help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on January 30, 2004, doi:10.1164/rccm.200308-1111OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200308-1111OCv1
169/11/1203    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ebina, M.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ebina, M.
Right arrow Articles by Nukiwa, T.
American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 1203-1208, (2004)
© 2004 American Thoracic Society

Original Article

Heterogeneous Increase in CD34-positive Alveolar Capillaries in Idiopathic Pulmonary Fibrosis

Masahito Ebina, Minoru Shimizukawa, Naoko Shibata, Yuichiro Kimura, Takashi Suzuki, Mareyuki Endo, Hironobu Sasano, Takashi Kondo and Toshihiro Nukiwa

Department of Respiratory Oncology and Molecular Medicine and Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University; and Department of Pathology, Tohoku University School of Medicine, Sendai, Japan

Correspondence and requests for reprints should be addressed to Masahito Ebina, M.D., Ph.D., Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Aoba-ku, Sendai 980-8575, Japan. E-mail: ebinam{at}idac.tohoku.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To elucidate the apparent contradictions in vascular remodeling in the lungs of patients with idiopathic pulmonary fibrosis, we evaluated alveolar vascularity in relation to the various degrees of fibrosis in surgically biopsied lungs of usual interstitial pneumonia. Alveolar capillary endothelial cells were intensely immunoreactive with CD34 but not with von Willebrand factor. Vascular density, that is, the relative ratio of capillary area to total area of alveolar walls, was significantly higher at low grades of fibrosis than in control lungs, whereas vascular density gradually decreased as the degree of fibrosis increased and was lower than that of control lungs in the most extensively fibrotic lesions. No vessels were observed inside fibroblastic foci. The potent angiogenic factors vascular endothelial growth factor and interleukin-8 were abundantly produced by capillary endothelial cells and alveolar epithelial cells in highly vascularized alveolar walls. In contrast, venules with CD34-negative but von Willebrand factor-positive endothelial cells localized in the center of the fibrotic lesions were slightly increased and identified as postcapillary venules by three-dimensional reconstructed images. These results indicate the presence of heterogeneous vascular remodeling in usual interstitial pneumonia.

Key Words: alveolar capillary • endothelial cells • idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, diffuse parenchymal lung disease of unknown etiology with significant morbidity and mortality despite aggressive therapy. IPF is associated with the histologic appearance of usual interstitial pneumonia (UIP) on surgical lung biopsy (1). A strikingly heterogeneous and nonuniform fibrosing process is one of the most characteristic features of UIP, with alternating zones of fibrosis, honeycomb change, and intervening patches of normal lung (2). Although apoptosis of the alveolar epithelial cells has been reported to be involved in the pathogenesis of IPF (3), there is only limited information about the alveolar capillaries in the UIP lungs of patients with IPF. Regarding vascular remodeling in IPF, there are contradictory reports of it being highly vascularized (4–6) or less vascularized (7, 8). We hypothesized that these discrepancies were due to the temporal heterogeneity of the lesions in UIP lungs. Therefore, we evaluated vascular density within the alveolar walls with variegated fibrosis in surgically biopsied lungs of seven patients diagnosed with IPF/UIP. As a pilot study, we compared alveolar capillary endothelial cells according to their immunoreactivity for CD34, von Willebrand factor (vWF), and thrombomodulin (TM) because of the phenotypic heterogeneity of pulmonary vascular endothelial cells (9, 10). We confirmed first that alveolar capillary endothelial cells were intensely immunoreactive with CD34, but rarely with vWF. Thrombomodulin immunoreactivity of alveolar capillary endothelial cells was decreased around fibrotic lesions. We applied double immunohistochemical staining for CD34 and vWF (CD34/vWF) with counterstaining by elastica-Goldner stain (11), which clarified the relation between vascularity and the degree of fibrosis. We observed that the alveolar capillaries with CD34-positive endothelial cells were remarkably increased in the nonfibrotic lesions of UIP lungs. A subset of these endothelial cells was immunoreactive with Ki-67, a marker of proliferation (12). Alveolar Type II epithelial cells in close contact with increased capillary endothelial cells were intensely immunoreactive with the potent angiogenic factors vascular endothelial growth factor (VEGF) (13) and interleukin-8 (IL-8) (14). In contrast, CD34-positive capillaries were decreased in fibrotic lesions where the venules with vWF-positive endothelial cells were localized in the center. Three-dimensional images demonstrated that these vWF-positive venules connected CD34-positive alveolar capillaries with pulmonary veins in the fibrotic lesions (10). The possible role of these increased CD34-positive alveolar capillaries in the regeneration of alveolar septa in IPF is discussed.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
For details, see the online supplement.

