CLB-08607; No. of pages: 8; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
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Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic bronchitis Seung Hyeun Lee a, Sang Hoon Lee b, Chul Hwan Kim c, Kyung Suk Yang d, Eun Joo Lee e, Kyung Hoon Min e, Gyu Young Hur e, Seung Heon Lee e, Sung Yong Lee e, Je Hyeong Kim e, Chol Shin e, Jae Jeong Shim e, Kwang Ho In e, Kyung Ho Kang e, Sang Yeub Lee e,⁎ a
Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, KEPCO Medical Center, Seoul, Republic of Korea Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea c Department of Pathology, Korea University College of Medicine, Seoul, Republic of Korea d Department of Biostatistics, Korea University College of Medicine, Seoul, Republic of Korea e Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 24 October 2013 Received in revised form 16 December 2013 Accepted 13 January 2014 Available online xxxx Keywords: Chronic bronchitis Chronic obstructive pulmonary disease Smoking Vascular endothelial growth factor
a b s t r a c t Objectives: Vascular endothelial growth factor (VEGF) seems to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its site-specific expression in lung tissue and the relationship with hypoxia inducible factor-1 alpha (HIF-1α) expression in chronic bronchitis (CB) type COPD have not been studied. Design and methods: We evaluated the expression of VEGF and its receptors in various compartments of lung tissue in three groups: non-smokers with normal lung function (non-smokers, n = 10), smokers without COPD (healthy smokers, n = 10) and smokers with CB (CB, n = 10), using immunohistochemical staining and Western blotting. The expression of HIF-1α was assessed by enzyme-linked immunosorbent assay. Results: Compared with healthy smokers, VEGF expression in CB was significantly increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle (p b 0.05). VEGF receptor (VEGFR)-2 expression in CB was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle compared with healthy smokers (p b 0.05). The level of HIF-1α was increased in CB compared with healthy smokers and positively correlated with those of VEGF (r = 0.64, p b 0.05). Conclusion: VEGF and VEGFR-2 expressions were up-regulated in CB and increased expression of VEGF was related with HIF-1α. HIF-1α-regulated VEGF overexpression may be a characteristic of chronic bronchitis. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is not fully reversible, usually progressive and associated with an abnormal inflammatory response of the lungs [1]. It is a major health problem and the fourth leading cause of death worldwide; its prevalence is expected to increase [2]. Despite the various influencing genetic and environmental factors, cigarette smoking is the most important risk factor for COPD. The pathologic changes of COPD are largely categorized as airway inflammation and parenchymal destruction, which are traditionally known as chronic bronchitis (CB) and emphysema, respectively. The relative contributions Abbreviations: CB, chronic bronchitis; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; HIF-1α, hypoxia inducible factor-1alpha; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea. Fax: +82 2 929 2045. E-mail address: [email protected] (S.Y. Lee).
of those two phenotypes to airflow obstruction vary from one person to another, and the mechanism of lung injury by cigarette smoke that results in the predominance of CB or emphysema remains unclear [3]. Vascular endothelial growth factor (VEGF) is one of the potent mediators of vascular regulation in angiogenesis and vascular permeability [4]. VEGF is also involved in lung development, maintenance of normal lung structure, thus it plays a crucial role in the lung homeostasis [5]. Several studies have raised the possibility that VEGF may be related with development of COPD. In an animal model, VEGF blockade with an inhibitor of VEGFR resulted in endothelial cell apoptosis and morphologic changes which is consistent with emphysema [6]. Moreover, the different expression patterns according to the different phenotypes of COPD were reported; VEGF level was increased in the induced sputum and bronchial biopsy specimen in CB patients, while being decreased in emphysema patients [7–10]. Hypoxia inducible factor-1alpha (HIF-1α) is a transcription factor that induces VEGF expression which is affected by both hypoxia and inflammation. Although VEGF expression is induced by variety of factors that include cytokines, chemokines and growth factors, HIF-1α is the major mechanism of VEGF regulation [11,12]. Taken together, VEGF
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Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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and HIF-1α seem to be involved in the pathogenesis of COPD but their expression in lung tissue of CB patients has not been studied. We therefore investigated the expression of VEGF, its receptors and HIF-1α in lung tissue of CB patients and evaluated the relationship between VEGF expression and that of HIF-1α as a possible mechanism of VEGF regulation in CB.
Enzyme-linked immunosorbent assay (ELISA) for HIF-1α
Materials and methods
Statistical analyses
Study population
Data are expressed as mean ± standard deviation. Data were not normally distributed. Therefore, comparisons among groups were made through Kruskal–Wallis test or Mann–Whitney U test. All tests were two-tailed, and p-values were corrected for the number of comparisons using the Bonferroni method. The correlation between the expression of HIF-1α and that of VEGF was assessed by Spearman's correlation analysis. A difference was defined as statistically significant if p b 0.05. Statistical analysis was carried out using SPSS version 13.0 for Windows (SPSS, Chicago, IL, USA).
We used non-tumorous lung tissues donated from the Korea Lung Tissue Bank which has been assigned and supported by the Korean Science and Engineering Foundation. The tissues were obtained from patients who underwent lobectomy or pneumonectomy and immediately frozen at − 80 °C until use. All patients gave their consent and the study protocol was approved by the Clinical Research Ethics Committee of the Korea University Medical Center (AN10139-001). Thirty subjects were enrolled: non-smokers with normal lung function (non-smokers, n = 10), smokers without COPD (healthy smokers, n = 10), and smokers with CB (CB, n = 10). The non-smokers group consisted of individuals who had never smoked tobacco. In contrast, smokers were defined as subjects who had a history of at least 20 pack-years. COPD was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [13]. The diagnosis of CB was made according to the following criteria [9]: cough with sputum expectoration for at least 3 months a year during a period of 2 consecutive years and no evidence of emphysema, based on high-resolution computed tomography (HRCT) scans of the lungs and diffusing capacity of the lung for carbon monoxide (DLCO). All CB patients had a moderate airflow limitation (post-bronchodilator forced expiratory volume in 1 s (FEV1) ≥50 to b80% of predicted, FEV1/functional vital capacity (FVC) b70%, GOLD stage 2). The lung function test results of the non-smokers and healthy smokers were normal. None of the subjects showed bronchodilator response on lung function test and had a history of asthma. Immunohistochemical staining and scoring Immunohistochemical staining was used to localize and compare VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 expressions in all subjects. Antibodies reactive to VEGF (Abcam, Cambridge, MA, USA) and its receptors (Abcam) were used. The procedure was performed according to the recommendations of the supplier. The details for the method are provided in the online Supplementary material. The expression of VEGF, VEGFR-1 and VEGFR-2 was analyzed semiquantitatively using a visual scoring method [14–16] in six different parts of lung tissue: alveolar epithelium, bronchiolar epithelium, bronchiolar smooth muscle, vascular endothelium, vascular smooth muscle and macrophage. The staining intensity was graded and expressed from 0 to 3 (0 = no staining; 1 = moderate staining, 2 = intense staining; 3 = very intense staining). After examining four different areas per each part of lung tissue in one slide, the mean score of the part was determined as staining score. The scoring was done by two observers (S.H. Lee and C. H. Kim) blinded to the clinical data of the subjects. The inter-observer agreement was assessed by weighted kappa statistics and the scores were highly agreed between two observers based on the kappa values ranging from 0.81 to 0.93. Western blot analysis for VEGF and VEGFR Total levels of VEGF and its receptors in lung tissues were analyzed by Western blotting. We used anti-VEGF monoclonal antibody (Abcam) and anti-VEGF receptor 2 polyclonal antibody (Thermo Fisher Scientific, Pittsburgh, PA, USA) and performed the procedure according to the recommendation of the supplier. The detailed procedure for Western blotting is provided in the online Supplementary material.
