Clinical Biochemistry 47 (2014) 552–559

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Clinical Biochemistry

j ourna l homepage: www.e lsev ie r .com/ locate /c l inb iochem

Increased expression of vascular endothelial growth factor and hypoxiainducible 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 Koreab Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Koreac Department of Pathology, Korea University College of Medicine, Seoul, Republic of Koread Department of Biostatistics, Korea University College of Medicine, Seoul, Republic of Koreae Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea

Abbreviations: CB, chronic bronchitis; COPD, chronicELISA, enzyme-linked immunosorbent assay; HIF-1α, hyVEGF, vascular endothelial growth factor.⁎ Corresponding author at: Division of Respiratory

Department of Internal Medicine, Korea University AnCollege of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-Korea. Fax: +82 2 929 2045.

E-mail address: pulsy@korea.ac.kr (S.Y. Lee).

0009-9120/$ – see front matter © 2014 The Canadian Sochttp://dx.doi.org/10.1016/j.clinbiochem.2014.01.012

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 24 October 2013Received in revised form 16 December 2013Accepted 13 January 2014Available online 21 January 2014

Keywords:Chronic bronchitisChronic obstructive pulmonary diseaseSmokingVascular endothelial growth factor

Objectives: Vascular endothelial growth factor (VEGF) seems to be involved in the pathogenesis of chronicobstructive pulmonary disease (COPD), but its site-specific expression in lung tissue and the relationship withhypoxia inducible factor-1 alpha (HIF-1α) expression in chronic bronchitis (CB) type COPD have not been studied.

Design andmethods:We evaluated the expression of VEGF and its receptors in various compartments of lungtissue 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 andWesternblotting. The expression of HIF-1αwas assessed by enzyme-linked immunosorbent assay.

Results: Compared with healthy smokers, VEGF expression in CB was significantly increased in bronchiolarepithelium, vascular endothelium and vascular smooth muscle (p b 0.05). VEGF receptor (VEGFR)-2 expressionin CB was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle com-

pared with healthy smokers (p b 0.05). The level of HIF-1α was increased in CB compared with healthy smokersand 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 wasrelated 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 bypersistent airflow limitation that is not fully reversible, usually progres-sive and associated with an abnormal inflammatory response of thelungs [1]. It is a major health problem and the fourth leading cause ofdeath worldwide; its prevalence is expected to increase [2]. Despitethe various influencing genetic and environmental factors, cigarettesmoking is the most important risk factor for COPD. The pathologicchanges of COPD are largely categorized as airway inflammation andparenchymal destruction, which are traditionally known as chronicbronchitis (CB) and emphysema, respectively. The relative contributions

obstructive pulmonary disease;poxia inducible factor-1alpha;

and Critical Care Medicine,am Hospital, Korea Universitygu, Seoul 136-705, Republic of

iety of Clinical Chemists. Published b

of those two phenotypes to airflow obstruction vary from one person toanother, and the mechanism of lung injury by cigarette smoke that re-sults in the predominance of CB or emphysema remains unclear [3].

Vascular endothelial growth factor (VEGF) is one of the potentmedi-ators of vascular regulation in angiogenesis and vascular permeability[4]. VEGF is also involved in lung development, maintenance of normallung structure, thus it plays a crucial role in the lung homeostasis [5].Several studies have raised the possibility that VEGF may be relatedwith development of COPD. In an animal model, VEGF blockade withan inhibitor of VEGFR resulted in endothelial cell apoptosis and mor-phologic changes which is consistent with emphysema [6]. Moreover,the different expression patterns according to the different phenotypesof COPD were reported; VEGF level was increased in the inducedsputum and bronchial biopsy specimen in CB patients, while being de-creased in emphysema patients [7–10].

Hypoxia inducible factor-1alpha (HIF-1α) is a transcription factorthat induces VEGF expression which is affected by both hypoxia and in-flammation. Although VEGF expression is induced by variety of factorsthat include cytokines, chemokines and growth factors, HIF-1α is themajor mechanism of VEGF regulation [11,12]. Taken together, VEGF

y Elsevier Inc. All rights reserved.

Table 1Clinical characteristics of the subjects.

