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Mast Cells Can Mediate Vascular Permeability through Regulation of the PI3K–HIF-1–VEGF Axis

¹ Department of Internal Medicine and Airway Remodeling Laboratory and ² Department of Anatomy, Chonbuk National University Medical School, Jeonju, South Korea

Correspondence and requests for reprints should be addressed to Yong Chul Lee, M.D., Ph.D., Department of Internal Medicine, Chonbuk National University Medical School, San 2-20, Geumam-dong, Deokjin-gu, Jeonju, 561-180, South Korea. E-mail: leeyc{at}chonbuk.ac.kr

	ABSTRACT

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Rationale: Bronchial inflammation is usually accompanied by increased vascular permeability. Mast cells release a number of mediators that act directly on the vasculature, resulting in vasodilatation, increased permeability, and subsequent plasma protein extravasation. Vascular endothelial growth factor (VEGF) has been implicated to contribute to asthmatic tissue edema through its effect on vascular permeability. However, the effects of mast cells on VEGF-mediated signaling in allergic airway disease are not clearly understood.

Objectives: An aim of the present study was to investigate the role of mast cells on VEGF-mediated signal transduction in allergic airway disease.

Methods: We used genetically mast cell–deficient WBB6F₁-Kit^W/Kit^W-v (W/W^v) mice and the congenic normal WBB6F₁^+/+ mouse model for allergic airway disease to investigate the role of mast cells on VEGF-mediated signal transduction in allergic airway disease, more specifically in vascular permeability.

Measurements and Main Results: Our present study, with ovalbumin (OVA)-sensitized without adjuvant and OVA-challenged mice, revealed the following typical pathophysiologic features of allergic airway diseases: increased inflammatory cells of the airways, airway hyperresponsiveness, increased vascular permeability, and increased levels of VEGF. However, levels of VEGF and plasma exudation in W/W^v mice after OVA inhalation were significantly lower than levels in WBB6F₁^+/+ mice. Moreover, mast cell–reconstituted W/W^v mice restored vascular permeability and VEGF levels similar to those of the WBB6F₁^+/+ mice. Our data also showed that VEGF expression was regulated by hypoxia-inducible factor-1 (HIF-1) activation through the phosphatidylinositol 3-kinase (PI3K)–HIF-1 pathway in allergic airway disease.

Conclusions: These results suggest that mast cells modulate vascular permeability by the regulation of the PI3K–HIF-1–VEGF axis.

Key Words: mast cells • vascular permeability • vascular endothelial growth factor • allergy • inflammation

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject Mast cells are believed to contribute to the development of allergic airway disease accompanied by increased vascular permeability and plasma exudation. What This Study Adds to the Field Mast cells can regulate vascular leakage, at least in part via modulation of the PI3K–HIF-1–VEGF axis in mice.

 
Bronchial asthma is an increasingly prevalent and often severe disease characterized by chronic inflammation of the airways, which is usually accompanied by increased vascular permeability, resulting in plasma exudation (1). Changes in vascular permeability are crucial to the development and perpetuation of the inflammatory response. Increased vascular permeability causes secretion of intravascular components, which contribute to airway obstruction and hyperresponsiveness (2, 3).

Vascular endothelial growth factor (VEGF) is an endothelial cell–specific mitogenic peptide and plays a key role in vasculogenesis and angiogenesis (4). VEGF also stimulates vascular permeability, which leads to airway inflammation (3, 5, 6). Recently, we demonstrated that VEGF is implicated in the pathogenesis of asthma, and thus the inhibition of VEGF receptor may be a good therapeutic strategy (3, 6, 7). In addition, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhance antigen sensitization and is crucial in adaptive T-helper type 2 (Th2) cell–mediated inflammation (5).

Mast cells are widely believed to contribute to the development of allergic airway disease. These cells are resident in tissues throughout the body and often are found in proximity to blood vessels, including capillaries and postcapillary venules (8–10). Mast cells can be activated to release potent mediators of inflammation by antibody-dependent mechanisms, and can respond to very low doses of specific antigen (11, 12). Although mast cells have been known to amplify inflammatory responses to antigen challenge, the molecular mechanism(s) by which these cells induce increased vascular permeability are not clearly understood.

In the present study, we used genetically mast cell–deficient and congenic normal mouse models of allergic airway disease to determine the role of mast cells, more specifically in the increase of vascular permeability. Some of the results of these studies have been previously reported in the form of an abstract (13).

	METHODS

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Additional detail on methods is provided in the online supplement.

