Study On Effect And Mechanism Of Oncostatin M In Calcific Aortic Valve Diseases

Part One Research on expression of Oncostatin M in Calcific aortic valve disease Objective:To investigate the expression and origin of OSM in calcified aortic valve disease(CAVD).Methods: Bioinformatics analysis database(GSE153555)was used to analyze the expression of OSM in normal and calcified valves.Approved by the Medical Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology,collecting aortic valve tissue from CAVD,aortic dissection and heart transplant recipients.The expressions of OSM were detected by PCR,the expressions of Runx2,ALP and OSM were detected by WB and immunohistochemistry.the expressions of CD3+ T cells,CD68+macrophages and OSM were detected by immunofluorescence double staining.The 10 x Genomics single-cell sequencing platform was used to perform high-throughput single-cell sequencing on healthy and calcified valve tissues.At the same time,the expressions of OSM,OSMR and LIFR in valvular interstitial cells,T cells,macrophages and DC cells were analyzed.Results: Bioinformatics analysis and q PCR detection showed that the expressions of OSM were up-regulated in calcific aortic valves;immunohistochemistry and WB detection showed that the expressions of OSM,Runx2 and ALP were up-regulated in calcific aortic valves.Single cell sequencing analysis showed that the expression of OSM was significantly increased in macrophages in human calcified valves,while that of OSMR and LIFR was significantly increased in valve interstitial cells.Immunofluorescence double staining indicated that OSM was mainly expressed in CD3+T cells and CD68+macrophages.Conclusion:These results suggest that OSM was highly expressed in calcified aortic valves,mainly from CD68+ macrophages.Part Two Activation of Oncostatin M / Oncostatin M receptor axis promotes osteoblastic differentiation of valvular Interstitial Cells Objective! To explore whether activation of the OSM/OSM receptor axis can promote osteoblastic differentiation of valvular Interstitial cells(VICs).Methods: With the approval of the Medical Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology,aortic valvular interstitial cells were extracted from heart transplant patients and aortic dissection patients.OSMR and LIFR in VICs were silenced by si RNA,the expression of Runx2 and ALP were detected by WB and PCR.RNA-seq was used for transcriptome sequencing analysis of OSM-treated VICs.Results: knockdown of OSMR,LIFR inhibited the OSM-induced expression of the osteoblastic markers ALP and Runx2,suppressed the induction of ALP and Runx2 m RNA in VICs,decreased calcium deposition.There were 985 differential genes(DEG)in the PBS group and OSM group,of which 498 DEG were up-regulated and 487 DEG were down-regulated.KEGG signaling pathway enrichment analysis Differential genes were highly enriched in protein digestion and absorption and mineral absorption,cytokine-cytokine receptor interactions,and JAK-STAT signaling pathways.OSMR,LIFR,JAK2,JAK3 and STAT3 genes were up-regulated in JAK-STAT signal.Conclusion!OSM promotes the expression of Runx2 and ALP of VICs by binding to OSMR and LIFR,and promote osteoblastic differentiation of VICs.Part Three Oncostatin M promotes osteoblastic differentiation of Valvular Interstitial Cells through JAK-STAT signaling Objective : To explore the molecular mechanism of OSM promoting osteoblastic differentiation of valvular Interstitial cells(VICs),and to provide theoretical basis for related drugs and targeted therapy.Methods: Human VICs were cultured and treated with OSM.Gene and protein expression levels of the osteoblastic markers ALP and Runx2,which can be blocked by specific JAK2/JAK3 inhibitors and/or si RNA of STAT3 and/or MCL,were measured by WB and q PCR.Apo E KO mice were fed MCL+ high-fat and high-cholesterol diet for 24 weeks.The flow velocity of aortic valve,calcium deposition,and p-STAT3 expression of valve were detected by Echocardiography,vonkossa staining,fluorescence staining.Results: JAK2 or JAK3 inhibitors decreased OSM-induced expression of the osteoblastic markers Runx2 and ALP,suppressed the expression of ALP and Runx2 m RNA in VICs,decreased calcium deposition.knockdown of STAT3 inhibited the OSM-induced expression of the osteoblastic markers ALP and Runx2.Meanwhile,suppressed the expression of ALP and Runx2 m RNA in VICs,decreased calcium deposition.MCL decreased OSM-induced expression of the osteoblastic markers Runx2 and ALP,Meanwhile,suppressed the expression of ALP and Runx2 m RNA in VICs,decreased calcium deposition.MCL can reduce the expression of p-STAT3 in Apo E KO mice aortic valve;Apo E KO +MCL group had significantly lower peak flow velocity and Von kossa staining than Apo E KO group.Conclusion:OSM promotes osteoblastic differentiation of VICs through the OSMR/LIFR-JAK2/JAK3-STAT3 pathway.MCL reduced aortic valve calcification in Apo E KO mice.