in parallel in the absence and presence of inhibitors, Student's t test for paired observations was used within treatment groups. Analysis of variance was used to compare means among the treatment groups. When a significant F value was obtained, a Scheffe's test was used to identify differences among means. Values were considered to be statistically different when p < 0.05. Histology. Sections oflungs were fixed in formalin, sectioned (6 ~m), and stained with hematoxylin and eosin. The degree of rejection was assessed according to the Working Formulation

754

The Journal of Thoracic and Cardiovascular Surgery

Nilsson. McGregor, Miller

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Fig. 1. Concentration-response curves to norepinephrinein canine pulmonary arteries with and without endothelium after lung transplantation. Left panel, Control, unoperated artery. Middle panel, Autotransplant group. Right panel, Rejecting group. Data are expressedas grams increase in tension and are shown as mean ± standard error of the mean. In control group, there was a significant difference between contractions of rings with and without endothelium (Student's t test for paired observationsof area under curve, p < 0.05).

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Fig. 2. Concentration-response curves to 5-HT in canine pulmonary arteries with and without endothelium from control (left panel), autotransplant (middle panel), and rejecting (right panel) groups. Data are shown as grams increase in tension and are expressedas mean ± standard error of the mean. In control and autotransplant groups, contractions were significantlyless in rings with compared with rings without endothelium (Student's t test of area under curve, p < 0.05). for the Standardization of Nomenclature in the Diagnosis of Heart and Lung Rejection." At the conclusion of the organ chamber studies, one pair of rings with and without endothelin was prepared for light microscopy to confirm the presence or absence of endothelial cells,

Results Physical examination, bacterial and fungal cultures of the blood and tracheal secretion, and histologic examination of the lungs from dogs receiving transplants were negative for infection. Histologic assessment in the allotransplanted group showed rejection in all animals varied

between mild (n = 4), moderate (n = 4), and severe (n = 4) rejection. Histologic observations in arteries used in organ chamber studies confirmed the absence of endothelium in those rings in which it had been deliberately removed. No infiltration of lymphocytes into the smooth muscle layer was observed in any of the rings. Plasma levels of leukocytes (Table I) were significantly elevated at the conclusion of the study both in dogs receiving allografts and in those receiving autotransplants. Lymphocytes and the number of platelets did not change significantly and were comparable between the

Volume 103 Number 4 April 1992

Pulmonary arterial reactivity after transplantation

with endothelium • without endothelium 0

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Fig. 3. Concentration-response curves to endothelin in canine pulmonary arteries with and without endothelium from unoperated (control, left panel), autotransplant (middle panel), and rejecting allotransplant (right panel) lungs. Data are shown as mean ± standard error of the mean and are expressed as grams increase in tension. Contractions in rings with and without endothelium were similar in rejecting arteries; in control and autotransplant groups, contractions were significantly less in rings with compared with rings without endothelium (area under curve by Student's t test for paired observations, p < 0.05). Maximal tensions were significantly reduced in autotransplant and rejecting arteries compared with control arteries (Scheffe's test for multiple comparisons, p < 0.05). control.o rejecting +0 OJ

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Fig. 4. Concentration-response curves to angiotensin I in canine pulmonary arteries with (left panel) and without endothelium (right panel) from unoperated (contro!), autotransplant, and rejecting autotransplanted lungs. Data are shown as mean ± standard error of the mean and are expressed as grams increase in tension. Sensitivity to contractile effectsof angiotensin I were lessin rings without compared with rings with endothelium in control and autotransplant groups. In rejecting group, maximal tension in rings with endothelium was significantly less than other groups (Scheffe's test for multiple comparisons,p < 0.05). EDsofor contractions in rings with endothelium was significantly less in autotransplant than in control arteries (-log mol/L, 6.8 ± 0.1 and 7.3 ± 0.1, respectively;one-way analysis of variance, p < 0.05). transplant groups. Concentrations of ACE in the allotransplant group were 11.4 ± 1.7 U jL preoperatively and did not change significantly after 4 days of immunosuppression (8.8 ± 1.0 U jL). Rejection resulted in a significant decline of serum levels of ACE (Table 11). The serum levels of ACE did not change in the autotransplant group. Serum concentration of renin increased signifi-

cantly in both transplant groups; concentrations of endothelin did not change significantly during the study (see Table II).