Mice
Six-week-old female genetically mast cell–deficient WBB6F₁-Kit^W/Kit^W–v (W/W^v) mice and the congenic normal WBB6F₁^+/+ mice were purchased from Japan SLC (Shizuoka, Japan). W/W^v mice ordinarily contain less than 1.0% of the number of dermal mast cells present in the skin of the congenic normal WBB6F₁^+/+ mice and have no detectable mature mast cells in the respiratory system or other anatomic sites (14–16). Mice were sensitized and challenged with ovalbumin (OVA) (Sigma-Aldrich Co., St. Louis, MO) as described previously with some modifications (17). Mice were sensitized on Days 1 and 14 by intraperitoneal injection of 20 µg OVA. On Days 21, 22, and 23 after the initial sensitization, the mice were challenged for 30 minutes with an aerosol of 3% (wt/vol) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). Bronchoalveolar lavage (BAL) was performed and smears of BAL cells were prepared with a cytospin (Thermo Electron, Waltham, MA).

Mast Cell Reconstitution
Selective reconstitution of mast cells in W/W^v mice was performed by the method described by Williams and Galli (18) with slight modifications. Bone marrow cells of WBB6F₁^+/+ mice were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 20% WEHI-3 condition medium (containing IL-3) for 4–5 weeks, at which time the cell populations were composed of more than 95% immature mast cells, as assessed by staining with May-Grünwald-Giemsa solution. Bone marrow–derived cultured mast cells (BMCMCs; (5 x 10⁶) were infused two times at 1-week intervals via the tail vein into each W/W^v mouse. Fourteen weeks later, these reconstituted mice were immunized with OVA.

Administration of SU5614, SU1498, 2-Methoxyestradiol, LY-294002, or Wortmannin
VEGF receptor inhibitor, SU5614 (2.5 mg/kg body weight per day; Calbiochem, San Diego, CA) or SU1498 (9 mg/kg body weight per day; Calbiochem) was administered intraperitoneally three times, beginning 1 hour before the first challenge. An inhibitor of hypoxia-inducible factor (HIF)-1, 2-methoxyestradiol (2ME2) (100 mg/kg body weight per day; Sigma-Aldrich, St. Louis, MO) was administered by oral gavage six times, beginning 2 days before the first challenge (19). LY-294002 (1.5 mg/kg body weight per day; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) or wortmannin (100 µg/kg body weight per day; Calbiochem) was administered intratracheally two times, beginning 1 hour before the first challenge.

Western Blot Analysis
Protein expression levels were analyzed by Western blot analysis, as described previously (17).

Measurement of Plasma Exudation
To assess lung permeability, Evans blue dye (EBD) was used as described previously (6).

Histology, Immunohistochemistry, and Immunocytochemistry
Histologic examination and immunohistochemistry or immunocytochemistry of VEGF were performed as described previously (6).

Measurements of Th2 Cytokines and VEGF in BAL Fluids
Levels of Th2 cytokines (IL-4, IL-5, and IL-13) and VEGF were quantified in the supernatants of BAL fluids by an enzyme immunoassay.

Determination of Airway Responsiveness
Airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine via airways, as described elsewhere (20). Under anesthesia, mice were tracheostomized and connected to a computer-controlled small-animal ventilator (flexiVent; SCIREQ, Montreal, PQ, Canada). Each mouse was challenged with methacholine aerosol in increasing concentrations (2.5–50 mg/ml in saline). After each methacholine challenge, data for respiratory system resistance (R_rs) were continuously collected. Maximum values of R_rs were selected to express changes in airway function, which was represented as a percentage change from baseline after saline aerosol.

Nuclear Protein Extractions for Analysis of HIF-1
Nuclear extraction for analysis of HIF-1 was performed as described previously (21).

Measurement of Phosphatidylinositol 3-Kinase Enzyme Activity in Lung Tissues
The amount of phosphatidylinositol-3,4,5-triphosphate (PIP3) produced was quantified by PIP3 competition enzyme immunoassays (17).

Densitometric Analyses and Statistics
All immunoreactive and phosphorylative signals were analyzed by densitometric scanning (Gel Doc XR; Bio-Rad Laboratories, Inc., Hercules, CA). Data are expressed as mean ± SEM. Statistical comparisons were performed using one-way analysis of variance followed by Scheffé's test. Pearson's correlation was calculated to assess the correlation between data. Significant differences between two groups were determined using the unpaired Student's t test. Statistical significance was set at P < 0.05.