HIF-1α protein level in lung tissues was measured by ELISA using Surveyor™ IC human/mouse total HIF-1α immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The detailed procedure is provided in the online Supplementary material.
Results Clinical characteristics The clinical data and lung functions of all subjects are shown in Table 1. The mean age was 67.8 years and no age difference was observed among groups. More females were enrolled in non-smokers. The smoking intensity between healthy smokers and CB patients was not significantly different. FEV1 and FEV1/FVC were significantly reduced in CB patients compared with healthy smokers (p = 0.032 and p = 0.028, respectively). The diffusing lung capacity for carbon monoxide (DLCO) in CB patients was also significantly reduced compared to healthy smokers (p = 0.040). Immunohistochemical staining for VEGF Representative examples of immunohistochemical staining for VEGF in alveoli, bronchiole and peripheral vessels are shown in Fig. 1A. In CB patients, many inflammatory cells in the alveolar space and thickened alveolar septum were more prominent compared with other groups. Hyperplasia of bronchiolar epithelial cells, goblet cells and vascular smooth muscle cells was also noted in CB patients. According to the semiquantitative scoring method, VEGF expression of smokers (healthy smokers and CB) was increased in bronchiolar epithelium (1.48 ± 0.27 versus 0.76 ± 0.25; p b 0.05), bronchiolar smooth muscle (1.79 ± 0.26 versus 0.82 ± 0.19; p b 0.05), vascular endothelium (0.67 ± 0.29 versus 0.24 ± 0.08; p b 0.05) and vascular smooth muscle (1.77 ± 0.23 versus 1.12 ± 0.25; p b 0.05) compared with non-smokers. Comparing healthy
Table 1 Clinical characteristics of the subjects.
Age, years Male/female Smoking, pack-years FEV1, % predictive FVC, % predictive FEV1/FVC, % DLCO, % predictive DLCO/VA, % predictive
Non-smokers (n = 10)
Healthy smokers (n = 10)
CB (n = 10)
p value
70.3 ± 12.6 6/4 0 100.9 ± 8.1 89.4 ± 11.5 80.9 ± 5.1 101.7 ± 26.6 114.5 ± 20.6
67.7 ± 5.8 8/2 33.3 ± 10.0 98.7 ± 18.5 89.8 ± 16.5 78.6 ± 4.4 99.7 ± 12.8 91.3 ± 8.6
62.1 ± 8.4 8/2 37.5 ± 9.1 78 ± 7.6 81.7 ± 8.1 66.3 ± 1.5 89.4 ± 15.4 83.1 ± 14.3
0.065 0.141a 0.032 0.107 0.028 0.040 0.025
Data are shown as mean ± standard deviation. CB, chronic bronchitis; FEV1, forced expiratory volume for 1 s; FVC, functional vital capacity; DLCO, diffusing capacity of lung for carbon monoxide; VA, volume of alveoli. a Comparing healthy smokers with CB.
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A a
b
c
B * *
* *
Fig. 1. (A) Representative photographs of immunohistochemical staining for VEGF in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGF is expressed (×200). (B) Staining scores of VEGF in six parts of the lung tissue. Comparing healthy smokers with non-smokers, VEGF expression was increased in bronchiolar epithelium. Comparing CB with healthy smokers, VEGF expression was increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle. Bars express standard deviation. CB, chronic bronchitis; black arrow and box, alveolar epithelium; arrow head, macrophage; red arrow, bronchiolar epithelium; red asterisk, bronchiolar smooth muscle; blue arrow, vascular endothelium; blue asterisk, vascular smooth muscle; *, p b 0.05; alv.epi., alveolar epithelium; br.epi., bronchiolar epithelium; br.sm., bronchiolar smooth muscle; v.endo., vascular endothelium; v.sm., vascular smooth; MØ, macrophage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
smokers with non-smokers, VEGF expression was only increased in bronchiolar smooth muscle (1.62 ± 0.26 versus 0.82 ± 0.19; p b 0.05). Comparing CB patients with healthy smokers, VEGF expression was increased in bronchiolar epithelium (1.80 ± 0.29 versus 1.13 ± 0.25; p b 0.05), vascular endothelium (0.84 ± 0.21 versus 0.42 ± 0.18; p b 0.05) and vascular smooth muscle (2.13 ± 0.22 versus 1.28 ± 0.24; p b 0.05) (Fig. 1B). Immunohistochemical staining for VEGFR-1 and VEGFR-2 Fig. 2A shows a representative example of immunohistochemical staining for VEGFR-1 in lung tissue. Comparing VEGFR-1 expression of smokers (healthy smokers and CB) with that of non-smokers, increased expression was found only in bronchiolar smooth muscle (1.51 ± 0.13
versus 1.01 ± 0.12; p b 0.05) and no significant differences were observed in other parts. Comparing healthy smokers with nonsmokers, the expression of VEGFR-1 was increased only in bronchiolar smooth muscle (1.41 ± 0.13 versus 1.01 ± 0.12; p b 0.05). The level of VEGFR-1 in CB did not show differences in any part of lung tissue compared with healthy smokers (Fig. 2B). Examples of immunohistochemical staining for VEGFR-2 are presented in Fig. 3A. Comparing smokers (healthy smokers and CB) with non-smokers, VEGFR-2 was more abundant in bronchiolar epithelium (2.11 ± 0.23 versus 0.76 ± 0.19; p b 0.05), bronchiolar smooth muscle (1.99 ± 0.29 versus 1.43 ± 0.24; p b 0.05) and vascular smooth muscle (1.09 ± 0.22 versus 0.45 ± 0.16; p b 0.05). Comparing healthy smokers with non-smokers, VEGFR-2 expression was only increased in bronchiolar epithelium (2.00 ± 0.29 versus 0.76 ± 0.19; p b 0.05). Comparing
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A a
b
c
B
*
Fig. 2. (A) Representative photographs of immunohistochemical staining for VEGFR-1 in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGFR-1 is expressed (×200). (B) Staining scores of VEGFR-1 in six parts of the lung tissue. Comparing healthy smokers with non-smokers, VEGFR-1 expression was increased in bronchiolar smooth muscle. Comparing CB with healthy smokers, VEGFR-1 expression was not different in any parts of the lung tissue. Bars express standard deviation. For arrows, asterisks and abbreviations, please refer to the legend of Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
CB patients with healthy smokers, VEGFR-2 expression was increased in bronchiolar smooth muscle (2.29 ± 0.32 versus 1.43 ± 0.37; p b 0.05), vascular endothelium (0.75 ± 0.23 versus 0.44 ± 0.16; p b 0.05) and vascular smooth muscle (1.24 ± 0.18 versus 0.62 ± 0.30; p b 0.05) (Fig. 3B). Western blot analysis for VEGF and VEGFR To validate the results of immunohistochemical staining, total VEGF and VEGFR-2 expressions were quantified in homogenized whole lung tissue of all subjects using Western blotting. For VEGF, one protein band was detected at the molecular weight of 45 kDa. Densitometric analysis revealed that VEGF expression was significantly increased in CB patients compared with healthy smokers (0.91 ± 0.30 versus 0.53 ± 0.18; p b 0.05) while it was not significantly different between