Non-smokers(n = 10)

Healthy smokers(n = 10)

CB(n = 10)

p value

Age, years 70.3 ± 12.6 67.7 ± 5.8 62.1 ± 8.4 0.065Male/female 6/4 8/2 8/2Smoking, pack-years 0 33.3 ± 10.0 37.5 ± 9.1 0.141a

FEV1, % predictive 100.9 ± 8.1 98.7 ± 18.5 78 ± 7.6 0.032FVC, % predictive 89.4 ± 11.5 89.8 ± 16.5 81.7 ± 8.1 0.107FEV1/FVC, % 80.9 ± 5.1 78.6 ± 4.4 66.3 ± 1.5 0.028DLCO, % predictive 101.7 ± 26.6 99.7 ± 12.8 89.4 ± 15.4 0.040DLCO/VA, % predictive 114.5 ± 20.6 91.3 ± 8.6 83.1 ± 14.3 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.

553S.H. Lee et al. / Clinical Biochemistry 47 (2014) 552–559

and HIF-1α seem to be involved in the pathogenesis of COPD but theirexpression in lung tissue of CB patients has not been studied.We there-fore investigated the expression of VEGF, its receptors and HIF-1α inlung tissue of CB patients and evaluated the relationship betweenVEGF expression and that of HIF-1α as a possible mechanism of VEGFregulation in CB.

Materials and methods

Study population

We used non-tumorous lung tissues donated from the Korea LungTissue Bank which has been assigned and supported by the KoreanScience and Engineering Foundation. The tissues were obtained frompatients who underwent lobectomy or pneumonectomy and immedi-ately frozen at −80 °C until use. All patients gave their consent andthe study protocol was approved by the Clinical Research EthicsCommittee 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 con-sisted of individualswho had never smoked tobacco. In contrast, smokerswere defined as subjects who had a history of at least 20 pack-years.COPD was defined according to the Global Initiative for ChronicObstructive Lung Disease (GOLD) [13]. The diagnosis of CB was madeaccording to the following criteria [9]: cough with sputum expectorationfor at least 3 months a year during a period of 2 consecutive years and noevidence of emphysema, based on high-resolution computed tomo-graphy (HRCT) scans of the lungs and diffusing capacity of the lung forcarbon monoxide (DLCO). All CB patients had a moderate airflow limita-tion (post-bronchodilator forced expiratory volume in 1 s (FEV1) ≥50to b80% of predicted, FEV1/functional vital capacity (FVC) b70%, GOLDstage 2). The lung function test results of the non-smokers and healthysmokers were normal. None of the subjects showed bronchodilatorresponse on lung function test and had a history of asthma.

Immunohistochemical staining and scoring

Immunohistochemical staining was used to localize and compareVEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 expressions in all sub-jects. Antibodies reactive to VEGF (Abcam, Cambridge, MA, USA) andits receptors (Abcam)were used. The procedurewas performed accord-ing to the recommendations of the supplier. The details for the methodare provided in the online Supplementary material. The expression ofVEGF, VEGFR-1 and VEGFR-2 was analyzed semiquantitatively using avisual scoring method [14–16] in six different parts of lung tissue:alveolar epithelium, bronchiolar epithelium, bronchiolar smoothmuscle,vascular endothelium, vascular smooth muscle and macrophage. Thestaining intensity was graded and expressed from 0 to 3 (0 = no stain-ing; 1 = moderate staining, 2 = intense staining; 3 = very intensestaining). After examining four different areas per each part of lung tissuein one slide, themean score of the part was determined as staining score.The scoring was done by two observers (S.H. Lee and C. H. Kim) blindedto the clinical data of the subjects. The inter-observer agreement wasassessed by weighted kappa statistics and the scores were highly agreedbetween two observers based on the kappa values ranging from 0.81 to0.93.

Western blot analysis for VEGF and VEGFR

Total levels of VEGF and its receptors in lung tissueswere analyzed byWestern blotting.Weused anti-VEGFmonoclonal antibody (Abcam) andanti-VEGF receptor 2 polyclonal antibody (Thermo Fisher Scientific,Pittsburgh, PA, USA) and performed the procedure according to therecommendation of the supplier. The detailed procedure for Westernblotting is provided in the online Supplementary material.

Enzyme-linked immunosorbent assay (ELISA) for HIF-1α

HIF-1α protein level in lung tissueswasmeasured by ELISA using Sur-veyor™ 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 Supplementarymaterial.

Statistical analyses

Data are expressed as mean ± standard deviation. Data were notnormally distributed. Therefore, comparisons among groups weremade through Kruskal–Wallis test or Mann–Whitney U test. All testswere two-tailed, and p-values were corrected for the number of com-parisons using the Bonferroni method. The correlation between the ex-pression of HIF-1α and that of VEGF was assessed by Spearman'scorrelation analysis. A difference was defined as statistically significantif p b 0.05. Statistical analysis was carried out using SPSS version 13.0for Windows (SPSS, Chicago, IL, USA).