Lung Tissues Examined
UIP lung tissues examined in this study were biopsied by video-assisted thoracoscopic surgery from seven patients with a definite diagnosis of IPF (1). Pathologically disease-free lung tissues of three never-smokers were also examined as normal controls. Informed consent for using these lung specimens in this study was obtained according to study protocols approved by the ethics committee of the Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan). These lung tissues were inflated by injection of 10% buffered formalin and embedded in paraffin wax.

Antibodies
The antibodies and optimal dilutions used in this study were as follows: monoclonal antibodies for CD34 (clone 4A1, diluted 1:100; Nichirei, Tokyo, Japan) and Ki-67 (MIB-1, diluted 1:100; Nichirei); monoclonal antibodies for thrombomodulin (TM) (TM1009, diluted 1:100; DakoCytomation, Glostrup, Denmark), SP-A (PE10, diluted 1:500; DakoCytomation), and a polyclonal antibody for vWF (diluted 1:2000; DakoCytomation); and a monoclonal antibody for VEGF (C-1, diluted 1:900; Santa Cruz Biotechnology, Santa Cruz, CA). The antibody against IL-8 was kindly provided by R. M. Strieter (University of California, Los Angeles School of Medicine) and used as described (6).

Immunohistochemistry
The antigen–antibody complex was visualized with Vector Red (Vector Laboratories, Burlingame, CA) or 1 mM 3,3'-diaminobenzidine (DAB). Hematoxylin stain or elastica-Goldner stain (11) was used for counterstaining. As positive controls, tissue sections of tonsils were used for CD34 and vWF, those of normal adult lung for TM, and those of breast cancer for VEGF according to a previous study (10).

Double Immunostaining
Double immunohistochemical staining for CD34/Ki-67 and CD34/vWF was performed as described previously (15). Cytoplasmic immunostaining for CD34 was revealed with DAB (brown) and nuclear staining for Ki-67 was revealed with Vector Red (red). Double immunohistochemical staining for CD34 and vWF (or PE10) showed CD34 in red (Vector Red) and vWF (or PE10) in brown (DAB).

Quantitative Evaluation of Vascularity
Quantitative analysis using the CAS 200 image analysis system (BD Biosciences, Lincoln Park, NJ) was performed according to previous studies (16). The CAS 200 computed image analysis system detects CAS red chromogen (BD Biosciences) from the 500-nm channel and methyl green from the 620-nm sensor. Using an imaging program (BD Biosciences), vascular density was represented by the percentage of the total area surrounded by CD34-positive endothelial cells per field of alveolar walls. The pathologic degrees of fibrosis of the fields were scored from 1 to 8 according to Ashcroft and coworkers (17).

Three-dimensional Reconstruction
Three-dimensional image reconstruction was based on previous studies (18, 19). From serial sections with double immunostaining for CD34 and vWF, images of CD34-positive capillaries and vWF-positive venules were captured with a digitizer (UD-1218-RE; Wacom, Saitama, Japan) and 3-D images were reconstructed (OZ95; Rise, Sendai, Japan) (15).

Statistical Analysis
For analysis of two unpaired samples, the nonparametric Mann–Whitney U test was used (StatView; SAS Institute, Cary, NC). A significant difference was defined as p < 0.05. All values were represented as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Heterogeneous Immunoreactivity of Endothelial Cells
The lung specimens from seven patients with IPF had a variegated appearance, with alternating zones of fibrosis and intervening patches of normal lung at low magnification. Fibroblastic foci were easily observed in these specimens, consistent with the characteristic features of UIP. To clarify the involvement of endothelial cells in UIP lesions, we examined the immunoreactivity for three endothelial markers: CD34, von Willebrand factor (vWF), and thrombomodulin (15). We observed heterogeneous immunolocalization of CD34 and vWF in endothelial cells of these UIP lungs. To compare the heterogeneous immunolocalization of these endothelial cells among lesions with various degrees of fibrosis, the sections for immunohistochemistry were counterstained with elastica-Goldner stain, which showed collagen tissues in green and elastic fibers in dark purple (11). Almost all the alveolar capillary endothelial cells were intensely positive for CD34, and these were distributed densely in the nonfibrotic lesions (Figure 1A) . In contrast, endothelial cells positive for vWF were localized mainly in the larger vessels, and little immunoreactivity for vWF was observed in alveolar capillaries (Figure 1B). Immunoreactivity for the thrombomodulin of endothelial cells was also examined in consecutive sections as a positive control (10). Variable immunoreactivity for thrombomodulin was observed in the endothelial cells of both CD34-positive alveolar capillaries and vWF-positive pulmonary veins (Figure 1C). Double immunohistochemical staining for CD34 and vWF (CD34/vWF) clearly showed the heterogeneous distribution of CD34-positive and vWF-positive endothelial cells (Figure 1D). Fibroblastic foci were free from any endothelial cells immunoreactive for CD34, vWF, or thrombomodulin (Figure 1).



