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Microvascular changes in small airways predispose to obliterative bronchiolitis after lung transplantation
LUNG REJECTION
Microvascular Changes in Small Airways Predispose to Obliterative Bronchiolitis After Lung Transplantation Heyman Luckraz, FRCS,a Martin Goddard, FRCPath,b Keith McNeil, FRACP,a Carl Atkinson, BSc,b Susan C. Charman, MSc,c Susan Stewart, FRCPath,b and John Wallwork, FRCSa Background: There is strong evidence that obliterative bronchiolitis (OB) in lung transplant recipients is related to acute rejection as graded by parenchymal perivascular infiltrates. OB (chronic rejection) is a small airways, rather than a parenchymal, scarring process. Moreover, there has been no study of the microcirculation in the small airways in lung transplantation. This study assesses the microvasculature around small airways (SA) in post-mortem lung allograft specimens. Methods: The microvasculature of SA (n ⫽ 19) from 5 patients who died within 24 hours of lung transplantation (Group A) and SA in OB lungs (11 patients, median post-transplant survival 1,371 days) was assessed by the use of monoclonal antibodies to the vascular endothelium, namely von Willebrand factor (vWF) and CD31. The second group was further sub-divided into Group B (airways not obliterated, n ⫽ 18), Group C (sub-total airways obliteration, n ⫽ 21) and Group D (airways with total luminal obstruction, n ⫽ 14). Results: The measured median circumference of the SA in the 4 groups was 2.1, 2.1, 2.5 and 2.3 mm, respectively (p ⫽ 0.66). Using CD31 as the endothelial marker, the median number of blood vessels per unit length of airway circumference (BVPL) was 3.5 vessels/mm for Group A, 0.8 for Group B, 1.3 for Group C and 2.8 for Group D, (p ⬍ 0.001). Large blood vessels (circumference ⬎0.20 mm) were present in 95%, 11%, 14% and 21% of each group, respectively (p ⬍ 0.001). Similar trends were confirmed with the vWF endothelial antibodies. Conclusions: OB after lung transplantation is associated with a decrease in microvascular supply to the small airway. This ischemic event may lead to airway damage or increase the tendency to repair by scarring. The small airways then appear to respond to this insult by angiogenesis, which may either occur too late to prevent permanent airway damage or be inadequate in restoring adequate blood supply to the airway. J Heart Lung Transplant 2004;23:527–31.
Over 15,000 lung transplantations have been carried out worldwide in the past 15 years.1 Establishment of lung transplant programs has been successful with most centers achieving an 85% and 75% 30-day and 1-year survival, respectively.1 This has been possible due to better recipient/donor selection criteria, meticulous and well-established surgical approaches, improved early post-operative management and state-of-the-art immunomodulating therapy. Unfortunately, the longterm outcome remains bleak with only 40% of patients surviving beyond 5 years. The most common cause of death in long-term survivors is obliterative bronchiolitis (OB); that is, chronic rejection, confirmed histologically in around 30% of patients.1 From the aTransplant Unit and bPathology Department, Papworth Hospital, Cambridge, UK and cMRC Biostatistics Unit, Cambridge, UK. Submitted January 30, 2003; accepted July 1, 2003. Reprint requests: Heyman Luckraz, FRCS, Transplant Unit, Papworth Hospital, Cambridge CB3 8RE, UK. Telephone: 00-44-1480-830541. Fax: 00-44-1480-364-610. E-mail: [email protected] Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter. doi:10.1016/ j.healum.2003.07.003
The 2 main risk factors for OB are acute rejection (AR) and cytomegalovirus (CMV) infection.2 CMV is known to infect endothelial cells and may initiate endothelialitis, whereas acute rejection is characterized and graded as parenchymal perivascular infiltrates with endothelialitis in higher grades and has associated airways involvement.3 Infiltrates around airway vessels are not included in the current acute rejection grading schema. OB represents an intraluminal scarring process that plugs the small airways. There is some evidence that OB is an immune-driven pathology,4,5 but so far no study explained the difference in anatomic distribution of AR and OB. Moreover, bronchial arterial revascularization has reduced the complications of the major airways (i.e., ischemia at level of airways anastomosis), but has failed to reduce the incidence of OB.6 These investigators ruled out ischemia as a cause of OB, as it was presumed that bronchial arterial revascularization would improve blood and nutrient delivery to the distal airways. However, they assessed only the macrocirculation, not the microvascular supply, of the distal airways. We hypothesized that the scarring seen in OB repre527
528
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sents the response of the small airways (SA) to repeated insults such as infections in the setting of local ischemia. The latter develops as a result of damage to the microvascular supply of the SA. The damaged airways then respond by repair, including angiogenesis, which may occur too late to prevent permanent small airways changes. Thus, we assessed the changes in morphology and density of microvessels around the SA in lung allografts that developed OB.