	RESULTS

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VEGF Protein Levels in Mice after OVA Inhalation
Western blot analysis revealed that levels of VEGF protein in the lung tissues were significantly increased at 48 hours after OVA inhalation compared with the levels in saline-sensitized and saline-challenged congenic normal WBB6F₁^+/+ mice, saline-sensitized and saline-challenged mast cell–deficient W/W^v mice, and saline-sensitized and saline-challenged mast cell–reconstituted W/W^v mice (hereinafter referred to as BMCMCW/W^v mice). The levels of VEGF protein in the lung tissues of W/W^v mice after OVA inhalation were significantly lower than the levels in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice and in OVA-sensitized and OVA-challenged BMCMCW/W^v mice (Figures 1A and 1B). The levels of VEGF in OVA-sensitized and OVA-challenged BMCMCW/W^v mice were similar to the levels in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice. Consistent with the results obtained from the Western blot analysis, enzyme immunoassays also showed that levels of VEGF protein in BAL fluids were significantly increased at 48 hours after OVA inhalation compared with levels in saline-sensitized and saline-challenged WBB6F₁^+/+ mice, saline-sensitized and saline-challenged W/W^v mice, and saline-sensitized and saline-challenged BMCMCW/W^v mice. The levels of VEGF protein in BAL fluids of W/W^v mice after OVA inhalation were also significantly lower than in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice and in OVA-sensitized and OVA-challenged BMCMCW/W^v mice (Figure 1C). The levels of VEGF in OVA-sensitized and OVA-challenged BMCMCW/W^v mice were similar to the levels in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice.

View larger version (19K): [in this window] [in a new window]   Figure 1. Vascular endothelial growth factor (VEGF) protein levels in lung tissues and bronchoalveolar lavage (BAL) fluids and plasma exudation of ovalbumin (OVA)-sensitized and OVA-challenged mice. Sampling was performed at 48 hours after the last challenge in saline-sensitized and saline-challenged, congenic normal WBB6F1+/+ mice (SAL+/+), saline-sensitized and saline-challenged, genetically mast cell–deficient WBB6F1-KitW/KitW-v (W/Wv) mice (SAL W/Wv), saline-sensitized and saline-challenged BMCMCW/Wv (mast cell–reconstituted W/Wv) mice (SAL BMCMCW/Wv), OVA-sensitized and OVA-challenged WBB6F1+/+ mice (OVA+/+), OVA-sensitized and OVA-challenged W/Wv mice (OVA W/Wv), and OVA-sensitized and OVA-challenged BMCMCW/Wv mice (OVA BMCMCW/Wv). (A) Western blotting of VEGF in lung tissues. (B) Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of VEGF in the lung tissues of SAL+/+ is arbitrarily presented as 1. (C) Enzyme immunoassay of VEGF in BAL fluids. (D) Evans blue dye assay in lung tissues. Bars represent mean ± SEM from 8 mice per group. #P < 0.05 versus SAL+/+, SAL W/Wv, and SAL BMCMCW/Wv; *P < 0.05 versus OVA+/+; P < 0.05 versus OVA W/Wv. (E) Correlation between VEGF levels in BAL fluids and plasma exudation.

Localization of Immunoreactive VEGF in Mice after OVA Inhalation
Immunohistochemical analysis showed localization of immunoreactive VEGF in inflammatory cells and epithelial layers around the bronchioles of OVA-sensitized and OVA-challenged +/+ mice (Figure 2D) and OVA-sensitized and OVA-challenged BMCMCW/W^v mice (Figure 2F), whereas in saline-sensitized and saline-challenged +/+ mice (Figure 2A), saline-sensitized and saline-challenged W/W^v mice (Figure 2B), saline-sensitized and saline-challenged BMCMCW/W^v mice (Figure 2C), and OVA-sensitized and OVA-challenged W/W^v mice (Figure 2E), VEGF was hardly detected in inflammatory cells around the bronchioles. Immunocytologic analysis of BAL fluids showed localization of immunoreactive VEGF in the BAL cells centrifuged out of solution from the OVA-sensitized and OVA-challenged +/+ mice (Figure 2J) and OVA-sensitized and OVA-challenged BMCMCW/W^v mice (Figure 2L). However, immunoreactive VEGF in BAL cells from saline-sensitized and saline-challenged +/+ mice (Figure 2G), saline-sensitized and saline-challenged W/W^v mice (Figure 2H), saline-sensitized and saline-challenged BMCMCW/W^v mice (Figure 2I), and OVA-sensitized and OVA-challenged W/W^v mice (Figure 2K) was less than that in the OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice and OVA-sensitized and OVA-challenged BMCMCW/W^v mice. Immunocytologic analysis of BAL fluids also revealed that immunoreactive VEGF staining was strong in macrophages, whereas immunoreactive VEGF was weakly stained in lymphocytes (see Figures E1A and E1B in the online supplement). When the expression of VEGF in BAL fluid eosinophils isolated by Percoll gradient centrifugation was examined, a low level of immunoreactive VEGF was observed (see Figure E1C).

