Expression of semaphorin 3A and neuropilin 1 in asthma

Abstract

Neuropilin 1 (NP1) is a part of essential receptor complexes mediating both semaphorin3A (SEMA3A) and vascular endothelial growth factor (VEGF) which is one of important mediators involved in the pathogenesis of asthma. Therefore, it is possible that SEMA3A plays a role in the pathogenesis of asthma through attenuation of VEGF-mediated effects. In the present study, we aimed to evaluate expression levels of SEMA3A and NP1 using induced sputum of asthmatics and a murine model of asthma. Firstly, SEMA3A and NP1 expressions in induced sputum of asthmatics and SEMA3A and NP1 expression on bronchoalveolar lavage (BAL) cells and lung homogenates of asthmatic mice were determined. Then we evaluated the immunolocalization of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), and NP1 expressions on asthmatic mice lung tissue and their subcellular distributions using fibroblast and BEAS2B cell lines. Sputum SEMA3A and NP1 expressions were significantly higher in asthmatics than controls. Similarly, SEMA3A and NP1 expressions on BAL cells and lung homogenates were significantly elevated in asthmatic mice compared to control mice. Immunohistochemical analysis showed that VEGFR1, VEGFR2, and NP1 expressions were also uniformly increased in asthmatic mice. Our observations suggest that SEMA3A and NP1 may play important roles in the pathogenesis of asthma.

Keywords: Asthma; Neuropilin; Semaphorin-3A (SEMA3A); Vascular Endothelial Growth Factor.

Fig. 1

Sputum SEMA3A and NP1 expressions of asthmatics. SEMA3A and NP1 mRNA in induced sputum was qualified using real-time quantitative polymerase chain reaction. Both values were normalized by beta-actin mRNA. Data indicate means±SEM. (A) SEMA3A expressions (asthmatics vs normal controls). (B) NP1 expressions (asthmatics vs normal controls). (C) SEMA3A expressions (severe asthmatics vs mild to moderate asthmatics).

Fig. 2

SEMA3A and NP1 expressions on BAL cells of asthmatic mice. Original magnification, × 400. blue (DAPI), nuclear staining. red (Cy3), SEMA3A and NP1. Representative images. (A) SEMA3. (B) NP1.

Fig. 3

SEMA3A, NP1, and VEGF expressions on lung homogenate of asthmatic mice. SEMA3A and NP1 mRNA in lung homogenate of asthmatic mice was qualified using real-time quantitative polymerase chain reaction. Both values were normalized by beta-actin mRNA. Data indicate means±SEM. (A) SEMA3A expressions (asthmatics [OVA/Alum] vs control mice [PBS]). (B) NP1 expressions (asthmatics vs control mice). (C) VEGF expressions (asthmatics vs control mice).

Fig. 4

Immunolocalization of VEGFR1, VEGFR2, and NP1 in the lung of asthmatic and control mice. Original magnification, × 400. Representative images. VEGFR1, VEGFR2, and NP1 expressions are uniformly increased in asthmatic mice compared to control mice. NP1 immunostaining on bronchial cells with membrane accentuation on basal cells is shown (arrow heads). (A) VEGFR1 (asthmatic vs control mice). (B) VEGFR2 (asthmatic vs control mice). (C) NP1 (asthmatic vs control mice).

Fig. 5

VEGFR1, VEGFR2, and NP1 subcellular distributions on fibroblast cells. Original magnification, × 100. blue (DAPI), nuclear staining. red (Cy3), VEGFR1, VEGFR2, and NP1. Left upper quadrant of each panel, DAPI staining image. Right upper quadrant of each panel, Cy3 staining image. Left lower quadrant of each panel, control image. Right lower quadrant of each panel, merged image. Representative images. VEGFR1 expressions are found predominantly in the cytoplasm with asymmetric and crescentic nature. VEGFR2 expressions are confined in the nucleus and co-localized with DAPI staining. NP1 expressions show predominantly membrane patterns associated with abundant granular cytoplasmic staining. (A) Control. (B) VEGFR1. (C) VEGFR2. (D) NP1.

Fig. 6

VEGFR1, VEGFR2, and NP1 subcellular distributions on BEAS2B cells. Original magnification, × 100. blue (DAPI), nuclear staining. red (Cy3), VEGFR1, VEGFR2, and NP1. Left upper quadrant of each panel, DAPI staining image. Right upper quadrant of each panel, Cy3 staining image. Left lower quadrant of each panel, control image. Right lower quadrant of each panel, merged image. Representative images. VEGFR2 and NP1 expressions on BEAS2B cells show similar patterns with those of fibroblast cells. The membrane staining of NP1 is often brighter at intercellular contacts (arrow head). (A) Control. (B) VEGFR1. (C) VEGFR2. (D) NP1.