Effects of Pranlukast Administration on Vascular Endothelial Growth Factor Levels in Asthmatic Patients

Effects of Pranlukast Administration on Vascular Endothelial Growth Factor Levels in Asthmatic Patients* Hiroshi Kanazawa, MD; Takahiro Yoshikawa, MD; Kazuto Hirata, MD; and Junichi Yoshikawa, MD

Study objectives: We have previously found that vascular endothelial growth factor (VEGF) levels in induced sputum were increased in asthmatic patients, and that its levels were closely associated with the degree of airway obstruction and microvascular permeability. Therefore, this study was designed to examine the effects of pranlukast, a selective leukotriene receptor antagonist, on VEGF levels in induced sputum from steroid-untreated or steroid-treated asthmatic patients. Design: Double-blind, randomized, placebo-controlled, crossover study. Setting: University hospital. Participants: Twenty-three asthmatic patients (steroid-untreated, 13 patients; steroid-treated, 10 patients) and 10 healthy control subjects. Interventions: All asthmatic patients received 4-weeks of therapy with pranlukast (225 mg bid), and sputum induction was performed before and after the 4-week treatment course. Measurements and results: In steroid-untreated asthmatic patients, the mean percentage of eosinophils (%EOS) and mean eosinophil cationic protein (ECP) levels in induced sputum were significantly decreased after 4 weeks of pranlukast administration (%EOS: before, 16.7% [SD, 7.1%]; after, 12.3% [SD, 4.0%]; p ⴝ 0.03; ECP levels: before, 774 ng/mL [SD, 258 ng/mL]; after, 564 ng/mL [SD, 204 ng/mL]; p ⴝ 0.034). Moreover, VEGF levels in the induced sputum and the airway vascular permeability index also were decreased after pranlukast administration (VEGF levels: before, 5,670 pg/mL [SD, 1,780 pg/mL]; after, 4,380 pg/mL [SD, 1,540 pg/mL]; p ⴝ 0.026; airway vascular permeability index: before, 0.032 [SD, 0.012]; after, 0.017 [SD, 0.006]; p ⴝ 0.01). In addition, the change in airway vascular permeability index from before to after pranlukast administration was significantly correlated with the change in VEGF levels (r ⴝ 0.782; p ⴝ 0.007). However, in steroid-treated asthmatic patients there was no significant difference in mean VEGF levels in induced sputum between placebo administration (before, 3,640 pg/mL [SD, 1,020 pg/mL]; after, 3,640 pg/mL [SD, 960 pg/mL] and pranlukast administration (before, 3,660 pg/mL [SD, 940 pg/mL]; after, 2,950 pg/mL [SD, 890 pg/mL]). Conclusions: Pranlukast administration decreased airway microvascular permeability through, at least in part, a decrease in airway VEGF levels in steroid-untreated asthmatic patients. However, it is likely that pranlukast administration added little efficacy to inhaled corticosteroid therapy for reduction in airway VEGF levels. (CHEST 2004; 125:1700 –1705) Key words: airway microcirculation; airway permeability; beclomethasone dipropionate; leukotriene antagonists Abbreviations: BDP ⫽ beclomethasone dipropionate; Cys-LT ⫽ cysteinyl-leukotriene; ECP ⫽ eosinophil cationic protein; %EOS ⫽ percentage of eosinophils; VEGF ⫽ vascular endothelial growth factor

of the cysteinyl-leukotrienes (CysT heLTs)importance [ie, LTC4, LTD4, and LTE4] as mediators of bronchial asthma is now widely recognized.1 They can be recovered in increased concentrations from

the BAL fluid and urine of patients with asthma. The administration of exogenous Cys-LTs causes airflow obstruction, increases mucus production, airway edema, and vascular permeability, and leads to the

*From the Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan. This work was supported by a Grant-in-Aid for Scientific Research (15590820) from the Japan Society for the Promotion of Science. Manuscript received August 14, 2003; revision accepted December 15, 2003.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Hiroshi Kanazawa, MD, Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, 1-4-3, Asahi-machi, Abenoku, Osaka, 545-8585, Japan; e-mail: [email protected]