View larger version (143K): [in this window] [in a new window]   Figure 2. Localization of immunoreactive vascular endothelial growth factor (VEGF) in lung tissues and bronchoalveolar lavage (BAL) fluids of ovalbumin (OVA)-sensitized and OVA-challenged mice. Sampling was performed at 48 hours after the last challenge in saline-sensitized and saline-challenged, congenic normal WBB6F1+/+ mice (A and G), saline-sensitized and saline-challenged W/Wv mice (B and H), saline-sensitized and saline-challenged BMCMCW/Wv (mast cell–reconstituted W/Wv) mice (C and I), OVA-sensitized and OVA-challenged WBB6F1+/+ mice (D and J), OVA-sensitized and OVA-challenged W/Wv mice (E and K), and OVA-sensitized and OVA-challenged BMCMCW/Wv mice (F and L). Representative light microscopy of VEGF-positive cells in the bronchioles (A–F) and in the BAL fluids (G–L). Brown indicates VEGF-positive cells. Bars indicate 50 µm. W/Wv = genetically mast cell–deficient WBB6F1-KitW/KitW-v.

View larger version (34K): [in this window] [in a new window]   Figure 3. Total cells and differential cellular components in bronchoalveolar lavage (BAL) fluids, pathologic changes in lung tissues, and airway responsiveness of ovalbumin (OVA)-sensitized and OVA-challenged mice. (A) Total cells and differential cellular components of BAL. (B–G) Representative hematoxylin-and-eosin–stained sections of the lungs. Sections were obtained in saline-sensitized and saline-challenged, congenic normal WBB6F1+/+ (+/+) mice (B), saline-sensitized and saline-challenged, genetically mast cell–deficient WBB6F1-KitW/KitW-v (W/Wv) mice (C), saline-sensitized and saline-challenged BMCMCW/Wv mice (D), OVA-sensitized and OVA-challenged +/+ mice (E), OVA-sensitized and OVA-challenged W/Wv mice (F), and OVA-sensitized and OVA-challenged BMCMCW/Wv mice (G). Bars indicate 50 µm. (H) Peribronchial and perivascular lung inflammation were measured, and total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. (I) Airway responsiveness in OVA-sensitized and OVA-challenged mice. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+, SAL W/Wv, and SAL BMCMCW/Wv; *P < 0.05 versus OVA+/+; P < 0.05 versus OVA W/Wv. SAL = saline.

Airway Responsiveness in Mice after OVA Inhalation
In OVA-sensitized and OVA-challenged mice, the dose–response curve of percent R_rs shifted to the left compared with that of saline-sensitized and saline-challenged mice (Figure 3I). In addition, the R_rs produced by methacholine administration (at doses ranging from 25 to 50 mg/ml) increased significantly in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice and OVA-sensitized and OVA-challenged BMCMCW/W^v mice compared with that in saline-sensitized and saline-challenged WBB6F₁^+/+ mice, saline-sensitized and saline-challenged W/W^v mice, and saline-sensitized and saline-challenged BMCMCW/W^v mice. The levels of R_rs in OVA-sensitized and OVA-challenged W/W^v mice were significantly lower at 50 mg/ml of methacholine inhalation than those in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice (Figure 3I). On the other hand, the levels of R_rs in BMCMCW/W^v mice after OVA inhalation were significantly higher at 50 mg/ml of methacholine inhalation than in OVA-sensitized and OVA-challenged W/W^v mice.

Th2 Cytokine Levels in Mice after OVA Inhalation
Western blot analysis revealed that IL-4, IL-5, and IL-13 protein levels in lung tissues were increased significantly at 48 hours after OVA inhalation compared with those in saline-sensitized and saline-challenged WBB6F₁^+/+ mice, saline-sensitized and saline-challenged W/W^v mice, and saline-sensitized and saline-challenged BMCMCW/W^v mice (Figures 4A and 4B). However, the levels of IL-4, IL-5, and IL-13 protein in the lung tissues of W/W^v mice after OVA inhalation were significantly lower than in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice. In contrast, the levels of IL-4, IL-5, and IL-13 protein in the lung tissues of BMCMCW/W^v mice after OVA inhalation were similar to those in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice. Consistent with these results, enzyme immunoassays also showed that levels of IL-4, IL-5, and IL-13 protein in BAL fluids were significantly increased after OVA inhalation compared with saline-sensitized and saline-challenged WBB6F₁^+/+ mice, saline-sensitized and saline-challenged W/W^v mice, and saline-sensitized and saline-challenged BMCMCW/W^v mice (Figure 4C). The levels of IL-4, IL-5, and IL-13 protein in BAL fluids of W/W^v mice after OVA inhalation were significantly lower than in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice. On the other hand, IL-4, IL-5, and IL-13 protein levels in BAL fluids of BMCMCW/W^v mice after OVA inhalation were significantly higher than in OVA-sensitized and OVA-challenged W/W^v mice.