Organ chamber experiments Contractile responses NOREPINEPHRINE. Norepinephrine caused concentration-dependent contractions in all rings. The contractions

The Journal of

756

Nilsson, McGregor, Miller

Thoracic and Cardiovascular Surgery

control • 0 rejecting +0

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Fig. 5. Concentration-dependent relaxations to isoproterenol in canine pulmonary arteries with (left panel) and without (right panel) endothelium after lung transplantation. Values are expressed as percent relaxations from contraction to phenylephrine 00-6 to 3 X 10-6 mol/L): 2.0 ± 0.3,2.9 ± 0.8, and 4.2 ± 0.8 gm in control, autotransplant, and rejecting arteries, respectively. Relaxations in autotransplant arteries were significantly greater than in rejecting group (Schetfe's test for multiple comparisons, p < 0.05).

Table III. Maximal tension developed to potassium chloride (60 mmoljL) in canine pulmonary arteries from control and transplanted lungs Endothelium

Group

With (gm)

Without (gm)

Control (n = 9) Autotransplanted (n = 5) Rejecting allotransplant in = 12)

4.0 ± 0.6 4.0 ± 0.6 4.9 ± 0.4

3.3 ± 0.7 3.3 ± 0.5 4.6 ± 0.5

Data are shown as mean ± standard error of the mean.

were significantly enhanced in rings without endothelium compared with rings with endothelium in the control group; this difference was not observed in the transplant groups (Fig. I). In the rejecting and autotransplant groups, contractions of rings with endothelium were significantly increased compared with rings with endothelium from the contractions of the control group. 5-HT. In control and autotransplant groups, 5-HT caused comparable concentration-dependent increases in tension that reached a maximum at 10-6 to 3 X 10-S mol/L and were greater in rings without compared with those with endothelium. At higher concentrations, relaxations were observed in these groups (Fig. 2). In the rejecting group there was a significantly increased sensitivity to the amine in rings with endothelium such that contractions of rings with and without endothelium were comparable. The maximal contraction in rings without endothelium was significantly greater in the rejecting (6.9 ± 1.1 gm) group than in autotransplant (4.0 ± 0.9 gm) and control (5.1 ± 0.9 gm) groups (see Fig. 2). ENDOTHELIN. Endothelin caused concentration-dependent contractions of arteries from all groups (Fig. 3).

These contractions were greater in rings without compared with those with endothelium in control and autotransplant arteries. This difference was not observed in rejecting arteries. The maximal contractions were significantly less than those of control arteries in the autotransplant and rejecting groups. ANGIOTENSIN 1. In rings with endothelium, angiotensin I caused concentration-dependent increases in tension that reached a maximum at 10-7 moljL (Fig. 4). In the rejecting group the contractions of rings with endothelium were significantly reduced compared with the other groups. However, the sensitivity to angiotensin I was comparable among all groups (EDso, -log mol/L; 7.5 ± 0.14, 7.5 ± 0.13, 7.6 ± 0.09, and 7.8 ± 0.11 in the rejecting, autotransplant, and control groups, respectively). In rings without endothelium, the maximal contraction was shifted to the right compared with rings with endothelium (see Fig. 4, right panel). The maximal contractions were comparable: 2.4 ± 0.7, 2.7 ± 0.4, and 4.3 ± 0.7 gm in rejecting, autotransplant, and control arteries, respectively. However, the sensitivity to the ago-

Volume 103 Number 4 April 1992

757

Pulmonary arterial reactivity after transplantation

Endothelium: with without autotx" '" rejecting. . <>

control autotx rejecting

0

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Adenosine diphosphate, -log M

Fig. 6. Concentration-dependent relaxations to histamine in canine pulmonary arteries from autotransplant and rejecting allotransplant lungs.Data are shownas mean ± standard error ofthe meanand are expressed as percentchangein tensionfrom a contraction to phenylephrine (10-6 to 3 X 10.6 mol/L), which did not differ between rings with and without endothelium. Contractions averaged 2.0 ± 0.3 gm in autotransplant and 2.2 ± 0.2 g.m in rejecting (n = 6) arteries. Relaxationsin control group were comparable to those of autotransplant group and are omitted for clarity. Relaxations of rejecting arteries were significantly greater than those of other groups (analysis of variance of maximal relaxations, p < 0.05).