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infiltration of eosinophils into asthmatic airways. Further evidence for the role of Cys-LTs in the pathogenesis of asthma has been provided by studies in which leukotriene antagonists have blunted the magnitude of the obstructive response following exercise, allergen challenge, cold-air hyperventilation, and administration of nonsteroidal anti-inflammatory drugs to sensitized subjects.2 In addition, leukotriene antagonists improve baseline pulmonary function both with short-term and long-term treatment, and reduce airway inflammation and bronchial hyperresponsiveness, as well as improve asthma control.3 Current guidelines4 recommend the use of inhaled corticosteroids as first-line control therapy in patients with asthma. However, the conditions of asthmatic patients cannot all be adequately controlled, despite the use of high-dose inhaled corticosteroids. Moreover, anti-inflammatory treatment options in these patients are limited to increasing the dose of inhaled corticosteroids or introducing systemic corticosteroids, with their increased risk of systemic side effects. Therefore, there has been a clinical interest in the use of leukotriene antagonists in patients who have symptoms despite treatment with inhaled corticosteroids. There is a reasonable expectation that leukotriene antagonists might have additive effects with inhaled corticosteroids for these patients since the overproduction of Cys-LTs in asthmatic airways is not inhibited by corticosteroids.5 Clinical trials6 have indeed yielded evidence of the efficacy of leukotriene antagonists in patients receiving inhaled corticosteroids. However, Robinson et al7 reported that leukotriene antagonists added little efficacy to more well-established agents such as inhaled corticosteroids, which their asthmatic patients were already receiving. We have emphasized in previous studies8,9 that the airway microcirculation has the potential to contribute to the pathophysiology of bronchial asthma. Vascular endothelial growth factor (VEGF) increases microvascular permeability,10 so that plasma proteins can leak into the extravascular space, leading to mucosal edema and thereby to the narrowing of airway diameters, which could amplify the effects of airway smooth muscle contraction. On the basis of these facts, we have hypothesized that leukotriene antagonists could have decreased microvascular permeability via a reduction in airway VEGF levels. However, it is important for our understanding of Cys-LTs in the pathogenesis of asthma and asthma control whether leukotriene antagonist is effective for the regulation of microvascular permeability in both steroid-untreated and steroid-treated asthmatic patients. Therefore, this study was designed to examine the effects of pranlukast, a selective Cys-LT1 www.chestjournal.org

receptor antagonist, on VEGF levels in induced sputum from steroid-untreated or steroid-treated asthmatic patients.

Materials and Methods Subjects Twenty-three asthmatic patients and 10 healthy control subjects were included in the study. All asthmatic patients were lifelong nonsmokers and met the American Thoracic Society standards for asthma.11 In short, they all had episodic cough, wheezing and dyspnea, and normal chest roentgenography results. They also exhibited reduced FEV1 during asthma attacks and an increase of ⱖ 20% in FEV1 in response to ␤2-adrenoceptor agonists. Healthy control subjects were healthy nonsmoking volunteers without a history of lung disease. Methacholine inhalation challenge testing was performed for all asthmatic patients. All challenge tests were performed at 1 pm to eliminate the effect of diurnal variation. Following baseline spirometry and the inhalation of diluent to establish the stability of FEV1, the subjects were instructed to take slow inspirations in each set of inhalations. All asthmatic patients in this study demonstrated airway hyperreactivity to methacholine. Medications for all patients were not changed for 1 month before the study and were withdrawn for at least 12 h before spirometric study and sputum induction. All patients were clinically stable, and none had a history of respiratory infection for at least the 4-week period preceding the study. All subjects gave their written informed consent for participation in this study, which was approved by the Ethics Committee of Osaka City University, Japan. Sputum Induction and Processing Asthmatic patients sometimes exhibited a significant decrease in FEV1 after the sputum induction procedure and experienced symptoms of breath shortness or chest tightness. Therefore, we treated inhaled salbutamol for both asthmatic patients and healthy control subjects in the same way. Spirometry was performed prior to the inhalation of 200 ␮g salbutamol via a metered-dose inhaler. All subjects were instructed to wash their mouths thoroughly with water. They then inhaled 3% saline solution at room temperature that was nebulized by an ultrasonic nebulizer (NE-U12; Omron; Tokyo, Japan) at maximum output. They were encouraged to cough deeply at 3-min intervals thereafter. After sputum induction, spirometry was repeated. If the FEV1 fell, the subjects were required to wait until it returned to baseline values. The sputum samples were kept at 4°C for ⱕ 2 h prior to further processing. The portion of the sample was diluted with phosphate buffer solution containing 10 mmol/L dithiothreitol (Sigma Chemical; St. Louis, MO) and was gently vortexed at room temperature. They then were centrifuged at 400g for 10 min, and the cell pellet was resuspended. The slides were made by using a cytospin (Cytospin3; Shandon; Tokyo, Japan) and were stained with May-Grunwald-Giemsa stain for differential cell counts. The results of differential cell counts of sputum samples were analyzed as the average of counts performed by at least two chest physicians on separate occasions in a blinded manner. The supernatant was stored at ⫺70°C for subsequent assays for albumin, VEGF, and eosinophil cationic protein (ECP). VEGF concentration was measured by using an enzyme-linked immunosorbent assay kit (R&D Systems; Minneapolis, MN). ECP concentration was measured by using a radioimmunoassay kit (Pharmacia Diagnostics; Uppsala, SweCHEST / 125 / 5 / MAY, 2004

