INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The development of bronchiolitis obliterans (BO) after lung transplantation limits the long-term success of this operation. Fifty percent of lung transplant patients develop this complication by postoperative Year 3, and it remains the major cause of late mortality following lung transplantation (1, 2). The pathogenesis of BO is not well understood, but it is thought to be a form of chronic rejection. Investigations done with experimental models of posttransplant airway obliteration have examined the roles of immune, infectious, and growth factors in this process (3). Soon after transplantation there is a prominent immune response involving endothelial and epithelial injury, which later gives rise to an ineffective reparative process, with fibroblast proliferation and collagen deposition in the airways (3, 6).

The angiotensin system has many roles in both physiologic and pathologic processes, such as stimulation of the sympathetic nervous system, vascular smooth-muscle contraction, stimulation of aldosterone and antidiuretic hormone release, and immune-mediated injury (7). Increasing evidence suggests that the angiotensin system plays a prominent role in the pathogenesis of fibrosis (10, 11). This system has been found to have growth-promoting effects through smooth-muscle hyperplasia, fibroblast proliferation and transformation to myofibroblasts, and extracellular matrix deposition (12). In a model of myocardial infarction and pericardial fibrosis, myofibroblasts were found at sites of fibrosis in both pathologies, and were positively labeled by antibody to angiotensin converting enzyme (ACE) (10).

Inhibition of the angiotensin system has prevented or lessened the development of fibrosis in many experimental situations. In a granuloma pouch model of tissue repair, ACE and angiotensin II receptor type 1 were expressed by myofibroblast cells, and inhibition of the angiotensin system decreased pouch weight and hydroxyproline concentration (13). Inhibitors of ACE have also effectively prevented radiation-induced pulmonary endothelial dysfunction and pulmonary fibrosis in a rat model (14).

The involvement of the angiotensin system in chronic rejection following transplantation has been examined in experimental heart and kidney transplantation. Captopril administration reduced graft rejection grades and prevented intimal and smooth-muscle proliferation in a rat heterotopic cardiac transplantation model (15, 16). Treatment with an angiotensin II receptor blocker following kidney transplantation in a rat model of chronic rejection significantly decreased proteinuria and preserved glomerular and tubulointerstitial structure (17).

We conducted a study in which we used a rat heterotopic tracheal transplant model to examine the involvement of the angiotensin system in the development of allograft-induced fibrotic airway obliteration (3). We describe the immunohistologic localization of ACE in isograft and allograft control and nontransplanted (NT) tracheas. We also report that captopril, an ACE inhibitor, significantly attenuated fibroproliferative airway obliteration in the allografted animals. This study implicates a role for the angiotensin system in the development of BO and suggests a novel therapeutic strategy for inhibiting fibrous airway obliteration.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Experimental Design

Heterotopic tracheal transplantations were performed from Brown Norway (BN) to Lewis (LW) rats (allografts), and from BN to BN rats (isografts). Five animals were allocated to each of five experimental groups as follows: (1) nontreated isograft controls; (2) nontreated allograft controls; (3) allograft-recipient animals treated with captopril 5 d before transplantation; (4) allograft-recipient animals treated with captopril beginning on postoperative Day 1; and (5) allograft-recipient animals treated with captopril beginning on postoperative Day 5. Captopril (generously provided by Bristol-Myers Squibb, Saint-Laurent, PQ, Canada) was administered in the animals' drinking water at 100 mg/kg/d. Once begun, captopril administration was continued until harvest of tracheal specimens. The drinking behavior of the rats was monitored, and the dosing was adjusted every 2 d to ensure that the animals received the total dose of captopril in their water. Control animals received water alone. The tracheal grafts were harvested on postoperative Day 21 for morphometric, histologic, and immunohistologic analyses.

Rats

Experiments were done with male (250 to 300 g) (BN rats aged 10 to 12 wk) (Harlan Sprague Dawley Inc., Indianapolis, IN) and with male (250 to 300 g) LW rats (aged 10 to 12 wk) (Charles River Canada Inc., St. Constant, PQ, Canada).

All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research, the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1985, U.S. Government Printing Office, Washington, DC), and the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care Committee of the Toronto General Hospital Research Institute.

Tracheal Transplants

A heterotopic tracheal transplant model was used as described (3). No immunosuppressive drugs were given with this model. The lesion that develops in allografts by Day 21 is composed of fibrous tissue, and closely resembles that seen in human patients with BO.

In the donor procedure, BN rats were anesthetized with 80 mg/kg pentobarbital sodium intraperitoneally. Using sterile technique, the trachea was exposed through a midline incision in the neck. The trachea was bluntly dissected and transected distal to the larynx and proximal to the carinal bifurcation. Each trachea was divided into two segments with nine cartilaginous rings in each segment.