View larger version (120K):
[in this window]
[in a new window]
  		Figure 1. Heterogeneity in the immunoreactions of endothelial cells in usual interstitial pneumonia (UIP) (scale bar, 200 µm). (A–D) Immunostaining of serial sections of the biopsied lungs of a patient with idiopathic pulmonary fibrosis (IPF) reveals the heterogeneous immunolocalization of endothelial cells. Almost all of the alveolar capillaries are positive for CD34 (red; A), but negative for von Willebrand factor (vWF) (B). In contrast, the endothelial cells of the venules (indicated by arrows) in the center of the lesions with massive fibrosis are CD34 negative (A) but vWF positive (red; B). Only some of these endothelial cells are immunoreactive for thrombomodulin (red; C). No capillaries are observed in the fibroblastic focus (indicated by an arrowhead; A–D). Double immunostaining for CD34 and vWF (D) also reveals heterogeneous endothelial cells positive for CD34 (red) and positive for vWF (dark brown). The sections were counterstained with elastica-Goldner stain. PV = pulmonary veins.

 
Remodeling of Alveolar Capillaries in UIP
We compared the relation between capillary remodeling and the distribution of alveolar Type II epithelial cells producing surfactant protein A (SP-A) in various degrees of fibrosis in UIP. The small fibrous tissues arising from nonvascularized alveolar septa contained no capillaries inside (Figure 2A) . Double staining for CD34 and SP-A of consecutive sections revealed the close contact of alveolar Type II cells with alveolar capillaries (Figure 2B). No capillaries were distributed in the side of fibrous tissues without alveolar Type II cells (Figure 2B). This tendency was also shown in massive fibrotic lesions (Figure 2C). In fibrotic lesions, CD34-positive capillaries were distributed only on the edges of fibrotic lesions, where there were alveolar Type II cells. In the walls of honeycomb lesions, venules with vWF-positive endothelial cells were sparsely distributed but dominant compared with CD34-positive capillaries (Figure 2D).



View larger version (100K):
[in this window]
[in a new window]
  		Figure 2. Heterogeneous distribution of alveolar capillaries in various degrees of fibrosis. (A) Double immunostaining for CD34 (red) and for vWF (brown), counterstained with elastica-Goldner stain (scale bar, 100 µm). Tiny fibrous tissues (arrow) develop from the alveolar septa without an increase in the number of capillaries. No endothelial cells are contained within the fibrous tissues. (B and C) Double immunostaining for CD34 (red) and for surfactant protein A (brown, arrowheads) in the consecutive section (B; scale bar, 100 µm) and in fibrous lesions (C; scale bar, 200 µm). Alveolar capillaries are distributed on the edges of fibrous tissues with alveolar Type II cells (arrowheads in C). (D) The walls of honeycomb lesions with immunostaining for CD34 (red, arrowheads) and for vWF (brown), counterstained with elastica-Goldner stain (scale bar, 200 µm). The vessels with CD34–vWF+ endothelial cells are sparsely distributed.

 
Proliferation of CD34-positive Endothelial Cells and Distribution of Angiogenic Factors in UIP
The proliferation of capillary endothelial cells was examined by double staining for CD34 and Ki-67, a cell proliferation-associated human nuclear antigen (12) (Figures 3A and 3B) . In contrast to the alveolar walls of normal control lungs (Figure 3A), alveolar capillaries in the nonfibrous lesions of UIP lungs were dilated and remarkably increased (Figure 3B). A subset of the endothelial cells of these increased alveolar capillaries was immunoreactive with Ki-67 (Figure 3B). The distribution of Ki-67–positive capillary endothelial cells in the area of a lung tissue section (cm2) was 2.6 ± 0.3 cells/cm2 in UIP lungs (n = 7) and 0 ± 0 cells/cm2 in control lungs (n = 3, p < 0.05). Augmented expression of both VEGF and IL-8 was detected in capillary endothelial cells and alveolar Type II epithelial cells in highly vascularized alveolar septa (Figures 3C–3F). In fibrotic lesions, fibroblasts and leukocytes were faintly immunoreactive with VEGF and IL-8 (Figures 3G and 3H), with few endothelial cells positive for Ki-67 (Figure 3I).



