help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on February 20, 2003, doi:10.1164/rccm.200210-1233OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200210-1233OCv1
167/9/1250    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 Santos, S.
Right arrow Articles by Barberà, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santos, S.
Right arrow Articles by Barberà, J. A.
American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1250-1256, (2003)
© 2003 American Thoracic Society

Enhanced Expression of Vascular Endothelial Growth Factor in Pulmonary Arteries of Smokers and Patients with Moderate Chronic Obstructive Pulmonary Disease

Salud Santos, Victor I. Peinado, Josep Ramírez, Jaime Morales-Blanhir, Ricardo Bastos, Josep Roca, Robert Rodriguez-Roisin and Joan A. Barberà

Departments of Pulmonary Medicine and Pathology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic
Universitat de Barcelona, Barcelona, Spain

Correspondence and requests for reprints should be addressed to Dr. Joan A. Barberà, Servei de Pneumologia, Hospital Clínic, Villarroel, 170, 08036 Barcelona, Spain. E-mail: jbarbera{at}clinic.ub.es


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic obstructive pulmonary disease (COPD) is associated with structural and functional changes in the pulmonary circulation that commence at an early stage. To investigate whether vascular endothelial growth factor (VEGF) might be implicated as a mediator in COPD-associated pulmonary vascular changes, we studied surgical specimens obtained from 19 nonsmokers, 21 smokers with normal lung function, 28 patients with moderate COPD, and 10 patients with severe emphysema. The expression of VEGF in pulmonary muscular arteries was evaluated by immunohistochemistry, its protein content in lung tissue by Western blot analysis, and VEGF mRNA and its isoforms were analyzed by reverse transcription-polymerase chain reaction. The immunohistochemical expression of VEGF was increased in pulmonary arteries of smokers (median, 68% [interquartile range, 60–88]) and patients with moderate COPD (77% [63–82]), compared with nonsmokers (53% [40–63]) (p < 0.05 each). The expression of VEGF in smooth muscle cells correlated with the thickness of the vessel wall (r = 0.38, p < 0.01). VEGF protein content in lung tissue was reduced in severe emphysema, where reverse transcription-polymerase chain reaction demonstrated a lower proportion of the VEGF189 isoform. In conclusion, the expression of VEGF varies according to the severity of COPD and might be involved in the pathogenesis of pulmonary vascular remodeling at early stages of the disease.

Key Words: chronic obstructive pulmonary disease • cigarette smoking • pulmonary artery • endothelium • vascular endothelial growth factor

Pulmonary vascular remodeling leading to pulmonary hypertension and cor pulmonale is a characteristic feature of chronic obstructive pulmonary disease (COPD) (1, 2). Hypoxia has been classically considered the major pathogenic mechanism of these changes. However, structural abnormalities of pulmonary arteries are not exclusive of advanced COPD, as they have been shown also in patients with mild COPD without arterial hypoxemia and even in smokers with normal lung function (3). In agreement with this notion, recent studies suggest that the natural history of pulmonary hypertension in COPD might commence at moderate degrees of disease severity (4).

In a previous study, we showed endothelial dysfunction in pulmonary arteries of patients with mild COPD, which was associated with an impaired release of endothelium-derived nitric oxide (NO) (5). Subjects with greater impairment in endothelial function were those with more pronounced vascular remodeling (intimal thickening) (5). Because the endothelium plays a central role in regulating vascular tone and controlling cell growth, we hypothesized that the impairment of endothelial function might promote pulmonary vessel remodeling at this early stage. At this time, it is not known which mediators are involved in this process.

Growth factors are intercellular signaling molecules that regulate cell proliferation by autocrine and paracrine mechanisms. Vascular endothelial growth factor (VEGF) is abundantly expressed in cells of lung tissue. This molecule is mainly implicated in the maintenance of vascular endothelial cell function and in vascular cell proliferation (6, 7). Some of these functions are performed through NO-dependent mechanisms. VEGF has been involved in the vascular remodeling of primary pulmonary hypertension, which is characterized by endothelial and smooth muscle cell (SMC) proliferation (8). Cigarette smoking may upregulate VEGF, as suggested by an acute increase of VEGF plasma levels during smoking (9). Furthermore, it has been shown that components of cigarette smoke enhance VEGF mRNA and protein expression in cultured endothelial cells of carotid artery (10). Accordingly, we hypothesized that VEGF could play a role in the pathogenesis of the intimal cell proliferation shown in pulmonary arteries of smokers and patients with mild COPD (11). Interestingly, it has been recently suggested that VEGF might be involved in the pathogenesis of emphysema through apoptotic mechanisms (12). This concept has promoted a growing interest in clarifying the role of VEGF at different stages of COPD.

With this background in mind, this study was addressed to investigate the potential role of VEGF in the pathogenesis of the vascular changes that take place in COPD. Accordingly, we evaluated the expression of VEGF protein and mRNA in pulmonary arteries and lung tissue of smokers and patients with different degrees of COPD severity. Preliminary results of the study have been previously reported in abstract form (13).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lung tissue specimens from 78 patients were studied: 68 from lung resection for localized lung carcinoma and 10 from lung volume reduction surgery performed in patients with severe emphysema. Part of these tissue samples has been used in previous studies from our group (5, 14).