In the recipient procedure, LW rats were anesthetized with a mixture of acepromazine (2.5 mg/kg) and ketamine (75 mg/kg) intraperitoneally. Before surgery, the animals received cefazolin sodium (5 mg) subcutaneously. Using sterile technique, we made two separate dorsal incisions in the neck, on the right and left sides. Two subcutaneous pouches were created with blunt dissection, and the tracheal segments from BN rats were placed into each of these cavities. The skin was closed with 5-0 Vicryl horizontal mattress sutures (Ethicon Inc., Johnson & Johnson, Peterborough, ON, Canada). After their recovery from anesthesia, animals received standard laboratory rat food.

Tissue Preparation

On postoperative Day 21, the right graft was fixed in 10% buffered formalin and embedded in paraffin, and 4-µm cross-sections were obtained from the center of each tracheal segment. The slides were stained with hematoxylin and eosin or elastic trichrome. The left graft was excised and the middle portion was embedded in ornithyl carbamyltransferase compound (Tissue-Tek; Sakura Finetek, CA), snap-frozen in liquid nitrogen, and stored at 70° C until used for immunohistologic examination. Tracheas from four NT BN rats were also processed for immunohistologic examination.

Histologic Evaluation

The tracheal transplants were examined with light microscopy (Nikon Labophot-2; Nikon Inc., Melville, NY). The percentage of airway covered by epithelium, and the differences in tissue architecture, were evaluated.

Morphometry

Video images of elastic trichrome-stained tracheal sections were taken with a microscope (Nikon Labophot) and an attached video camera (Model TMC-7I; Pulnix America Inc., Sunnyvale, CA) and transferred to a computer-imaging 1280 morphometric system (Compix Inc., Cranberry Township, PA). We modified the method used by Reichenspurner and coworkers to quantitate the percentage of luminal obliteration (18). Briefly, the inner surface of the cartilage ring was outlined with the cursor. A line drawn through the membranous portion of the tracheal tissue connected the two ends of the cartilage. The area within this circle was calculated (inner tracheal ring area). Next, the cursor was used to outline the luminal area. This area was then calculated (luminal area). The percentage of obliteration of the inner tracheal ring was then calculated as follows: 100  (luminal area/inner tracheal ring area × 100). The inner tracheal ring area contained the epithelium and submucosa, and the average percentage of obliteration in the NT trachea was therefore approximately 5% (data not shown).

Immunohistochemistry

Serial frozen sections (4 to 6 µm) were air-dried onto silane-coated slides and were fixed in acetone at 20° C for 15 min. The fixed slides were washed in phosphate-buffered saline (PBS). After being blocked with 10% bovine serum albumin (BSA) in PBS for 15 min, the frozen sections were incubated with mouse monoclonal anti-ACE Clone 9B9 IgG antibody (100 µg per 0.1 ml) (Research Diagnostics Inc., Flanders, NJ) at a dilution of 1:100 at room temperature (RT) for 1 h. The primary antibody was diluted in PBS with 0.1% BSA. With intervening washes in PBS, the following steps were performed: exposure to biotinylated antimouse immunoglobulins and biotinylated antirabbit immunoglobulins at RT for 10 min; exposure to strepavidin-conjugated alkaline phosphatase at RT for 10 min (LSAB 2 Kit Alkaline Phosphatase for Use on Rat Specimens; Dako Corp., Carpinteria, CA); and detection of a positive reaction with the Fast Red Substrate-Chromagen System (Dako) with levamisole (Dako), which yields a red reaction product. The specimens were counterstained with hematoxylin, and coverslips were mounted on the specimens (Glycergel; Dako). For negative controls, the primary antibody was excluded and the staining procedure was otherwise performed as it was with antibody. In addition, sections were treated with mouse IgG in place of the primary antibody, in order to evaluate primary antibody specificity.

Immunohistochemical Semiquantification

The intensity of staining was assigned by consensus of two observers in a blinded review, and was scored from 0 to 4 as follows: 0 = no staining; 1 = detectable staining; 2 = light staining; 3 = intermediate staining; and 4 = heavy staining. The following areas of each specimen were examined: obliterated portion; epithelium; lamina propria: tissue and vessels; and serosa: tissue and vessels. If any of these structures were not present, this was noted as such.

Statistical Analyses

Data are expressed as mean ± SEM. Parametric data were analyzed through one-way analysis of variance followed by Dunnett's test. A value of p < 0.05 was considered significant. Data analysis was performed with SigmaStat version 1.0 statistical software (Jandel Scientific, San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

One animal in the allograft control group died approximately 1 h after the transplant operation. All other animals survived the study period, and no side effects were observed.