View larger version (133K):
[in this window]
[in a new window]
  		Figure 3. Proliferation of CD34-positive endothelial cells and distribution of angiogenic factors (scale bar, 100 µm). (A and B) Double immunostaining for CD34 (brown) and Ki-67 (red) in a control lung (A) and in a UIP lung (B), counterstained with hematoxylin (scale bars, 100 µm). Nuclear staining of endothelial cells for Ki-67 was not observed in control lung but was observed in a subset of endothelial cells (arrowheads) in UIP (B). A Ki-67–positive nucleus of a capillary endothelial cell is clearly shown in the inset of (B) (arrowhead). (C and D) Immunoreactivity for vascular endothelial growth factor (VEGF) was faint in control (C), but intense in the increased capillary lesions in UIP lungs. Type II alveolar epithelial cells (arrowheads) and capillary endothelial cells (arrows) were positive for VEGF (D). (E and F) Cells immunoreactive with IL-8 were also rarely found in control lungs (E) but were abundant among Type II alveolar epithelial cells (arrowheads) and capillary endothelial cells (arrows) in UIP lungs (F). (G and H) In serial sections of fibrotic lesions, fibroblasts, epithelial cells, and leukocytes were faintly immunoreactive for VEGF (G, arrowheads) or IL-8 (H, arrowheads). (I) Immunoreactivity for Ki-67 was occasionally detected in fibroblasts (arrowhead), leukocytes, and epithelial cells, but rarely in endothelial cells.

 
Reverse Correlation between Vascular Density and the Degree of Fibrosis in UIP
To examine the heterogeneous remodeling of CD34-positive capillary endothelial cells in UIP by morphometric analysis, we quantified the vascular density of alveolar walls with various degrees of fibrosis, using an image analyzer system. Figure 4A shows a field of thickening alveolar septum in UIP lung tissues, monitored with a CAS 200 image analysis system (15). Vascular density was determined as the percentage of the total area surrounded by CD34-positive capillary endothelial cells per area of alveolar septa in each field, scored according to the degree of fibrosis from 1 (minimal) to 8 (severe) (17). The relation between vascular density and the fibrotic degree in each field of one patient with IPF/UIP is shown in Figure 4B. The vascular density of minimally fibrotic lesions, scored as 1 or 2, was 25 ± 2.6% (mean ± SEM, n = 10), and decreased gradually toward more severe fibrosis. In the moderately fibrotic area, scored as 5 or 6, vascular density was 11 ± 2.2% (n = 8), which was lower than that of minimally fibrotic lesions (p < 0.01). The vascular density of more severe lesions, scored as 7 or 8, was the lowest (6.8 ± 1.6%, n = 8, p < 0.01).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 4. Relation between vascular density and the scores of fibrosis. (A) A thick alveolar wall in the lung of a patient with IPF, monitored with an image analysis system, reveals a capillary area surrounded by CD34-positive endothelial cells (green), and the field of the alveolar wall is contoured in red (scale bar, 20 µm). (B) Relation between the vascular density of alveolar walls and the fibrosis scores of lung tissues of a patient with IPF. The degree of fibrosis is scored from 1 (minimal fibrosis) to 8 (severe fibrosis) (*p < 0.01). (C) Mean values of the vascular density of each degree of fibrosis in the lung tissues of seven patients with IPF were pooled. The fibrotic degree of three control lungs was scored as 0 (*p < 0.01).

 
This tendency toward high vascularity in minimally fibrotic lesions and low vascularity in severely fibrotic lesions was also apparent when the mean values of vascular density at each fibrotic score of the seven patients with IPF were pooled (Figure 4C). The control level of vascular density at score 0 (12 ± 1.6%) was obtained from the mean values of vascular density of three control lungs without lung disease. The value of vascular density at a score of 1 (22 ± 1.7%) was remarkably higher than that of the control level (p < 0.01), and the vascular density was the highest at score 2 (25 ± 1.6%, p < 0.01). As the fibrosis score increased to more than 3, however, the vascular density gradually decreased. A significant difference in the decrease from the control level of vascular density was revealed at scores of 6 (8.0 ± 0.5%, p < 0.05), 7 (5.7 ± 0.6%, p < 0.01), and 8 (3.1 ± 0.7%, p < 0.01).

