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Ann Thorac Surg 2006;82:1212-1218
© 2006 The Society of Thoracic Surgeons
a Transplant Unit, Papworth Hospital, Papworth Everard, Cambridge, United Kingdom
b Pathology Department, Papworth Hospital, Papworth Everard, Cambridge, United Kingdom
c MRC Biostatistics Unit, Cambridge, United Kingdom
Accepted for publication March 20, 2006.
* Address correspondence to Dr Luckraz, Transplant Unit, Papworth Hospital, Cambridge CB3 8RE, United Kingdom (Email: heymanluckraz{at}aol.com).
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Abstract |
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METHODS: Microvasculature of 695 small airways (99 patients) was assessed in post-mortem lung allograft specimens using monoclonal antibodies for von Willebrand factor and CD31. Group A consisted of 343 small airways from 58 patients with no evidence of OB. The remaining 41 patients had histological evidence of OB in some of their small airways and grouped as B, C, and D with some patients contributing to all three groups ie, their lung specimen had some small airways which were completely obliterated with OB, some airways which were partially obliterated and some small airways without any histological evidence of OB development. Thus group B consisted of 145 small airways (34 patients) without OB. Group C consisted of 171 small airways with partial luminal obstruction (36 patients). Group D consisted of 36 small airways (14 patients) with complete luminal obliteration.
RESULTS: Airway circumference (mean ± standard deviation) was 2.36 ± 0.37, 2.41 ± 0.51, 2.49 ± 0.51, and 2.57 ± 0.79 mm, respectively (p = 0.40). Mean number of blood vessels per unit length of airway circumference was 4.12 ± 1.1, 1.58 ± 0.61, 2.42 ± 1.06, and 4.42 ± 1.46 vessels/mm, respectively (p < 0.001). Blood vessels with circumference greater than 0.2 mm were present in 100%, 64%, 39%, and 7% of small airways, respectively (p < 0.001). Univariate and multivariate analyses (donor and recipient age, sex, and cytomegalovirus status, recipient pretransplant diagnosis, ischemic times, acute rejection and infective episodes, postoperative survival days, recipient group [A to D], blood vessels per unit length, and airway circumference) confirmed that reduction in blood vessels per unit length was associated with the development of OB and was time-independent.
CONCLUSIONS: Obliterative bronchiolitis was preceded by a decrease in microvascular supply to the small airways (group B). The subsequent onset of airway scarring (groups C and D) was associated with an increased number of significantly smaller vessels, suggestive of neovascularization.
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Introduction |
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Obliterative bronchiolitis is an intraluminal scarring process that occludes small airways. It has been suggested that OB results from an airway-directed immune process [3, 4]; however, no specific immune mechanism or pathway has been identified. Furthermore, despite increasing knowledge of the risks for, and natural history of, this condition, there have been no satisfactory explanations as to how an acute immune-mediated vascular insult (acute rejection) leads to the development of small airway scarring and obliteration (OB).
Ischemia as a cause for OB had been ruled out in the past on the basis that bronchial arterial revascularization only reduced major airways complications (ie, ischemia at the level of airways anastomosis) and not the incidence of OB [5]. However, the authors only assessed the macrocirculation to the proximal airways, and not the microvascular supply of the distal small airways.
A pilot study was carried out involving 11 patients who died of OB, with 5 patients dying of acute lung allograft failure used as control subjects [6]. This study showed that the earlier stages of OB were associated with a decrease in the microvascular blood supply to the small airways, with some attempt to repair through neovascularization as the small airways become completely obliterated.
The purpose of the current study is to assess a larger number of the microvasculature of the small airways in the setting of OB in an attempt to confirm our previous findings and to assess the factors influencing changes in the microvasculature of small airways.
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Material and Methods |
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Specimen
One hundred seventy patients were identified from the postmortem database, with survival from transplantation ranging from 0 to 4,535 days. From the tissue specimens available, small airways and their blood supply were not assessable in 71 patients because of severe consolidation process, incomplete small airways wall, or airways too large to be measured accurately with the light microscope or damage to the specimen during the staining process. The remaining 99 patients contributed a total of 695 small airways, which formed the basis of this study.
Stains
Lung tissue was fixed in 10% phosphate-buffered formalin, processed to paraffin, and then sectioned at 4 µm. 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
Immunocytochemistry techniques were used to assess the microvasculature around the small airways. Cells of endothelial lineage were identified using the monoclonal anti-endothelial antibodies to CD31 (1:30, Dako, Cambridge, UK) and von Willebrand factor (1:20, Dako). Paraffin sections were antigen-retrieved using a microwave (Euroserv 750 W) and 0.01 mol/L sodium bicarbonate antigen retrieval solution for CD31 and proteinase K enzyme retrieval for 10 minutes for von Willebrand factor. Endogenous peroxidase activity was quenched by treatment with hydrogen peroxide (Dako). Sections were then rinsed in phosphate-buffered saline solution and 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 counterstained with Carazzi's hematoxylin. Normal lung tissue obtained at lung resection surgery was used as a 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.