METHODS Lung Tissue Patients who underwent successful lung transplantation at Papworth Hospital, surviving ⬎1,000 days postoperatively and who died of OB as confirmed by histologic examination of post-mortem lung, were selected for study (n ⫽ 11). Five lung transplant recipients who died within 24 hours of surgery were used as controls. The Huntingdon local research ethics committee approved the study. The lung material was fixed in 10% phosphate-buffered formalin, processed to paraffin, and then sectioned at 4 m. Each sample was stained with hematoxylin and eosin and elastic van Gieson stain to evaluate the presence of OB in the airways and for morphometric analysis of the airway circumference. Immunocytochemistry To assess the microvasculature around both the SA and within the luminal scar, characteristic of OB an immunocytochemistry technique was employed. To identify cells of endothelial lineage, the monoclonal anti-endothelial antibodies CD31 (1:30 Dako, UK) and vWF (1:20 Dako, UK) were used. Paraffin sections were antigen retrieved using a microwave (Euroserv 750 W) and 0.01 mol/liter sodium bicarbonate antigen-retrieval solution for CD31 and proteinase K enzyme retrieval for 10 minutes for vWF. Endogenous peroxidase activity was quenched by treatment with hydrogen peroxide (Dako, UK). Sections were then rinsed in phosphate-buffered saline (PBS) and then incubated with primary antibody for 1 hour. Antigens were visualized with a labeled streptavidin– biotin complex and visualized with 3,3⬘ diaminobenzidine tetrahydrochloride, producing a brown reaction product. Sections were then counterstained with Carazzi’s hematoxylin. Normal lung tissue obtained at lung resection surgery was used as positive control for each antibody. Specificity of the antibodies was confirmed by omission of primary antibody. All staining was carried out using the Dako Chemate 500 autostainer to maintain consistency in the staining process.
Figure 1. (a) High-power magnification (original magnification ⫻20) of part of the small airway (SA) to demonstrate the morphology and density of microvessels on the muscular layer of the normal SA. The specimen has been doubly stained with vWF and smooth muscle actin antibodies. (b) Annotated image of the SA in (a) showing the muscle layer (brown) of the SA and the blood vessels (red) supplying the SA.
Image Analysis The microvascular density around the SA was quantified using a computerized image analysis package (AEQUITAS IA). Figure 1 shows a normal SA with the smooth muscle layer and blood vessels supplying that airway. Although the blood vessels within the obliterated lumen of the SA were assessed, this was not included in the analysis as it was difficult to differentiate between vessels within the intraluminal OB scar and those due to intraluminal granulation tissue. Statistical Analysis Data are expressed as mean ⫾ standard deviation, median (interquartile range) and percentage. Non-parametric data were analyzed by the Mann–Whitney U-test and Kruskal–Wallis test. The presence of large blood vessels around the SA in the 4 groups was compared by