Three-dimensional Reconstruction of Capillaries and Vessels
The connection between capillaries with CD34-positive endothelial cells and venules with vWF-positive endothelial cells in fibrotic lesions was confirmed in serial sections of UIP lungs (Figures 5A–5C) . Figure 5D shows the orifices of CD34-positive alveolar capillaries inside pulmonary veins with vWF-positive endothelial cells.



View larger version (128K):
[in this window]
[in a new window]
  		Figure 5. The connection between capillaries with CD34-positive endothelial cells and venules with vWF-positive endothelial cells. (A–C) Serial sections with double staining for CD34 and vWF reveal that alveolar capillaries connect to venules with vWF-positive endothelial cells (arrowheads; scale bars, 100 µm). (D) Orifices of alveolar capillaries with CD34-positive endothelial cells can be observed within a venule (indicated by arrowheads; scale bar, 100 µm).

 
We reconstructed three-dimensional images of CD34-positive capillaries and vWF-positive venules to compare the relation between alveolar capillaries and vWF-positive venules. Each three-dimensional image was reconstructed from serial two-dimensional images with double immunostaining for CD34 and vWF (Figures 6B and 6C) , on the basis of our previous studies (18, 19). Figure 6A shows a three-dimensionally reconstructed image of fibrotic lesions in UIP. CD34-positive alveolar capillaries appear in red, pulmonary veins and venules with vWF-positive endothelial cells are light green, and the contours of fibrotic lesions are blue. This reconstructed image revealed the distribution of CD34-positive capillary endothelial cells on the edges of fibrotic lesions and the connection of vWF-positive venules between CD34-positive capillaries and pulmonary veins located in the center of fibrotic lesions. These small vWF-positive venules, seen as green dots in Figure 6A, were slightly increased compared with those venules shown in the three-dimensional image of a control lung (Figure 6D).



View larger version (44K):
[in this window]
[in a new window]
  		Figure 6. Three-dimensional image of CD34-positive and vWF-positive endothelial cells in fibrotic lesions of UIP. (A and B) A 3-D image of CD34-positive alveolar capillary endothelial cells (red), vWF-positive endothelial cells of pulmonary veins and venules (green), and the contours of fibrotic lesions (blue) was reconstructed from the serial sections of a UIP lung with double immunostaining for CD34 and vWF (B; scale bar, 200 µm). (C and D) From the serial sections of a control lung as well, CD34-positive capillaries and vWF-positive vessels of a section (C; scale bar, 200 µm) were reconstructed into a three-dimensional image (D).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to show the remarkable increase in alveolar capillaries with CD34+vWF– endothelial cells in nonfibrotic lesions in UIP. The highly vascular nature of the lesions in pulmonary fibrosis was noted first by Golden and Bronk in 1953 (4). The vascular supply was found to derive from the systemic circulation through systemic–pulmonary anastomoses in the neighborhood of the vascular proliferation (5). However, few studies have identified clearly the vessels increased in UIP lungs with heterogeneous endothelial cells. In this study, we demonstrated the proliferation of CD34-positive alveolar capillaries in nonfibrotic lesions in UIP. In close contact with these increased CD34-positive alveolar capillaries, alveolar Type II cells produced the potent angiogenic factors VEGF (13) and IL-8 (14). These angiogenic factors were also detected in increased endothelial cells in nonfibrotic lesions. In fibrotic lesions, CD34-positive capillaries were distributed only on the edges where there were alveolar Type II cells. No capillaries were observed in fibrotic tissues without the attachment of alveolar cells. The important role of VEGF for maintaining alveolar structures (20) would account for the increased expression of VEGF by these alveolar epithelial cells. In this context, increased CD34-positive capillaries may contribute to the regeneration of alveolar walls lost in IPF. A report showing that transforming growth factor-ß1 and hypoxia increased the production of VEGF by epithelial cells also supports the possible role of VEGF in the endothelial repair and angiogenesis that follow lung injury (21).