healthy smokers and non-smokers (Fig. 4A). For VEGFR-2, one protein band was detected at the molecular weight of 150 kDa. VEGFR-2 expression was also increased in CB patients compared with healthy smokers (1.20 ± 0.27 versus 0.70 ± 0.35; p b 0.05) (Fig. 4B). The results of Western blots were consistent with those of the immunohistochemical staining. HIF-1α expression and correlation with VEGF expression To evaluate whether HIF-1α level is affected by smoking status or presence of CB, we compared the HIF-1α levels among groups. HIF-1α expression did not show any difference between nonsmokers and healthy smokers. In contrast, its expression was significantly increased in CB patients compared to healthy smokers (0.54 ± 0.28
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A a
b
c
B * *
* *
Fig. 3. (A) Representative photographs of immunohistochemical staining for VEGFR-2 in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGFR-2 is expressed (×200). (B) Staining scores of VEGFR-2 in six parts of the lung tissue. Comparing healthy smoker with non-smokers, VEGFR-2 expression was increased in bronchiolar epithelium. Comparing CB with healthy smokers, VEGFR-2 was increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle. Bars express standard deviation. For arrows, asterisks and abbreviations, please refer to the legend of Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
versus 0.39 ± 0.19; p b 0.05) (Fig. 5A). In addition, we performed correlation analysis between VEGF and HIF-1α levels to assess whether VEGF expression is related with HIF-1α in all subjects. A significant positive correlation was found between these expressions (r = 0.64, p b 0.05) (Fig. 5B). Discussion In the present study, CB was associated with increased expression of VEGF in the bronchiolar epithelium, bronchiolar smooth muscle and vascular smooth muscle. VEGFR-2 was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle. In addition, VEGF expression was correlated with HIF-1α expression. To the best of our knowledge, this is the first study to document the increased expression of VEGF and VEGFR in different compartments of
lung tissues in patients with CB. More importantly, our data suggest that HIF-1α is involved in the increased expression of VEGF in those patients. Although the molecular and cellular mechanisms that are responsible for the development and progress of COPD are not fully understood, anti-protease/protease activity, oxidative stress, apoptosis and influx of inflammatory cells including neutrophils, macrophages and CD8+ T lymphocytes are involved [3]. COPD has two different phenotypes: emphysema and CB. The former is characterized by the destruction of the alveolar septum, the resulting decrease of elastic recoil and increased airway resistance. The latter is characterized by mucus gland enlargement and goblet cell hyperplasia leading to cough and sputum. The relative contribution of these phenotypes to airflow limitation differs from patient to patient [2]. The mechanism concerning how individual subjects have emphysema- or CB-dominant phenotype from the same
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A 45 kDa
Relative density of VEGF
37 kDa
B 150 kDa
Relative density of VEGFR-2
37 kDa
Fig. 4. Western blot analysis results of VEGF and VEGFR-2. Protein bands for VEGF and VEGFR-2 and individual densitometry data are shown. (A) Compared to healthy smokers, the expression of VEGF was significantly increased in CB. (B) The expression of VEGFR-2 was also increased in CB. Bars express mean and standard deviation. *, p b 0.05; CB, chronic bronchitis.
insult – cigarette smoking – is largely unknown. Several studies have suggested that VEGF signaling may be involved in the phenotypic expression of the disease based on different expression levels in emphysema and CB [7–9,17]. However, previous studies have limitations in that they used only sputum or bronchial biopsy specimen instead of whole lung tissue and lacked healthy smokers as a control. Moreover, VEGFR expression has not been investigated in CB patients. Presently, we successfully demonstrated the increased VEGF and VEGFR-2 expressions in CB patients compared with those of healthy smokers in various parts of lung tissue and the correlation between HIF-1α and VEGF expressions. Cigarette smoking is a potent inducer of VEGF expression and production [9,18,19]. In this study, VEGF expression in bronchial smooth muscle was increased in healthy smokers compared with nonsmokers reflecting that the augmented VEGF expression is linked to cigarette smoking. However, there was no difference in the expression of HIF-1α between non-smokers and healthy smokers, while increased expression was noted in CB patients compared with healthy smokers. Taking the previous and current data together, we can suggest that VEGF overexpression in CB is not a result of smoking, but may be an intrinsic characteristic of the disease itself. The clinical implication of increased VEGF expression can be explained by two points of view. First,
increased expression of VEGF and its receptor may be a result of the unique ongoing inflammatory process of CB, which is different from that induced by cigarette smoke only. Previous studies showed the abundance of neutrophils, interleukin-8 and tumor necrosis factoralpha in CB patients compared with healthy smokers, all of which induce VEGF expression [9,20]. In addition, VEGF itself can act as a chemokine for neutrophils [21]. Thus, the inflammation could be worsened in a vicious cycle with the interaction between VEGF and inflammatory mediators participating in CB pathogenesis. Second, because VEGF and its receptor system are essential in lung homeostasis, increased VEGF expression may partly indicate an attempt to repair damage in airway and lung parenchyma to maintain normal lung structure and function [22]. This adaptive process may contribute to adverse consequences, such as pulmonary bronchial or vascular remodeling [23]. In this study, the expression of VEGFR-2 was increased in various compartments of lung tissue of CB patients, while that of VEGFR-1 showed no increase compared with healthy smokers. Both VEGFR-1 and VEGFR-2 are the main receptors in VEGF signaling, but they have different functions in vivo. VEGFR-2 is the active receptor involved in the mediation of major growth and permeability actions of VEGF, whereas VEGFR-1 inhibits the action of VEGF by acting as a modulating decoy to VEGFR-2 [24]. The former allows the excessive proliferation
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A
Relative density of VEGF
B
Fig. 5. HIF-1α levels in each group and relationship between VEGF and HIF-1α expressions. (A) HIF-1α level was significantly increased in CB compared with healthy smokers. (B) A significant positive correlation was shown between HIF-1α and VEGF levels in all patients. Bars express mean and standard deviation. *, p b 0.05; CB, chronic bronchitis.