View larger version (24K): [in this window] [in a new window]   Figure 4. IL-4, IL-5, and IL-13 levels in lung tissues and in bronchoalveolar lavage (BAL) fluids of ovalbumin (OVA)-sensitized and OVA-challenged mice. (A) Western blotting of IL-4, IL-5, and IL-13 in lung tissues. (B) Densitometric analyses are presented as the relative ratio of each molecule to actin. The relative ratio of each molecule in the lung tissues of saline-challenged, congenic normal WBB6F1+/+ (SAL+/+) is arbitrarily presented as 1. (C) Enzyme immunoassay of IL-4, IL-5, and IL-13 in BAL fluids. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+, SAL W/Wv (saline-challenged, genetically mast cell–deficient WBB6F1-KitW/KitW-v), and SAL BMCMCW/Wv (saline-sensitized and saline-challenged mast-cell–reconstituted W/Wv mice); *P < 0.05 versus OVA+/+; P < 0.05 versus OVA W/Wv.

Effects of SU1498 on Changes in Cell Number and Th2 Cytokine Levels in BMCMCW/W^v Mice after OVA Inhalation

View larger version (8K): [in this window] [in a new window]   Figure 5. Effects of SU1498 on total cells, differential cellular components, and the T-helper type 2 (Th2) cytokine levels in bronchoalveolar lavage fluids of ovalbumin (OVA)-sensitized and OVA-challenged BMCMCW/Wv (saline-sensitized and saline-challenged mast-cell–reconstituted W/Wv) mice. Sampling was performed at 48 hours after the last challenge in OVA-sensitized and OVA-challenged BMCMCW/Wv mice administered drug vehicle (OVA BMCMCW/Wv + VEH) and OVA-sensitized and OVA-challenged BMCMCW/Wv mice administered SU1498 (OVA BMCMCW/Wv + SU1498). Bars represent mean ± SEM for 6 mice per group. #P < 0.05 versus OVA BMCMCW/Wv. VEH = vehicle.

View larger version (19K): [in this window] [in a new window]   Figure 6. Phosphorylated Akt (p-Akt) and Akt protein expression, phosphatidylinositol 3-kinase (PI3K) enzyme activity in lung tissues (A–C), and hypoxia-inducible factor (HIF)-1 levels in nuclear protein extracts from lung tissues (D and E) of ovalbumin (OVA)–sensitized and OVA-challenged mice. (A) Western blotting of p-Akt and Akt in lung tissues. (B) Densitometric analyses are presented as the relative ratio of p-Akt to Akt. (C) Enzyme immunoassay of phosphatidylinositol-3,4,5-triphosphate (PIP3) generation by PI3K in lung tissue extracts. (D) Western blotting of HIF-1 in lung tissues. (E) Densitometric analyses are presented as the relative ratio of HIF-1 to HIF-1β. The relative ratio of p-Akt or HIF-1 in the lung tissues of saline-challenged mice (SAL+/+) is arbitrarily presented as 1. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+, SAL W/Wv, and SAL BMCMCW/Wv (saline-sensitized and saline-challenged mast-cell–reconstituted W/Wv mice); *P < 0.05 versus OVA+/+; P < 0.05 versus OVA W/Wv.

HIF-1 Protein Levels in Mice after OVA Inhalation

Effect of SU5614 or SU1498 on VEGF Levels, Akt Phosphorylation, and Plasma Extravasation in Mice after OVA Inhalation
Western blot analysis revealed that the protein levels of VEGF and p-Akt in lung tissues of WBB6F₁^+/+ mice were increased significantly at 48 hours after OVA inhalation compared with levels after saline inhalation, and that administration of SU5614 or SU1498 dramatically reduced the increased levels of the proteins in lung tissues (Figures 7A, 7B, 7D, and 7E). Consistent with these results, enzyme immunoassay showed that the administration of SU5614 or SU1498 reduced significantly the increased levels of VEGF in BAL fluids of WBB6F₁^+/+ mice after OVA inhalation (Figure 7C). In addition, EBD assay revealed that plasma extravasation was significantly increased at 48 hours after the last challenge. The increase in plasma extravasation was significantly reduced by the administration of SU5614 or SU1498 in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice (Figure 7F). These data suggest that vascular permeability is modulated by VEGF at least in part through PI3K signaling in allergic airway disease.

View larger version (42K): [in this window] [in a new window]   Figure 7. Effects of SU5614 or SU1498 on protein levels of vascular endothelial growth factor (VEGF) and phosphorylated Akt (p-Akt) in lungs and on plasma exudation of ovalbumin (OVA)-sensitized and OVA-challenged mice. Sampling was performed at 48 hours after the last challenge in saline-sensitized and saline-challenged, congenic normal WBB6F1+/+ mice administered saline (SAL+/+ + SAL), OVA-sensitized and OVA-challenged WBB6F1+/+ mice administered saline (OVA+/+ + SAL), OVA-sensitized and OVA-challenged WBB6F1+/+ mice administered drug vehicle (OVA+/+ + VEH), OVA-sensitized and OVA-challenged WBB6F1+/+ mice administered SU5614 (OVA+/+ + SU5614), and OVA-sensitized and OVA-challenged WBB6F1+/+ mice administered SU1498 (OVA+/+ + SU1498). (A) Western blotting of VEGF protein. (B) Densitometric analyses are presented as the relative ratio of VEGF to actin. (C) Enzyme immunoassay of VEGF in bronchoalveolar lavage fluids. (D) Western blotting of p-Akt protein. (E) Densitometric analyses are presented as the relative ratio of p-Akt to Akt. (F) Measurement of plasma exudation using Evans blue dye. The relative ratio of VEGF or p-Akt in the lung tissues of SAL+/+ + SAL is arbitrarily presented as 1. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+ + SAL; *P < 0.05 versus OVA+/+ + SAL.