In contrast to the increase in capillaries in nonfibrotic lesions, we observed a decrease of CD34-positive capillary endothelial cells in fibrotic lesions in UIP. Gracey and coworkers pointed out the striking feature, as revealed by microscopy, of a decrease in the number of capillaries within the alveolar septa and the total absence of septal capillaries in the lungs of patients with pulmonary fibrosis (7). The decrease in lumenal size of vessels within remodeled alveolar walls has also been shown in biopsied specimens of pulmonary fibrosis (8). Decreased vessel density was also reported in cryptogenic fibrosing alveolitis (22). Given the lack of vascularization in fibroblastic foci, the process of fibrosis appears not to require neovascularization. The development of small fibrous tissues from nonfibrotic septa without an increase in capillaries suggested that these fibrous tissues were early lesions in UIP. In this regard, the increase in alveolar capillaries in nonfibrotic lesions is thought to represent a secondary change following the fibrotic process. A reduction in the microvasculature of intralumenal fibromyxoid lesions in UIP compared with that in organizing pneumonia was also reported (23).

Keane and coworkers demonstrated an increase in vessels with vWF-positive endothelial cells in fibrous lesions and the regulatory roles of IL-8 and IP-10 in the angiogenesis and fibrosis of IPF (6). These results are consistent in part with our results that showed the dominant distribution of venules with vWF-positive endothelial cells in the centers of fibrous lesions. These vWF-positive venules are assumed to be postcapillary venules because these venules connect CD34-positive alveolar capillaries with pulmonary veins. Our three-dimensionally reconstructed images suggested that there was a slight increase in vWF-positive venules in fibrotic lesions. Because most of the endothelial cells in fibrotic lesions were not immunoreactive with Ki-67, a marker of proliferation, this increase in vWF-positive venules may have been caused by the increased immunoreactivity of vWF in CD34-positive endothelial cells, as reported in injured endothelial cells (24). In this sense, the apparent vascular density, based on the immunoreactivity with CD34 for endothelial cells, may have been biased in these fibrotic lesions. Another possible mechanism would be the contribution of circulating bone marrow–derived endothelial cells (25) to the increase in these vWF-positive venules or even to the increase in CD34-positive capillaries.

In conclusion, we demonstrated a remarkable increase in CD34-positive alveolar capillaries in nonfibrotic lesions and a corresponding decrease in fibrotic lesions. vWF-positive venules, identified as postcapillary venules, were slightly increased in fibrotic lesions. These results account for the apparent contradictions concerning vascularity in IPF. The increase in CD34-positive capillaries in nonfibrotic lesions appears to be a subsequent event in the fibrotic process, which may contribute to the regeneration of alveolar walls damaged by fibrosis in IPF.


   FOOTNOTES
 
Supported by a grant for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to M.E.).

This study was presented in part at the annual meeting of the American Thoracic Society in 2003.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: cM.E. has no declared conflict of interest; M.S. has no declared conflict of interest; N.S. has no declared conflict of interest; Y.K. has no declared conflict of interest; T.S. has no declared conflict of interest; M.E. has no declared conflict of interest; H.S. has no declared conflict of interest; T.K. has no declared conflict of interest; T.N. has no declared conflict of interest.

