Fig 2: Oscillatory shear enhances M1 polarization by SEMA3E-PLXND1. (A) Immunofluorescence staining of bifurcation lesions was performed with iNOS (red), F4/80 (green), PLXND1 (pink), SEMA3E (green blue) and DAPI (blue). Representative images are shown on the left (scale bar, 100 μm) and enlarged images of inserts are shown on the right (scale bar, 20 μm). (B) THP-1-derived macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear in treatment with or without 500 ng/mL recombinant SEMA3E (rSEMA3E). Immunoblot analysis of iNOS are shown. (C) Quantitative analysis showed SEMA3E enhanced M1 polarization under oscillatory shear. (D) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to oscillatory shear in treatment with rSEMA3E. The expression of iNOS was analyzed by WB. (E) Quantitative analysis showed iNOS expression was significantly lower in sh-PLXND1 cells. Data are shown as the mean ± SEM. n = 4. **P < 0.01, ****P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests or unpaired two-tailed Student's t tests. LS, laminar shear. OS, oscillatory shear. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Sema3E ameliorated the airway hyperresponsiveness, collagen deposition and mucus production in AS model. (a) Protocol outline. Mice were sensitized intraperitoneally with 10 µg OVA (or NS as control) on Days 0, 7, and 14. On Days 21–23, mice were challenged i.n. with 200 µg OVA (or NS as control), mice were exposed intranasally to Sema3E 1 hr before challenge in AS + Sema3E group. (b) Lung resistance in response to inhaled methacholine 24 hr after the last OVA challenge. (c) The total inflammatory cell counts in the BALF of mice for all groups. (d) Representative lung sections stained with hematoxylin and eosin (HE), periodic acid–Schiff (PAS), and Masson staining. (e) Differential cell counts in BALF. (f) Scoring of leukocyte infiltration in HE-stained lung tissue sections. (g) Quantification of collagen deposition. (h) Quantification of mucus-producing goblet cells. Data in (b)–(h) were expressed as mean ± standard deviation (n = 5). Compared with the NS group, ∗: p < 0.05; ∗∗: p < 0.01, ∗∗∗: p < 0.001, compared with AS group, #: p < 0.05, ##: p < 0.01, ###: p < 0.001. HE staining: magnification = ×100, scale bar = 100 µm, PAS and Masson staining: magnification = ×200, scale bar = 100 µm. (NS: nasal saline group, AS: asthma group, EB: eosinophilic bronchitis group, DXM: dexamethasone group, Sema3E: Semaphorin3E, OVA: ovalbumin, MCh: methacholine, Eos: eosinophil, Neu: Neutrophil, Mac: macrophage, and Lym: lymphocyte).

Fig 4: The expression of Sema3E in mouse vagal ganglion and lung tissue. (a) Representative image of Sema3E immunofluorescence staining of mouse vagal ganglion. DNA staining was blue, and Sema3E staining was green original magnifications: ×200, scale bar = 75 μm. (b) The expression level of Sema3E in the mouse vagal ganglion. (c) Representative image of Sema3E immunofluorescence staining of mouse lung tissue. Original magnifications: ×200, scale bar = 75 μm. (d) Sema3E expression level in mouse lung tissue. (e) Western blot analysis of Sema3E levels in lung tissue. (f) Western blot statistical results of Sema3E in mouse lung. (Compared with NS group, ∗: p < 0.05; ∗∗: p < 0.01; compared with EB group, #: p < 0.05; ##: p < 0.01). Data were expressed as mean ± standard deviation (n = 6). (NS: nasal saline group, AS: asthma group, EB: eosinophilic bronchitis group, DXM: dexamethasone group, Sema3E: Semaphorin3E).