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den), and albumin concentration was measured by laser nephelometry. We calculated the airway vascular permeability index (ie, the ratio of albumin concentrations in induced sputum and serum) as we have previously described.9 All subjects produced an adequate specimen of sputum. A sample was considered to be adequate if the patient was able to expectorate at least 2 mL of sputum and, if on differential cell counting, the slides contained ⬍ 10% squamous cells. Study for Steroid-Untreated Asthmatic Patients Thirteen asthmatic patients were not receiving oral or inhaled corticosteroids. Their regular medication consisted of a metereddose inhaler of ␤2-agonist salbutamol on demand and theophylline. Patients receiving any leukotriene antagonists or chemical mediator inhibitors such as cromolyn sodium were excluded from the study. All medications were not changed for a 1-month period preceding the study and were withdrawn for at least 12 h before the methacholine challenge test and pulmonary function test. After the first sputum induction, all asthmatic patients received 4 weeks of therapy with pranlukast (225 mg bid) [Ono Pharmaceuticals; Osaka, Japan], and then sputum induction was repeated in the same way.

Table 1—Clinical Characteristics of Study Subjects*

Characteristics Subjects, No. Gender, No. Male Female Age, yr FEV1, % predicted PC20†, mg/mL Sputum %EOS ECP, ng/mL

Healthy Control Subjects

Steroid Untreated

Steroid Treated

10

13

10

6 4 32.3 (7.1) 107.4 (5.4) ⬎ 10.0 0.8 (0.5) 121 (63)

Asthmatic Patients

8 5 31.5 (5.3) 90.4 (4.6) 2.75 (0.66)‡ 16.7 (7.1)‡ 774 (258)‡

6 4 28.3 (12.1) 87.0 (12.0) 2.88 (0.25)‡ 6.3 (4.6)‡ 317 (161)‡

*Values given as mean (SD), unless otherwise indicated. PC20 ⫽ provocative concentration of methacholine causing a 20% fall in FEV1. †Geometric mean. ‡p ⬍ 0.01, asthmatic patients vs normal control subjects.

Study for Steroid-Treated Asthmatic Patients Ten asthmatic patients received inhaled beclomethasone dipropionate (BDP) [800 ␮g/d] for ⬎ 1 month. This protocol was performed in a double-blind, randomized, placebo-controlled, crossover manner. There was an initial 2-week run-in period during which patients continued to take the same medications. In the next 4-week double-blind treatment period, the daily medications were continued in all patients, while they received either pranlukast (225 mg bid) or placebo. After a washout period of 2 weeks, the subjects crossed over to receive the alternative treatment for 4 weeks. Patients were allowed to take salbutamol if supplemental medication was needed, but all other treatment remained unchanged. All patients visited the outpatient clinic before and after each 4-week treatment period, and sputum induction was performed before and after each 4-week treatment course. Statistical Analysis All values are presented as the mean (SD). Multiple comparisons among groups were analyzed by one-way analysis of variance. When analysis of variance revealed a significant difference, the Bonferroni correction was applied. A p value of ⬍ 0.05 was considered to be significant.

Results Baseline pulmonary function and airway hyperreactivity to methacholine in the 10 healthy control subjects, 13 steroid-untreated asthmatic patients, and 10 steroid-treated asthmatic patients are shown in Table 1. All three groups were well-matched with respect to age. However, the percentage of eosinophils (%EOS) and the levels of ECPs in induced sputum were significantly higher in asthmatic patients who both did not received steroids and received steroids than in healthy control subjects. In steroid-untreated asthmatic patients, the mean 1702

