The Role Of Matrix Remodeling Associated 7 In And Effects Of Advanced Glycation End Products On The Development Of Megakaryocytes And Hematopoiesis

Hematopoietic stem cells(HSCs)are consisted of multipotent primitive hematopoietic cells with self-renewal and differentiation ability,growth and physiological functions of which depend on hematopoietic microenvironment.The interaction between hematopoietic cells and their microenvironment determines the fate of the hematopoietic cells and the host overall hematopoietic functions.Hematopoietic microenvironment regulates the self-renewal,directional differentiation,mobilization,proliferation and migration of HSCs via various microenvironmental signals.Hematopoietic microenvironment mainly consists of matrix cells,extracellular matrix and various hematopoietic factors in hematopoietic tissues.Megakaryocytes(Mks)are one of the decedents HSCs and are responsible for building up the platelet lineage.Apart from generating and releasing platelets,Mks also participate in the establishment and maintenance of BM niches under physiological and pathological conditions.In bone marrow,Mks interact with other cellular and extracellular components to maintain the homeostasis and physiology of hematopoiesis and,as a result,of peripheral blood.Interestingly,Mks are involved in extracellular matrix(ECM)deposition and reconstruction.On the other side,both HSCs and Mks functions depends on the participation of extracellular matrix.Matrix remodeling associated 7(MXRA7)is a newly-surfaced gene,which was first noted to co-express with a group of matrix remodeling related genes(such as MMPs,TIMPs and BM40),indicating that MXRA7 may be involved in matrix remodeling.Public data and domestic studies in this lab showed that MXRA7 was expressed abundantly in bone marrow stromal cells,strongly suggesting that MXRA7 proteins might contribute to the ECM system as wellIn addition,in view of the pathological processes affecting hematopoietic function and circulation system in condition of diabetes,we hypothesized that Mks were among the targets of the pathogenic factors in diabetes.Since excessive accumulation of advanced glycation endproducts(AGEs)is a typical feature of diabetes beside high blood glucose in blood,we assumed that these two factors account for the damage of diabetes to the microenvironment of bone marrow.In this project,the role of MXRA7 in and the effects of AGEs on megakaryocyte development and hematopoietic system were studied.Part I.The role of matrix-remodeling associated 7 in the development of megakaryocytes and hematopoiesisObjective:Identification of molecules that regulate HSCs is important for dissecting the networks of hematopoietic mechanisms,and has significance to guide clinical practice(such as hematopoietic disease therapy,hematopoietic stem cell transplantation and hematopoietic reconstitution).The biological functions of the gene MXRA7 had remained unknown.The public databases showed that MXRA7 is phase-dependently expressed in hematopoietic cells,suggesting that it may be involved in hematopoiesis.So we proposed that MXRA7 may play an important role in regulation of hematopoietic process.Here we investigated the function of MXRA7 in the hematopoietic system.Methods:1.Compare the hematopoietic function between the WT mice and MXRA7-/-mice:Peripheral blood of mice was collected and the numbers of red blood cells,white blood cells and platelets were measured by hemocytometer.Single cell suspension of spleen and bone marrow were obtained for cell counting.Bone marrow tissues were collected for histological H&E staining.Bone marrow smears were stained by Wright-Giemsa to classify megakaryocytes.The differences of megakaryocytes in bone barrow were also analyzed by flow cytometry.2.Investigate the effectiveness of MXRA7 in the differentiation of hematopoietic stem/progenitor cells(HSPCs):The proliferation and differentiation capacity of HSPCs was identified by colony forming cells assay and induced differentiation assay.3.Compare the functions of platelets between WT mice and MXRA7-/-mice:by using tail bleeding time,the aggregation of platelet,the clot retraction,the survival time of platelet,and the activation and apoptosis of platelet.4.Investigate the effectiveness of MXRA7 in the biological function of megakaryocyte progenitor cell line MEG-01:We constructed sh-MXRA7 lentiviral system and transfected MEG-01 cells.Cell proliferation of MEG-01 RNAi cells was analyzed by CCK8.Differentiation,polyploidy and platelet-like particles of megakaryocyte were detected by flow cytometry.Then MEG-01 cells were cultured in medium in the presence of 10 ng/ml rhTPO to induce differentiation.The expression of megakaryocyte development related genes was performed by real-time qPCR.Proplatelet formation(PPF)from MEG-01 was captured by phase contrast imaging.?