Fig. 7. Relaxations to adenosine diphosphate in canine pulmonaryarteries withand withoutendotheliumafter lung transplantation. Values are expressed as percent relaxations from a contraction to phenylephrine (10-6 to 3 X 10-6 mol/L): 3.0 ± 0.5 gm, 2.0 ± 0.3 gm, 2.4 ± 0.3 gm in control, autotransplant and rejectingarteries,respectively. Responses of rings with endotheliumdid not differ betweengroups and were combinedfor clarity. These were significantly less than relaxations of rings with endothelium. Data are given as mean ± standard error of the mean. Relaxationsin rings with endotheliumfrom rejectinggroup are significantly lessthan thoseof control group (Scheffe's test for multiple comparisons, area under curve, p < 0.05).

nist was reduced in autotransplant and rejecting compared with control arteries (ED50 [-log M]: 6.8 ± 0.1 [n = 4];6.9 ± 0.1 [n = 7];and 7.3 ± 0.1 [n = 6],respectively). In the presence of the ACE inhibitor captopril (l0-5 mol/L), concentration-dependent contractions in rejecting (n = 6), autotransplant (n = 5), and control (n = 5) arteries with endothelium were comparable (data not shown). POTASSIUM CHLORIDE AND PHENYLEPHRINE. Potassium chloride (60 mmol/L) caused comparable contractions in pulmonary arteries with and without endothelium among groups (Table III). Phenylephrine caused concentration-dependent contractions in all groups. The contractions to phenylepinephrine were comparable among groups in rings with endothelium. There were no significant differences between the groups when the rings were contracted with a single submaximal concentration of phenylephrine (10- 6 to 3 X 10-6 mol/L) to study the relaxations of the arteries.These contractions were 66% ± 2% in rings with and 86% ± 9% in rings without endothelium of the maximal contraction to KCI (60 mmoljL).

Relaxations Endothelium-independent relaxations ISOPROTERENOL. Relaxations to isoproterenol were significantly greater in rings with endothelium than in those without endothelium in all groups. The autotransplant group showed greater sensitivity to isoproterenol than the control and rejecting groups (Fig. 5). HISTAMINE. Histamine caused concentration-dependent relaxations in all rings. In the control and autotransplant groups (Fig. 6), relaxations were comparable. Relaxations in arteries of the rejecting lungs were significantly increased compared with the other groups (Fig. 6). NITRIC OXIDE. Nitric oxide, an endothelium-derived relaxing factor, caused concentration-dependent relaxations in rings without endothelium. There were no significant differences among groups (Table IV). Endothelium-dependent relaxations ADENOSINE DIPHOSPHATE. Adenosine diphosphate caused significant concentration-dependent relaxations only in rings with endothelium. The relaxations were significantly less in arteries of the rejecting group than in the other groups. The control and autotransplant groups were comparable (ED50, -log mol/L, in rings with endotheli-

758

The Journal of Thoracic and Cardiovascular Surgery

Nilsson, McGregor, Miller

Table IV. Maximal relaxation and concentration causing 50% relaxation (ED50) to calcium ionophore A23187 (with endothelium) and nitric oxide (without endothelium) in canine pulmonary arteries Nitric oxide

A23I87 Group Control (n = 6) Autotransplant (n Rejecting (n = 6)

= 5)

Maximum (%)

ED50 (-log mol/L)

Maximum (%)

ED50 (-log mol/L)

93 ± 2 98 ± I 80 ± 7

7.0 ± 0.1 7.0 ± 0.2 6.8 ± 0.2

76 ± 5 85 ± 5 89 ± 5

6.0 ± 0.1 6.4 ± 0.3 6.4 ± 0.1

Data are presented as percent change in tension from a contraction to phenylephrine. All data are shown as mean ± standard error of the mean.