Received in original form August 11, 2003; accepted in final form January 26, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International Consensus Statement. Am J Respir Crit Care Med 2000;161:646–664.[Free Full Text]
  2. Katzenstein AL, Myers JL. Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am J Respir Crit Care Med 1998;157:1301–1315.[Free Full Text]
  3. Kuwano K, Hagimoto N, Kawasaki M, Yatomi T, Nakamura N, Nagata S, Suda T, Kunitake R, Maeyama T, Miyazaki H, et al. Essential roles of the Fas–Fas ligand pathway in the development of pulmonary fibrosis. J Clin Invest 1999;104:13–19.[Medline]
  4. Golden A, Bronk TT. Diffuse interstitial fibrosis of lungs. Arch Intern Med 1953;92:606–614.[Abstract/Free Full Text]
  5. Turner-Warwick M. Precapillary systemic–pulmonary anastomoses. Thorax 1963;18:225–237.
  6. Keane MP, Arenberg DA, Lynch JP, III, Whyte RI, Iannettoni MD, Burdick MD, Wilke CA, Morris SB, Glass MC, DiGiovine B, et al. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 1997;159:1437–1443.[Abstract]
  7. Gracey DR, Divertie MB, Brown AL, Jr. Alveolar–capillary membrane in idiopathic interstitial pulmonary fibrosis. Am Rev Respir Dis 1968;98:16–21.[Medline]
  8. Coalson JJ. The ultrastructure of human fibrosing alveolitis. Virchows Arch 1982;395:181–199.
  9. Yamamoto M, Shimokata K, Nagura H. An immunohistochemical study on phenotypic heterogeneity of human pulmonary vascular endothelial cells. Virchows Arch A 1988;412:479–486.
  10. Ebina M, Shimizukawa M, Kimura Y, Kondo T, Suzuki T, Nukiwa N. Three-dimensional reconstruction of alveolar septa in usual interstitial pneumonia (UIP) reveals remodeling patterns of alveolar capillaries [abstract]. Am J Respir Crit Care Med 2003;167:A983.
  11. Goldner J. A modification of the Goldner trichrome technique for routine laboratory purposes. Am J Pathol 14:237–243.
  12. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody. J Immunol 1984;133:1710–1715.[Abstract]
  13. Ferrara N, Houck K, Jakeman L, Winer J, Leung D. The vascular endothelial growth factor family of polypeptides. J Cell Biochem 1991;47:211–218.[CrossRef][Medline]
  14. Strieter RM, Polverini PJ, Kunkel SL, Arenberg DA, Burdick MD, Kasper J, Dzuiba J, Van Damme J, Walts A, Marriott D, et al. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995;270:27348–27357.[Abstract/Free Full Text]
  15. Maeda S, Suzuki S, Suzuki T, Endo M, Moriya T, Chida M, Kondo T, Sasano H. Analysis of intrapulmonary vessels and epithelial–endothelial interactions in the human developing lung. Lab Invest 2002;82:293–301.[CrossRef][Medline]
  16. Sasano H, Ohashi Y, Suzuki T, Nagura H. Vascularity in human adrenal cortex. Mod Pathol 1998;11:329–333.[Medline]
  17. Ashcroft T, Simpson J, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988;41:467–470.[Abstract/Free Full Text]
  18. Ebina M, Yaegashi H, Takahashi T, Motomiya M, Tanemura M. Distribution of small muscles along bronchial tree: a morphometric study of ordinary autopsy lungs. Am Rev Respir Dis 1990;141:1322–1326.[Medline]
  19. Ebina M, Yaegashi H, Chiba R, Takahashi T, Motomiya M, Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles: a morphometric study. Am Rev Respir Dis 1990;141:1327–1332.[Medline]
  20. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 2000;106:1311–1319.[Medline]
  21. Boussat S, Eddahibi S, Coste A, Fataccioli V, Gouge M, Housset B, Adnot S, Maitre B. Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2000;279:L371–L378.[Abstract/Free Full Text]
  22. Renzoni EA, Walsh DA, Salmon M, Wells AU, Sestini P, Nicholson AG, Veeraraghavan S, Bishop AE, Romanska HM, Pantelidis P, et al. Interstitial vascularity in fibrosing alveolitis. Am J Respir Crit Care Med 2003;167:438–443.[Abstract/Free Full Text]
  23. Lappi-Blanco E, Kaarteenaho-Wiik R, Soini Y, Risteli J, Paakko P. Intraluminal fibromyxoid lesions in bronchiolitis obliterans organizing pneumonia are highly capillarized. Hum Pathol 1999;30:1192–1196.[CrossRef][Medline]
  24. Reidy MA, Chopek M, Chao S, McDonald T, Schwartz SM. Injury induces increase of von Willebrand factor in rat endothelial cells. Am J Pathol 1989;34:857–864.
  25. Asahara T, Murohara T, Sullivian A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ERRHome page
M. P. Keane
The role of chemokines and cytokines in lung fibrosis
Eur. Respir. Rev., December 1, 2008; 17(109): 151 - 156.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Garantziotis, E. Zudaire, C. S. Trempus, J. W. Hollingsworth, D. Jiang, L. H. Lancaster, E. Richardson, L. Zhuo, F. Cuttitta, K. K. Brown, et al.
Serum Inter-{alpha}-Trypsin Inhibitor and Matrix Hyaluronan Promote Angiogenesis in Fibrotic Lung Injury
Am. J. Respir. Crit. Care Med., November 1, 2008; 178(9): 939 - 947.
[Abstract] [Full Text] [PDF]