Preoperative functional measurements were performed in all patients. Patients who underwent lung resection for lung carcinoma were divided according to their smoking habit and pulmonary function into three groups: nonsmokers, smokers with normal lung function, and moderate COPD. An additional fourth group included 10 patients with emphysema who underwent lung volume reduction surgery (15, 16). None of the patients with severe emphysema had pulmonary hypertension, as documented by right heart catheterization. General characteristics and lung function measurements in the four groups of patients are shown in Table 1 . The study was approved by the Committee on Human Research at our institution.


View this table:
[in this window]
[in a new window]
  		TABLE 1. General characteristics and lung function measurements

 
Morphometric characteristics of pulmonary muscular arteries with an external diameter of less than 500 µm and with complete elastic laminas were analyzed in sections stained with orcein using a computerized image analyzer, as previously reported (3). The areas occupied by the muscular layer, the intima and the lumen, were expressed as a percentage of the total area.

Formalin-fixed paraffin-embedded tissue sections were immunostained with a mouse monoclonal antibody antihuman VEGF using the avidin–biotin complex/horseradish peroxidase method. A mean of 46 ± 26 arteries was analyzed in each subject. The number of pulmonary muscular arteries with positive immunoreactivity to VEGF in the vessel wall (either in endothelial or SMCs) was expressed as the percentage of the total number of arteries. The intensity of the immunoreaction was additionally graded as absent, 0; mild, 1; moderate, 2; or intense, 3. An average grading score was computed in each patient.

Western blot analysis was performed from frozen samples of 36 patients (9 in each group). Some of these frozen tissues were collected after performing the immunohistochemical studies. Briefly, 100 µg of protein were loaded in each lane of a sodium dodecyl sulfate-polyacrylamide gradient gel (from 10 to 15%) and submitted to electrophoresis. Subsequently, proteins were transferred to PVDF membranes and incubated with an anti-VEGF antibody at a 1:200 dilution. After incubation with the secondary antibody, specific immunoreactivity was detected with the avidin–biotin complex/horseradish peroxidase method. Protein bands were visualized by enhanced chemiluminescence. Band intensity was quantitated by densitometry and was normalized for the expression of two housekeeping proteins and the mean density of all bands, as previously reported (14).

Total RNA was extracted from frozen fresh lung tissue of 30 cases using Trizol Reagent. Reverse transcription-polymerase chain reaction (RT-PCR) for VEGF was performed as previously described (17). Under the exponential phase of PCR amplification (30 cycles), products of 403, 535, and 607 bp long were detected, corresponding to the 121-, 165-, and 189-amino acid VEGF isoforms, respectively.

Data were expressed as mean ± SD or as medians and interquartile range according to whether the variable followed a normal distribution (Kolmogorov-Smirnov test). Comparisons between groups were performed using the analysis of variance or the Kruskall-Wallis test, when appropriate. Post hoc comparisons were performed using the Student's t test or the Mann-Whitney U-test for parametric and nonparametric tests, respectively. Correlations between variables were analyzed with Pearson's coefficient. Probability values lower than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Functional and Morphometric Measurements
The functional characteristics of the groups are reported in Table 1. Patients in the four groups were matched by age. Smokers were heavy cigarette consumers with a mean of 50 pack-years (range, 10 to 90 pack-years) without differences among them. Fifty-three percent (n = 31) were ex-smokers, and 47% (n = 28) were current smokers. In the moderate COPD group, arterial PO2 was normal or only slightly altered. Patients with severe emphysema had severe airflow obstruction, hyperinflation, and air trapping. All patients in this group were hypoxemic, and three had chronic respiratory failure.

Morphometric measurements of pulmonary muscular arteries are summarized in Table 2 . We examined a similar number of arteries per patient in each group. According to morphometric analysis, the intimal layer was enlarged in smokers and in patients with moderate COPD, as compared with nonsmokers, in agreement with previous studies (3, 5). In the severe emphysema group, intimal enlargement was greater than in the smoker and moderate COPD groups. There were no differences in the thickness of the medial layer between groups.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Morphometric characteristics of pulmonary arteries

 
Immunohistochemical Analysis
In nonsmoker control cases, positive immunoreaction to VEGF antibody was observed in both endothelial and medial SMCs of the arterial wall, macrophages, and airway epithelial cells. Intense immunoreactivity to VEGF was observed in pulmonary arteries of the smoker and moderate COPD groups. In these patients, positive immunoreaction was shown in the endothelium, the medial layer, and also the cells (SMCs) proliferating in the enlarged intimas (Figure 1) (11). Compared with nonsmokers, semiquantitative analysis of the immunohistochemical data revealed that the proportion of pulmonary arteries showing positive immunoreaction to VEGF in the arterial wall was greater and the intensity of the immunoreaction stronger in both the smoker and the moderate COPD groups (Figure 2 and Table 3) . It should be noted that differences between groups were mainly determined by the expression in SMCs (Table 3). In severe emphysema, the expression of VEGF showed a trend to be lower than in moderate COPD.





