Semaphorin 7A promotes endothelial permeability and inflammation via plexin C1 and integrin β1 in Kawasaki disease

Abstract

Background: Kawasaki disease (KD) is a pediatric systemic vasculitis characterized by endothelial cell dysfunction. Semaphorin 7A (Sema7A) has been reported to regulate endothelial phenotypes associated with cardiovascular diseases, while its role in KD remains unknown. This study aims to investigate the effect of Sema7A on endothelial permeability and inflammatory response in KD conditions.

Methods: Blood samples were collected from 68 KD patients and 25 healthy children (HC). The levels of Sema7A and A Disintegrin and Metalloprotease 17 (ADAM17) in serum were measured by enzyme-linked immunosorbent assay (ELISA), and Sema7A expression in blood cells was analyzed by flow cytometry. Ex vivo monocytes were used for Sema7A shedding assays. In vitro human coronary artery endothelial cells (HCAECs) were cultured in KD sera and stimulated with Sema7A, and TNF-α, IL-1β, IL-6, and IL-18 of HCAECs were measured by ELISA and qRT-PCR. HCAECs monolayer permeability was measured by FITC-dextran.

Results: The serum level of Sema7A was significantly higher in KD patients than in HC and correlated with disease severity. Monocytes were identified as one of the source of elevated serum Sema7A, which implicates a process of ADAM17-dependent shedding. Sera from KD patients induced upregulation of plexin C1 and integrin β1 in HCAECs compared to sera from HC. Sema7A mediated the proinflammatory cytokine production of HCAECs in an integrin β1-dependent manner, while both plexin C1 and integrin β1 contributed to Sema7A-induced HCAEC hyperpermeability.

Conclusions: Sema7A is involved in the progression of KD vasculitis by promoting endothelial permeability and inflammation through a plexin C1 and integrin β1-dependent pathway. Sema7A may serve as a potential biomarker and therapeutic target in the prognosis and treatment of KD.

Keywords: Endothelial cell; Inflammation; Kawasaki disease; Semaphorin 7A.

Fig. 1

sSema7A is increased in serum of KD patients and correlated with disease severity. a Levels of serum sSema7A in HC, KD-nCAL and KD-CAL. b Changes of serum sSema7A at the same KD patient at the acute, subacute and convalescent phase (n = 39). c Relationship between sSema7A and Z-score (n = 22), CRP and albumin (n = 68). *P < 0.05; ***P < 0.001; ****P < 0.0001. Sema7A: Semaphorin 7A; HC: health control; KD: Kawasaki disease; CAL: coronary artery lesions; nCAL: no CAL; CRP: C reaction protein; Alb: albumin

Fig. 2

Monocyte mSema7A shedding mediated by ADAM17 may be one of the reasons of increased serum sSema7A in KD. a Percentage of Sema7A⁺ monocytes in CD14⁺ cells. The result shown is representative of FCM findings from KD-nCAL (n = 6), KD-CAL (n = 6) and HC (n = 6). b Percentage of Sema7A⁺ cells in CD3⁺ T cells and CD15⁺ granulocytes from KD-nCAL (n = 6), KD-CAL (n = 6) and HC (n = 6). c The relationship between serum sSema7A concentration and monocyte counts in KD (n = 68). d Concentration of sSema7A in the culture supernatant of healthy donor monocytes treated with MMP9, ADAM17 and TAPI-1 (a specific ADAM17 inhibitor). Data are expressed as mean ± standard error of mean (M ± SEM) from 3 separate experiments. e The relationship of sSema7A with ADAM17 and MMP9 in the sera of KD patients (n = 68). *P < 0.05; **P < 0.01; n.s.: not significant. Sema7A: Semaphorin 7A; HC: health control; KD: Kawasaki disease; CAL: coronary artery lesions; nCAL: no CAL; MMP9: matrix metalloproteinase 9; ADAM17: A disintegrin and metalloproteinase domain 17

Fig. 3

KD sera upregulate the expression of Sema7A receptors in HCAECs. a The proinflamamtory cytokine mRNA expression and permeability of HCAECs cultured in 10% FBS-containing RPMI 1640 and subsequently stimulated with 10 µg/ml rhSema7A or not. b The proinflamamtory cytokine mRNA expression and permeability of HCAECs cultured in 20% KD sera-containing or 20% HC sera-containing RPMI 1640 and subsequently stimulated with 10 µg/ml rhSema7A or not. c The mRNA expression levels of plexin C1 and integrin β1 in HCAECs treated with 20% KD sera or HC sera. Data are expressed as M ± SEM from 3 separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s.:not significant. Sema7A: Semaphorin 7A; FBS: fetal bovine serum; PBS: phosphate buffered saline; KDS: sera from KD patients (RPMI 1640 containing 20% KD sera (pooled from 10 KD patients) ); HCS: sera from healthy controls (RPMI 1640 containing 20% healthy control sera (pooled from 10 healthy controls) ); KD: Kawasaki disease

Fig. 4

Sema7A mediates cytokine production of HCAECs by binding to integrin β1. a The mRNA expression levels and culture supernatant concentrations of TNF-α, IL-1β, IL-6 and IL-18 of HCAECs stimulated with different dosage of rhSema7A. b The mRNA expression levels of TNF-α, IL-1β, IL-6 and IL-18 of HCAECs pretreated with anti-plexin C1 antibody and anti-integrin β1 antibody, respectively, followed by 10 µg/ml rhSema7A stimulation. Data are expressed as M ± SEM from 3 separate experiments. *P < 0.05; **P < 0.01; n.s.:not significant. Sema7A: Semaphorin 7A; TNF: tumor necrosis factor; IL: interleukin

Fig. 5

Sema7A induces HCAEC hyperpermeability via both plexin C1 and integrin β1. a Induction of HCAEC monolayer permeability by medium containing 20% untreated KD sera or 20% sSema7A-neutralized KD sera. b HCAEC permeability assay by rhSema7A stimulation upon blockade of plexin C1 and integrin β1, respectively. Data are expressed as M ± SEM from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Sema7A: Semaphorin 7A; FITC: fluorescein isothiocyanate; KDS: sera from KD patients (medium containing 20% KD sera (pooled from 10 KD patients) ); KD: Kawasaki disease