Molecular Mechanism Study Of VEGF-C Mediated Lymphangiogenesis Through Src/eNoS Pathway In Silicosis

Objectives This experiment explores the effect of SiO2 stimulated human mononuclear macrophages U937 producing vascular endothelial growth factor C(VEGF-C)on the tube formation of human lymphatic endothelial cells(HLEC)and the mediation of downstream signaling pathway Src/e NOS,and illustrates the molecular mechanism of pulmonary lymphangiogenesis during the pathogenesis of silicosis.Methods 1 Bronchoalveolar lavage fluid(BALF)and alveolar macrophages(AM)were collected from silicosis patients(n=16)and healthy controls(n=4),and VEGF-C levels were detected by ELISA.2 U937 cells were stimulated with SiO2 at different concentrations(0,25,50,100,200 μg/m L)for different time(12,24,48 h).Western blot and ELISA were used to detect VEGF-C protein levels in U937 cells and culture supernatant,respectively.The combination of SiO2 stimulation concentration and time generating the highest VEGF-C protein expression level was selected for further experiment,and the cell culture supernatant was collected as conditioned medium(CM).3HLEC were divided into negative control group,CM control group,CM treatment group,VEGF-C treatment group and VEGF-Cm Ab treatment group.The CM treatment group was treated with CM stimulated by SiO2,the VEGF-C treatment group with a medium containing 500 ng/m L human VEGF-C recombinant protein,the VEGF-Cm Ab treatment group was treated with U937 cell culture supernatant stimulated by SiO2 and VEGF-Cm Ab,the CM control group with cell culture supernatant without SiO2 stimulation,and the negative control group without any treatment.The cell proliferation of HLEC was detected by MTS method,cell chemotaxis and tube formation were detected by Transwell chemotaxis assay and matrix tube formation assay.Western blot assay detected protein expression of vascular cell adhesion molecule 1(VCAM-1).4 Src inhibitor PP2 pretreated HLEC,which were divided into CM group,CM-PP2 group,VEGF-C group,and VEGF-CPP2 group.The protein expressions of VEGFR-3,LYVE-1,Src and e NOS were detected by western blot,and the tube formation was detected by matrix tube formation assay.5Protein contents of hyaluronic acid(HA),basic fibroblast growth factor(b FGF)and hepatocyte growth factor(HGF)in CM were detected by ELISA.Results 1 The level of VEGF-C in AM in silicosis patients was significantly higher than that in healthy controls [(263.53±66.37)and(119.10±32.04)ng/L,respectively].2 The SiO2 stimulation at 50 μg/m L for 24 h produced the highest VEGF-C protein level in both U937 cells and cell culture supernatant compared with the 0 μg/m L group [(1.12±0.08)and(5335.33±208.66)ng/L,respectively].3 Compared with the control group,the proliferation of HLEC [(200.33±13.61)and(167.00±9.54),respectively],the number of HLEC chemotaxis [(101.40±13.83)and(93.40±9.61),respectively],VCAM-1 protein expression[(0.97±0.05)and(0.87±0.05),respectively],number of tubes [(32.20±7.26)and(25.00±6.25),respectively],area of tube coverage [(46.96±6.93)and(35.22±2.61),respectively],number of tube branches [(77.20±6.80)and(84.60±7.90),respectively] and total length of tube branches [(12869.80±549.20)and(10538.60±482.50),respectively]were significantly increased in the CM group and VEGF-C group.The above indicators of HLEC were significantly reduced in the VEGF-Cm Ab group compared with the CM group.4 After treatment with CM and VEGF-C,the protein expressions of VEGFR-3 [(0.75±0.13)and(0.88±0.06),respectively]),LYVE-1 [(0.85±0.09)and(0.88±0.13),respectively],pSrc [(0.96±0.14)and(1.05±0.10),respectively],and p-e NOS [(1.12±0.20)and(1.05±0.13)],respectively] were significantly increased compared with the control group.After inhibition of Src,p-e NOS expression was significantly reduced compared to the CM group and VEGF-C group.Compared with VEGF-C group,the number of tubes of VEGFC-PP2 group(5.40±1.67)was significantly reduced;there was no significant change in the number of tubes between CM-PP2 group(22.40±4.51)and CM group(23.40±3.36).5 The levels of b FGF and HA were increased in CM compared with control group [(47.40±4.35)ng/L and(13.37±3.14)μg/L,respectively].But the concentration of HGF was almost equal in the two groups.Conclusions 1 Lymphangiogenesis in silicosis is associated with SiO2 stimulation of macrophages to secrete various pro-lymphangiogenic growth factors,in which VEGF-C plays a major role.2 SiO2-induced activation of lung macrophages to secrete VEGF-C may further mediate the downstream signaling pathway Src/e NOS to promote lymphangiogenesis in silicosis.Figure 11;Table 14;Reference 139...