Image Analysis
The microvasculature around the small airways was quantified using a computerized image analysis package (Aequitas IA; Dynamic Data Links, Cambridge, UK). This was carried out by measuring the circumference of the small airway and counting the number of blood vessels supplying the airway. The number of blood vessel per unit length of airway circumference (BVPL) was then calculated (total number of blood vessels supplying small airway divided by the circumference of that airway). The circumference of the blood vessels was also measured, and the blood vessels were arbitrarily categorized as large blood vessels if their circumference was equal to or greater than 0.20 mm. The presence of large blood vessels around the small airway circumference was also noted.
Statistical Analysis
Data are expressed as mean (standard deviation), median (interquartile range) and percentages. Nonparametric data were analyzed by Kruskall-Wallis test and the analysis of variance test was used to analyze parametric data. The presence of large blood vessels around the small airways in the four groups was compared by Pearson's 
2 test.
Univariate and multivariate analyses were used to define the factors associated with the blood supply of the small airways. The factors evaluated were donor and recipient age, sex, CMV status, recipient pretransplant diagnosis, ischemic times, acute rejection and infective episodes, postoperative survival days, recipient group (A to D), BVPL, and airway circumference. A probability value of less than 0.05 was accepted as statistically significant.
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Results |
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Group A (343 small airways, 58 patients) represented small airways in patients who never developed OB. Group B (145 small airways, 34 patients) represented those small airways in which no evidence of OB was present but in which OB was seen in other small airways of the same lung. Group C (171 small airways, 36 patients) represented small airways in which there was partial luminal obliteration with OB, whereas the small airways in group D (36 small airways, 14 patients) showed complete luminal obliteration. Summary statistics for the four groups appear in Table 1.
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The median survival for the respective groups was 21 (interquartile range, 0 to 1,927), 919 (interquartile range, 50 to 3,293), 1,001 (interquartile range, 60 to 3,786), and 1,239 (interquartile range, 198 to 3,293) days (p < 0.001). There were no significant differences in the rates of rejection, chest infection, or CMV pneumonitis in the first year (Table 2). However, follow-up times for the patients without OB (group A) were shorter, and the event rates are not constant, even within the first year.
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Figure 3 demonstrates the normal blood supply around the small airway (group A). These microvessels are of good caliber and are more or less evenly distributed around the small airway. Figure 4 shows the scarcity of blood vessels around a small airway that has not yet developed OB but in which OB was seen in other small airways of the same lung specimen (group B). In Figure 5, the changes of the small airway microvasculature comprised alterations in the caliber of the vessels and also the uniformity of vessel distribution. These changes characterized group C in the study. Finally, when OB completely obliterates the small airway lumen (group D), the vessels look very different from those in group A (Fig 6 compared with Fig 3).
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In a small subgroup of patients in group A (ie, no OB, n = 10) who survived more than 100 days (range, 100 to 1,927 days), large blood vessels were present around all small airways irrespective of postoperative day. Moreover, their mean BVPL was 4.4 ± 0.8 mm compared with 4.0 ± 1.1 mm for the early deaths (postoperative day < 100 days) in that same group (group A). Thus, in patients who did not develop OB, blood vessels were not damaged solely as a consequence of time after transplantation.
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Comment |
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The aim of our study was to assess the microvascular supply to small airways and its association with the histopathologic outcome in OB. Several studies, including our own work at Papworth, have identified acute rejection, lymphocytic bronchitis or bronchiolitis, and CMV pneumonitis as the main risk factors for developing OB [2, 11, 12]. Of these, acute rejection has been consistently identified as the most powerful predictor of the development of OB. Acute rejection is, however, a vascular disease, whereas OB represents (small) airway damage.
Our data confirm that the microvasculature of the small airways in patients who do not develop OB (group A) is similar to that of normal patients described by Reid and Meyrick [7], irrespective of the time after transplantation. However, before the development of OB (group B airways), there is a significant decrease in the number of blood vessels supplying the small airways. Yousem and colleagues [13] described the chronic vascular changes in lung allografts as an immune-mediated injury directed against vascular endothelial cells, resulting in endothelialitis and fibrointimal narrowing of arterioles and venules. This process would account for the significant reduction in BVPL in the unobstructed (group B) and partially obstructed (group C) airways compared with the control group. This process may therefore lead not only to direct ischemic airway injury through compromise of the microvascular blood supply, but may also affect the reparative responses in the small airways to insults such as infection. The end result is the scarring characteristic of OB.