um;5.5 ± 0.22 and 5.5 ± 0.43inthecontrol(n=6)and autotransplant (n = 5) groups, respectively) (Fig. 7). BRADYKININ. Bradykinin caused significantly greater relaxations in rings with than in those without endothelium in all groups. The maximal relaxation to bradykinin was significantly decreased in the rejecting group compared with the other groups (Fig. 8; 75% ± 9.4%, 99% ± 1.2%, and 99% ± 1.1% in the rejecting, autotransplant, and control groups, respectively). Indomethacin did not increase the maximal relaxation to bradykinin in the rejecting arteries. However, incubating the vessels with a combination indomethacin (10-5 mol/L) plus captopril (10-6 mol/L) resulted in significantly increased sensitivity to bradykinin in all groups (data not shown, n = 5 to 6 in all groups). A23187. The calcium ionophore A23187 caused concentration-dependent relaxations in rings with endothelium in all groups. There were no significant differences among groups in the relaxing effect of the ionophore (see Table IV). Discussion This study demonstrates that transplantation of a lung alters the reactivity of both the endothelium and the smooth muscle of intralobar pulmonary arteries, and that rejection results in functional changes different from those of denervation per se. There was not a generalized inhibition or augmentation of responses to all agonists tested, but rather there were selective changes in function associated with mediators derived from nerve endings (norepinephrine, isoproterenol), blood cells and platelets (5-HT, adenosine diphosphate, histamine, bradykinin), and the endothelium (angiotensin II, endothelin, nitric oxide). Denervatiou/autotransplantation. The blood vessels of all solid transplanted organs are denervated at the time of transplantation. In the present study, at 8 days after transplantation, changes in reactivity of the pulmonary arteries of transplanted lungs cannot be explained only by a loss of smooth muscle or contractile proteins because contractions to potassium chloride were not diminished.

Further, maximal tensions developed to some agonists were diminished (angiotensin I and endothelin) whereas others were unchanged (norepinephrine and 5-HT). Canine pulmonary arteries are innervated by adrenergic nerves.i-? Chronic sympathetic denervation may increase the sensitivity of the smooth muscle to adrenergic transmitter (norepinephrine) by altering the disposition of the transmitter or by increasing the responsiveness of the postjunctional adrenergic receptors. to, II Contractions to norepinephrine were increased with denervation only in rings with endothelium. These data suggest that the disposition of the transmitter by the endothelial cells is altered after denervation. Alternatively, release of inhibitory vasoactive factors from endothelial cells may be reduced after denervation. Decreased sensitivity to contractile agents, particularly to adrenergic agonists in blood vessels with compared with those without endothelium, is attributed to basal or stimulated (or both) release of endothelium-derived relaxing factor.! The absence of a difference in contractions of rings with and without endothelium to norepinephrine is consistent with the notion that release of endothelium-derived relaxing factor is reduced after chronic denervation of blood vessels.F After autotransplantation, however, there was not a generalized impairment of all endothelium-mediated responses because differences in sensitivity between rings with and without endothelium to 5-HT, angiotensin I, and endothelin were still observed. These data indicate then that there is a decrease in a-adrenergic receptor-eoupled release of endothelium-derived relaxing factor in blood vessels of denervated/autotransplanted lungs. Alternatively, there could be an increased release of an endothelium-derived contractile factor(s) after autotransplantation. Endothelin is one such endothelium-derived contractile factor. Serum levels of endothelin did not increase after transplantation. However, serum levelsof the peptide may not be an accurate measure of its concentration at the levelof the smooth muscle. The response of the smooth muscle (rings without endothelium) to norepinephrine reflects stimulation of

Volume 103 Number 4 April 1992

a-adrenergic and ,a-adrenergic receptors. The former cause contractions and were not affected by denervation because contractions to phenylephrine were similar among groups. Stimulation of ,a-adrenergic receptors causes relaxations. Relaxations to the selective ,a-adrenergic agonist isoproterenol were enhanced after denervation, perhaps indicating postjunctional supersensitivity of ,a-adrenergic receptors. The sensitivity of the smooth muscleto nitric oxidedid not change without transplantation. Therefore, denervation may not affect cyclicguanosine monophosphate-related? relaxations of the smooth muscle. A similar conclusion wasreached in chronicallydenervatedear arteries of rabbits.12 Endothelindecreases releaseof neurotransmitter from adrenergicnerveendings.13 However,this cannot explain differences between the control and autotransplant groups because contractions to endothelin were depressed. Therefore, the procedure of autotransplantation may alter either the number of endothelinreceptorsor the specific receptor-coupled activation of contractile proteins. Angiotensin I is converted to angiotensin II by ACE, a surface' enzyme on the endothelial cells and in the smoothmuscle. 14 Decreasedsensitivity to angiotensinI of the smooth muscle after denervation may therefore reflectdecreasedconversion to angiotensinII or a change in angiotensin II receptor-coupled events. Autotransplanted lungs, in addition to being denervated,werealsosubjectedto preservationand reperfusion during the transplantation procedure. Perfusion of the lungs with Euro-Collins solution reduces endotheliumdependentrelaxationsto adenosinediphosphate.15 While it cannot be excludedthat the differences betweencontrol and autotransplanted arteries may be the result of changes due to the preservative, this is unlikely because relaxations to adenosine diphosphate were similar between these two groups. Changes in oxygen tension associated with reperfusion injury can affect activity of ACE,16 release a contracting factor from pulmonary endothelial cells,17-19 and release oxygen-derived free radicals.P However, it is not known how these factors might modify responses of the pulmonary arteries days after the initial insult. Rejection of allografts. The allografted lungs were exposed to the same handling procedures as the autotransplanted lungs,and yet responses of the arteries were different between the transplanted groups. Therefore selective responses of the endothelium and smooth muscle are altered by the rejection process. It can be argued that a differencebetween the autotransplant and rejecting autograft groups was the presenceand withdrawal of