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Pearson’s chi-square test p ⬍ 0.05 was considered statistically significant. RESULTS The mean recipient and donor ages for the OB and control groups were 33.7 ⫾ 11.6 and 26.2 ⫾ 13.9 years, and 37 ⫾ 9.9 and 33.8 ⫾ 11 years, respectively (p ⫽ 0.61 and 0.28, respectively). The median ischemic times were 164 (13 to 234) minutes for the OB group and 255 (210 to 309) minutes for the control group (p ⫽ 0.03). Median post-operative survival for the OB patients was 1,371 (1,190 to 2,895) days. For that group, the mean number of chest infections during that period was 8.5 episodes. CMV pneumonitis was identified in the lung biopsies in 3 patients and the mean number of clinically significant acute rejection episodes per 100 patient-days was 2.7. Nineteen small airways were identified and assessed in the control group (Group A). The SA of patients in the OB category were grouped according to the degree of luminal obliteration: Group B, no obliteration (n ⫽ 18); group C, sub-total obliteration (n ⫽ 21); and group D, complete obliteration (n ⫽ 14). The measured median external circumference of the SA using elastic van Gieson was 2.1 (1.7 to 2.9), 2.1 (1.5 to 2.7), 2.5 (1.8 to 3.2) and 2.3 (1.7 to 3.2) mm for Groups A, B, C and D, respectively (p ⫽ 0.66). Using immunohistochemical marker CD31, the median number (interquartile range) of blood vessels (supplying the SA) per unit length of airway circumference (BVPL) was 3.5 (2.4 to 5.2) vessels/mm for Group A and 0.8 (0.4 to 1.5), 1.3 (1.3 to 2.2) and 2.8 (1.4 to 4.6) for Group B, C and D, respectively (p ⬍ 0.001) (Figure 2a). Large blood vessels (LBV) (circumference ⬎0.20 mm) were present in 95%, 11%, 14% and 21% of the groups, respectively (p ⬍ 0.001). With the vWF endothelial antibodies, the median BVPL were 3.1 (2.6 to 3.9), 0.7 (0.5 to 1.3), 0.8 (0.5 to 2.1) and 3.1 (1.1 to 4.4) (p ⬍ 0.001), and the presence of LBV was noted in 100%, 12.5%, 20% and 0% for Groups A, B, C and D, respectively (p ⬍ 0.001). DISCUSSION The microvasculature of the distal airways was investigated previously by Reid et al over 3 decades ago.7,8 Their description of the blood supply to normal SA was similar to that of the control group (Group A) in our study. However, they reported mainly on the microvascular changes that occurred during the lifespan of a normal individual with a brief assessment of the microvasculature in patients with primary pulmonary hypertension and congenital cardiac defects. Changes in the microvasculature of the airways in chronic inflammatory lung diseases have been reported only recently.9,10 The present study has enabled us to link the clinical and histologic pathogeneses involved in the develop-
Figure 2. Median number of blood vessels per unit length of SA circumference (BVPL) using (a) CD31 and (b) vWF for the 4 groups.
ment of OB. Several studies, including our own work at Papworth, have identified acute rejection and cytomegalovirus pneumonitis as the main culprits in developing OB.2,11,12 In the past, an ischemic pathology was not favored as bronchial arterial revascularization (BAR) failed to prevent OB.6 However, the investigators studied only the macrocirculation associated with BAR, not comment the microcirculation. Moreover, there is an extensive anastomotic network between pulmonary and bronchial circulation at the level of the SA and thus BAR does not add any benefit in terms of SA blood supply. From these data, it appears that during the lifespan of the lung allograft, there is damage to the microcirculation of the SA, and hence a reduction in BVPL in the non-obstructed and partially obstructed airways as compared with the control group. As ischemia sets in, due to the reduction in blood supply, the SA responds to insults such as chest infections and rejection, by a
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Figure 3. Four-fold magnification of an OB-affected small airway to demonstrate the altered morphology and increased density of microvessels on the muscular layer. The specimen has been doubly stained with vWF and smooth muscle actin antibodies.
