Effect Of G-CSF And HIF On Bone Metabolism And Bone Repair

Granulocyte-Colony Stimulating Factor(G-CSF)is an important drug for mobilizing bone marrow transplanted hematopoietic stem and progenitor cells.It can also be used to treat neutropenia caused by diseases such as chemotherapy and bone marrow transplantation.The recombinant form of human G-CSF is known by its generic name Filgrastim.In addition,elevated levels of G-CSF are also associated with the condition of some diseases,such as patients with myeloproliferative diseases or bacterial infections,serum levels of G-CSF are significantly elevated.However,elevated levels of G-CSF have a detrimental effect on bone health.Previous studies have shown that long-term treatment with G-CSF can significantly reduce bone mineral density and induce compression fractures of the vertebral body.Overexpression of mouse G-CSF can result in reduced trabecular bone and cortical bone thickness.Therefore,it is necessary to understand the mechanism by which G-CSF affects the characteristics of bone cells.Interestingly,studies have shown that osteoblasts or primary osteoblasts do not express G-CSF receptors(G-CSFR).This suggests that the osteopenic effect of G-CSF is mediated by non-osteoblast types.In addition,G-CSF receptors are expressed on many other cell types,including hematopoietic cells,endothelial cells,neurons and muscle cells.Bone marrow transplantation studies have clearly shown that the inhibitory effect of G-CSF on osteoblasts is mediated by hematopoietic cells.In hematopoietic cells,a similar effect was observed in lymphoblast-deficient mice,and thus the inhibition of osteoblasts by G-CSF was not caused by lymphocytes.The role of G-CSF in inhibiting osteoblasts has been shown to be mediated by neutrophils,as neutrophil depletion may abolish the inhibitory effect of G-CSF on osteoblasts.G-CSF-induced osteoblast inhibition is achieved by inhibiting osteoblast differentiation,osteoblast apoptosis,or transformation of mature osteoblasts into resting bone cells.G-CSF treatment also enhances the formation and activity of mouse and human osteoclasts.In addition,osteoclasts derived from myeloid lineage cells of the hematopoietic lineage also express G-CSF,and thus osteoclasts are direct targets of G-CSF action.Under in vitro conditions,osteoclast formation and activity increased with increasing G-CSF.Although G-CSF has made these advances in understanding the effects of osteoblasts and osteoclasts,it remains unclear how G-CSF affects bone cellsHere,we found that long-term treatment with G-CSF reduced the number of osteoblasts and newly formed bone cells.Therefore,gene markers for osteoblast differentiation are significantly reduced,and gene markers for bone cell activity are also reduced.Our study shows that the decrease in osteoblast numbers is due to increased osteoblast apoptosis,which is mediated by increased nitric oxide secretion by neutrophils.Nonunion or delayed bone healing is a common complication of bone fracture,which not only brought to the patient great pain physically and mentally,and also because the patient direct medical costs and indirect has brought enormous economic burden.The vascular injury during the fracture occurred or post treatment is main cause resulting in nonunion or delayed union.Now,more stduies find vessle plays important role in the process of bone healing.The vessle injury or blood flow obstruction is a important reason for nonunion or delayed bone healing.The research usually gets rid of the soft tissue in the fracture site in order to reach the effect of nonunion or delayed bone healing in the animal models.Clinically,we can find the blood-flow shortage areas in the calluses of nonunion or delayed bone healing models.So the discovery of new drugs to stimulate blood vessel growth to prevent fracture nonunion or delayed healing is an important proposition in Bone science and related fieldsClinically,the fracture healing of osteotic fracture models often delayed because of the loss of bone or the change of bone micro-envioronment.At present,a great deal basic research reports about osteoporosis after ovariectomy had been published.The study of osteoporotic fractures are more concentrated in the morphological and biochemical changes,but the study about blood supply after osteoporotic fracture is rare.The establishment of ovariectomized mouse fracture model,confirmed the delay in fracture healing in ovariectomized mice,meanwhile,the amount of new blood vessels also showed a visible decline.HIF is the most important sense-regulate factor depending on the oxide density in tissues finded by now.HIF is activated in the process of bone healing.HIF is a heterologous dimer including two subunit ???.In the normal physiology condition,the proline pro residue of HIF-1? protein is hydroxylated by PHDs,hydroxylated HIF-1? can be rapidly degradated by proteases in cytoplasm.The activity of PHDs depends on the introcellular oxide density,also needs Fe2+and 2-oxyglutarate.In the hypoxia condition or lack of Fe2+ and 2-oxyglutarate,the activity of PHDs is repressed or even completely lost.Because of this,HIF-1? accummulate in the cytoplasm.The accummulated HIF-1? transfer to the nucleus.In the nucleus,HIF-1? combine with HIF-1? becoming a dimer,then activated HIF-1 sensitive target genes.The expression of these gene produtions such as EPOn Glut or VEGF,plays important role in vessle remolding?erythrocyte generation?cell proliferation or apoptosis.HIF-1 is the master regulator of response to low oxygen,and has been shown to be activated in skeletal repair.HIFs are