Effects of LY-294002 or Wortmannin on HIF-1 Levels in Mice after OVA Inhalation

View larger version (34K): [in this window] [in a new window]   Figure 8. Effects of LY-294002 or wortmannin on hypoxia-inducible factor (HIF)-1 levels in nuclear protein extracts from lung tissues of ovalbumin (OVA)-sensitized and OVA-challenged mice. Sampling was performed in saline-challenged mice administered saline (SAL+/+ + SAL), OVA-sensitized and OVA-challenged mice administered saline (OVA+/+ + SAL), OVA-sensitized and OVA-challenged mice administered vehicle (OVA+/+ + VEH), OVA-sensitized and OVA-challenged WBB6F1+/+ mice administered LY-294002 (OVA+/+ + LY294002), and OVA-sensitized and OVA-challenged, congenic normal WBB6F1+/+ mice administered wortmannin (OVA+/+ + wortmannin). (A) Western blotting of HIF-1 protein. (B) Densitometric analyses are presented as the relative ratio of HIF-1 to HIF-1β. The relative ratio of HIF-1 in the lung tissues of SAL+/+ + SAL is arbitrarily presented as 1. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+ + SAL; *P < 0.05 versus OVA+/+ + SAL.

Effect of 2ME2 on HIF-1 and VEGF Protein Levels in Mice after OVA Inhalation

View larger version (19K): [in this window] [in a new window]   Figure 9. Effect of 2-methoxyestradiol (2ME2) on protein levels hypoxia-inducible factor (HIF)-1 and vascular endothelial growth factor (VEGF) in lungs of ovalbumin (OVA)-sensitized and OVA-challenged mice. Sampling was performed at 48 hours after the last challenge in saline-challenged mice administered saline (SAL+/+ + SAL), OVA-sensitized and OVA-challenged mice administered saline (OVA+/+ + SAL), OVA-sensitized and OVA-challenged mice administered vehicle (OVA+/+ + VEH), and OVA-sensitized and OVA-challenged, congenic normal WBB6F1+/+ mice administered 2ME2 (OVA+/+ + 2ME2). (A) Western blotting of HIF-1 protein. (B) Densitometric analyses are presented as the relative ratio of HIF-1 to HIF-1β. (C) Western blotting of VEGF protein. (D) Densitometric analyses are presented as the relative ratio of VEGF to actin. (E) Enzyme immunoassay of VEGF in bronchoalveolar lavage fluids. The relative ratio of HIF-1 or VEGF in the lung tissues of SAL+/+ + SAL is arbitrarily presented as 1. Bars represent mean ± SEM for 8 mice per group. #P < 0.05 versus SAL+/+ + SAL; *P < 0.05 versus OVA+/+ + SAL.

Effects of PI3K Inhibitors and HIF-1 Inhibitor on Plasma Exudation in Mice after OVA Inhalation

Relationship between VEGF Protein in BAL Fluids and Plasma Exudation
The changes of VEGF levels and plasma exudation were analyzed using an enzyme immunoassay and the EBD assay at 6, 12, 36, 24, 48, and 72 hours after a single OVA inhalation in OVA-sensitized WBB6F₁^+/+ mice. The levels of VEGF protein in BAL fluids were increased significantly at 36, 48, and 72 hours after a single OVA inhalation compared with prechallenge levels (see Figure E3A). EBD assay revealed that plasma exudation was significantly increased at 24, 36, 48, and 72 hours after a single OVA inhalation compared with prechallenge levels (see Figure E3B). The levels of VEGF protein in BAL fluids correlated significantly with the levels of plasma exudation in OVA-sensitized WBB6F₁^+/+ mice with a single OVA challenge (r = 0.608, P < 0.05) (see Figure E3C).

	DISCUSSION

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Inflammation of the asthmatic airway is usually accompanied by increased vascular permeability and plasma exudation. Mast cells release a number of mediators that act directly on the vasculature to produce vasodilatation, increased permeability, and subsequent plasma protein extravasation into surrounding tissue (22). VEGF has been implicated to contribute to asthmatic tissue edema through its effect on vascular permeability (23, 24). However, the roles of mast cells in VEGF-mediated signaling in allergic airway disease are not clearly understood. We have used genetically mast cell–deficient mice to determine the role of mast cells, more specifically in the increase of vascular permeability.