scarring process, OB. This is supported by the work of Adams et al,13 who demonstrated that, in the setting of a reduction in the blood supply to the tracheal isograft in rats, damage to the respiratory epithelium healed by a scarring process similar to OB. Thus, repeated pulmonary infective and or inflammatory processes in the ischemic setting, as confirmed by our study, may lead to the development of OB. This mechanism is further supported by joint work published by the Stanford and Helsinki group.14 In their model, they showed that airway ischemia induced by transplanting lung fragments of 1 cm3 is reversible in autografts (in the absence of an immune-mediated injury), as compared with lung allografts. In the latter, the ischemic insult of the transplant coupled with the persistence of acute rejection (a vascular-based pathology) led to the development of OB in their swine model. There is evidence that fibroblasts in the OB scar release nitric oxide (NO),15,16 a potent angiogenetic factor.17–19 NO upregulates the transcription of vascular endothelial cell growth factor (VEGF), which increases vascular permeability,20 and along with NO (a vasodilator) causes extravasation of plasma proteins. Among these are proteinases that degrade the extracellular matrix and metalloproteinases that promote new vessel growth via VEGF, basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1).21 Thus, new vessels sprout around the ischemic SA and within its scarred lumen (Figures 1 and 3). This explains the differences seen in BVPL between non-obliterated and partially and completely obliterated airways (Groups B, C and D). It also accounts for the changes in vessels morphology as visualized by the endothelial markers used in this study. The angiogenetic vessels are smaller
The Journal of Heart and Lung Transplantation May 2004
compared with the native microvasculature around the SA (Figure 1). This confirmed pathologic process explains the clinical links between the risk factors for OB and its subsequent development. The patients included in this study developed OB at a later stage, probably because they had a small number of acute rejection episodes. We now intend to perform a longitudinal study to assess the effect of infection and acute rejection episodes on the BVPL of the SA. This may lead to a reappraisal of our current grading of acute rejection in lung transplantation to include peri-bronchial blood vessels. In addition, it will provide potential targets for both prevention and treatment of OB. In conclusion, long-term success in lung transplantation has so far been hampered by the early, seemingly irreversible development of the chronic rejection process, namely OB. This study proposed and confirmed the link between microvascular damage and development of OB. Further studies will identify the critical timing of this damage and therapeutic approaches could then be formulated to reduce or even prevent OB. REFERENCES 1. Hertz MI, Taylor DO, Trulock EP, et al. The registry of the International Society for Heart and Lung Transplantation: nineteenth Official Report—2002. J Heart Lung Transplant 2002;21:950 –70. 2. Heng D, Sharples L, McNeil K, Stewart S, Wreghitt T, Wallwork J. Bronchiolitis obliterans syndrome: incidence, natural history, prognosis and risk factors. J Heart Lung Transplant 1998;17:1255–63. 3. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1–15. 4. Duncan SR, Valentine V, Roglic M, et al. T-cell receptor biases and clonal proliferations among lung transplant recipients with obliterative bronchiolitis. J Clin Invest 1996;97:2642–50. 5. Al-Dossari GA, Kshettry VR, Jessurun J, Bolman RM, III. Experimental large animal model of obliterative bronchiolitis after lung transplantation. Ann Thorac Surg 1994; 53:34 –40. 6. Norgaard MA, Andersen CB, Pettersson G. Does bronchial artery revascularization influence results concerning bronchiolitis obliterans syndrome and/or obliterative bronchiolitis after lung transplantation? Eur J CTS 1998; 14:311–8. 7. Reid L, Meyrick B. Microcirculation: definition and organization at tissue level. Ann NY Acad Sci 1982:3–20. 8. Reid LM. The pulmonary circulation: remodelling in growth and disease. Am Rev Respir Dis 1979;119:531–46. 9. McDonald D. Angiogenesis and remodelling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001;164(suppl):S39 –S45.
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10. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 2001;56:902–6. 11. Paradis I. Bronchiolitis obliterans: pathogenesis, prevention and management. Am J Med Sci 1998;315(3):161–78. 12. Girgis RE, Tu I, Berry GJ, et al. Risk factors for the development of obliterative bronchiolitis after lung transplantation. J Heart Lung Transplant 1996;15:1200 –8. 13. Adams BF, Brazelton T, Berry GJ, Morris RE. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000;69:661–3. 14. Ikonen T, Uusitalo M, Taskinen E, et al. Small airway obliteration in a new swine heterotopic lung and bronchial allograft model. J Heart Lung Transplant 1998;17: 945–53. 15. Gabbay E, Walters EH, Orsida B, et al. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide levels and epithelial inducible nitric oxide synthase. Am J Crit Care Med 2000;162(6):2182–7. 16. Mason NA, Springall DR, Pomerance A, Evans TJ, Yacoub
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18.
19. 20.
21.
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MH, Polak JM. Expression of inducible nitric oxide synthase and formation of peroxynitrite in post-transplant obliterative bronchiolitis. J Heart Lung Transplant 1998; 17:710 –4. Conway EM, Collen D, Carmeliat P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2001;49: 507–21. Thompson WD, Li WW, Maragoudakis M. The clinical manipulation of angiogenesis: pathology, side-effects, surprises and opportunities with novel human therapies. J Pathol 1999;187:503–10. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res 2000;87:434 –9. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000;95:189 –97. Nelson AR, Fingleton B, Rothengerg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135–49.