Pulmonary arterial reactivity after transplantation

759

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Fig. 8.' Concentration-dependent responses to bradykinin in canine pulmonary arteries from unoperated (control), autotransplant, and rejecting allotransplant lungs. Data are shown asmean ± standard errorofthemean andareexpressed as percent change in tension from a contraction to phenylephrine(10-6 to 3 X 10-6 mol/L), which didnotdiffer between rings with and without endothelium. Contractions averaged 3.8 ± 0.7 gm in control (n = 6), 2.0 ± 0.2 gm in autotransplant (n = 5), and 2.8 ± 0.7 gm in rejecting (n = 6) arteries. Relaxations in rings with endothelium were greater than those without endothelium in each group (Student's t test for paired observations, p < 0.05). Relaxations of rings without endothelium didnotdiffer among groups andare combined forclarity. Maximal relaxations were significantly less in rejecting compared with other groups with endothelium (Scheffe's test for multiple comparisons, p < 0.05). immunosuppressive agents. However, this cannot be the only explanationbecause responses of the rejecting arteries were different from those of blood vessels from dogs that had been exposed to the same immunosuppressive regimen (unpublished observations). In addition, the effectsof rejectiondo not represent lossof endothelialcells because this was not evident with light microscopic examination. Likearteries from the autotransplant group, arteries of the rejecting group did not show differencesin sensitivity to norepinephrine between rings with and without endothelium. However, unlike the autotransplanted arteries, the rejecting arteries with and without endothelium also showedsimilar sensitivity to 5-HT and endothelin.There were no differences between rings with and without endotheliumto severalcontractileagents, suggestingthat, with rejection, there may be a generalized decrease in basal/stimulated releaseof endothelium-derivedrelaxing factors. Consistent with this conclusion are the observa-

760

The Journai 01 Thoracic and Cardiovascular Surgery

Nilsson, McGregor, Miller

tions that endothelium-dependent relaxations to adenosine diphosphate and bradykinin were also reduced with rejection. However, relaxations to an agonist that does not activate receptors (calcium ionophore A23187) were not altered with rejection. Therefore, during rejection, decreases in release of endothelium-derived relaxing factors are associated with receptor activation. Further, since sensitivity of the smooth muscle to nitric oxide, an endothelium-derived relaxing factor, was not altered, activation of soluble guanylate cyclase probably does not change with rejection. An alternative explanation for impaired endotheliumdependent relaxations in arteries of the rejecting group is that there is an increased release of endothelium-derived contractile factors, for example, thromboxane A2 or endothelin. This is unlikely because circulating levels of endothelin were not increased in rejecting animals (of course, this does not rule out that endothelin may increase in the tissue as a paracrine hormone). Further, if contractile prostanoids were produced, endothelium-dependent relaxations would increase in the presence of indomethacin; this was not observed, at least in response to bradykinin. Endothelium-derived relaxing factors (nitric oxide) and prostacyclin are potent inhibitors of platelet adhesion and aggregation.I! 22 Therefore, a dysfunctional pulmonary endothelium may favor the occurrence of platelet aggregation and platelet-induced vasospasm. In rejecting arteries, endothelium-dependent relaxations to two platelet products (adenosine diphosphate and 5_HT23) were reduced. Long-term impairment of endothelium-dependent relaxations to aggregating platelets and platelet products occurs after reperfusion injury.i" However, it is unlikely that this is the cause of the altered responses to adenosine diphosphate and 5-HT in the rejecting arteries. Responses to these agents were not different between the autotransplanted arteries (which saw the same ischemic time and reperfusion procedure as the rejecting arteries) and unmanipulated control arteries. Histamine is an important mediator of the acute edematous response that accompanies inflammatory reactions.P The rejection process affects the response of the smooth muscle to histamine because the relaxations to this agonist of rings without endothelium were augmented with rejection. In some tissues histamine receptors may be coupled to a guanine nucleotide regulatory protein that inhibits cyclic adenosine monophosphate (cAMP).25,26 Therefore the results of the present study provide indirect evidence that the rejection process may selectively augment responses mediated by guanine nucleotide regulatory proteins that inhibit cAMP. Consistent with this notion, in addition to the augmented relaxations to histamine, are the observations that relax-