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involved in two chronic airway diseases including asthma and COPD, although they are developed via different pathobiologic mechanisms. Our study has several limitations. First, the sample size of study subjects was relatively small and we did not investigate the expression in emphysema-dominant patients simultaneously. However, our aim was to evaluate VEGF expression in CB patients, and we verified the significantly increased VEGF expression in CB patients compared with healthy smokers even with small subject numbers. Second, the enrolled subjects were all Korean. It could limit the applicability of our results to the general population. Third, more females were enrolled in nonsmokers compared with other groups. Gortner et al. have suggested the possibility of the different regulation of VEGF between genders, thus the unequal gender distribution among groups could influence our results [35]. Finally, other possible regulators other than HIF-1α that could be involved in VEGF expression were not evaluated simultaneously. Although VEGF is regulated by numerous cytokines and other molecules, HIF-1α-induced transcriptional control is considered as a major mechanism of VEGF expression [11]. We hypothesized that HIF-1α would also play a certain role in VEGF expression in CB and successfully demonstrated the significant association between VEGF expression and that of HIF-1α, which is comparable to the results of a previous study on patients with emphysema [29]. In conclusion, VEGF and VEGFR-2 expressions were up-regulated in various compartments of lung tissues and VEGF expression was correlated with the increased expression of HIF-1α in CB patients. Our findings add important evidence that HIF-1α-regulated VEGF expression could be one of the mechanisms to determine the predominance between CB and emphysema in smokers. Although further studies are needed to verify our results, the current data provide important clues for understanding the pathogenesis of COPD and form the theoretical basis for the treatment of the disease. Acknowledgments
and migration of vascular endothelial cells, while the latter inhibits the process [25]. Thus, we can suggest that the exaggerated inflammation in CB may be the result of the enhanced interaction mainly between VEGF and VEGFR-2. Although, increased expression of VEGF, VEGFR-1 and VEGFR-2 was demonstrated in the bronchiolar epithelium and smooth muscle of COPD patients [26], VEGFR expression has not been investigated in CB patients. More studies are needed to support our data and elucidate the role of each receptor in the pathogenesis of the disease. VEGF expression is regulated by various factors such as hypoxia, growth factors and inflammatory cytokines. Especially, hypoxia plays a key role in VEGF gene expression both in vitro and in vivo [27]. HIF1α is considered as a major and important factor involved in control of VEGF expression by inducing VEGF transcription [11,28]. Presently, HIF-1α was increased in CB patients compared with healthy smokers and its expression was positively correlated with that of VEGF. Interestingly, a recent study reported that the expression of VEGF is reduced and associated with that of HIF-1α in emphysema patients [29]. Because emphysema and CB can co-exist in the same patient, we excluded emphysema-dominant patients based on thorough review of HRCT scan and DLCO. The mean DLCO of enrolled CB patients was over 80% of predictive value suggesting that they are CB dominant. Therefore, our results add important evidence that HIF-1α may be an important regulator of VEGF expression in both emphysema and CB. In asthma, VEGF, VEGFR-1 and VEGFR-2 have been shown to be increased in sputum, bronchoalveolar lavage fluid and lung tissue [30–32]. Moreover, a recent study has demonstrated that the increased expression of VEGF in asthmatics is related with high level of hypoxia inducible factor-1α (HIF-1α) as in CB patients of the present study [33]. Lee et al. hypothesized that the VEGF excess contributes to the Th2 response as in asthma, and deficiency of VEGF contributes to the Th1 response as in emphysema [34]. Collectively, HIF-1α-regulated VEGF expression seems to be
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inflammation in healthy smokers and in patients with bronchitis type of chronic obstructive pulmonary disease? Respir Res 2007;8:53. Santos S, Peinado VI, Ramirez J, Morales-Blanhir J, Bastos R, Roca J, et al. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:1250–6. Semenza GL. Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology (Bethesda) 2009;24:97–106. Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, et al. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 2008;453:807–11. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007;176:532–55. de Boer WI, Schuller AG, Vermey M, van der Kwast TH. Expression of growth factors and receptors during specific phases in regenerating urothelium after acute injury in vivo. Am J Pathol 1994;145:1199–207. de Boer WI, van Schadewijk A, Sont JK, Sharma HS, Stolk J, Hiemstra PS, et al. Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;158:1951–7. Kranenburg AR, De Boer WI, Van Krieken JH, Mooi WJ, Walters JE, Saxena PR, et al. Enhanced expression of fibroblast growth factors and receptor FGFR-1 during vascular remodeling in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2002;27:517–25. Suzuki M, Betsuyaku T, Nagai K, Fuke S, Nasuhara Y, Kaga K, et al. Decreased airway expression of vascular endothelial growth factor in cigarette smoke-induced emphysema in mice and COPD patients. Inhal Toxicol 2008;20:349–59. Wright JL, Tai H, Churg A. Cigarette smoke induces persisting increases of vasoactive mediators in pulmonary arteries. Am J Respir Cell Mol Biol 2004;31:501–9. Volpi G, Facchinetti F, Moretto N, Civelli M, Patacchini R. Cigarette smoke and alpha, beta-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts. Br J Pharmacol 2011;163: 649–61. Takanashi S, Hasegawa Y, Kanehira Y, Yamamoto K, Fujimoto K, Satoh K, et al. Interleukin-10 level in sputum is reduced in bronchial asthma, COPD and in smokers. Eur Respir J 1999;14:309–14. Tetley TD. Inflammatory cells and chronic obstructive pulmonary disease. Curr Drug Targets Inflamm Allergy 2005;4:607–18.