Microvascular Changes in Small Airways Predispose to Obliterative Bronchiolitis After Lung Transplantation Heyman Luckraz, FRCS,a Martin Goddard, FRCPath,b Keith McNeil, FRACP,a Carl Atkinson, BSc,b Susan C. Charman, MSc,c Susan Stewart, FRCPath,b and John Wallwork, FRCSa Background: There is strong evidence that obliterative bronchiolitis (OB) in lung transplant recipients is related to acute rejection as graded by parenchymal perivascular infiltrates. OB (chronic rejection) is a small airways, rather than a parenchymal, scarring process. Moreover, there has been no study of the microcirculation in the small airways in lung transplantation. This study assesses the microvasculature around small airways (SA) in post-mortem lung allograft specimens. Methods: The microvasculature of SA (n ⫽ 19) from 5 patients who died within 24 hours of lung transplantation (Group A) and SA in OB lungs (11 patients, median post-transplant survival 1,371 days) was assessed by the use of monoclonal antibodies to the vascular endothelium, namely von Willebrand factor (vWF) and CD31. The second group was further sub-divided into Group B (airways not obliterated, n ⫽ 18), Group C (sub-total airways obliteration, n ⫽ 21) and Group D (airways with total luminal obstruction, n ⫽ 14). Results: The measured median circumference of the SA in the 4 groups was 2.1, 2.1, 2.5 and 2.3 mm, respectively (p ⫽ 0.66). Using CD31 as the endothelial marker, the median number of blood vessels per unit length of airway circumference (BVPL) was 3.5 vessels/mm for Group A, 0.8 for Group B, 1.3 for Group C and 2.8 for Group D, (p ⬍ 0.001). Large blood vessels (circumference ⬎0.20 mm) were present in 95%, 11%, 14% and 21% of each group, respectively (p ⬍ 0.001). Similar trends were confirmed with the vWF endothelial antibodies. Conclusions: OB after lung transplantation is associated with a decrease in microvascular supply to the small airway. This ischemic event may lead to airway damage or increase the tendency to repair by scarring. The small airways then appear to respond to this insult by angiogenesis, which may either occur too late to prevent permanent airway damage or be inadequate in restoring adequate blood supply to the airway. J Heart Lung Transplant 2004;23:527–31.
Over 15,000 lung transplantations have been carried out worldwide in the past 15 years.1 Establishment of lung transplant programs has been successful with most centers achieving an 85% and 75% 30-day and 1-year survival, respectively.1 This has been possible due to better recipient/donor selection criteria, meticulous and well-established surgical approaches, improved early post-operative management and state-of-the-art immunomodulating therapy. Unfortunately, the longterm outcome remains bleak with only 40% of patients surviving beyond 5 years. The most common cause of death in long-term survivors is obliterative bronchiolitis (OB); that is, chronic rejection, confirmed histologically in around 30% of patients.1 From the aTransplant Unit and bPathology Department, Papworth Hospital, Cambridge, UK and cMRC Biostatistics Unit, Cambridge, UK. Submitted January 30, 2003; accepted July 1, 2003. Reprint requests: Heyman Luckraz, FRCS, Transplant Unit, Papworth Hospital, Cambridge CB3 8RE, UK. Telephone: 00-44-1480-830541. Fax: 00-44-1480-364-610. E-mail: [email protected] Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter. doi:10.1016/ j.healum.2003.07.003
The 2 main risk factors for OB are acute rejection (AR) and cytomegalovirus (CMV) infection.2 CMV is known to infect endothelial cells and may initiate endothelialitis, whereas acute rejection is characterized and graded as parenchymal perivascular infiltrates with endothelialitis in higher grades and has associated airways involvement.3 Infiltrates around airway vessels are not included in the current acute rejection grading schema. OB represents an intraluminal scarring process that plugs the small airways. There is some evidence that OB is an immune-driven pathology,4,5 but so far no study explained the difference in anatomic distribution of AR and OB. Moreover, bronchial arterial revascularization has reduced the complications of the major airways (i.e., ischemia at level of airways anastomosis), but has failed to reduce the incidence of OB.6 These investigators ruled out ischemia as a cause of OB, as it was presumed that bronchial arterial revascularization would improve blood and nutrient delivery to the distal airways. However, they assessed only the macrocirculation, not the microvascular supply, of the distal airways. We hypothesized that the scarring seen in OB repre527
528
Luckraz, et al.