Home page
 Hum Exp ToxicolHome page
J. Herseth, M Refsnes, M Lag, G Hetland, and P. Schwarze
IL-1{beta} as a determinant in silica-induced cytokine responses in monocyte-endothelial cell co-cultures
Human and Experimental Toxicology, May 1, 2008; 27(5): 387 - 399.
[Abstract] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. Marchal-Somme, Y. Uzunhan, S. Marchand-Adam, M. Kambouchner, D. Valeyre, B. Crestani, and P. Soler
Dendritic Cells Accumulate in Human Fibrotic Interstitial Lung Disease
Am. J. Respir. Crit. Care Med., November 15, 2007; 176(10): 1007 - 1014.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
H. Kanazawa and T. Yoshikawa
Up-Regulation of Thrombin Activity Induced by Vascular Endothelial Growth Factor in Asthmatic Airways
Chest, October 1, 2007; 132(4): 1169 - 1174.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
N. M. Patel, D. J. Lederer, A. C. Borczuk, and S. M. Kawut
Pulmonary Hypertension in Idiopathic Pulmonary Fibrosis
Chest, September 1, 2007; 132(3): 998 - 1006.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
D. A. Zisman, A. S. Karlamangla, D. J. Ross, M. P. Keane, J. A. Belperio, R. Saggar, J. P. Lynch III, A. Ardehali, and J. Goldin
High-Resolution Chest CT Findings Do Not Predict the Presence of Pulmonary Hypertension in Advanced Idiopathic Pulmonary Fibrosis
Chest, September 1, 2007; 132(3): 773 - 779.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. D. Nathan, P. W. Noble, and R. M. Tuder
Idiopathic Pulmonary Fibrosis and Pulmonary Hypertension: Connecting the Dots
Am. J. Respir. Crit. Care Med., May 1, 2007; 175(9): 875 - 880.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
E. Roger Parra, R. Adib Kairalla, C. R. R. de Carvalho, and V. L. Capelozzi
Abnormal deposition of collagen/elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias
Thorax, May 1, 2007; 62(5): 428 - 437.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
N. F. Voelkel, I. S. Douglas, and M. Nicolls
Angiogenesis in Chronic Lung Disease
Chest, March 1, 2007; 131(3): 874 - 879.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
K. M. Antoniou, A. Tzouvelekis, M. G. Alexandrakis, K. Sfiridaki, I. Tsiligianni, G. Rachiotis, N. Tzanakis, D. Bouros, J. Milic-Emili, and N. M. Siafakas
Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.
Chest, October 1, 2006; 130(4): 982 - 988.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
Q. Ye, B. Chen, Z. Tong, S. Nakamura, R. Sarria, U. Costabel, and J. Guzman
Thalidomide reduces IL-18, IL-8 and TNF-{alpha} release from alveolar macrophages in interstitial lung disease
Eur. Respir. J., October 1, 2006; 28(4): 824 - 831.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
D. W. Visscher and J. L. Myers
Histologic spectrum of idiopathic interstitial pneumonias.
Proceedings of the ATS, January 1, 2006; 3(4): 322 - 329.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
A. L. Mora, C. R. Woods, A. Garcia, J. Xu, M. Rojas, S. H. Speck, J. Roman, K. L. Brigham, and A. A. Stecenko
Lung infection with {gamma}-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice
Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L711 - L721.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
R. M. Strieter
Pathogenesis and Natural History of Usual Interstitial Pneumonia: The Whole Story or the Last Chapter of a Long Novel
Chest, November 1, 2005; 128(5_suppl_1): 526S - 532S.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
J. C. Grutters and R. M. du Bois
Genetics of fibrosing lung diseases
Eur. Respir. J., May 1, 2005; 25(5): 915 - 927.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. D. Bradley, Y. E. Miller, F. J. Martinez, D. C. Angus, W. MacNee, and E. Abraham
Interstitial Lung Disease, Lung Cancer, Lung Transplantation, Pulmonary Vascular Disorders, and Sleep-disordered Breathing in AJRCCM in 2004
Am. J. Respir. Crit. Care Med., April 1, 2005; 171(7): 675 - 685.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. D. Burdick, L. A. Murray, M. P. Keane, Y. Y. Xue, D. A. Zisman, J. A. Belperio, and R. M. Strieter
CXCL11 Attenuates Bleomycin-induced Pulmonary Fibrosis via Inhibition of Vascular Remodeling
Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 261 - 268.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
G. P. Cosgrove, K. K. Brown, W. P. Schiemann, A. E. Serls, J. E. Parr, M. W. Geraci, M. I. Schwarz, C. D. Cool, and G. S. Worthen
Pigment Epithelium-derived Factor in Idiopathic Pulmonary Fibrosis: A Role in Aberrant Angiogenesis
Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 242 - 251.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
E. A. Renzoni
Neovascularization in Idiopathic Pulmonary Fibrosis: Too Much or too Little?
Am. J. Respir. Crit. Care Med., June 1, 2004; 169(11): 1179 - 1180.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200308-1111OCv1
169/11/1203    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ebina, M.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ebina, M.
Right arrow Articles by Nukiwa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society