Lysyl-oxidase-like2 Stimulates MSC Activating CAF To Promote Myelofibrosis Of Myeloproliferative Neoplasms Under Hypoxic Niche

BackgroundChronic myeloid leukemia(CML),polycythemia vera(PV),essential thrombocythemia(ET)and primary myelofibrosis(PMF),a classic of myeloproliferative neoplasms,often accompanied by myeloid fibrosis in the process of disease progression.Progressive myelofibrosis is also a high risk factor for the translation of MPN into leukemia and bone marrow failure.Therefore,studing the specific cytokines in myelofibrosis may provide a new entry point for exploring the pathogenesis of MPN and searching for more effective targeted therapies.Gene expression profiles revealed that the dynamic evolution of the bone marrow microenvironment was involved in the process of myelofibrosis in MPN.Bone marrow microenvironment in the oxygen concentration of about 1%of the hypoxic state,which can promote tumor resistance,relapse and progression by inducing angiogenesis,suppressing immune surveillance and immune response,and inducing cancer stem cells in tumor.Cancer associated fibroblasts(CAF)are the major constituents of stromal cells in tumor microenvironment and play a vital role in tumor biological behavioral characteristics.Bone marrow mesenchymal stem cells(BMMSC)are important sources of CAF,which are the main components of stromal cells in tumor microenvironment.Recent studies have shown that CAF is closely related to the degree of MPN fibrosis,but CAF activation in simple hypoxic environment is decreased,which indicated that the activation of CAF and the role of tumor development and tumorigenesis need to have the "crosstalk" with cancer cells or their secretory factors.However,it is not clear which factors contribute to the activation of CAF to promote myelofibrosis.Lysyl oxidase-like protein 2(LOXL2)is a member of the family of lysyl oxidases.It is involved in the formation of aggressive biological characteristics of many malignant tumors through activating FAK/Src,Erk signaling pathway,mediating epithelial mesenchymal transformation and other means,also closely related to the degree of fibrosis in MPN.In hypoxic microenvironment,tumor cell-derived LOXL2 could activate fibroblasts into CAF through FAK/Src signal pathway,which secrete a variety of cytokines to regulate cancer cells proliferation and metastasis.But its mechanism of mediating bone marrow fibrosis is unclear.Therefore,we speculated that BMMSC in MPN patients could obtain CAF phenotype to promote myelofibrosis;and to test whether LOXL2 was involved in promoting the activation of BMMSCs into CAF,further promote the process of myelofibrosis under hypoxic niche.Objective:Confirm that CAF and LOXL-2 were closely related to the progress of MPN and myelofibrosis;BMMSCs of MPN obtaine CAF phenotype to promote fibrosis process;verify the ability of CAF activation could be reduced in long-term hypoxic environment;search whether LOXL-2 as a promoter to stimulate the activation of BMMSC into CAF promoting myelofibrosis,whether this effect can be blocked by LOX inhibitors.Method:Clinical specimen level:1.a-SMA,FAP and LOXL2 expression are detected by RT-PCR and Western-blot in bone marrow mononuclear cells from normal and MPN patients.2.Collect the serum of normal people and MPN patients,detect the difference of LOXL2 expression by ELISA.3.Immunohistochemistry is used to detect the expression of net-stained proteins,CAF markers(FAP,a-SMA)and LOXL2 in bone marrow biopsy specimens.Cell level:1.BMMSCs are isolated from bone marrow of normal and MPN patients by informed consent.Mor:phology of cells is observed and the biological characteristics of MSC are detected:osteogenic and adipogenic differentiation.The expression of a-SMA and FAP in normal and MPN patients were detected by RT-PCR and Western blot respectively.2.BMMSCs are cultured for 24 h,48 h,72 h and 96 h respectively under normoxia and 1%oxygen concentration.Morphological changes of cells are observed and the expression of a-SMA,FAP and p-FAK?p-SRC?FAK?SRC signal pathway protein are detected.3.BMMSCs are stimulated by recombinant human LOXL2(rhLOXL2),and the expression of a-SMA,FAP and protein of signal pathway are detected.4.The above-mentioned groups are treated with LOX inhibitor of?-aminopropionitrile.The expression of ?-SMA,FAP and protein of signal pathway are detected.Result:1.The expression of a-SMA,FAP and LOXL2 in MPN patients was significantly higher than that in normal controls(P<0.05),and the expression of LOXL2 in MPN serum of bone marrow or peripheral blood was significantly higher than that in normal contrals(P<0.05).2.Immunohistochemical results of bone marrow biopsy showed that a-SMA,FAP and LOXL2 were not expressed or expressed lowly in normal subjects,expressed in different degrees in MPN patients,and gradually increased with the increase of fibrosis,and the most significant increase in patients with severe fibrosis(+++?++++)of PMF.3.BMMSCs of MPN biological characteristics were different from normal subjects,its osteogenic differentiation ability increased,adipogenic differentiation ability weakened.The expression of a-SMA and FAP in BMMSCs of MPN patients was significantly higher than normal controls.4.a-SMA and FAP were significantly lower in the group of MPN BMMSC under hypoxic niche(P<0.05),and was increased significantly after treated with rh-LOXL2,especially at 96h(P<0.05);a-SMA and FAP were significantly decreased by the addition of LOX inhibitor ?-aminopropionitrile(P<0.05);Relative pathway protein tests showed that,p-FAK and p-Src were up-regulated by rhLOXL2,and BAPN could weaken the expression of path protein.BMMSCs cultured in normal oxygen were insensitive to rhLOXL2 stimulation,and the expression of a-SMA and FAP were not significant.Conclusion:1.Cancer associated fibroblasts(a-SMA,FAP),and LOXL2 were closely related to MPN and myelofibrosis process;2.BMMSCs in patients with MPN have different biological characteristics than normal subjects,and CAF phenotype was obtained in MPN bone marrow microenvironment.3.LOXL-2 stimulates BMMSCs activation into CAFs probable through FAK/Src signal pathway under hypoxic conditions,which may be a key factor in the process of promoting myelofibrosis,and this effect could be blocked by the LOX inhibitor aminopropionitrile.