ations to isoproterenol are reduced in rejected arteries and the contractions to 5-HT are enhanced. The former are associated with increases in cAMP,27 and the latter are sensitive to inhibition by pertussis toxin (therefore, potentially inhibition of cAMp28). Decreased endothelium-dependent contractions to angiotensin I in the rejected group suggest a decreased amount or activity of the converting enzyme. That circulating levels of the enzyme were reduced and that the contractions to angiotensin I were normalized in the presence of converting enzyme inhibitor supports this conclusion. Activity of ACE decreases in hypoxic lungs.i? and measurement of this enzyme may be a useful marker for microvascular injury.l" To our knowledge serum levels of ACE have not been reported in patients after lung transplantation. Changes in the circulating concentrations of the enzyme must be affected by rejection alone because there were no differences observed among groups while the immunosuppressive agents were administered (at 4 days postoperatively) or after autotransplantation. Changes in circulating enzymes suggest that the rejection process may affect vascular function at sites distant from the rejecting organ. It is not known how circulating levels of ACE may affect function of the endothelium in the general circulation or whether damaged endothelial cells may release a factor/metabolite that could affect other cells. Alternatively, circulating activated leukocytes may affect the function of the endothelium in the general circulation.'! In conclusion, rejection of allotransplanted lungs results in selective changes in reactivity of the vascular endothelium and smooth muscle independent of denervation alone. These changes may not only affect pulmonary vascular resistance but may also result in systemic manifestations due to changes in the metabolic activity of the pulmonary endothelium. These changes may not be characteristic only of transplanted lungs but may occur also in the endothelium of other transplanted organs. Furthermore, such endothelial dysfunction may contribute to chronic vascular changes that limit the long-term prognosis of organ grafts. Wethank Mr. RonaldLee,Mr. Arland Hildestaad, and Mrs. MarilynOltejenand the veterinary technicians for their expert technical assistance; Mrs. Denise Huebleinfor performing the endothelin assay; Mr. Robert Lorenz for drawing the figures; and Ms. Ellen Gladwell for typing the manuscript. REFERENCES I. PearsonFG. Lungtransplantation: the Torontoexperience. Eur J CardiothoracSurg 1989;3:6-11. 2. McGregorCGA, Jamieson SW, Baldwin JC, et al. Combined heart-lung transplantation for end stage Eisenmenger's syndrome. J THORAC CARDIOVASC SURG 1986; 91:443-50.