[22] Tuder RM, Chacon M, Alger L, Wang J, Taraseviciene-Stewart L, Kasahara Y, et al. Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis. J Pathol 2001;195:367–74. [23] Healy AM, Morgenthau L, Zhu X, Farber HW, Cardoso WV. VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung. Dev Dyn 2000;219:341–52. [24] Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature 2000;407:242–8. [25] Voelkel NF, Vandivier RW, Tuder RM. Vascular endothelial growth factor in the lung. Am J Physiol Lung Cell Mol Physiol 2006;290:L209–21. [26] Kranenburg AR, de Boer WI, Alagappan VK, Sterk PJ, Sharma HS. Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease. Thorax 2005;60:106–13. [27] Boussat S, Eddahibi S, Coste A, Fataccioli V, Gouge M, Housset B, et al. Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2000;279:L371–8. [28] Jung YJ, Isaacs JS, Lee S, Trepel J, Neckers L. IL-1beta-mediated up-regulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis. FASEB J 2003;17:2115–7. [29] Yasuo M, Mizuno S, Kraskauskas D, Bogaard HJ, Natarajan R, Cool CD, et al. Hypoxia inducible factor-1alpha in human emphysema lung tissue. Eur Respir J 2011;37: 775–83. [30] Asai K, Kanazawa H, Kamoi H, Shiraishi S, Hirata K, Yoshikawa J. Increased levels of vascular endothelial growth factor in induced sputum in asthmatic patients. Clin Exp Allergy 2003;33:595–9. [31] 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;107:1034–8. [32] Lee SY, Kwon S, Kim KH, Moon HS, Song JS, Park SH, et al. Expression of vascular endothelial growth factor and hypoxia-inducible factor in the airway of asthmatic patients. Ann Allergy Asthma Immunol 2006;97:794–9. [33] Kim SR, Lee KS, Park HS, Park SJ, Min KH, Moon H, et al. HIF-1alpha inhibition ameliorates an allergic airway disease via VEGF suppression in bronchial epithelium. Eur J Immunol 2010;40:2858–69. [34] Lee CG, Ma B, Takyar S, Ahangari F, Delacruz C, He CH, et al. Studies of vascular endothelial growth factor in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 2011;8:512–5. [35] Gortner L, Shen J, Tutdibi E. Sexual dimorphism of neonatal lung development. Klin Padiatr 2013;225:64–9.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic bronchitis Seung Hyeun Lee a, Sang Hoon Lee b, Chul Hwan Kim c, Kyung Suk Yang d, Eun Joo Lee e, Kyung Hoon Min e, Gyu Young Hur e, Seung Heon Lee e, Sung Yong Lee e, Je Hyeong Kim e, Chol Shin e, Jae Jeong Shim e, Kwang Ho In e, Kyung Ho Kang e, Sang Yeub Lee e,⁎ a
Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, KEPCO Medical Center, Seoul, Republic of Korea Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea c Department of Pathology, Korea University College of Medicine, Seoul, Republic of Korea d Department of Biostatistics, Korea University College of Medicine, Seoul, Republic of Korea e Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 24 October 2013 Received in revised form 16 December 2013 Accepted 13 January 2014 Available online xxxx Keywords: Chronic bronchitis Chronic obstructive pulmonary disease Smoking Vascular endothelial growth factor
a b s t r a c t Objectives: Vascular endothelial growth factor (VEGF) seems to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its site-specific expression in lung tissue and the relationship with hypoxia inducible factor-1 alpha (HIF-1α) expression in chronic bronchitis (CB) type COPD have not been studied. Design and methods: We evaluated the expression of VEGF and its receptors in various compartments of lung tissue in three groups: non-smokers with normal lung function (non-smokers, n = 10), smokers without COPD (healthy smokers, n = 10) and smokers with CB (CB, n = 10), using immunohistochemical staining and Western blotting. The expression of HIF-1α was assessed by enzyme-linked immunosorbent assay. Results: Compared with healthy smokers, VEGF expression in CB was significantly increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle (p b 0.05). VEGF receptor (VEGFR)-2 expression in CB was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle compared with healthy smokers (p b 0.05). The level of HIF-1α was increased in CB compared with healthy smokers and positively correlated with those of VEGF (r = 0.64, p b 0.05). Conclusion: VEGF and VEGFR-2 expressions were up-regulated in CB and increased expression of VEGF was related with HIF-1α. HIF-1α-regulated VEGF overexpression may be a characteristic of chronic bronchitis. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is not fully reversible, usually progressive and associated with an abnormal inflammatory response of the lungs [1]. It is a major health problem and the fourth leading cause of death worldwide; its prevalence is expected to increase [2]. Despite the various influencing genetic and environmental factors, cigarette smoking is the most important risk factor for COPD. The pathologic changes of COPD are largely categorized as airway inflammation and parenchymal destruction, which are traditionally known as chronic bronchitis (CB) and emphysema, respectively. The relative contributions Abbreviations: CB, chronic bronchitis; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; HIF-1α, hypoxia inducible factor-1alpha; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea. Fax: +82 2 929 2045. E-mail address: [email protected] (S.Y. Lee).
of those two phenotypes to airflow obstruction vary from one person to another, and the mechanism of lung injury by cigarette smoke that results in the predominance of CB or emphysema remains unclear [3]. Vascular endothelial growth factor (VEGF) is one of the potent mediators of vascular regulation in angiogenesis and vascular permeability [4]. VEGF is also involved in lung development, maintenance of normal lung structure, thus it plays a crucial role in the lung homeostasis [5]. Several studies have raised the possibility that VEGF may be related with development of COPD. In an animal model, VEGF blockade with an inhibitor of VEGFR resulted in endothelial cell apoptosis and morphologic changes which is consistent with emphysema [6]. Moreover, the different expression patterns according to the different phenotypes of COPD were reported; VEGF level was increased in the induced sputum and bronchial biopsy specimen in CB patients, while being decreased in emphysema patients [7–10]. Hypoxia inducible factor-1alpha (HIF-1α) is a transcription factor that induces VEGF expression which is affected by both hypoxia and inflammation. Although VEGF expression is induced by variety of factors that include cytokines, chemokines and growth factors, HIF-1α is the major mechanism of VEGF regulation [11,12]. Taken together, VEGF
0009-9120/$ – see front matter © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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and HIF-1α seem to be involved in the pathogenesis of COPD but their expression in lung tissue of CB patients has not been studied. We therefore investigated the expression of VEGF, its receptors and HIF-1α in lung tissue of CB patients and evaluated the relationship between VEGF expression and that of HIF-1α as a possible mechanism of VEGF regulation in CB.