-Tubulin was detected by immunofluorescence staining and western blot.Expression of Bax,phosphorylation of ERK and AKT were detected by western blot.5.Study the function of MXRA7 in hematopoietic recovery and reconstitution:The blood cell lineages were analyzed by flow cytometry in non myeloablative model and bone marrow cells transplantation model.Results:1.There was little change of red blood cells and white blood cells in the wild-type mice and MXRA7-/-mice,while the number of platelets was significantly reduced in MXRA7-/-mice than that in WT mice.2.The number of megakaryocytes in the bone marrow was slightly reduced in MXRA7-/-mice than wild-type mice,as analyzed by flow cytometry and H&E staining.3.MXRA7 deficiency didn't change the morphology and number of CFU-GEMM,CFU-GM and BFU-E significantly.While MXRA7 deficiency reduced the percent of Mks differentiated from HSPCs.4.In the platelet function assay,MXRA7-/-mice showed decreased platelet activation,increased early apoptosis,increased aggregation and clot retraction,but there was no significant difference in tail bleeding time and life span between MXRA7-/-mice and WT mice.5.Knocked down MXRA7 in MEG-01 cells by transfection of sh-MXRA7 MXRA7 knock-down inhibited the proliferation,differentiation and polyploid formation in MEG-01,as well as the inhibition of proplatelet formation(PPF).There was no significant change in the expression level of genes related with megakaryocyte development(i.e.GATA1,FOG1,PU-1 and NF-E2).Further results indicated that MXRA7 knock-down mainly inhibited the phosphorylation of ERK signaling and the expression of Bax.6.Moreover,MXRA7 deficiency changed neither the number of peripheral blood,splenocytes or bone marrow cells;nor the various lineages of hematopoietic cells in non myeloablative model and bone marrow cells transplantation model.Conclusions:1.Our results demonstrated that MXRA7 deficiency reduced the number of platelets by inhibiting the development of megakaryocytes and promoting platelet apoptosis.2.In vitro,MXRA7 knock-down could inhibit the cell proliferation,and suppress Mk differentiation through inhibiting the expression of p-ERK and the rearrangement of?-Tubulin.3.MXRA7 had no significant effects on the recovery and reconstitution of hematopoietic cells.Part ?.The effects of advanced glycation end products on the development of megakaryocyte and hematopoiesisObjectives:Mks have long been as progenitor cells of platelet,however,Mks have recently been identified as regulatory cells adjusting proliferation and quiescence of hematopoietic stem cells,which can adjust the hematopoietic microenvironment by interacting with marrow stromal cells and other components in bone marrow.Diabetes is a serious and widespread systemic disease,whether it would affect hematopoiesis and bone marrow microenvironment remains unclear.The excessive accumulation of advanced glycation end products(AGEs)is a typical sign of diabetes.However,the AGEs are necessary for the maintenance of homeostasis and tissue reconstruction.Therefore,AGEs were used as a representative to study the effects of diabetes on the development and differentiation of megakaryocyte and regulation of hematopoietic microenvironment.Methods:1.BSA-AGEs were prepared by incubating BSA(50mg/mL)with D-glucose(0.5M)in phosphate buffer(0.2M,pH 7.4)for 8 weeks.After incubation,unbound sugar was removed by extensive dialysis against PBS(0.2M,pH 7.4).All incubations were performed under sterile conditions.2.The effect of AGEs on the biological function of megakaryocyte progenitor cell line MEG-01:BSA-AGEs,BSA controls or high concentration glucose was added to the culture.Cell proliferation,apoptosis and cell cycle were used to study the effect of AGEs on the biological function of MEG-01 cells.Induction of MEG-01 differentiation was achieved by phorbol myristate acetate(PMA),and flow cytometry was used to detect Mk differentiation markers and platelet-like particles.The real-time quantitative PCR was used to detect mRNA expression level of both megakaryocyte development related genes(NF-E2,GATA1 and PU1)and hematopoietic microenvironment regulation related genes(IL-6,VEGFa,PDGFa,PF4 and OPG).3.The effectiveness of AGEs on differentiation of human umbilical blood mononuclear cells toward megakaryocytes induced by TPO/SCF/IL-3:Flow cytometry was used to detect Mk differentiation markers and cell cycle.The real-time quantitative PCR was used to detect mRNA expression level of megakaryocyte development related genes.4.Ob/ob and STZ-induced mice were used for in vivo studying the development of megakaryocytes and hemopoiesis:Platelets,red blood cells,white blood cells and bone marrow nucleated cells were determined by hematometer.Megakaryocyte surface markers in bone marrow were determinated by FACS.Bone marrow smears were stained by Wright's staining,and the different stages of megakaryocytes were classified under the microscope.The real-time quantitative PCR was used to detect mRNA expression levels of both megakaryocyte development related genes and hematopoietic microenvironment regulation related genes.5.The effect of AGEs on proliferation of hematopoietic progenitor cells was evaluated by using colony forming cell experiment.Results:1.BSA-AGEs were prepared successfully.Pilot studies determined 200 ?g/ml was as an optimal concentration for studying the effect of BSA-AGEs on MEG-01.2.AGEs stimulated the cell growth of MEG-01 while high glucose inhibited the progression of the cell growth.3.AGEs improved the mRNA expression level of RAGE presented a time-dependent increase,but not resulting in the change of ROS level.4.In ob/ob mice(increased AGEs),increased AGEs could increase the number of platelets and Mks,and increase the expression of Mk development related genes.In STZ induced mice,the number of platelets had no obvious change,the percent of Mks decreased.The trend change of Mk development related genes in STZ induced mice was different from that in ob/ob mice.5.In addition,the colony forming experiment showed that AGEs promoted the formation of GEMM colony.Conclusions:1.Current study demonstrated that AGEs change Mks behavior in vitro,it also partially effect the hematopoietic process by regulating hematopoietic microenvironment related genes.2.AGEs didn't show significant effects on the differentiation of single nuclear cells from umbilical cord blood into megakaryocytes induced by TPO/SCF/IL-3.3.In vivo experiments showed that AGEs could promote the differentiation of megakaryocytes and increased the amounts of platelets.