%EOS and ECP levels in induced sputum were significantly decreased after 4 weeks of pranlukast administration (%EOS: before, 16.7% [SD, 7.1%]; after, 12.3% [SD, 4.0%]; p ⫽ 0.03; ECP levels: before, 774 ng/mL [SD, 258 ng/mL]; after, 564 ng/mL [SD, 204 ng/mL]; p ⫽ 0.034) [Fig 1]. Moreover, VEGF levels in induced sputum and airway vascular permeability index also were decreased after pranlukast administration (VEGF levels: before, 5,670 pg/mL [SD, 1,780 pg/mL]; after, 4,380 pg/mL [SD, 1,540 pg/mL]; p ⫽ 0.026; airway vascular permeability index: before, 0.032 [SD, 0.012]; after, 0.017 [0.006]; p ⫽ 0.01) [Fig 2]. In addition, the change in the airway vascular permeability index from before to after pranlukast administration was significantly correlated with the change in VEGF levels (r ⫽ 0.782; p ⫽ 0.007) [Fig 3]. In steroidtreated asthmatic patients, we found that there was no significant difference in VEGF levels in induced sputum between placebo administration (before, 3,640 pg/mL [SD, 1,020 pg/mL]; after, 3,640 pg/mL [SD, 960 pg/mL]) and pranlukast administration (before, 3,660 pg/mL [SD, 940 pg/mL]; after, 2,950 pg/mL [890 pg/mL]; p ⫽ 0.11) [Fig 4]. After the 4 weeks of treatment with pranlukast, none of the subjects reported any side effects. Discussion In steroid-untreated asthmatic patients, we found that the %EOS and ECP levels in induced sputum were significantly decreased after pranlukast administration. Moreover, VEGF levels in induced sputum and airway microvascular permeability were also Clinical Investigations

Figure 1. Comparison of the %EOS and ECP levels in induced sputum from healthy control subjects and steroid-untreated asthmatic patients before and after the administration of pranlukast.

decreased after pranlukast administration. In addition, a significant correlation was found between the change in airway vascular permeability and the change in VEGF levels from before to after pranlukast administration. These findings suggest that pranlukast administration has a significant effect on the reduction in eosinophilic airway inflammation, airway VEGF levels, and microvascular permeability in asthmatic patients who were not receiving inhaled corticosteroids. In contrast, pranlukast administration did not have a significant effect on the reduction in airway VEGF levels in asthmatic patients who were already receiving inhaled BDP therapy. Cys-LTs contribute to tissue edema by widening endothelial gap junctions in postcapillary venules.12 In an animal model, Cys-LT–induced microvascular permeability was reduced to near basal levels by montelukast administration, suggesting that airway

microvascular leakage appears to be predominantly mediated via the activation of Cys-LT1 receptors. In addition, our previous study13 indicated that VEGF levels in induced sputum from asthmatic patients were significantly higher than those in healthy control subjects, and that VEGF levels were closely correlated with the degree of airway microvascular permeability. Therefore, it is likely that pranlukast administration induces the reduction in airway microvascular permeability through the mechanisms of both Cys-LT1 receptor blockade and a decrease in airway VEGF levels. Also, it has previously been reported that the transcription of VEGF messenger RNA and the secretion of VEGF protein were down-regulated by corticosteroid therapy.14 Moreover, in our earlier study,15 we determined that VEGF levels in induced sputum from asthmatic patients were decreased by inhaled BDP therapy.

Figure 2. Comparison of VEGF levels in induced sputum and airway vascular permeability index from healthy control subjects and steroid-untreated asthmatic patients before and after the administration of pranlukast. www.chestjournal.org

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Figure 3. Correlation between the change in airway vascular permeability index (⌬ airway vascular permeability index) and the change in VEGF levels (⌬ VEGF) from before to after pranlukast administration in steroid-untreated asthmatic patients.

However, the present findings suggest that pranlukast administration does not have an additive effect on reduction in VEGF levels in the airways of BDP-treated asthmatic patients. Bronchial asthma is a chronic airway inflammatory disease that is associated with airway wall remodel-

ing, and these structural alterations in the airway wall include the growth and proliferation of new blood vessels. It has been reported16 that both the number and percentage of vessels in biopsy specimens taken from asthmatic patients were increased compared with healthy control subjects. Moreover, it has been

Figure 4. Comparison of VEGF levels in induced sputum from steroid-treated asthmatic patients before and after the administration of placebo or pranlukast. NS ⫽ not significant. 1704