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3. Griffith BP, Hardesty RL, Trento A, et al. Heart-lung transplantation: lessons learned and future hopes. Ann Thorac Surg 1987;43:6-16. 4. Reitz BA. Heart-lung transplantation: a review. Heart Transplantation 1982;1:291-8. 5. Yousem SA, Berry GJ, Brunt EM, et aI. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: lung rejection study group. J Heart Transplant 1990;9:593-601. 6. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEBJ 1989;3:2007-18. 7. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of the endothelium-derived relaxing factor. Nature 1987;327:524-6. 8. Miller VM, Vanhoutte PM. Endothelial a'2-adrenoceptors in canine pulmonary and systemic blood vessels. Eur J Pharmacol 1985;118:123-9. 9. Rorie D, Tyee G, Edwards W, Sittipong R, Kaye M. Chronic hypoxia alters structure and transmitter dynamics in dog pulmonary artery. Respir PhysioI1988;74:211-27. 10. Fleming WW, Westfall DP. Altered resting membrane potential in the supersensitive vas deferens of the guineapig. J Pharmacol Exp Ther 1975;192:381-9. 11. Westfall DP. Supersensitivity of smooth muscle. In: Bulbring E, Brading AF, Jones A W, Tomita T, eds. Smooth muscle. Austin: University of Texas Press, 1981, pp 285311. 12. Mangiarua EI, Bevan RD. Altered endothelium-mediated relaxation after denervation of growing rabbit ear artery. Eur J PharmacoI1986;122:149-52. 13. Wiklund NP, Ohlen A, Cederqvist B. Inhibition of adrenergic neuroeffector transmission by endothelin in the guinea-pig femoral artery. Acta Physiol Scand 1988;134:311-2. 14. Ryan US, Ryan JW, Chiu A. Kinase II (angiotensin converting enzyme) and endothelial cells in culture. Adv Exp Med Bioi 1976;70:217-27. 15. Struber M, McGregor CGA, Locke TJ, Miller VM. Effect of flush-perfusionwith Euro-Collins solution on pulmonary arterial function. Transplant Proc 1990;22:2206-11. 16. Krulewitz AH, Fanburg BL. The effect of oxygen tension on the in vitro production and release of angiotensinconverting enzyme by bovine pulmonary artery endothelial cells. Am Rev Respir Dis 1984;130:866-9. 17. DeMey JG, Vanhoutte PM. Heterogeneous behavior of the canine arterial and venous wall. Circ Res 1982;51:439-47. 18. Vanhoutte PM, Auch-Schwelk W, Boulanger C, et al. Does endothelin-l mediate endothelium-dependent contractions during anoxia? J Cardiovasc PharmacoI1989;13:S124-8. 19. Rakugi H, Tabuchi Y, Nakamuru M, et aI. Evidence for endothelin-l release from resistance vessels of rats in response to hypoxia. Biochem Biophys Res Commun 1990; 169:973-7. 20. Rhoades RA, Packer CS, Meiss RA. Pulmonary vascular smooth muscle contractility: effect of free radicals. Chest 1988;93:94-5. 21. Azuma H, Ishikawa M, Sekizaki S. Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol 1986;88:441-5.

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22. Thiemermann C, May GR, Page CP, Vane JR. Endothelin-l inhibits platelet aggregation in vivo: a study with Illindium-labelled platelets. Br J Pharmacol 1990;99: 303-8. 23. Houston DS, Shepherd JT, Vanhoutte PM. Adenine nucleotides, serotonin, and endothelium-dependent relaxations to platelets. Am J Physiol 1985;248:H389-95. 24. Pearson PJ, SchaffHV, VanhouttePM.Long-termimpairment of endothelium-dependent relaxations to aggregating platelets after reperfusion injury in canine coronary arteries. Circulation 1990;81:1921-7. 25. Carson MR, Shasby SS, Shasby DM. Histamine and inositol phosphate accumulation in endothelium: cAMP and a G protein. Am J Physiol 1989;257:L259-64. 26. Weinheimer G, Osswald H. Pertussis toxin and N-ethylmaleimide inhibit histamine- but not calcium ionophoreinduced endothelium-dependent relaxation. Naunyn Schmiedebergs Arch PharmacoI1989;339:14-8. 27. Karnushina IL, Spatz M, Bembry J. Cerebral endothelial cell culture. 1. The presence of {32 and a'2-adrenergic receptors linked to adenylate cyclase activity. Life Sci 1982; 30:849-58. 28. Hohlfeld J, Liebau S, Forstermann U. Pertussis toxin inhibits contractions but not endothelium-dependent relaxations of rabbit pulmonary artery in response to acetylcholine and other agonists. J Pharmacol Exp Ther 1990; 252:260-4. 29. Leuenberger PJ, Stalcup SA, Mellins RB, Greenbaum LM, Turino GM. Decrease in angiotensin I conversion by acute hypoxia in dogs. Proc Soc Exp Bioi Med 1978; 158:586-9. 30. Stalcup SA, Turino GM, Mellins RB. Critical issues in the use of vasoactive substances to assess lung microvascular injury. Ann NY Acad Sci 1982;384:435-57. 31. Shaddy RE, Mak C, Zimmerman GA. Evidence that the serum of moderate-to-severely rejecting heart transplant patients induces peripheral blood mononuclear cells to injure endothelial cells. Transplantation 1990;49:1013-5.