View larger version (264K):
[in this window]
[in a new window]
  		Figure 1. Photomicrographs of pulmonary muscular arteries from a nonsmoker (A), a patient with moderate COPD (B), and a patient with severe emphysema (C), immunostained with antibody against VEGF. Positive cells (brown) were located in endothelial and SMCs of the arterial wall. Immunoreactivity to VEGF was greater in pulmonary arteries of patients with moderate COPD. Original magnification x200. a = pulmonary muscular artery; br = bronchiole.

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Individual data of the immunohistochemical expression of VEGF in pulmonary muscular arteries. Results are expressed as percentage of arteries with positive immunoreaction to VEGF (left panel) and intensity of the immunoreaction (right panel). Horizontal bars indicate median values.

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Expression of vascular endothelial growth factor in lung tissue

 
The expression of VEGF was unrelated to the current smoking status, as the number of positive arteries in ex-smokers and current smokers was similar (median 71% [interquartile range, 56–91%] and 77% [59–86%], respectively; p = 0.64).

The immunohistochemical expression of VEGF in SMCs correlated with the arterial wall thickness (r = 0.38, p < 0.01) (Figure 3) .



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Relationship between the number of arteries showing positive immunohistochemical expression of VEGF in SMCs and the thickness of the arterial wall (r = 0.38, p < 0.01).

 
Western Blot Analysis of VEGF Protein Levels
Protein extracts from lung tissue samples were examined for VEGF protein content by Western blot analysis. The monoclonal antibody to VEGF detected two protein bands at a molecular mass of 23 and 29 kD (Figure 4) that correspond to the VEGF165 and VEGF189 isoforms, respectively. Densitometric analysis of protein bands revealed a reduced VEGF protein content in patients with severe emphysema, which was due to a reduction in the VEGF189 isoform (Figure 4 and Table 3). No statistical differences were found among nonsmokers, smokers, and patients with moderate COPD. Protein signal of the VEGF165 isoform was similar in all groups. Total VEGF protein content was positively correlated with arterial PO2 (r = 0.43, p < 0.05).




View larger version (50K):
[in this window]
[in a new window]
  		Figure 4. (A) Western blot analysis for VEGF in homogenized lung tissue of nonsmokers, smokers with normal lung function, moderate COPD, and severe emphysema. Bands at 23 kD and 29 kD are consistent with the size of VEGF165 and VEGF189 isoforms, respectively. MW = molecular weight. (B) Quantification of the Western blot analysis for total VEGF protein content. VEGF expression is significantly reduced in patients with severe emphysema compared with nonsmokers. Horizontal bars represent median values.

 
VEGF mRNA Analysis by RT-PCR
Total VEGF mRNA in lung tissue, measured by RT-PCR, did not differ between groups. Nevertheless, the relative proportion among VEGF isoforms differed in patients with severe emphysema (Figure 5 and Table 4) . The proportional contribution of the VEGF189 isoform to total VEGF mRNA was reduced in the severe emphysema group compared with the other three groups. This finding is consistent with the reduced expression of the 29-kD band in the severe emphysema group shown in the Western blot analysis. No differences in the PCR products of the VEGF121 and the VEGF165 isoforms were observed among the four groups (Table 4).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 5. RT-PCR analysis of VEGF mRNA. Representative RT-PCR of mRNA for VEGF189 (607 bp long), VEGF165 (535 bp long), and VEGF121 (403 bp long) isoforms in lung tissue of nonsmokers, smokers, moderate COPD, and severe emphysema are shown. Amplification products for ß-actin, a constitutively expressed gene, served as control (285 bp long). Note the weak expression of VEGF189 isoform in severe emphysema as compared with the other three subsets of patients. MW = molecular weight.

 

View this table:
[in this window]
[in a new window]
  		TABLE 4. Vascular endothelial growth factor mRNA analysis by reverse transcription-polymerase chain reaction

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study show an increased expression of VEGF in pulmonary muscular arteries of patients with moderate COPD and also in smokers with normal lung function, as compared with nonsmokers, and that this expression is associated with the enlargement of the arterial wall. In contrast, in patients with severe emphysema, the immunohistochemical expression of VEGF in pulmonary arteries and its protein content in lung tissue tends to be low, despite intense vascular remodeling.