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recognized that the airway mucosa is edematous, and contains dilated and congested blood vessels, even in patients with mild asthma. VEGF is known as a vascular permeability factor. It was previously reported that VEGF induced fenestration in endothelial cells both in vitro and in vivo.16,17 In our earlier study,18 we clearly showed that increased levels of VEGF in asthmatic airways were closely associated with exercise-induced bronchoconstriction via an airway hyperpermeability-dependent mechanism. Thus, VEGF is thought to cause leakage of the mucosal and submucosal capillary beds and to induce airway wall thickness. The airway microcirculation could exert an important influence on airway geometry. Vascular engorgement, capillary leakage, and edema formation could induce airway narrowing. Cys-LTs thought to cause the constriction of bronchial smooth muscle also can cause dilation and leakage of the mucosal and submucosal capillary beds, and can induce airway wall thickness. A previous study19 suggested that small increases in wall thickness induced by airway inflammation could produce striking changes in airway hyperresponsiveness to various stimuli, even when there was a trivial increase in resting muscle tone. Thus, capillary leakage and airway mucosal edema formation induced by both Cys-LTs and VEGF may contribute to airway narrowing. In conclusion, we found that pranlukast administration decreased airway microvascular permeability via reduction of airway VEGF levels in steroiduntreated asthmatic patients. In contrast, it is likely that pranlukast administration adds little efficacy to inhaled corticosteroids therapy in the reduction of airway VEGF levels. However, it is possible that the trends toward reduction in airway VEGF levels after pranlukast administration in steroid-treated asthmatic patients might have reached statistical significance if more patients had been included. It will be important to examine in future studies whether pranlukast administration improves airway microvascular permeability and induces clinically a significant improvement of asthma control in larger samples of steroid-treated asthmatic patients. ACKNOWLEDGMENT: We thank Miss Yukari Matsuyama for her help in the preparation and editing of the manuscript.

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References 1 Holgate ST, Sampson AP. Antileukotriene therapy: future directions. Am J Respir Crit Care Med 2000; 161:S147–S153 2 Drazen JM. Clinical pharmacology of leukotriene receptor

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antagonists and 5-lipoxygenase inhibitors. Am J Respir Crit Care Med 1998; 157:S233–S237 Hui KP, Barnes NC. Lung function improvement in asthma with a cysteinyl-leukotriene receptor antagonist. Lancet 1991; 337:1062–1063 National Heart, Lung, and Blood Institute, National Institutes of Health. Global initiative for asthma. Bethesda, MD: National Institutes of Health, 1995; NIH Publication No. 95–3659 Pavord ID, Ward R, Woltmann G, et al. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med 1999; 160:1905–1909 Christian VJ, Prasse A, Naya I, et al. Zafirlukast improves asthma control in patients receiving high-dose inhaled corticosteroids. Am J Respir Crit Care Med 2000; 162: 578 –585 Robinson DS, Campbell D, Barnes PJ. Addition of leukotriene antagonists to therapy in chronic persistent asthma: a randomised double-blind placebo controlled trial. Lancet 2001; 357:2007–2011 Kanazawa H, Hirata K, Yoshikawa J. Role of endogenous nitric oxide in exercise-induced airway narrowing in patients with bronchial asthma. J Allergy Clin Immunol 2000; 106: 1081–1087 Kanazawa H, Asai K, Hirata K, et al. Vascular involvement in exercise-induced airway narrowing in patients with bronchial asthma. Chest 2002; 122:166 –170 Monacci WT, Merrill MJ, Oldfield EH. Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol 1993; 264:C994 –C1002 American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987; 136:225–244 Hele DJ, Birrell MA, Webber SE, et al. Mediator involvement in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitized Brown Norway rats. Br J Pharmacol 2001; 132:481– 488 Asai K, Kanazawa H, Kamoi H, et al. Increased levels of vascular endothelial growth factor in induced sputum in asthmatic patients. Clin Exp Allergy 2003; 33:595–599 Nauck M, Roth M, Tamm M, et al. Induction of vascular endothelial growth factor by platelet-activating factor and platelet-derived growth factor is down-regulated by corticosteroids. Am J Respir Cell Mol Biol 1997; 16:398 – 406 Asai K, Kanazawa H, Otani K, et al. Imbalance between vascular endothelial growth factor and endostatin in induced sputum in asthmatics. J Allergy Clin Immunol 2002; 110:571– 575 Esser S, Wolburg K, Wolburg H, et al. Vascular endothelial growth factor induces endothelial fenestrations in vitro. J Cell Biol 1998; 140:947–959 Roberts WG, Palade GE. Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res 1997; 57:765–772 Kanazawa H, Hirata K, Yoshikawa J. Involvement of vascular endothelial growth factor in exercise-induced bronchoconstriction in asthmatic patients. Thorax 2002; 57:885– 888 Laitinen LA, Laitinen A, Widdicombe J. Effects of inflammatory and other mediators on airway vascular beds. Am Rev Respir Dis 1987; 135:567–570

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