| Part one: Objective: To investigate the role of PGRN(progranulin)and its degradation in phenotypic transformation and osteogenic differentiation of aortic VICs(valve interstitial cells),which could provide a theoretical basis for early intervention and treatment of CAVD(calcific aortic valve disease).Methods: Three non-calcified aortic valves and six calcified aortic valves were taken.The expressions of PGRN,its degradation,myofibroblast-like phenotypic markers(α-SMA,Collage I),early osteogenic indicator(Runx2,Runt-related transcription factor 2)and late osteogenic indicators(OPN,OCN)were detected by immunohistochemistry staining and Western blot.Moreover,the expressions of pro-inflammatory factor TNF-α and apoptotic indicators(C Caspase3,Bax)were detected by Western blot.VICs were isolated by continuous collagenase digestion,their morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining.The expressions of PGRN and its degradation in osteogenic medium(OM)-induced human VICs were detected by Western blot.Calcification of VICs was induced by OM or TNF-α,and recombinant protein PGRN or Ad-45 GRN were used to up-regulate the levels of PGRN and 45-GRN.The expressions of myofibroblast-like/osteogenic markers,apoptotic indicators and TNF-α were detected by qPCR and Western blot.The expression of α-SMA was detected by immunofluorescence staining.ALP(Alkaline phosphatase)staining and Alizarin red S staining were used to evaluate the cell early and late osteogenic differentiation abilities.Results: PGRN and 45-GRN were increased in calcified human aortic valve(AV)tissues as shown by upregulation of α-SMA,collage I,Runx2,OPN,OCN.The band of approximately 45 KD was much stronger than PGRN.In addition,TNF-α,C Caspase3 and Bax were increased in calcified AVs.VICs were successfully isolated,the staining of α-SMA and Vimentin were positive,the staining of vWF was negative.45-GRN was upregulated whereas PGRN was reduced in human VICs under calcifying conditions which is induced by OM.The ALP activity and deposition of calcium salts of VICs were significantly decreased by PGRN.The mRNA and protein levels of myofibroblast-like/osteogenic markers,apoptotic indicators and TNF-α were reduced.However,45-GRN overexpression increased them.Conclusion: PGRN effectively attenuates apoptosis,phenotypic transformation and osteogenic differentiation of VICs during calcifying conditions whereas 45-GRN plays a positive regulatory role.Part two: Objective: To explore the mechanism of PGRN/45-GRN affecting phenotypic transformation and osteogenic differentiation of VICs.Methods: VICs were treated with recombinant protein PGRN or Ad-45 GRN to up-regulate the levels of PGRN or 45-GRN under calcifying conditions which is induced by OM.The protein level of p-Smad1/5/8,p-NF-κB and p-AKT was determined by Western blot.SC-79,an activator of the AKT,was used for reverse verification.VICs transfected with Ad-45 GRN were additionally treated with LDN193189(an inhibitor of the Smad1/5/8 pathway),BAY11-7082(an inhibitor of the NF-κB pathway)or LY294002(an inhibitor of the AKT pathway).The expressions of α-SMA and Runx2 were detected by Western blot.Results: The phosphorylation level of AKT was down-regulated by PGRN.The phosphorylation levels of p-Smad1/5/8,p-NF-κB and p-AKT were up-regulated by 45-GRN.Conclusion: PGRN may inhibit phenotypic transformation and osteogenic differentiation of VICs through the inhibition of AKT signal.45-GRN overexpression promoted OM-induced calcification through activating the Smad1/5/8,NF-κB and AKT signaling pathways. |