Discussion Dr. Severi Mattila (Helsinki, Finland). Twenty years ago we studied the effectsof norepinephrine and isoproterenol on the distribution of blood flowafter unilateral pulmonary autotransplantation in dogs with use of radiographic xenon to measure the distribution between the intact and the transplanted lung (Scand J Thorac Cardiovasc Surg 1973;7:49-52). It appeared that norepinephrine, which is a vasoconstrictor, caused a shift of blood flow toward the transplanted lung, whereas after administration of isoproterenol, which is a vasodilating agent, a shift of blood flow toward the intact lung was noted. Our findings confirm in vivo the results of Dr. McGregor's group with a diminished response of denervated pulmonary vascular wall to these sympathomimetic agents. Dr. Craig R. Smith (New York, N.y.). I think we would all be excited to think that we can find a biochemical marker for rejection, something that has been missing from all of solid organ transplantation. This study makes an excellent start toward defining the sensitivity of this method. Although the mechanisms are obscure in terms of what is stimulating

7 6 2 Nilsson, McGregor, Miller

angiotensin, that is only part of the story, and it is worth remembering that in the lung specificity is another important part of the problem. We would have to be confident that a model that includes viral infection, for example, does not show similar changes. Do you have any data that would define specificity? Dr. Thomas M. Egan (Chapel Hill, N. C.). Several years ago it was noted that ACE levels in blood were elevated with a variety of inflammatory conditions of the lung, including sarcoidosis. I wonder how you might explain this paradox of a reduction in ACE level when we have a condition that we know is associated with perivascular inflammation. Along those lines, you mention that rejection was confirmed histologically. Was that the case in all of your animals that demonstrated the reduction in ACE levels? Finally, did you have any histologic data that might suggest that the number of viable endothelial cells was reduced, and could that explain the reduction in ACE levelsthat you observed? Dr. John R. Benfield (Sacramento, Calif). I am concerned about the fact that you are studying excised segments of intralobar pulmonary arteries. Do you have any evidence that the reactivity or lack of reactivity of an intralobar pulmonary artery has some meaningful relationship to the reactivity of intraparenchymal arteries of the lung? There is, of course, the opportunity to pursue your line of investigation by leaving certain portions of the lung in vivo and in situ, excising another portion of the same lung with both of the portions having been denervated before your measurements. Studies done in that way could ascertain differences, if there are any, between intralobar arteries and intraparenchymal portions of the artery. Dr. Hanni Shennib (Montreal, Quebec, Canada). Regarding your suggestion or conclusion, perhaps there are less relaxing factors that may be contributing. We have observed an increase in pulmonary vascular resistance early on, immediately after transplantation, but we have observed that endothelin levels

The Journal of Thoracic and Cardiovascular Surgery

actually go up. Perhaps one of the reasons for your observation is actually that you have more removal of vasoactive substances rather than removal of relaxing factors. Dr. Nilsson. We studied intralobar pulmonary arteries because there are data regarding function of these arteries in unoperated animals. It would be interesting to study the reactivity of smaller caliber resistance arteries. At this time, however, we are not equipped to study this size of blood vessels. An alternative explanation for reduced endothelium-dependent relaxations in arteries of rejecting dogs could be an increased release of endothelium-derived contractile factors, for example, endothelin or thromboxane A 2. It is unlikely that production of prostanoids, such as thromboxane A2, was increased because endothelium-dependent relaxations would increase in the presence of inhibitors of cyclooxygenase such as indomethacin; this was not observed in the present study. The role of endothelin is not clear. Circulating levels of endothelin were comparable among the groups. However, the maximal contractions to endothelin were less both in groups receiving autotransplants and in rejecting groups compared with control animals. Other techniques that measure local production, clearance, and binding of endothelin will be necessary to define its role in transplantation/rejection. Perfusion-fixed preparations for electron microscopy were not prepared. Examination of the arteries with use of light microscopy did not demonstrate infiltration of lymphocytes in the media or endothelial layer of the arteries used in organ chamber experiments. Not all endothelium-dependent mechanisms were affected by rejection. For example, the relaxations to the calcium ionophore A23187, which releases endothelium-derived relaxing factor by a mechanism independent of receptor activation, were comparable among groups. Therefore it is unlikely that a diffuse loss of endothelial cells is the only explanation for decreases in endothelial function associated with acute rejection.