A similar pathologic pathway has been proposed in rat trachea [14] and lung fragment [15] allograft models. After the initial damage caused by the immune and nonimmune inflammatory response, the epithelial integrity is further disrupted by persistence of the inflammatory process. This generates transforming growth factor-ß [16–18], which, in turn, stimulates extracellular matrix deposition. Transforming growth factor-ß increases the transcription of fibronectin and procollagen and downregulates collagenases and proteases. Other profibrotic mediators, such as platelet-derived growth factor, have also been described in the remodeling process after acute lung injury and in patients with bronchiolitis obliterans syndrome after lung transplantation [16]. There is further evidence that the fibroblasts in the OB scar release nitric oxide [19, 20], a potent angiogenic factor [21, 22]. Nitric oxide upregulates the transcription of vascular endothelial growth factor, which increases vascular permeability and along with nitric oxide (a vasodilator) results in extravasation of plasma proteins into the lung interstitium. Among these proteins are metalloproteinases, which promote new vessel growth through a number of signaling mechanisms, including vascular endothelial growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 [23]. Interestingly, Krebs and associates [24] recently reported on the dual role of vascular endothelial growth factor in the setting of OB. In their model, vascular endothelial growth factor provided some protection to the epithelial integrity but at the same time enhanced luminal occlusion by chemotaxis. Thus, new vessels develop both around the ischemic small airways and within the luminal scarring, accounting for the morphometric and morphologic difference seen in BVPL among unobstructed, partially obstructed, and completely obliterated airways (groups B, C, and D, respectively; Figs 3–6).
In the past, an ischemic etiology of OB was not favored, as bronchial arterial revascularization failed to prevent OB [5]. However, the authors only investigated the macrocirculation associated with bronchial arterial revascularization and did not comment on the microcirculation, ie, the microvessels around the small airways. Bronchial arterial revascularization did not influence the development of OB, possibly because the "ischemic insult" is at a local microvascular level.
The changes described in the vascular supply of the small airways of the lung allograft have also been reported in the cardiac allograft setting. Atkinson and coworkers [25] have recently shown that the adventitial blood supply to coronary arteries is affected in a similar manner in patients who develop cardiac allograft vasculopathy. In this study, the changes in the microvasculature correlated with the degree of coronary artery luminal obstruction. It was postulated that local ischemia contributed to the proliferation of smooth muscle cells, leading to the deposition of collagen and progressive luminal occlusion as part of an adaptive remodeling process.
On the basis of our study, we confirmed the loss of the microvascular supply to the airways in patients with OB. This microvascular change was directly associated with OB.
Further studies are needed to identify the critical timing of the microvascular damage and to confirm the causes (immunologic and nonimmunologic) of the microvascular changes. Strategies to prevent the loss of microvascular blood supply (better control of acute rejection, CMV status, and so forth), to reduce scarring (antiproliferative agents), or to induce earlier or more effective angiogenesis may all be necessary to prevent the development of OB after lung transplantation.
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Online Discussion Forum |
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Once the article selected for discussion is published in the online version of The Annals, we will post a notice on the CTSNet home page (http://www.ctsnet.org) with a FREE LINK to the full-text article. Readers wishing to comment can post their own commentary in the discussion forum for that article, which will be informally moderated by The Annals Internet Editor. We encourage all surgeons to participate in this interesting exchange and to avail themselves of the other valuable features of the CTSNet Discussion Forum and Web site.
For October, the article chosen for discussion under the Pediatric Cardiac Dilemma Section of the Discussion forum is: Bovine Jugular Vein Conduit for Right Ventricular Outflow Tract Reconstruction: Evaluation of Risk Factors for Mid-Term Outcome
Ardawan J. Rastan, MD, Thomas Walther, MD, PhD, Ingo Daehnert, MD, Jörg Hambsch, MD, Friedrich W. Mohr, MD, PhD, Jan Janousek, MD, PhD, and Martin Kostelka, MD, PhD
Tom R. Karl, MD
The Annals Internet Editor
UCSF Children's Hospital
Pediatric Cardiac Surgical Unit
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San Francisco, CA
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e-mail: karlt{at}surgery.ucsf.edu
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  P. A. Corris and J. D. Christie Update in Transplantation 2007 Am. J. Respir. Crit. Care Med., May 15, 2008; 177(10): 1062 - 1067. [Full Text] [PDF]
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A. R. Glanville, C. L. Aboyoun, A. Havryk, M. Plit, S. Rainer, and M. A. Malouf Severity of Lymphocytic Bronchiolitis Predicts Long-Term Outcome after Lung Transplantation Am. J. Respir. Crit. Care Med., May 1, 2008; 177(9): 1033 - 1040. [Abstract] [Full Text] [PDF]
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P. A. Corris and J. D. Christie Update in Transplantation 2006 Am. J. Respir. Crit. Care Med., March 1, 2007; 175(5): 432 - 435. [Full Text] [PDF]
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