The Journal of Heart and Lung Transplantation May 2004
sents the response of the small airways (SA) to repeated insults such as infections in the setting of local ischemia. The latter develops as a result of damage to the microvascular supply of the SA. The damaged airways then respond by repair, including angiogenesis, which may occur too late to prevent permanent small airways changes. Thus, we assessed the changes in morphology and density of microvessels around the SA in lung allografts that developed OB.
METHODS Lung Tissue Patients who underwent successful lung transplantation at Papworth Hospital, surviving ⬎1,000 days postoperatively and who died of OB as confirmed by histologic examination of post-mortem lung, were selected for study (n ⫽ 11). Five lung transplant recipients who died within 24 hours of surgery were used as controls. The Huntingdon local research ethics committee approved the study. The lung material was fixed in 10% phosphate-buffered formalin, processed to paraffin, and then sectioned at 4 m. Each sample was stained with hematoxylin and eosin and elastic van Gieson stain to evaluate the presence of OB in the airways and for morphometric analysis of the airway circumference. Immunocytochemistry To assess the microvasculature around both the SA and within the luminal scar, characteristic of OB an immunocytochemistry technique was employed. To identify cells of endothelial lineage, the monoclonal anti-endothelial antibodies CD31 (1:30 Dako, UK) and vWF (1:20 Dako, UK) were used. Paraffin sections were antigen retrieved using a microwave (Euroserv 750 W) and 0.01 mol/liter sodium bicarbonate antigen-retrieval solution for CD31 and proteinase K enzyme retrieval for 10 minutes for vWF. Endogenous peroxidase activity was quenched by treatment with hydrogen peroxide (Dako, UK). Sections were then rinsed in phosphate-buffered saline (PBS) and then incubated with primary antibody for 1 hour. Antigens were visualized with a labeled streptavidin– biotin complex and visualized with 3,3⬘ diaminobenzidine tetrahydrochloride, producing a brown reaction product. Sections were then counterstained with Carazzi’s hematoxylin. Normal lung tissue obtained at lung resection surgery was used as positive control for each antibody. Specificity of the antibodies was confirmed by omission of primary antibody. All staining was carried out using the Dako Chemate 500 autostainer to maintain consistency in the staining process.
Figure 1. (a) High-power magnification (original magnification ⫻20) of part of the small airway (SA) to demonstrate the morphology and density of microvessels on the muscular layer of the normal SA. The specimen has been doubly stained with vWF and smooth muscle actin antibodies. (b) Annotated image of the SA in (a) showing the muscle layer (brown) of the SA and the blood vessels (red) supplying the SA.
Image Analysis The microvascular density around the SA was quantified using a computerized image analysis package (AEQUITAS IA). Figure 1 shows a normal SA with the smooth muscle layer and blood vessels supplying that airway. Although the blood vessels within the obliterated lumen of the SA were assessed, this was not included in the analysis as it was difficult to differentiate between vessels within the intraluminal OB scar and those due to intraluminal granulation tissue. Statistical Analysis Data are expressed as mean ⫾ standard deviation, median (interquartile range) and percentage. Non-parametric data were analyzed by the Mann–Whitney U-test and Kruskal–Wallis test. The presence of large blood vessels around the SA in the 4 groups was compared by