Mast cells play a central role in the development of allergic respiratory disorders. IgE-dependent activation of mast cells can induce these cells to release a panel of preformed or newly synthesized mediators, including histamine, tryptase, prostaglandins, leukotrienes, platelet activating factor, and cytokines such as IL-4, IL-5, IL-13, and tumor necrosis factor-, which can induce allergic reactions in the lung including airway obstruction, airway microvascular leakage, and mucosal edema, as well as mucus gland hypersecretion (25, 26). Consistent with these findings, our data revealed that levels of Th2 cytokines, the number of inflammatory cells of the airways, and bronchial hyperresponsiveness in W/W^v mice were significantly lower than those in WBB6F₁^+/+ mice after OVA inhalation. Moreover, reconstitution of mast cells in W/W^v mice resulted in increases in levels of Th2 cytokines, the number of inflammatory cells of the airways, and bronchial hyperresponsiveness, which were similar to those of the WBB6F₁^+/+ mice. Mast cells can also produce and secrete VEGF upon stimulation through fragment crystallizable epsilon (Fc) receptor I or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore A23187. The stimulated mast cells can rapidly release VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein (27). In addition, VEGF can be produced by a wide variety of cells, which can be increased and recruited in airways by allergenic stimulation, including macrophages, neutrophils, eosinophils, and lymphocytes (3, 5, 6, 28, 29). We have found that levels of VEGF in W/W^v mice were significantly lower after OVA inhalation than the levels in OVA-induced WBB6F₁^+/+ mice. Moreover, reconstitution of mast cells in W/W^v mice resulted in an increase in VEGF that was similar to those of the WBB6F₁^+/+ mice. Immunocytologic analysis has shown the localization of immunoreactive VEGF on macrophages, lymphocytes, and eosinophils in BAL cells from WBB6F₁^+/+ mice after OVA inhalation. Our present data also showed that the administration of VEGF receptor inhibitor SU1498 significantly reduced the increased numbers of inflammatory cells and the increased Th2 cytokines after OVA inhalation in BMCMCW/W^v mice. Taken together, these findings indicate that the mast cells may regulate airway inflammation and bronchial hyperresponsiveness through orchestrating VEGF expression in mast cells as well as the other inflammatory cells in allergic airway inflammation.

VEGF is a potent stimulator of inflammation, airway remodeling, and physiologic dysregulation that augments antigen sensitization and Th2 cell–mediated inflammation (5). The major role of VEGF in asthma appears to be the enhancement of vascular permeability (3, 4, 6). The mechanism of VEGF-mediated induction of the vascular permeability seems to be the enhanced functional activity of vesicular/vacuolar organelles (4, 30). Several studies have shown that overproduction of VEGF causes an increase in vascular permeability, which results in leakage of plasma proteins, including inflammatory mediators, and inflammatory cells into the extravascular space and allows migration of inflammatory cells into the airways (3, 6, 27). Consistent with these observations, we have found that VEGF expression was up-regulated and vascular permeability was increased in OVA-induced allergic airway disease. Interestingly, plasma exudation in W/W^v mice, which produce low levels of VEGF, was significantly lower after OVA inhalation than the levels in WBB6F₁^+/+ mice after OVA inhalation. Supporting the observations, BMCMCW/W^v mice exhibited increased levels of VEGF and increased vascular permeability that were similar to those of the WBB6F₁^+/+ mice after OVA inhalation. In addition, we have found that the levels of VEGF protein in BAL fluids correlated significantly with the levels of plasma exudation in OVA-sensitized and OVA-challenged mice and that the inhibition of VEGF activity reduced the plasma exudation in OVA-sensitized and OVA-challenged WBB6F₁^+/+ mice. Taken together, these results suggest that the levels of VEGF are closely associated with vascular permeability, although the role of other contributing factors such as histamine cannot be excluded.

Several studies have indicated that PI3K plays a crucial role in VEGF-mediated signaling (31, 32). In the present study, the administration of SU5614 or SU1498, which reduces the increased levels of VEGF in lungs, reduced dramatically the increased levels of p-Akt protein in lung tissues of WBB6F₁^+/+ mice after OVA inhalation. Previous reports have shown that increase of PI3K/Akt activity enhances the HIF signaling pathway (33–35). Li and colleagues have also reported that activation of Akt turns on HIF-1 independently of hypoxia (36). In the present study, levels of p-Akt protein and PI3K activity in the lung tissues were increased after OVA inhalation. On the other hand, levels of p-Akt and PI3K activity in W/W^v mice were significantly lower after OVA inhalation than those in OVA-induced WBB6F₁^+/+ mice. BMCMCW/W^v mice exhibited increased p-Akt protein and PI3K activity that were similar to those of WBB6F₁^+/+ mice. In addition, the increased HIF-1 protein levels as well as plasma exudation were reduced significantly by the administration of PI3K inhibitor, LY-294002, or wortmannin in WBB6F₁^+/+ mice after OVA inhalation.