Enzyme-linked immunosorbent assay (ELISA) for HIF-1α
Materials and methods
Statistical analyses
Study population
Data are expressed as mean ± standard deviation. Data were not normally distributed. Therefore, comparisons among groups were made through Kruskal–Wallis test or Mann–Whitney U test. All tests were two-tailed, and p-values were corrected for the number of comparisons using the Bonferroni method. The correlation between the expression of HIF-1α and that of VEGF was assessed by Spearman's correlation analysis. A difference was defined as statistically significant if p b 0.05. Statistical analysis was carried out using SPSS version 13.0 for Windows (SPSS, Chicago, IL, USA).
We used non-tumorous lung tissues donated from the Korea Lung Tissue Bank which has been assigned and supported by the Korean Science and Engineering Foundation. The tissues were obtained from patients who underwent lobectomy or pneumonectomy and immediately frozen at − 80 °C until use. All patients gave their consent and the study protocol was approved by the Clinical Research Ethics Committee of the Korea University Medical Center (AN10139-001). Thirty subjects were enrolled: non-smokers with normal lung function (non-smokers, n = 10), smokers without COPD (healthy smokers, n = 10), and smokers with CB (CB, n = 10). The non-smokers group consisted of individuals who had never smoked tobacco. In contrast, smokers were defined as subjects who had a history of at least 20 pack-years. COPD was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [13]. The diagnosis of CB was made according to the following criteria [9]: cough with sputum expectoration for at least 3 months a year during a period of 2 consecutive years and no evidence of emphysema, based on high-resolution computed tomography (HRCT) scans of the lungs and diffusing capacity of the lung for carbon monoxide (DLCO). All CB patients had a moderate airflow limitation (post-bronchodilator forced expiratory volume in 1 s (FEV1) ≥50 to b80% of predicted, FEV1/functional vital capacity (FVC) b70%, GOLD stage 2). The lung function test results of the non-smokers and healthy smokers were normal. None of the subjects showed bronchodilator response on lung function test and had a history of asthma. Immunohistochemical staining and scoring Immunohistochemical staining was used to localize and compare VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 expressions in all subjects. Antibodies reactive to VEGF (Abcam, Cambridge, MA, USA) and its receptors (Abcam) were used. The procedure was performed according to the recommendations of the supplier. The details for the method are provided in the online Supplementary material. The expression of VEGF, VEGFR-1 and VEGFR-2 was analyzed semiquantitatively using a visual scoring method [14–16] in six different parts of lung tissue: alveolar epithelium, bronchiolar epithelium, bronchiolar smooth muscle, vascular endothelium, vascular smooth muscle and macrophage. The staining intensity was graded and expressed from 0 to 3 (0 = no staining; 1 = moderate staining, 2 = intense staining; 3 = very intense staining). After examining four different areas per each part of lung tissue in one slide, the mean score of the part was determined as staining score. The scoring was done by two observers (S.H. Lee and C. H. Kim) blinded to the clinical data of the subjects. The inter-observer agreement was assessed by weighted kappa statistics and the scores were highly agreed between two observers based on the kappa values ranging from 0.81 to 0.93. Western blot analysis for VEGF and VEGFR Total levels of VEGF and its receptors in lung tissues were analyzed by Western blotting. We used anti-VEGF monoclonal antibody (Abcam) and anti-VEGF receptor 2 polyclonal antibody (Thermo Fisher Scientific, Pittsburgh, PA, USA) and performed the procedure according to the recommendation of the supplier. The detailed procedure for Western blotting is provided in the online Supplementary material.
HIF-1α protein level in lung tissues was measured by ELISA using Surveyor™ IC human/mouse total HIF-1α immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The detailed procedure is provided in the online Supplementary material.
Results Clinical characteristics The clinical data and lung functions of all subjects are shown in Table 1. The mean age was 67.8 years and no age difference was observed among groups. More females were enrolled in non-smokers. The smoking intensity between healthy smokers and CB patients was not significantly different. FEV1 and FEV1/FVC were significantly reduced in CB patients compared with healthy smokers (p = 0.032 and p = 0.028, respectively). The diffusing lung capacity for carbon monoxide (DLCO) in CB patients was also significantly reduced compared to healthy smokers (p = 0.040). Immunohistochemical staining for VEGF Representative examples of immunohistochemical staining for VEGF in alveoli, bronchiole and peripheral vessels are shown in Fig. 1A. In CB patients, many inflammatory cells in the alveolar space and thickened alveolar septum were more prominent compared with other groups. Hyperplasia of bronchiolar epithelial cells, goblet cells and vascular smooth muscle cells was also noted in CB patients. According to the semiquantitative scoring method, VEGF expression of smokers (healthy smokers and CB) was increased in bronchiolar epithelium (1.48 ± 0.27 versus 0.76 ± 0.25; p b 0.05), bronchiolar smooth muscle (1.79 ± 0.26 versus 0.82 ± 0.19; p b 0.05), vascular endothelium (0.67 ± 0.29 versus 0.24 ± 0.08; p b 0.05) and vascular smooth muscle (1.77 ± 0.23 versus 1.12 ± 0.25; p b 0.05) compared with non-smokers. Comparing healthy
Table 1 Clinical characteristics of the subjects.
Age, years Male/female Smoking, pack-years FEV1, % predictive FVC, % predictive FEV1/FVC, % DLCO, % predictive DLCO/VA, % predictive
Non-smokers (n = 10)
Healthy smokers (n = 10)
CB (n = 10)
p value
70.3 ± 12.6 6/4 0 100.9 ± 8.1 89.4 ± 11.5 80.9 ± 5.1 101.7 ± 26.6 114.5 ± 20.6
67.7 ± 5.8 8/2 33.3 ± 10.0 98.7 ± 18.5 89.8 ± 16.5 78.6 ± 4.4 99.7 ± 12.8 91.3 ± 8.6
62.1 ± 8.4 8/2 37.5 ± 9.1 78 ± 7.6 81.7 ± 8.1 66.3 ± 1.5 89.4 ± 15.4 83.1 ± 14.3
0.065 0.141a 0.032 0.107 0.028 0.040 0.025
Data are shown as mean ± standard deviation. CB, chronic bronchitis; FEV1, forced expiratory volume for 1 s; FVC, functional vital capacity; DLCO, diffusing capacity of lung for carbon monoxide; VA, volume of alveoli. a Comparing healthy smokers with CB.