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Pearson’s chi-square test p ⬍ 0.05 was considered statistically significant. RESULTS The mean recipient and donor ages for the OB and control groups were 33.7 ⫾ 11.6 and 26.2 ⫾ 13.9 years, and 37 ⫾ 9.9 and 33.8 ⫾ 11 years, respectively (p ⫽ 0.61 and 0.28, respectively). The median ischemic times were 164 (13 to 234) minutes for the OB group and 255 (210 to 309) minutes for the control group (p ⫽ 0.03). Median post-operative survival for the OB patients was 1,371 (1,190 to 2,895) days. For that group, the mean number of chest infections during that period was 8.5 episodes. CMV pneumonitis was identified in the lung biopsies in 3 patients and the mean number of clinically significant acute rejection episodes per 100 patient-days was 2.7. Nineteen small airways were identified and assessed in the control group (Group A). The SA of patients in the OB category were grouped according to the degree of luminal obliteration: Group B, no obliteration (n ⫽ 18); group C, sub-total obliteration (n ⫽ 21); and group D, complete obliteration (n ⫽ 14). The measured median external circumference of the SA using elastic van Gieson was 2.1 (1.7 to 2.9), 2.1 (1.5 to 2.7), 2.5 (1.8 to 3.2) and 2.3 (1.7 to 3.2) mm for Groups A, B, C and D, respectively (p ⫽ 0.66). Using immunohistochemical marker CD31, the median number (interquartile range) of blood vessels (supplying the SA) per unit length of airway circumference (BVPL) was 3.5 (2.4 to 5.2) vessels/mm for Group A and 0.8 (0.4 to 1.5), 1.3 (1.3 to 2.2) and 2.8 (1.4 to 4.6) for Group B, C and D, respectively (p ⬍ 0.001) (Figure 2a). Large blood vessels (LBV) (circumference ⬎0.20 mm) were present in 95%, 11%, 14% and 21% of the groups, respectively (p ⬍ 0.001). With the vWF endothelial antibodies, the median BVPL were 3.1 (2.6 to 3.9), 0.7 (0.5 to 1.3), 0.8 (0.5 to 2.1) and 3.1 (1.1 to 4.4) (p ⬍ 0.001), and the presence of LBV was noted in 100%, 12.5%, 20% and 0% for Groups A, B, C and D, respectively (p ⬍ 0.001). DISCUSSION The microvasculature of the distal airways was investigated previously by Reid et al over 3 decades ago.7,8 Their description of the blood supply to normal SA was similar to that of the control group (Group A) in our study. However, they reported mainly on the microvascular changes that occurred during the lifespan of a normal individual with a brief assessment of the microvasculature in patients with primary pulmonary hypertension and congenital cardiac defects. Changes in the microvasculature of the airways in chronic inflammatory lung diseases have been reported only recently.9,10 The present study has enabled us to link the clinical and histologic pathogeneses involved in the develop-
Figure 2. Median number of blood vessels per unit length of SA circumference (BVPL) using (a) CD31 and (b) vWF for the 4 groups.
ment of OB. Several studies, including our own work at Papworth, have identified acute rejection and cytomegalovirus pneumonitis as the main culprits in developing OB.2,11,12 In the past, an ischemic pathology was not favored as bronchial arterial revascularization (BAR) failed to prevent OB.6 However, the investigators studied only the macrocirculation associated with BAR, not comment the microcirculation. Moreover, there is an extensive anastomotic network between pulmonary and bronchial circulation at the level of the SA and thus BAR does not add any benefit in terms of SA blood supply. From these data, it appears that during the lifespan of the lung allograft, there is damage to the microcirculation of the SA, and hence a reduction in BVPL in the non-obstructed and partially obstructed airways as compared with the control group. As ischemia sets in, due to the reduction in blood supply, the SA responds to insults such as chest infections and rejection, by a
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Figure 3. Four-fold magnification of an OB-affected small airway to demonstrate the altered morphology and increased density of microvessels on the muscular layer. The specimen has been doubly stained with vWF and smooth muscle actin antibodies.