VEGF expression is regulated through HIF-1 (37, 38). Previous reports have demonstrated that HIF-1 plays a critical role in immune and inflammatory responses (39, 40). Determination of HIF-1 protein level in nuclear extracts has revealed that this protein level was substantially increased in our present OVA-induced model of allergic airway disease, suggesting that HIF-1 was activated. However, levels of HIF-1 in W/W^v mice were significantly lower after OVA inhalation than in OVA-challenged WBB6F₁^+/+ mice. To show the effects of HIF-1 inhibition on VEGF expression and vascular permeability, an HIF-1 inhibitor, 2ME2, was used. This inhibitor has been shown to inhibit HIF-1 activity through molecular mechanisms that suppress the HIF-1 translation and its nuclear translocation (19). A previous study has demonstrated that the increased HIF-1 protein levels after allergen inhalation were inhibited by the administration of 2ME2 (41). In our study, the increased VEGF, vascular permeability, and HIF-1 protein levels after OVA inhalation were significantly decreased by administration of 2ME2. Taken together, we suggest that mast cell regulates HIF-1 action through a PI3K/Akt signaling pathway, resulting in increased VEGF expression in a murine model of allergic airway disease.

In this study, inhibitors of VEGF receptor tyrosine kinase, SU5614 and SU1498, were used to inhibit VEGF activity. Previous studies have demonstrated that administration of VEGF receptor inhibitors inhibits VEGF activity in a murine model of asthma (3, 6). The increased leakage of plasma proteins and migration of inflammatory cells into the airways can be caused by the overproduction of VEGF (3, 6, 23). In our previous study, the plasma leakage and the migration of inflammatory cells, which are the sources of VEGF production, were blocked by administration of SU5614 or SU1498, thereby reducing VEGF production (42). In addition, there is a positive feedback loop with VEGF enhancing Th2 sensitization, inflammation, and cytokine elaboration and IL-13 subsequently enhancing VEGF production (5, 43). Our previous data have also shown that inhibition of VEGF activity by administration of VEGF receptor inhibitors decreased Th2 inflammation, cytokine elaboration, and VEGF production in OVA-sensitized and OVA-challenged mice (42). Consistent with these data, the increased numbers of inflammatory cells and the increased levels of Th2 cytokines after OVA inhalation were inhibited by the administration of SU1498 in our present study. Moreover, the autocrine action of VEGF has also been reported (44). These findings suggest that VEGF receptor inhibitors can decrease VEGF production through inhibition of migration and inflammatory response of VEGF-producing cells or blocking of an autoinductive VEGF pathway.

SU5614 is a potent inhibitor of VEGF and platelet-derived growth factor (PDGF) receptor tyrosine kinases. It does not have any effect on the epidermal growth factor (EGF) and insulin-like growth factor (IGF) receptor tyrosine kinases. Previous studies have demonstrated that SU5614 also has inhibitory activity on other receptors such as c-kit and Flt-3 (45–47). SU1498 is potent and selective inhibitor of Flk-1 kinase, a VEGF receptor kinase. It has only a weak inhibitory effect on PDGF receptor, EGF receptor, and human epidermal growth factor receptor-2 kinase. Therefore, the use of these two VEGF receptor inhibitors may rule out the inhibitory effects of these chemical inhibitors on other receptors.

In summary, we examined the role of mast cells in a murine model of allergic airway disease, more specifically in the increase of vascular permeability. By using genetically mast cell–deficient mice, we have shown that mast cells may regulate vascular leakage, at least in part, via modulation of the PI3K–HIF-1–VEGF axis in mice.

	Acknowledgments

 
The authors thank Professor Stephen J. Galli (Stanford University School of Medicine, Stanford, CA) and Professor Mie-Jae Im (Chonbuk National University Medical School, Jeonju, South Korea) for critical readings of the manuscript.

	FOOTNOTES

 
Supported by a grant from the Korean Science and Engineering Foundation through the National Research Lab. Program funded by the Ministry of Science and Technology (R0A-2005-000-10052–0[2007]), by a Korean Research Foundation grant funded by the Korean Government (Ministry of Education and Human Resource Development, Basic Research Promotion Fund) (KRF-2005-201-E00014), by a grant from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A060169), and by a grant from the Korean Health 21 R&D Project (0412-CR03-0704-0001).

^* These authors contributed equally to this article.

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

Originally Published in Press as DOI: 10.1164/rccm.200801-008OC on July 31, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 3, 2008; accepted in final form July 29, 2008

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