FUT8-mediated Core Fucosylation Promotes The Pulmonary Vascular Remodeling In Pulmonary Arterial Hypertension

Objective: Pulmonary arterial hypertension(PAH)is a cardiovascular system disease with a high mortality rate and poor prognosis that is defined by its pathological feature of vascular remodeling.The most important factor in pulmonary vascular remodeling is disordered proliferation and apoptosis resistance of pulmonary artery smooth muscle cells(PASMC)in the middle membrane,which is similar to tumor cells.Fucosyltransferase 8(FUT8)is an enzyme that mediates the core fucosylation level of protein,which has been found to promote the occurrence and development in a number of tumor and immune system disease studies.However,there was no reports about the effect of FUT8 or core fucosylation in PAH.In this experiment,we explored the relationship between FUT8 and its mediated core fucosylation and PAH,aiming to provide new ideas for the mechanism study and clinical diagnosis and treatment of PAH.Methods:(1)we built a control or MCT treated rats model and used right catheterization,HE staining and immunofluorescence to evaluate hemodynamics and vascular remodeling;Lectin staining and lectin immunofluorescence were applied to determinate the changes of core fucosylation in lung tissues of PAH rats.Then we built 2-fluorofucose(2FF)treated PAH rat model to verify the biological effect of 2FF in vivo,and to detect its improvement effect on hemodynamics and vascular remodeling by right heart catheterization and histological staining;Lectin staining,immunofluorescence and western blot were applied to detect the level of core fucosylation and the abilities of cell proliferation in rat lung tissues,which could reveal the potential therapeutic value of 2FF in MCT-induced PAH rats.(2)Primary PASMCs were extracted and stimulated by platelet-derived growth factor BB(PDGF-BB)to construct cell model and detect changes of core fucosylation level by lectin blot and fluorescence;CCK8,western blot,immunofluorescence,flow cytometry,scratch assay were applied to assess the abilities of PASMC proliferation,migration,apoptosis resistence,and phenotypic transformation from contractile to synthetic,after inhibitting overall core fucosylation of cells by 2FF.(3)ELISA was used to detect the FUT8 concentration changes in the blood samples of PAH patients and rats;western blot,q RT-PCR,immunofluorescence were applied to determinate the expression of FUT8 in PAH rats lung tissue and PDGF-stimulatied PASMC;CCK8,western wlot,immunofluorescence,flow cytometry,scratch assay were applied to assess the abilities of PASMC proliferation,migration,apoptosis resistence,and phenotypic transformation,after knocking down FUT8 by si RNA.(4)The level of core fucosylation in PASMC was detected by lectin staining after knocking down FUT8 by si RNA;western blot and rescue experiments were used to assess activation of AKT pathway after knocking down FUT8,and immunoprecipitation and lectin blotting were used to detect changes of core fucosylation in VEGFR2;Knocking down FUT8 and VEGFR2 to explore the effct of FUT8 on VEGFR2/AKT pathway.Results:(1)We found the level of core fucosylation was up-regulated in our PAH rat lung tissues.And in vivo experiments found that intraperitoneal injection of 2FF could reduce the level of core fucosylation in lung tissue of PAH rats,and improve the hemodynamics and pulmonary vascular remodeling.(2)Core fucosylation level in PAH PASMC models was up-regulated and 2FF could inhibit the proliferation and migration ability after reducing the level of core fucosylation of PASMC,as well as the ability of apoptosis resistance and its transformation into fibroblasts.(3)The concentration of FUT8 in the blood samples of patients with PAH and PAH rats was increased compared with the control group;FUT8 expression was increased in PAH rat lung tissues and PASMC models.After knocking down the expression of FUT8,the abilities of proliferation,migration and apoptosis resistance were weakened,and the tendency from contractile to synthetic type was curbed.(4)After knocking down FUT8,the level of core fucosylation of protein in PASMC was decreased,including VEGFR2 and inhibited the activation of AKT pathway.Conclusion:(1)The level of core fucosylation increased in PAH rats and PASMC models,and promoted PASMC proliferation,migration,switching from contractile to synthetic type,but inhibited apoptosis,eventually leading to pulmonary vascular remodeling.(2)The content of FUT8 in the blood samples of patients and rats with PAH was increased.The expression of FUT8 in PAH-PASMC models was increased,leading to the phenotypic changes of PASMC.(3)FUT8 affected the core fucosylation modification of VEGFR2 and activated AKT pathway to promote the occurrence and development of PAH in rats.