scarring process, OB. This is supported by the work of Adams et al,13 who demonstrated that, in the setting of a reduction in the blood supply to the tracheal isograft in rats, damage to the respiratory epithelium healed by a scarring process similar to OB. Thus, repeated pulmonary infective and or inflammatory processes in the ischemic setting, as confirmed by our study, may lead to the development of OB. This mechanism is further supported by joint work published by the Stanford and Helsinki group.14 In their model, they showed that airway ischemia induced by transplanting lung fragments of 1 cm3 is reversible in autografts (in the absence of an immune-mediated injury), as compared with lung allografts. In the latter, the ischemic insult of the transplant coupled with the persistence of acute rejection (a vascular-based pathology) led to the development of OB in their swine model. There is evidence that fibroblasts in the OB scar release nitric oxide (NO),15,16 a potent angiogenetic factor.17–19 NO upregulates the transcription of vascular endothelial cell growth factor (VEGF), which increases vascular permeability,20 and along with NO (a vasodilator) causes extravasation of plasma proteins. Among these are proteinases that degrade the extracellular matrix and metalloproteinases that promote new vessel growth via VEGF, basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1).21 Thus, new vessels sprout around the ischemic SA and within its scarred lumen (Figures 1 and 3). This explains the differences seen in BVPL between non-obliterated and partially and completely obliterated airways (Groups B, C and D). It also accounts for the changes in vessels morphology as visualized by the endothelial markers used in this study. The angiogenetic vessels are smaller
The Journal of Heart and Lung Transplantation May 2004
compared with the native microvasculature around the SA (Figure 1). This confirmed pathologic process explains the clinical links between the risk factors for OB and its subsequent development. The patients included in this study developed OB at a later stage, probably because they had a small number of acute rejection episodes. We now intend to perform a longitudinal study to assess the effect of infection and acute rejection episodes on the BVPL of the SA. This may lead to a reappraisal of our current grading of acute rejection in lung transplantation to include peri-bronchial blood vessels. In addition, it will provide potential targets for both prevention and treatment of OB. In conclusion, long-term success in lung transplantation has so far been hampered by the early, seemingly irreversible development of the chronic rejection process, namely OB. This study proposed and confirmed the link between microvascular damage and development of OB. Further studies will identify the critical timing of this damage and therapeutic approaches could then be formulated to reduce or even prevent OB. REFERENCES 1. Hertz MI, Taylor DO, Trulock EP, et al. The registry of the International Society for Heart and Lung Transplantation: nineteenth Official Report—2002. J Heart Lung Transplant 2002;21:950 –70. 2. Heng D, Sharples L, McNeil K, Stewart S, Wreghitt T, Wallwork J. Bronchiolitis obliterans syndrome: incidence, natural history, prognosis and risk factors. J Heart Lung Transplant 1998;17:1255–63. 3. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1–15. 4. Duncan SR, Valentine V, Roglic M, et al. T-cell receptor biases and clonal proliferations among lung transplant recipients with obliterative bronchiolitis. J Clin Invest 1996;97:2642–50. 5. Al-Dossari GA, Kshettry VR, Jessurun J, Bolman RM, III. Experimental large animal model of obliterative bronchiolitis after lung transplantation. Ann Thorac Surg 1994; 53:34 –40. 6. Norgaard MA, Andersen CB, Pettersson G. Does bronchial artery revascularization influence results concerning bronchiolitis obliterans syndrome and/or obliterative bronchiolitis after lung transplantation? Eur J CTS 1998; 14:311–8. 7. Reid L, Meyrick B. Microcirculation: definition and organization at tissue level. Ann NY Acad Sci 1982:3–20. 8. Reid LM. The pulmonary circulation: remodelling in growth and disease. Am Rev Respir Dis 1979;119:531–46. 9. McDonald D. Angiogenesis and remodelling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001;164(suppl):S39 –S45.
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10. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 2001;56:902–6. 11. Paradis I. Bronchiolitis obliterans: pathogenesis, prevention and management. Am J Med Sci 1998;315(3):161–78. 12. Girgis RE, Tu I, Berry GJ, et al. Risk factors for the development of obliterative bronchiolitis after lung transplantation. J Heart Lung Transplant 1996;15:1200 –8. 13. Adams BF, Brazelton T, Berry GJ, Morris RE. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000;69:661–3. 14. Ikonen T, Uusitalo M, Taskinen E, et al. Small airway obliteration in a new swine heterotopic lung and bronchial allograft model. J Heart Lung Transplant 1998;17: 945–53. 15. Gabbay E, Walters EH, Orsida B, et al. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide levels and epithelial inducible nitric oxide synthase. Am J Crit Care Med 2000;162(6):2182–7. 16. Mason NA, Springall DR, Pomerance A, Evans TJ, Yacoub
Luckraz, et al.
17.
18.
19. 20.
21.
531
MH, Polak JM. Expression of inducible nitric oxide synthase and formation of peroxynitrite in post-transplant obliterative bronchiolitis. J Heart Lung Transplant 1998; 17:710 –4. Conway EM, Collen D, Carmeliat P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2001;49: 507–21. Thompson WD, Li WW, Maragoudakis M. The clinical manipulation of angiogenesis: pathology, side-effects, surprises and opportunities with novel human therapies. J Pathol 1999;187:503–10. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res 2000;87:434 –9. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000;95:189 –97. Nelson AR, Fingleton B, Rothengerg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135–49.