This study was mainly addressed to investigate the potential role of VEGF in the pathogenesis of pulmonary vascular remodeling that takes place in the initial stage of COPD (3, 5). At this stage, remodeling is characterized by SMCs proliferation and extracellular matrix deposition in the intimal layer of pulmonary muscular arteries (11). A major finding of this investigation was an enhanced expression of VEGF in the wall of pulmonary arteries in patients with moderate COPD and in smokers with normal lung function, who also had enlarged intimas. In pulmonary vessels, VEGF can be expressed in both endothelial cells and SMCs (18, 19). In our study, differences among groups were essentially due to the expression of VEGF in SMCs, located either in the medial or in the intimal layer, whereas the expression in endothelial cells did not differ significantly (Table 3). Interestingly, the expression of VEGF in SMCs correlated with the thickening of the arterial wall (Figure 3), suggesting a potential role of VEGF in the pathogenesis of pulmonary vascular remodeling. The role of VEGF on SMC activity remains unsettled. In vitro studies indicate that VEGF may induce and modulate vascular SMC proliferation and migration through the upregulation of matrix metalloproteinases (7, 20) and that the VEGF receptors flt-1 and flk-1 are expressed in vascular SMCs (7, 21). Experimental studies in animal models show that VEGF may promote SMC proliferation and neointimal formation after vascular injury (22) and that recombinant VEGF enhances the progression of the atherosclerotic plaque (23, 24). In humans, VEGF has been related with the progression of coronary atherosclerosis (25). Consequently, we hypothesize that in the early stages of COPD, VEGF might contribute to the structural remodeling of pulmonary arteries, presumably by enhancing the proliferation and intimal migration of SMCs. Nevertheless, the expression and function of VEGF receptors involved in this process need further clarification to confirm the potential role of VEGF in mediating these vascular changes.

Some of the biologic actions of VEGF are related to NO-dependent mechanisms (26). The inhibition of NO synthesis with N-nitro-L-arginine methyl ester has been shown to upregulate the gene expression of VEGF and VEGF receptor in a model of isolated perfused lung (27). It has been recently reported that long-term inhibition of NO by administering N-nitro-L-arginine methyl ester, induces coronary vascular arteriosclerosis (28). In this animal model, N-nitro-L-arginine methyl ester produced a significant increase of VEGF gene expression (29). Furthermore, when VEGF receptors were blocked, there was a decrease in the proliferative changes of the coronary artery (29), hence implicating this growth factor in the process. These results suggest that endothelial dysfunction and decreased NO synthesis may facilitate the upregulation of VEGF. In previous studies, we have shown endothelial dysfunction and decreased endothelial NO synthase expression in pulmonary arteries of smokers and patients with mild COPD (5, 14), changes that may likely promote VEGF activity. Indeed, in 25 cases of this series, in which we also had data on endothelium-dependent vascular relaxation (5), we have found an inverse relationship between VEGF expression and endothelial function, as assessed by the maximal relaxation of pulmonary artery rings induced by adenosine diphosphate (r = -0.52, p < 0.01). Accordingly, endothelial dysfunction and impaired NO synthesis, which are present in smokers and patients with mild COPD, may facilitate the upregulation of VEGF in pulmonary vessels.

It could be speculated that upregulation of VEGF could be beneficial for preventing the development of pulmonary hypertension, as previously reported by Partovian and colleagues (30) in an hypoxic rat model. Nevertheless, it seems unlikely that VEGF could play a "vasodilator" role in our series because we found an inverse relationship between VEGF expression and endothelium-dependent vascular relaxation, suggesting that upregulation of VEGF occurred in the context of a vasoconstrictor/vasodilator imbalance that favors pulmonary hypertension.

Hypoxia is a major factor involved in the induction of VEGF gene expression, being considered one of the major stimuli accounting for pulmonary vascular remodeling in chronic lung diseases (27, 31). However, the potential mechanisms that implicate VEGF in the early vascular remodeling of COPD do not seem to be related to hypoxia, as the majority of the patients in our series had normal arterial oxygen tension. Indeed, we found a weak although significant correlation between PaO2 values and protein content of VEGF in lung tissue. Therefore, it is necessary to consider other potential triggers for the increased VEGF activity at early stages of COPD.

Inflammatory cell (CD8+ T-lymphocytes) infiltrate has been shown in the adventitial layer of pulmonary arteries in both smokers and patients with mild COPD (32). The degree of lymphocytic infiltrate correlated with the extent of intimal thickening (32). It is unknown, however, to what extent these recruited inflammatory cells are a cause or a consequence of vascular remodeling. Conceivably, an inflammatory stimulus could be a potential trigger for VEGF upregulation at initial stages of the process. Many inflammatory mediators, including prostaglandin E2, interleukin-6, and interleukin-1 have been shown to induce VEGF mRNA and protein expression (33, 34). In addition, it has been shown that macrophages and T-lymphocytes express VEGF receptors (35) and that VEGF induces monocyte activation and migration (36, 37). Accordingly, it might be speculated that increased VEGF activity in pulmonary arteries at early stages of COPD could be related, to some extent, with an underlying inflammatory process.

It is conceivable that cigarette smoke products could be potential triggers for VEGF because its expression was increased in pulmonary arteries of smokers with normal lung function. This suggestion is supported by the findings of Conklin and colleagues (10), who demonstrated that nicotine and cotinine, in doses similar to those seen in the plasma of current smokers, upregulate VEGF expression in endothelial cells. Furthermore, a recent study by Wright and colleagues (38) demonstrates upregulation of VEGF gene expression and its receptor (flk-1) in pulmonary arteries of rats exposed to cigarette smoke.