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A a
b
c
B * *
* *
Fig. 1. (A) Representative photographs of immunohistochemical staining for VEGF in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGF is expressed (×200). (B) Staining scores of VEGF in six parts of the lung tissue. Comparing healthy smokers with non-smokers, VEGF expression was increased in bronchiolar epithelium. Comparing CB with healthy smokers, VEGF expression was increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle. Bars express standard deviation. CB, chronic bronchitis; black arrow and box, alveolar epithelium; arrow head, macrophage; red arrow, bronchiolar epithelium; red asterisk, bronchiolar smooth muscle; blue arrow, vascular endothelium; blue asterisk, vascular smooth muscle; *, p b 0.05; alv.epi., alveolar epithelium; br.epi., bronchiolar epithelium; br.sm., bronchiolar smooth muscle; v.endo., vascular endothelium; v.sm., vascular smooth; MØ, macrophage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
smokers with non-smokers, VEGF expression was only increased in bronchiolar smooth muscle (1.62 ± 0.26 versus 0.82 ± 0.19; p b 0.05). Comparing CB patients with healthy smokers, VEGF expression was increased in bronchiolar epithelium (1.80 ± 0.29 versus 1.13 ± 0.25; p b 0.05), vascular endothelium (0.84 ± 0.21 versus 0.42 ± 0.18; p b 0.05) and vascular smooth muscle (2.13 ± 0.22 versus 1.28 ± 0.24; p b 0.05) (Fig. 1B). Immunohistochemical staining for VEGFR-1 and VEGFR-2 Fig. 2A shows a representative example of immunohistochemical staining for VEGFR-1 in lung tissue. Comparing VEGFR-1 expression of smokers (healthy smokers and CB) with that of non-smokers, increased expression was found only in bronchiolar smooth muscle (1.51 ± 0.13
versus 1.01 ± 0.12; p b 0.05) and no significant differences were observed in other parts. Comparing healthy smokers with nonsmokers, the expression of VEGFR-1 was increased only in bronchiolar smooth muscle (1.41 ± 0.13 versus 1.01 ± 0.12; p b 0.05). The level of VEGFR-1 in CB did not show differences in any part of lung tissue compared with healthy smokers (Fig. 2B). Examples of immunohistochemical staining for VEGFR-2 are presented in Fig. 3A. Comparing smokers (healthy smokers and CB) with non-smokers, VEGFR-2 was more abundant in bronchiolar epithelium (2.11 ± 0.23 versus 0.76 ± 0.19; p b 0.05), bronchiolar smooth muscle (1.99 ± 0.29 versus 1.43 ± 0.24; p b 0.05) and vascular smooth muscle (1.09 ± 0.22 versus 0.45 ± 0.16; p b 0.05). Comparing healthy smokers with non-smokers, VEGFR-2 expression was only increased in bronchiolar epithelium (2.00 ± 0.29 versus 0.76 ± 0.19; p b 0.05). Comparing
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A a
b
c
B
*
Fig. 2. (A) Representative photographs of immunohistochemical staining for VEGFR-1 in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGFR-1 is expressed (×200). (B) Staining scores of VEGFR-1 in six parts of the lung tissue. Comparing healthy smokers with non-smokers, VEGFR-1 expression was increased in bronchiolar smooth muscle. Comparing CB with healthy smokers, VEGFR-1 expression was not different in any parts of the lung tissue. Bars express standard deviation. For arrows, asterisks and abbreviations, please refer to the legend of Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
CB patients with healthy smokers, VEGFR-2 expression was increased in bronchiolar smooth muscle (2.29 ± 0.32 versus 1.43 ± 0.37; p b 0.05), vascular endothelium (0.75 ± 0.23 versus 0.44 ± 0.16; p b 0.05) and vascular smooth muscle (1.24 ± 0.18 versus 0.62 ± 0.30; p b 0.05) (Fig. 3B). Western blot analysis for VEGF and VEGFR To validate the results of immunohistochemical staining, total VEGF and VEGFR-2 expressions were quantified in homogenized whole lung tissue of all subjects using Western blotting. For VEGF, one protein band was detected at the molecular weight of 45 kDa. Densitometric analysis revealed that VEGF expression was significantly increased in CB patients compared with healthy smokers (0.91 ± 0.30 versus 0.53 ± 0.18; p b 0.05) while it was not significantly different between
healthy smokers and non-smokers (Fig. 4A). For VEGFR-2, one protein band was detected at the molecular weight of 150 kDa. VEGFR-2 expression was also increased in CB patients compared with healthy smokers (1.20 ± 0.27 versus 0.70 ± 0.35; p b 0.05) (Fig. 4B). The results of Western blots were consistent with those of the immunohistochemical staining. HIF-1α expression and correlation with VEGF expression To evaluate whether HIF-1α level is affected by smoking status or presence of CB, we compared the HIF-1α levels among groups. HIF-1α expression did not show any difference between nonsmokers and healthy smokers. In contrast, its expression was significantly increased in CB patients compared to healthy smokers (0.54 ± 0.28
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A a
b
c
B * *
* *
Fig. 3. (A) Representative photographs of immunohistochemical staining for VEGFR-2 in aveoli and macrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicate where VEGFR-2 is expressed (×200). (B) Staining scores of VEGFR-2 in six parts of the lung tissue. Comparing healthy smoker with non-smokers, VEGFR-2 expression was increased in bronchiolar epithelium. Comparing CB with healthy smokers, VEGFR-2 was increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle. Bars express standard deviation. For arrows, asterisks and abbreviations, please refer to the legend of Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
versus 0.39 ± 0.19; p b 0.05) (Fig. 5A). In addition, we performed correlation analysis between VEGF and HIF-1α levels to assess whether VEGF expression is related with HIF-1α in all subjects. A significant positive correlation was found between these expressions (r = 0.64, p b 0.05) (Fig. 5B). Discussion In the present study, CB was associated with increased expression of VEGF in the bronchiolar epithelium, bronchiolar smooth muscle and vascular smooth muscle. VEGFR-2 was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle. In addition, VEGF expression was correlated with HIF-1α expression. To the best of our knowledge, this is the first study to document the increased expression of VEGF and VEGFR in different compartments of
lung tissues in patients with CB. More importantly, our data suggest that HIF-1α is involved in the increased expression of VEGF in those patients. Although the molecular and cellular mechanisms that are responsible for the development and progress of COPD are not fully understood, anti-protease/protease activity, oxidative stress, apoptosis and influx of inflammatory cells including neutrophils, macrophages and CD8+ T lymphocytes are involved [3]. COPD has two different phenotypes: emphysema and CB. The former is characterized by the destruction of the alveolar septum, the resulting decrease of elastic recoil and increased airway resistance. The latter is characterized by mucus gland enlargement and goblet cell hyperplasia leading to cough and sputum. The relative contribution of these phenotypes to airflow limitation differs from patient to patient [2]. The mechanism concerning how individual subjects have emphysema- or CB-dominant phenotype from the same
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A 45 kDa
Relative density of VEGF
37 kDa
B 150 kDa
Relative density of VEGFR-2
37 kDa
Fig. 4. Western blot analysis results of VEGF and VEGFR-2. Protein bands for VEGF and VEGFR-2 and individual densitometry data are shown. (A) Compared to healthy smokers, the expression of VEGF was significantly increased in CB. (B) The expression of VEGFR-2 was also increased in CB. Bars express mean and standard deviation. *, p b 0.05; CB, chronic bronchitis.