ACE Expression in Isografts, Allografts, and NT Tracheas

We examined immunohistochemical staining for ACE in isograft and allograft control tracheas at Day 21 after heterotopic transplantation, and also in NT tracheas (Table 1). The epithelium of both the isografts and NT tracheas showed detectable ACE staining. Both the NT and isograft tracheas had ACE staining on the vessels in the lamina propria, although the staining in the isograft tracheas is less than in the NT tracheas (the difference was not statistically significant). As compared with NT and isograft tracheas, allograft control tracheas failed to show ACE-positive vessels in the lamina propria. This is also the portion of the trachea that is most heavily infiltrated with inflammatory cells and is more severely affected by ischemic and necrotic processes early in the time course of fibrotic obliteration (3). The serosal tissue and vessels in the isografted, allografted, and NT tracheas showed a similar extent of ACE staining. The allografts showed an obliterated portion with heavy ACE staining (Figure 1). This area was not present in NT or isograft tracheas. No background staining was present when the primary antibody was omitted or replaced with mouse IgG.

View larger version (74K): [in this window] [in a new window]   Figure 1.   Photomicrographs of obliterated portions of Day-21 allograft control tracheas: (A) Hematoxylin and eosin-stained section, showing fibroblasts and cellular infiltration. (B) Immunohistochemical staining for ACE shows ACE-positive fibroblasts. (C ) Negative control does not reveal background staining (original magnification: ×400).

Epithelium and Tissue Architecture in Isograft Controls, Allograft Controls and Captopril-Treated Allografts

At Day 21, the respiratory epithelium was well preserved and histologically normal in all isografts, whereas the epithelium had disappeared from allograft controls. Captopril treatment beginning at the three timepoints relative to transplantation had no protective effect on the loss of airway epithelium in the allografts. Isograft controls retained normal tissue architecture. Captopril-treated and control allografts lost this architecture as the tissue was infiltrated by fibroblasts and inflammatory cells.

Morphometric Analysis of Percentage Obliteration of Control and Captopril-Treated Allografts

The allograft control tracheas were 83.3 ± 10.0% obliterated by fibroproliferative tissue by Day 21 after transplantation (Figures 2 and 3). In rats in which captopril treatment was begun on preoperative Day 5 or on postoperative Day 1, the extent of tracheal luminal obliteration showed a statistically significant decrease at Day 21 (45.1 ± 6.9% and 25.9 ± 2.5%, respectively) from that of allograft controls (p < 0.05). No protective effect was observed when captopril administration was begun on postoperative Day 5. 

View larger version (15K): [in this window] [in a new window]   Figure 2.   Percent obliteration of tracheal lumen by fibroproliferative tissue, as measured with computerized morphometry for allograft control and captopril-treated tracheas. Captopril started on preoperative Day 5 or postoperative Day 1 resulted in significantly less obliteration than in allograft controls (p < 0.05).

View larger version (74K): [in this window] [in a new window]   Figure 3.   Photomicrographs of (A) allograft control (B) captopril-treated tracheas with treatment from preoperative Day 5 (Preop), (C) postoperative Day 1 (Pod1), or (D) postoperative Day 5 (Pod5). (Elastic trichrome staining; original magnification: ×100.) Captopril administration from preoperative Day 5 (A) or postoperative Day 1 (B) to postoperative Day 21 significantly attenuated luminal obliteration. No restorative effect was seen for the epithelium or lamina propria for any of the treatment groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we showed immunohistochemically the presence of ACE in the fibroproliferative tissue of allografts in a rat heterotopic tracheal transplant model of posttransplantation airway obliteration. We also provided evidence by morphometric analysis of the antifibrotic potential of captopril administration in this model. To our knowledge, this is the first report to suggest a role for the angiotensin system in the development of posttransplantation airway obliteration.

The rat heterotopic tracheal transplant model of posttransplantation airway obliteration shares pathologic features with BO (3). In rats, at 21 d after tracheal transplantation, isografts fail to develop the fibrotic obliterative lesion typical of BO, whereas allografts do. This suggests that the fibroproliferative response is in part immune mediated or allogeneic in nature. In humans, the lesion begins with epithelial damage followed by obliteration with granulation tissue composed of mainly type I and III collagen and fibroblasts (19). This chronologic sequence of epithelial and fibroproliferative changes has been demonstrated in the rat model (3). It should be noted that the failure of this model to include immunosuppression limits the resemblance of the model to the clinical setting in which BO develops.

The immunohistochemically heavy ACE staining observed in the fibroproliferative tissue in this rat model provides a target for study and manipulation in the hope of better understanding the complexity of BO. The angiotensin system has been implicated in the pathogenesis of chronic rejection in other organ systems, such as coronary arteriopathy following heart transplantation (15, 16). A role for the angiotensin system has also been demonstrated in the development of pulmonary fibrosis (20).