Finally, it can be argued that in our series the presence of a neoplasm could be a potential trigger for VEGF. However, we consider this unlikely because differences in VEGF expression were observed between nonsmokers, smokers, and patients with moderate COPD, all of them having a neoplasic process. Moreover, for the purposes of this study, we sampled areas of lung tissue with normal appearance, far from the neoplasic lesion, hence being doubtful the influence of the neoplasic lesion in these areas.

Compared with patients with moderate COPD, the immunohistochemical expression of VEGF and its protein content was lower in patients with severe emphysema. These findings are consistent with recent observations by Kasahara and colleagues (12) demonstrating decreased VEGF expression in alveolar septal cells in emphysema. In our study, a quantitative analysis of VEGF expression in alveolar structures was not performed. Nevertheless, the fact that VEGF content in protein extracts of lung tissue was significantly reduced in patients with emphysema suggests that VEGF expression was diminished not only in pulmonary arteries but also in other lung structures, thus supporting the observation by Kasahara and colleagues (12). The reduced immunohistochemical expression of VEGF in pulmonary arteries occurred despite that patients with severe emphysema had marked remodeling in these vessels. A potential explanation for this finding could be related to the heterogeneous phenotype of SMCs proliferating in the intimal layer. In a recent study, we observed that a significant proportion of SMCs proliferating in the intimal layer of remodeled arteries of smokers and patients with mild COPD did not express contractile filaments, suggesting a less differentiated state and a synthetic phenotype (11). Inoue and colleagues (25) have suggested that nondifferentiated SMCs (synthetic phenotype) express more VEGF than differentiated SMCs (normal contractile phenotype). Thus, it can be speculated that pulmonary vascular remodeling in COPD might be an evolving process. At an early stage of the disease, vascular remodeling is produced by poorly differentiated SMCs with a high expression of VEGF, whereas at advanced stages, remodeling is mainly conformed by differentiated SMCs expressing less VEGF.

RT-PCR studies revealed a reduced proportion of VEGF189 mRNA isoform in patients with severe emphysema. This finding is consistent with a reduced protein signal of the 29-kD molecular weight band in the Western blot analysis. The existence of multiple VEGF isoforms, which vary in localization, accessibility, receptor binding, and activity, raises the possibility that individual VEGF isoforms might mediate different aspects of vascular pathophysiology (39). The VEGF189 isoform appears to be involved in vessel development (40) and maintenance (41). Thus, a reduction of this isoform might be implicated in the apoptotic changes that take place in the alveolar septa of emphysematous lungs, as suggested by Kasahara and colleagues (12). In addition, VEGF189 is completely retained on the cell surface and in the extracellular matrix, from where it can be released as a result of the activity of proteases (6, 39). As a consequence, the decrease of the VEGF189 isoform might also be related to protease–antiprotease imbalance and/or lung tissue destruction.

In summary, the results of this study show that the expression of VEGF in pulmonary arteries of patients with COPD may vary according to the severity of the disease. At an early stage, VEGF might be an important signaling molecule linking changes in endothelial function with vessel remodeling. In contrast, in patients with severe emphysema the vascular expression of VEGF decreases in agreement with changes that take place in the underlying lung parenchyma.


   Acknowledgments
 
The authors thank M. Sánchez and M. Cerrillo for their technical work in immunohistochemical studies and the Department of Thoracic Surgery for their help in the collection of the lung specimens.


   FOOTNOTES
 
Supported by grants from the Fondo de Investigación Sanitaria (FIS 99/188 and 00/922) and the Sociedad Española de Neumología y Cirugía Torácica (SEPAR-1999). Dr. Santos is the recipient of a Research Fellowship award from SEPAR. Dr. Peinado is supported by the Comissió Interdepartamental de Recerca i Innovació Tecnològica and the Fundació Clínic per a la Recerca Biomèdica. Dr. Morales-Blanhir is the recipient of a Research Fellowship award from the European Respiratory Society.