Results

Clinical characteristics

The clinical data and lung functions of all subjects are shown inTable 1. The mean age was 67.8 years and no age difference wasobserved among groups. More females were enrolled in non-smokers.The smoking intensity between healthy smokers and CB patients wasnot significantly different. FEV1 and FEV1/FVC were significantly re-duced in CB patients compared with healthy smokers (p = 0.032 andp= 0.028, respectively). The diffusing lung capacity for carbonmonoxide(DLCO) in CB patients was also significantly reduced compared to healthysmokers (p= 0.040).

Immunohistochemical staining for VEGF

Representative examples of immunohistochemical staining for VEGFin alveoli, bronchiole and peripheral vessels are shown in Fig. 1A. In CBpatients, many inflammatory cells in the alveolar space and thickenedalveolar septum were more prominent compared with other groups.Hyperplasia of bronchiolar epithelial cells, goblet cells and vascularsmooth muscle cells was also noted in CB patients. According to thesemiquantitative scoringmethod, VEGF expression of smokers (healthysmokers and CB) was increased in bronchiolar epithelium (1.48 ± 0.27versus 0.76 ± 0.25; p b 0.05), bronchiolar smooth muscle (1.79 ± 0.26versus 0.82± 0.19; p b 0.05), vascular endothelium(0.67±0.29 versus0.24 ± 0.08; p b 0.05) and vascular smooth muscle (1.77± 0.23 versus1.12± 0.25; p b 0.05) comparedwith non-smokers. Comparing healthy

a

b

c

*

*

**

A

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 indicatewhere VEGF is expressed (×200). (B) Staining scores of VEGF in six parts of the lung tissue. Comparing healthy smokerswith non-smokers, VEGF expression was increased in bronchiolarepithelium. Comparing CB with healthy smokers, VEGF expression was increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle. Bars express standarddeviation. CB, chronic bronchitis; black arrow and box, alveolar epithelium; arrow head, macrophage; red arrow, bronchiolar epithelium; red asterisk, bronchiolar smooth muscle; bluearrow, vascular endothelium; blue asterisk, vascular smoothmuscle; *,p b 0.05; alv.epi., alveolar epithelium; br.epi., bronchiolar epithelium; br.sm., bronchiolar smoothmuscle; v.endo., vas-cular endothelium; v.sm., vascular smooth; MØ, macrophage.

554 S.H. Lee et al. / Clinical Biochemistry 47 (2014) 552–559

smokerswith non-smokers, VEGF expressionwas only increased in bron-chiolar smooth muscle (1.62 ± 0.26 versus 0.82 ± 0.19; p b 0.05). Com-paring CB patients with healthy smokers, VEGF expressionwas increasedin 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) andvascular 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 immunohistochemicalstaining for VEGFR-1 in lung tissue. Comparing VEGFR-1 expression ofsmokers (healthy smokers and CB) with that of non-smokers, increasedexpression was found only in bronchiolar smooth muscle (1.51 ± 0.13

versus 1.01 ± 0.12; p b 0.05) and no significant differences wereobserved in other parts. Comparing healthy smokers with non-smokers, the expression of VEGFR-1 was increased only in bronchiolarsmooth muscle (1.41 ± 0.13 versus 1.01 ± 0.12; p b 0.05). The levelof VEGFR-1 in CB did not show differences in any part of lung tissuecompared with healthy smokers (Fig. 2B).

Examples of immunohistochemical staining for VEGFR-2 are pre-sented in Fig. 3A. Comparing smokers (healthy smokers and CB) withnon-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 smokerswith non-smokers, VEGFR-2 expression was only increased in bronchio-lar epithelium (2.00 ± 0.29 versus 0.76 ± 0.19; p b 0.05). Comparing

a

b

c

*

A

B

Fig. 2. (A) Representative photographs of immunohistochemical staining for VEGFR-1 in aveoli andmacrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicatewhere 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 inbronchiolar smoothmuscle. Comparing CBwith healthy smokers, VEGFR-1 expression was not different in any parts of the lung tissue. Bars express standard deviation. For arrows, asterisksand abbreviations, please refer to the legend of Fig. 1.

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CB patients with healthy smokers, VEGFR-2 expression was increased inbronchiolar 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 vas-cular smoothmuscle (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 VEGFand VEGFR-2 expressions were quantified in homogenized whole lungtissue of all subjects using Western blotting. For VEGF, one proteinband was detected at the molecular weight of 45 kDa. Densitometricanalysis revealed that VEGF expression was significantly increasedin CB patients compared with healthy smokers (0.91 ± 0.30 versus0.53 ± 0.18; p b 0.05) while it was not significantly different between

healthy smokers and non-smokers (Fig. 4A). For VEGFR-2, one proteinbandwas detected at themolecularweight of 150 kDa. VEGFR-2 expres-sion 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 ofWestern blots were consistent with those of the immunohistochemicalstaining.