insult – cigarette smoking – is largely unknown. Several studies have suggested that VEGF signaling may be involved in the phenotypic expression of the disease based on different expression levels in emphysema and CB [7–9,17]. However, previous studies have limitations in that they used only sputum or bronchial biopsy specimen instead of whole lung tissue and lacked healthy smokers as a control. Moreover, VEGFR expression has not been investigated in CB patients. Presently, we successfully demonstrated the increased VEGF and VEGFR-2 expressions in CB patients compared with those of healthy smokers in various parts of lung tissue and the correlation between HIF-1α and VEGF expressions. Cigarette smoking is a potent inducer of VEGF expression and production [9,18,19]. In this study, VEGF expression in bronchial smooth muscle was increased in healthy smokers compared with nonsmokers reflecting that the augmented VEGF expression is linked to cigarette smoking. However, there was no difference in the expression of HIF-1α between non-smokers and healthy smokers, while increased expression was noted in CB patients compared with healthy smokers. Taking the previous and current data together, we can suggest that VEGF overexpression in CB is not a result of smoking, but may be an intrinsic characteristic of the disease itself. The clinical implication of increased VEGF expression can be explained by two points of view. First,
increased expression of VEGF and its receptor may be a result of the unique ongoing inflammatory process of CB, which is different from that induced by cigarette smoke only. Previous studies showed the abundance of neutrophils, interleukin-8 and tumor necrosis factoralpha in CB patients compared with healthy smokers, all of which induce VEGF expression [9,20]. In addition, VEGF itself can act as a chemokine for neutrophils [21]. Thus, the inflammation could be worsened in a vicious cycle with the interaction between VEGF and inflammatory mediators participating in CB pathogenesis. Second, because VEGF and its receptor system are essential in lung homeostasis, increased VEGF expression may partly indicate an attempt to repair damage in airway and lung parenchyma to maintain normal lung structure and function [22]. This adaptive process may contribute to adverse consequences, such as pulmonary bronchial or vascular remodeling [23]. In this study, the expression of VEGFR-2 was increased in various compartments of lung tissue of CB patients, while that of VEGFR-1 showed no increase compared with healthy smokers. Both VEGFR-1 and VEGFR-2 are the main receptors in VEGF signaling, but they have different functions in vivo. VEGFR-2 is the active receptor involved in the mediation of major growth and permeability actions of VEGF, whereas VEGFR-1 inhibits the action of VEGF by acting as a modulating decoy to VEGFR-2 [24]. The former allows the excessive proliferation
Please cite this article as: Lee SH, et al, Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic..., Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012
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A
Relative density of VEGF
B
Fig. 5. HIF-1α levels in each group and relationship between VEGF and HIF-1α expressions. (A) HIF-1α level was significantly increased in CB compared with healthy smokers. (B) A significant positive correlation was shown between HIF-1α and VEGF levels in all patients. Bars express mean and standard deviation. *, p b 0.05; CB, chronic bronchitis.
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involved in two chronic airway diseases including asthma and COPD, although they are developed via different pathobiologic mechanisms. Our study has several limitations. First, the sample size of study subjects was relatively small and we did not investigate the expression in emphysema-dominant patients simultaneously. However, our aim was to evaluate VEGF expression in CB patients, and we verified the significantly increased VEGF expression in CB patients compared with healthy smokers even with small subject numbers. Second, the enrolled subjects were all Korean. It could limit the applicability of our results to the general population. Third, more females were enrolled in nonsmokers compared with other groups. Gortner et al. have suggested the possibility of the different regulation of VEGF between genders, thus the unequal gender distribution among groups could influence our results [35]. Finally, other possible regulators other than HIF-1α that could be involved in VEGF expression were not evaluated simultaneously. Although VEGF is regulated by numerous cytokines and other molecules, HIF-1α-induced transcriptional control is considered as a major mechanism of VEGF expression [11]. We hypothesized that HIF-1α would also play a certain role in VEGF expression in CB and successfully demonstrated the significant association between VEGF expression and that of HIF-1α, which is comparable to the results of a previous study on patients with emphysema [29]. In conclusion, VEGF and VEGFR-2 expressions were up-regulated in various compartments of lung tissues and VEGF expression was correlated with the increased expression of HIF-1α in CB patients. Our findings add important evidence that HIF-1α-regulated VEGF expression could be one of the mechanisms to determine the predominance between CB and emphysema in smokers. Although further studies are needed to verify our results, the current data provide important clues for understanding the pathogenesis of COPD and form the theoretical basis for the treatment of the disease. Acknowledgments
and migration of vascular endothelial cells, while the latter inhibits the process [25]. Thus, we can suggest that the exaggerated inflammation in CB may be the result of the enhanced interaction mainly between VEGF and VEGFR-2. Although, increased expression of VEGF, VEGFR-1 and VEGFR-2 was demonstrated in the bronchiolar epithelium and smooth muscle of COPD patients [26], VEGFR expression has not been investigated in CB patients. More studies are needed to support our data and elucidate the role of each receptor in the pathogenesis of the disease. VEGF expression is regulated by various factors such as hypoxia, growth factors and inflammatory cytokines. Especially, hypoxia plays a key role in VEGF gene expression both in vitro and in vivo [27]. HIF1α is considered as a major and important factor involved in control of VEGF expression by inducing VEGF transcription [11,28]. Presently, HIF-1α was increased in CB patients compared with healthy smokers and its expression was positively correlated with that of VEGF. Interestingly, a recent study reported that the expression of VEGF is reduced and associated with that of HIF-1α in emphysema patients [29]. Because emphysema and CB can co-exist in the same patient, we excluded emphysema-dominant patients based on thorough review of HRCT scan and DLCO. The mean DLCO of enrolled CB patients was over 80% of predictive value suggesting that they are CB dominant. Therefore, our results add important evidence that HIF-1α may be an important regulator of VEGF expression in both emphysema and CB. In asthma, VEGF, VEGFR-1 and VEGFR-2 have been shown to be increased in sputum, bronchoalveolar lavage fluid and lung tissue [30–32]. Moreover, a recent study has demonstrated that the increased expression of VEGF in asthmatics is related with high level of hypoxia inducible factor-1α (HIF-1α) as in CB patients of the present study [33]. Lee et al. hypothesized that the VEGF excess contributes to the Th2 response as in asthma, and deficiency of VEGF contributes to the Th1 response as in emphysema [34]. Collectively, HIF-1α-regulated VEGF expression seems to be
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