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

Received in original form October 29, 2002; accepted in final form February 17, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wilkinson M, Langhorne CA, Heath D, Barer GR, Howard P. A pathophysiological study of 10 cases of hypoxic cor pulmonale. Q J Med 1988;66:65–85.
  2. Wright JL, Petty T, Thurlbeck WM. Analysis of the structure of the muscular pulmonary arteries in patients with pulmonary hypertension and COPD: National Institutes of Health nocturnal oxygen therapy trial. Lung 1992;170:109–124.[Medline]
  3. Barberà JA, Riverola A, Roca J, Ramírez J, Wagner PD, Ros D, Wiggs BR, Rodriguez-Roisin R. Pulmonary vascular abnormalities and ventilation-perfusion relationships in mild chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1994;149:423–429.[Abstract]
  4. Kessler R, Faller M, Weitzenblum E, Chaouat A, Aykut A, Ducoloné A, Ehrhart M, Oswald-Mammosser M. Natural history of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease. Am J Respir Crit Care Med 2001;164:219–224.[Abstract/Free Full Text]
  5. Peinado VI, Barberà JA, Ramírez J, Gómez FP, Roca J, Jover L, Gimferrer JM, Rodriguez-Roisin R. Endothelial dysfunction in pulmonary arteries of patients with COPD. Am J Physiol 1998;274:L908–L913.
  6. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25.[Abstract/Free Full Text]
  7. Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells. Circ Res 1998;83:832–840.[Abstract/Free Full Text]
  8. Tuder RM, Chacon M, Alger L, Wang J, Taraseviciene-Stewart L, Kasahara Y, Cool CD, Bishop AE, Geraci M, Semenza G, et al. Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis. J Pathol 2001;195:367–374.[CrossRef][Medline]
  9. Wasada T, Kawahara R, Katsumori K, Naruse M, Omori Y. Plasma concentration of immunoreactive vascular endothelial growth factor and its relation to smoking. Metabolism 1998;47:27–30.[CrossRef][Medline]
  10. Conklin BS, Zhao W, Zhong D-S, Chen CH. Nicotine and cotinine up-regulate vascular endothelial growth factor expression in endothelial cells. Am J Pathol 2002;160:413–418.[Abstract/Free Full Text]
  11. Santos S, Peinado VI, Ramirez J, Melgosa T, Roca J, Rodriguez-Roisin R, Barberà JA. Characterization of pulmonary vascular remodeling in smokers and patients with mild COPD. Eur Respir J 2002;19:632–638.[Abstract/Free Full Text]
  12. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 2001;163:737–744.[Abstract/Free Full Text]
  13. Barberà JA, Santos S, Peinado VI, Ramírez J, Morales J, Gimferrer JM, Roca J, Rodriguez-Roisin R. Expression of vascular endothelial growth factor (VEGF) in pulmonary muscular arteries of COPD spectrum [abstract]. Am J Respir Crit Care Med 2001;163:A123.
  14. Barberà JA, Peinado VI, Santos S, Ramirez J, Roca J, Rodriguez-Roisin R. Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med 2001;164:709–713.[Abstract/Free Full Text]
  15. Brenner M, Yusen R, McKenna R, Sciurba F, Gelb AF, Fischel R, Swain J, Chen JC, Kafie F, Lefrak SS. Lung volume reduction surgery for emphysema. Chest 1996;110:205–218.[Abstract/Free Full Text]
  16. Retamales I, Elliott WM, Meshi B, Coxson HO, Pare PD, Sciurba FC, Rogers RM, Hayashi S, Hogg JC. Amplification of inflammation in emphysema and its association with latent adenoviral infection. Am J Respir Crit Care Med 2001;164:469–473.[Abstract/Free Full Text]
  17. Cejudo-Martín P, Ros J, Navasa M, Fernández J, Fernández-Varo G, Ruiz del Arbol L, Rivera F, Arroyo V, Rodés J, Jiménez W. Increased production of vascular endothelial growth factor in peritoneal macrophages of cirrhotic patients with spontaneous bacterial peritonitis. Hepatology 2001;34:487–493.[CrossRef][Medline]
  18. Shifren JL, Doldi N, Ferrara N, Mesiano S, Jaffe RB. In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action. J Clin Endocrinol Metab 1994;79:316–322.[Abstract]
  19. Shehata SM, Mooi WJ, Okazaki T, El-Banna I, Sharma HS, Tibboel D. Enhanced expression of vascular endothelial growth factor in lungs of newborn infants with congenital diaphragmatic hernia and pulmonary hypertension. Thorax 1999;54:427–431.[Abstract/Free Full Text]
  20. Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman BI, Zetter B, D'Amore PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvasc Res 1999;58:128–136.[CrossRef][Medline]
  21. Ishida A, Murray J, Saito Y, Kanthou C, Benzakour O, Shibuya M, Wijelath ES. Expression of vascular endothelial growth factor receptors in smooth muscle cells. J Cell Physiol 2001;188:359–368.[CrossRef][Medline]
  22. Lazarous DF, Shou M, Scheinowith M, Hodge E, Thirumurti V, Kitsiou AN, Stiber JA, Lobo AD, Hunsberger S, Guetta E, et al. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation 1996;94:1074–1082.[Abstract/Free Full Text]
  23. Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, Dake MD. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med 2001;7:425–429.[CrossRef][Medline]
  24. Celletti FL, Hilfiker PR, Ghafouri P, Dake MD. Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque. J Am Coll Cardiol 2001;37:2126–2130.[Abstract/Free Full Text]