transcription factors whose activity is regulatedthrough oxygen-dependent proteolysis of the asubunit.Because of the recognition of importance of vascular supply to tissue repair in general and to skeletal repair more specifically,studies have focused on pharmacologic interventions to improve blood supply in these settings.Vascular endothelial growth factor(VEGF),which has been used to increase healing of nonunions and criticalsized defects in rabbits.Therapeutic application of VEGF may proveproblematic and expensive,because of the need for recombinant protein or gene therapy approaches.As an alternative approach we explored the use of small molecules that target the hypoxia inducible factor(HIF)pathway to activate angiogenesis more globally.The small molecule drugs inhibiting PHDs can interupt the degradation of HIF-1 a,and then activate the HIF pathway.DFO is a kind of Fe2+complexing agent,can be used in treat of mental ion poisoning clinically.In the process of HIF-1 a degradation,DFO can inhibit the activity of PHDs through the merging with Fe2+in cells,resulting in the accummulation of HIF-1?.Our study before has finded DFO can promote the expression of HIF-1? and VEGF of MSCs.In vivo,Local injection at the fracture site with DFO(200 mM),can accelerate the bone healing by increasing callus formation and angiogenesis.In our study,we found that locally applied DFO has good effct upon fracture healing in OVX mice by increasing angiogenesis and callus formation.Part I G-CSF inhibits growths of osteoblasts and osteocytesin mice by upregulating nitric oxide production inneutrophilsObjective:Elevated levels of G-CSF has a detrimental effect on bone health.The researches before have testified the effect of G-CSF on osteoblsts and osteoclasts,but the specific mechanism and how to affect osteocytes is still unclear.Our study aims to analyse the effect of G-CSF on osteocytes and osteoblastsMethods:1.8-10 weeks old Female C57BL/6 mice were divided into control group,granulocyte colony-stimulating factor(G-CSF)group and G-CSF+N?-nitro-L-arginine methyl ester(L-NAME)group.Each group of mice was intramuscularly injected with solvent,G-CSF and G-CSF+L-NAME.Animals were sacrificed 10 days after treatment.2.The peripheral blood of each group of mice was collected in a heparinized blood collection tube for whole blood count detection.3.The femur of each group of mice were collected for tissue section and immunohistochemistry.4.The femur bone marrow of each group of mice was washed out,and RNA was obtained by the Trizol method for real-time quantitative PCR detection.Results:1.G-CSF treatment significantly reduced the number of osteoblasts and newly formed bone cells.2.The decrease in the number of cells in the osteoblast lineage caused by G-CSF is due to an increase in apoptosis.3.G-CSF-induced osteoblast apoptosis is due to increased secretion of nitric oxide from neutrophils.Conclusions:The present study suggests that dyslipidaemia may be associated with a higher risk of developing lumbar disc herniation.Serum lipid levels could be a useful predictor for intervertebral disc degeneration in Chinese population.Part II The preliminary study of impaired early fracturehealing in osteoporotic fracture miceObjective:To investigate the mechanism of delay in osteoporotic fracture healing in mice.Methods:60 mice were randomly divided into OVX group(N=30),SHAM group(N=30).Four weeks after ovariectomy,the right femur fracture healing of ovariectomized mouse models was eatablished,and callus samples were collected 2 weeks after fracture.The development of callus and new bone formation were analysed with X-ray film,Micro-CT,histological,immunohistochemical,and vascular perfusion testing.Results:Micro-CT and the histological results confirmed the delayed fracture healing in OVX mice;Immunohistochemistry showed that new bone callus in OVX group had decreased expression of VEGF;vascular perfusion showed decreased vessel number in ovx group 2 weeks after fracture.Conclusion:The reduction of the expression of VEGF in OVX mice callus may lead to the bring down of angiogenesis,and because of this the early period of fracture healing in OVX group was delayedPart ? Locally applied DFO activating HIF pathway promotes fracture healing in ovariectomized miceObjective:Desferoxamine(DFO)solution was injected to the site of fractured femur of ovariectomized(OVX)mice,and then analyse whether the local apply of desferoxamine affect angiogenesis and callus formation.Methods:Ninety female C57BL/6 mice,aged 7 weeks old,were randomized into OVX+SALINE;OVX+DFO,and SHAM+SALINE groups(n=30,respectively).Femoral shaft fracture was created 4 weeks after ovariectomy.Local injection at the fracture site with 20 mL of DFO(200 mM),or saline was performed every other day for five doses except for the SHAM group.At different stages(2?4?8 weeks after fracture)of fracture healing,X-ray,microcomputed tomography,histology,immunohistochemistry and biomechanical analyses were performed.Results:Compared with the SALINE group,the callus cross-section area in DFO group was significantly enlarged at 2 and 4 weeks,but both were smaller than the SHAM group;In DFO group,the vascularity was increased at 2 weeks compared with the saline group,but both were still less than the SHAM group.Biomechanical testing confirmed the beneficial effects of DFO on restoration of biomechanical competence,but which is still poorer in the DFO gurop in contrast with the SHAM group.Conclusion:DFO(PHD inhibitors)could increase callus formation in ovariectomized fracture mice model by increasing the angiogenesis.