The angiotensin system has a complex role in the development of fibrous tissue. The system has two main effector peptides: ACE and angiotensin II. In the classic description of the angiotensin system, circulating angiotensin I is cleaved by ACE to angiotensin II. ACE is found in its highest concentration on pulmonary endothelial cells (21). However, investigators have shown that in fibrosis, this relationship is complicated by the influence of other peptides and cells. For instance, ACE has been localized to macrophages, smooth muscle, epithelial cells, and myofibroblasts (10, 22, 23).

The antifibrotic effect of ACE inhibition can occur through various mediators. ACE inhibition leads to a decrease in the generation of angiotensin II and a resultant downregulation of transforming growth factor (TGF)-₁ production. Fewer fibroblasts are transformed to myofibroblasts, and less collagen is therefore deposited (11). As shown by Linz and coworkers, ACE can also act as a kininase II and degrade bradykinin (24). Linz and coworkers examined the effect of ACE inhibitor treatment on cardiac remodeling by pretreating kinin-replete and kinin-deficient rats with an ACE inhibitor. The ACE inhibitor attenuated infarct size and resulted in lower end-diastolic pressure in kinin-replete rats but not in kinin-deficient rats. The beneficial effect of ACE inhibition in their model was therefore likely to have occurred through the prevention of bradykinin degradation. Ward and coworkers showed that captopril administration in a rat model of lung radiation injury decreased the accumulation of hydroxyproline and mast cells (25). Crawford and coworkers showed that ACE inhibition suppresses platelet activating factor (PAF) synthesis in a rat heterotopic cardiac transplant model. This hypothesis was based on the ability of angiotensin II to activate phospholipase A, which is an important enzyme in the synthesis of PAF (16).

Enzymes other than ACE are capable of generating angiotensin II, and this ability appears to be tissue- and species-dependent. Chymase (on mast cells), and cathepsin G (expressed on neutrophils) are both angiotensin II-generating systems (26, 27). Voors and coworkers examined the effect of inhibition of ACE and chymase on angiotensin II formation in in vitro segments of human internal mammary arteries. They found that the inhibition was greatest when inhibitors of both enzymes were administered (28). However, in a rat model of glomerulonephritis, the effect of blocking both ACE and one type of angiotensin II receptor (type 1) on the expression of TGF-₁ was not superior to that of either ACE inhibition or of angiotensin II receptor blocking alone (29).

We chose a dose of captopril that has been shown to be effective in inhibiting the proliferation of fibrous tissue in other models (30). It is possible that the antifibrotic effect of captopril is dose dependent, and this needs to be studied. We selected three time points from which to begin captopril administration. Five days before transplantation was chosen because the fibroproliferative lesion that follows transplantation has been shown to develop from mesenchymal cells resident in the area in which the trachea is implanted (31). This was therefore an attempt to "pretreat" these cells. Postoperative Day 1 was chosen as a clinically relevant timepoint. At 24 h after a lung transplant procedure, the majority of patients would be able to either begin oral or intravenous ACE inhibition. Postoperative Day 5 was selected because of the effect of the angiotensin system on TGF-₁. Investigators have described the ability of angiotensin II to upregulate TGF-₁ expression, with the resultant transformation of fibroblasts into myofibroblasts and the deposition of collagens. TGF-₁ has been shown to have early immunosuppressive effects and later fibroproliferative effects (32, 33). We hypothesized that if this system works through TGF-₁, then it might be better to downregulate this pathway after its initial beneficial immunosuppressive effects have occurred.

Captopril administration did not help preserve the airway epithelium. Captopril did, however, decrease luminal obliteration by fibrous tissue. This result was expected, as inhibition of ACE has been shown to inhibit the fibroproliferative process rather than prevent the ischemic loss of epithelium. When captopril administration was started either 5 d preoperatively or on postoperative Day 1, and was continued until postoperative Day 21, we noted a significant attenuation of tracheal luminal obliteration. The same effect did not occur when captopril administration began on postoperative Day 5. It is possible that the fibroproliferative effects of angiotensin II have already started by this time, and that the administration of captopril at this point is simply too late to be beneficial. It is also possible that by postoperative Day 5 the number of cells that are capable of producing ACE has increased beyond the capacity of the dose of captopril delivered to inhibit them, and that this is why early inhibition is more effective. On the other hand, perhaps an enzyme other than ACE generates angiotensin II at this point in the time course of rejection. This could also explain why ACE inhibition was not 100% effective in attenuating fibrous airway obliteration.

The work presented here is the first examination of the angiotensin system in a model of posttransplantation airway obliteration. We found that oral captopril administration starting either 5 d before transplantation or from postoperative Day 1 to postoperative Day 21 was effective in preventing fibrous airway obliteration in the rat model used in our study. The mechanisms by which captopril was effective in attenuating fibrous obliteration in this model are the focus of ongoing studies in our laboratory. It is important to ultimately address, the clinical relevance of our findings and their therapeutic implications.