  25. Inoue M, Itoh H, Ueda M, Naruko T, Kojima A, Komatsu R, Doi K, Ogawa Y, Tamura N, Takaya K, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation 1998;98:2108–2116.[Abstract/Free Full Text]
  26. He H, Venema VJ, Gu X, Venema RC, Marrero MB, Caldwell RB. Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through Flk-1/KDR activation of c-Src. J Biol Chem 1999;274:25130–25135.[Abstract/Free Full Text]
  27. Tuder RM, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. J Clin Invest 1995;95:1798–1807.
  28. Kitamoto S, Egashira K, Kataoka C, Koyanagi M, Katoh M, Shimokawa H, Morishita R, Kaneda Y, Sueishi K, Tadeshita A. Increased activity of nuclear factor-kappa B participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats. Circulation 2000;102:806–812.[Abstract/Free Full Text]
  29. Zhao Q, Egashira K, Inoue S, Usui M, Kitamoto S, Ni W, Ishibashi M, Hiasa K, Ichiki T, Shibuya M, et al. Vascular endothelial growth factor is necessary in the development of arteriosclerosis by recruiting/activating monocytes in a rat model of long-term inhibition of nitric oxide synthesis. Circulation 2002;105:1110–1115.[Abstract/Free Full Text]
  30. Partovian C, Adnot S, Raffestin B, Louzier V, Levame M, Mavier IM, Lemarchand P, Eddahibi S. Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hipoxic pulmonary hypertension in rats. Am J Respir Cell Mol Biol 2000;23:762–771.[Abstract/Free Full Text]
  31. Minchenko A, Bauer T, Salceda S, Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vivo and in vitro. Lab Invest 1994;71:374–379.[Medline]
  32. Peinado VI, Barberà JA, Abate P, Ramírez J, Roca J, Santos S, Rodriguez-Roisin R. Inflammatory reaction in pulmonary muscular arteries of patients with mild chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;159:1605–1611.[Abstract/Free Full Text]
  33. Li J, Perrella MA, Tsai JC, Yet SF, Hsieh CM, Yoshizumi M, Patterson C, Endege WO, Zhou F, Lee ME. Induction of vascular endothelial growth factor gene expression by interleukin-1ß in rat aortic smooth muscle cells. J Biol Chem 1995;270:308–312.[Abstract/Free Full Text]
  34. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin-6 induces the expression of vascular endothelial growth-factor. J Biol Chem 1996;271:736–741.[Abstract/Free Full Text]
  35. Hoshino M, Nakamura Y, Hamid QA. Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma. J Allergy Clin Immunol 2001;107:1034–1038.[CrossRef][Medline]
  36. Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan CE, Olander JV, Connolly DT, Stern D. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 1990;72:1535–1545.
  37. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the receptor Flt-1. Blood 1996;87:3336–3343.[Abstract/Free Full Text]
  38. Wright JL, Tai H, Dai J, Churg A. Cigarette smoke induces rapid changes in gene expression in pulmonary arteries. Lab Invest 2002;82:1391–1398.[Medline]
  39. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9–22.[Abstract/Free Full Text]
  40. Galambos C, Ng Y-S, Ali A, Noguchi A, Lovejoy S, D'Amore PA, deMello DE. Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. Am J Respir Cell Mol Biol 2002;27:194–203.[Abstract/Free Full Text]
  41. Ng Y-S, Rohan R, Sunday ME, deMello DE, D'Amore PA. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dyn 2001;220:112–121.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
P. M. Hassoun, L. Mouthon, J. A. Barbera, S. Eddahibi, S. C. Flores, F. Grimminger, P. L. Jones, M. L. Maitland, E. D. Michelakis, N. W. Morrell, et al.
Inflammation, growth factors, and pulmonary vascular remodeling.
J. Am. Coll. Cardiol., June 30, 2009; 54(1 Suppl): S10 - S19.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
V. I. Peinado, S. Pizarro, and J. A. Barbera
Pulmonary Vascular Involvement in COPD
Chest, October 1, 2008; 134(4): 808 - 814.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
I. Edirisinghe, S.-R. Yang, H. Yao, S. Rajendrasozhan, S. Caito, D. Adenuga, C. Wong, A. Rahman, R. P. Phipps, Z.-G. Jin, et al.
VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction
FASEB J, July 1, 2008; 22(7): 2297 - 2310.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
J. A. Falk, S. Kadiev, G. J. Criner, S. M. Scharf, O. A. Minai, and P. Diaz
Cardiac Disease in Chronic Obstructive Pulmonary Disease
Proceedings of the ATS, May 1, 2008; 5(4): 543 - 548.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
R. G. Barr, S. Mesia-Vela, J. H. M. Austin, R. C. Basner, B. M. Keller, A. P. Reeves, D. Shimbo, and L. Stevenson
Impaired Flow-mediated Dilation Is Associated with Low Pulmonary Function and Emphysema in Ex-smokers: The Emphysema and Cancer Action Project (EMCAP) Study
Am. J. Respir. Crit. Care Med., December 15, 2007; 176(12): 1200 - 1207.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
T. Yoshida and R. M. Tuder
Pathobiology of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease
Physiol Rev, July 1, 2007; 87(3): 1047 - 1082.
[Abstract] [Full Text] [PDF]