HIF-1α expression and correlation with VEGF expression

To evaluate whether HIF-1α level is affected by smoking statusor presence of CB, we compared the HIF-1α levels among groups.HIF-1α expression did not show any difference between non-smokers and healthy smokers. In contrast, its expressionwas significantlyincreased in CB patients compared to healthy smokers (0.54 ± 0.28

a

b

c

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A

B

Fig. 3. (A) Representative photographs of immunohistochemical staining for VEGFR-2 in aveoli andmacrophage (a), bronchiole (b) and peripheral vessel (c). The brownish areas indicatewhere 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 inbronchiolar epithelium. Comparing CBwith healthy smokers, VEGFR-2was increased in bronchiolar smoothmuscle, vascular endotheliumandvascular smoothmuscle. Bars express standarddeviation. For arrows, asterisks and abbreviations, please refer to the legend of Fig. 1.

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versus 0.39 ± 0.19; p b 0.05) (Fig. 5A). In addition, we performed corre-lation analysis between VEGF and HIF-1α levels to assess whether VEGFexpression is related with HIF-1α in all subjects. A significant positivecorrelation was found between these expressions (r = 0.64, p b 0.05)(Fig. 5B).

Discussion

In the present study, CBwas associated with increased expression ofVEGF in the bronchiolar epithelium, bronchiolar smooth muscle andvascular smooth muscle. VEGFR-2 was also increased in bronchiolarsmooth muscle, vascular endothelium and vascular smooth muscle. Inaddition, VEGF expression was correlated with HIF-1α expression. Tothe best of our knowledge, this is the first study to document theincreased expression of VEGF and VEGFR in different compartments of

lung tissues in patients with CB. More importantly, our data suggestthat HIF-1α is involved in the increased expression of VEGF in thosepatients.

Although the molecular and cellular mechanisms that are responsi-ble for the development and progress of COPD are not fully understood,anti-protease/protease activity, oxidative stress, apoptosis and influx ofinflammatory cells including neutrophils, macrophages and CD8+ Tlymphocytes are involved [3]. COPD has two different phenotypes: em-physema and CB. The former is characterized by the destruction of thealveolar septum, the resulting decrease of elastic recoil and increasedairway resistance. The latter is characterized by mucus gland enlarge-ment and goblet cell hyperplasia leading to cough and sputum. Therelative contribution of these phenotypes to airflow limitation differsfrom patient to patient [2]. The mechanism concerning how individualsubjects have emphysema- or CB-dominant phenotype from the same

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45 kDa

37 kDa

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-2

150 kDa

37 kDa

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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, theexpression of VEGFwas significantly increased in CB. (B) The expression of VEGFR-2was also increased in CB. Bars expressmean and standard deviation. *, p b 0.05; CB, chronic bronchitis.

557S.H. Lee et al. / Clinical Biochemistry 47 (2014) 552–559

insult – cigarette smoking – is largely unknown. Several studies havesuggested that VEGF signaling may be involved in the phenotypicexpression of the disease based on different expression levels in emphy-sema and CB [7–9,17]. However, previous studies have limitations inthat they used only sputum or bronchial biopsy specimen instead ofwhole 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 expres-sions in CB patients compared with those of healthy smokers in variousparts of lung tissue and the correlation between HIF-1α and VEGFexpressions.

Cigarette smoking is a potent inducer of VEGF expression and pro-duction [9,18,19]. In this study, VEGF expression in bronchial smoothmuscle was increased in healthy smokers compared with nonsmokersreflecting that the augmented VEGF expression is linked to cigarettesmoking. However, there was no difference in the expression ofHIF-1α between non-smokers and healthy smokers, while increasedexpression was noted in CB patients compared with healthy smokers.Taking the previous and current data together, we can suggest thatVEGF overexpression in CB is not a result of smoking, but may be an in-trinsic characteristic of the disease itself. The clinical implication of in-creased VEGF expression can be explained by two points of view. First,

increased expression of VEGF and its receptor may be a result of theunique ongoing inflammatory process of CB, which is different fromthat induced by cigarette smoke only. Previous studies showed theabundance of neutrophils, interleukin-8 and tumor necrosis factor-alpha in CB patients comparedwith healthy smokers, all ofwhich induceVEGF expression [9,20]. In addition, VEGF itself can act as a chemokinefor neutrophils [21]. Thus, the inflammation could be worsened in avicious cycle with the interaction between VEGF and inflammatorymediators participating in CB pathogenesis. Second, because VEGF andits receptor systemare essential in lunghomeostasis, increasedVEGF ex-pressionmay partly indicate an attempt to repair damage in airway andlung parenchyma to maintain normal lung structure and function [22].This adaptive process may contribute to adverse consequences, such aspulmonary bronchial or vascular remodeling [23].