Home page
 The OncologistHome page
S. Senan and E. F. Smit
Design of Clinical Trials of Radiation Combined with Antiangiogenic Therapy
Oncologist, April 1, 2007; 12(4): 465 - 477.
[Abstract] [Full Text] [PDF]

Home page
 ERRHome page
M. Diez, V. I. Peinado, E. Ferrer, J. Ramirez, J. Roca, R. Rodriguez-Roisin, and J. A. Barbera
Plasticity of vascular progenitor cells: Implications in pulmonary vascular remodelling in COPD
Eur. Respir. Rev., December 1, 2006; 15(101): 209 - 210.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
H. A. Ghofrani, R. Voswinckel, F. Reichenberger, N. Weissmann, R. T. Schermuly, W. Seeger, and F. Grimminger
Hypoxia- and non-hypoxia-related pulmonary hypertension - Established and new therapies
Cardiovasc Res, October 1, 2006; 72(1): 30 - 40.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
B. Beghe, E. Bazzan, S. Baraldo, F. Calabrese, F. Rea, M. Loy, P. Maestrelli, R. Zuin, L. M. Fabbri, and M. Saetta
Transforming growth factor-{beta} type II receptor in pulmonary arteries of patients with very severe COPD
Eur. Respir. J., September 1, 2006; 28(3): 556 - 562.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. A. Marwick, C. S. Stevenson, J. Giddings, W. MacNee, K. Butler, I. Rahman, and P. A. Kirkham
Cigarette smoke disrupts VEGF165-VEGFR-2 receptor signaling complex in rat lungs and patients with COPD: morphological impact of VEGFR-2 inhibition
Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L897 - L908.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
V. I. Peinado, J. Ramirez, J. Roca, R. Rodriguez-Roisin, and J. A. Barbera
Identification of Vascular Progenitor Cells in Pulmonary Arteries of Patients with Chronic Obstructive Pulmonary Disease
Am. J. Respir. Cell Mol. Biol., March 1, 2006; 34(3): 257 - 263.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
K. M. Oltmanns, H. Gehring, S. Rudolf, B. Schultes, C. Hackenberg, U. Schweiger, J. Born, H. L. Fehm, and A. Peters
Acute hypoxia decreases plasma VEGF concentration in healthy humans
Am J Physiol Endocrinol Metab, March 1, 2006; 290(3): E434 - E439.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J.-H. Lee, D.-S. Lee, E.-K. Kim, K.-H. Choe, Y.-M. Oh, T.-S. Shim, S.-E. Kim, Y.-S. Lee, and S.-D. Lee
Simvastatin Inhibits Cigarette Smoking-induced Emphysema and Pulmonary Hypertension in Rat Lungs
Am. J. Respir. Crit. Care Med., October 15, 2005; 172(8): 987 - 993.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
T. L. Petty
Are COPD and Lung Cancer Two Manifestations of the Same Disease?
Chest, October 1, 2005; 128(4): 1895 - 1897.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. Chaouat, A.-S. Bugnet, N. Kadaoui, R. Schott, I. Enache, A. Ducolone, M. Ehrhart, R. Kessler, and E. Weitzenblum
Severe Pulmonary Hypertension and Chronic Obstructive Pulmonary Disease
Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 189 - 194.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
J L Wright, R D Levy, and A Churg
Pulmonary hypertension in chronic obstructive pulmonary disease: current theories of pathogenesis and their implications for treatment
Thorax, July 1, 2005; 60(7): 605 - 609.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
K. Nagai, T. Betsuyaku, Y. Ito, Y. Nasuhara, and M. Nishimura
Decrease of vascular endothelial growth factor in macrophages from long-term smokers
Eur. Respir. J., April 1, 2005; 25(4): 626 - 633.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
R. Naeije
Pulmonary Hypertension and Right Heart Failure in Chronic Obstructive Pulmonary Disease
Proceedings of the ATS, April 1, 2005; 2(1): 20 - 22.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
E. F. M. Wouters
Local and Systemic Inflammation in Chronic Obstructive Pulmonary Disease
Proceedings of the ATS, April 1, 2005; 2(1): 26 - 33.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
P. J. Barnes
Mediators of Chronic Obstructive Pulmonary Disease
Pharmacol. Rev., December 1, 2004; 56(4): 515 - 548.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
J. L. Wright, H. Tai, and A. Churg
Cigarette Smoke Induces Persisting Increases of Vasoactive Mediators in Pulmonary Arteries
Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 501 - 509.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
K. Tang, H. B. Rossiter, P. D. Wagner, and E. C. Breen
Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice
J Appl Physiol, October 1, 2004; 97(4): 1559 - 1566.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
P.-N. Tsao, Y.-N. Su, H. Li, P.-H. Huang, C.-T. Chien, Y.-L. Lai, C.-N. Lee, C.-A. Chen, W.-F. Cheng, S.-C. Wei, et al.
Overexpression of Placenta Growth Factor Contributes to the Pathogenesis of Pulmonary Emphysema
Am. J. Respir. Crit. Care Med., February 15, 2004; 169(4): 505 - 511.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Chronic Obstructive Pulmonary Disease, Pollution, Pulmonary Vascular Disease, Transplantation, Pleural Disease, and Lung Cancer in AJRCCM 2003
Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 301 - 313.
[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:
200210-1233OCv1
167/9/1250    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 Santos, S.
Right arrow Articles by Barberà, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santos, S.
Right arrow Articles by Barberà, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2003 American Thoracic Society