In this study, the expression of VEGFR-2 was increased in variouscompartments of lung tissue of CB patients, while that of VEGFR-1showed no increase compared with healthy smokers. Both VEGFR-1and VEGFR-2 are the main receptors in VEGF signaling, but they havedifferent functions in vivo. VEGFR-2 is the active receptor involved inthe mediation of major growth and permeability actions of VEGF,whereas VEGFR-1 inhibits the action of VEGF by acting as a modulatingdecoy to VEGFR-2 [24]. The former allows the excessive proliferation

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GF

Fig. 5. HIF-1α levels in each group and relationship between VEGF and HIF-1α expres-sions. (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 allpatients. Bars express mean and standard deviation. *, p b 0.05; CB, chronic bronchitis.

558 S.H. Lee et al. / Clinical Biochemistry 47 (2014) 552–559

and migration of vascular endothelial cells, while the latter inhibits theprocess [25]. Thus, we can suggest that the exaggerated inflammation inCB may be the result of the enhanced interaction mainly between VEGFand VEGFR-2. Although, increased expression of VEGF, VEGFR-1 andVEGFR-2 was demonstrated in the bronchiolar epithelium and smoothmuscle of COPD patients [26], VEGFR expression has not been investi-gated in CB patients. More studies are needed to support our data andelucidate 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 playsa key role in VEGF gene expression both in vitro and in vivo [27]. HIF-1α is considered as a major and important factor involved in controlof VEGF expression by inducing VEGF transcription [11,28]. Presently,HIF-1α was increased in CB patients compared with healthy smokersand its expression was positively correlated with that of VEGF. Interest-ingly, a recent study reported that the expression of VEGF is reducedand associatedwith that of HIF-1α in emphysemapatients [29]. Becauseemphysema and CB can co-exist in the same patient, we excludedemphysema-dominant patients based on thorough review of HRCTscan and DLCO. The mean DLCO of enrolled CB patients was over 80% ofpredictive value suggesting that they are CB dominant. Therefore, ourresults add important evidence that HIF-1αmay be an important regu-lator 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, arecent study has demonstrated that the increased expression of VEGFin 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. hypothe-sized that the VEGF excess contributes to the Th2 response as in asthma,and deficiency of VEGF contributes to the Th1 response as in emphyse-ma [34]. Collectively, HIF-1α-regulated VEGF expression seems to be

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 sub-jects was relatively small and we did not investigate the expression inemphysema-dominant patients simultaneously. However, our aimwas to evaluate VEGF expression in CB patients, andwe verified the sig-nificantly increased VEGF expression in CB patients compared withhealthy smokers evenwith small subject numbers. Second, the enrolledsubjects were all Korean. It could limit the applicability of our results tothe general population. Third, more females were enrolled in non-smokers compared with other groups. Gortner et al. have suggestedthe possibility of the different regulation of VEGF between genders,thus the unequal gender distribution among groups could influenceour results [35]. Finally, other possible regulators other than HIF-1αthat could be involved in VEGF expression were not evaluated simulta-neously. Although VEGF is regulated by numerous cytokines and othermolecules, HIF-1α-induced transcriptional control is considered as amajor mechanism of VEGF expression [11]. We hypothesized thatHIF-1α would also play a certain role in VEGF expression in CB andsuccessfully demonstrated the significant association between VEGFexpression and that of HIF-1α, which is comparable to the results of aprevious study on patients with emphysema [29].

In conclusion, VEGF and VEGFR-2 expressions were up-regulated invarious compartments of lung tissues and VEGF expression was corre-lated with the increased expression of HIF-1α in CB patients. Our find-ings add important evidence that HIF-1α-regulated VEGF expressioncould be one of the mechanisms to determine the predominance be-tween CB and emphysema in smokers. Although further studies areneeded to verify our results, the current data provide important cluesfor understanding the pathogenesis of COPD and form the theoreticalbasis for the treatment of the disease.

Acknowledgments

This work was supported by grant from the National ResearchResource Bank Program of the Korea Science & Engineering Foundationin the Ministry of Science & Technology (No. R21-2007-000-10058-0)and by the Korea University Grant (Nos. K1032051 and R1205861).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clinbiochem.2014.01.012.

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