Deterioration Of Hematopoietic Autophagy Is Linked To Osteoporosis

Part I Hematopoietic autophagy is deteriorated in osteoporotic patientsObjective: To explore cellular connection between hematopoietic function and osteoporosis.Methods: Physical examination results of 4979 healthy individuals,which from Physical Examination Center,the Second Affiliated Hospital of Soochow University,from 2013 to 2018,were analyzied for blood count and bone mineral density.Subjects with malignancy diseases were excluded.30 samples of human femur bone marrow samples were collected from hip replacement surgery and knee replacement surgery.Hematopoietic cells were separated from bone marrow to detect autophagy function.Patients who had fracture caused by falling without obvious violence were included in the study(inclusive criteria).Subjects with secondary osteoporosis,anti-osteoporosis drug intervention,disrupted hematopoietic system,malignancy,diabetes,or other severe diseases in the previous 5 years were excluded from the study(exclusive criteria).Results: Red blood cell count was significantly related to hip bone mineral density in 4979 healthy individuals.Red blood cell count was reduced in both male and female osteoporotic patients(p<0.05),with no obvious differences between two sexes.Expression of autophagy essential genes,including Atg7,Atg5,Atg12,Lc3 b,Lamp2a and P62 in the primary human hematopoietic stem cells were downregulated in osteoporosis group(p<0.05).Consistent with the decreased genes expression,the co-locolization of LC3 and lysosomes was reduced in human osteoporotic bone marrow CD45 hematopoietic cells and bone marrow CD34-enriched hematopoietic stem cells.Conclusion: Red blood cell count is decreased in osteoporotic patients.Bone marrow hematopoietic autophagy is decreased in osteoporotic patients.Part II Induced bone loss is associated with hematopoietic abnormality in mice Objective: In order to support the observation in patients that osteoporosis is associated with dysfunction of hematopoietic autohpagy,bone marrow hematopoietic function of osteoporosis mouse models were evaluated.Methods: Bone marrow was collected from ovariectomized mice and control mice.The proportion and cell apoptosis of hematopoietic stem and progenitor cells were measured by flow cytometry.P62 and LC3 expression of hematopoietic stem and progenitor cells were detected by western blotting.Hematopoietic stem and progenitor cells autophagy function was observed by image flow cytometry double staining.Bone marrow was also collected from iron accumulation mice and control mice.Femur was collected for micro-CT.The proportion and cell apoptosis of hematopoietic stem and progenitor cells were measured by flow cytometry.Hematopoietic stem and progenitor cells autophagy function was detected by image flow cytometry.Hematopoietic stem and progenitor cells autophagy function was observed by image flow cytometry double staining.Results: The proportion of hematopoietic stem and progenitor cells was increased in ovariectomized mice(p<0.05).However,the apoptosis of hematopoietic stem and progenitor cells was also increased(p<0.05).Bone marrow hematopoietic stem cells displayed a reduced basal autophagy activity in ovariectomized mice.Hematopoietic stem cells frequency in hematopoietic stem and progenitor cells was also reduced in ovariectomized mice(p<0.05).Micro-CT showed bone loss in the iron accumulation mice.Consistent with that in the ovariectomized mice model,the frequency of hematopoietic stem and progenitor cells increased in iron accumulation mice(p<0.05),accompanied with increased cell apoptosis(p<0.05).Imagestream measurement suggested formation of autolysosomes was significantly reduced in hematopoietic cells of iron accumulation mice.Conclusion: Osteoporosis animal models generated by either ovariectomy or iron accumulation have negative impacts on hematopoietic stem cells or hematopoietic progenitor cells,suggesting that hematopoietic autophagy dysfunction and osteoporosis are linked.Part III Hematopoietic autophagy defect caused severe bone loss Objective: To explain the observation that hematopoietic autophagy is deficient in osteoporotic patients and osteoporotic mice models,we used conditional mouse model Atg7flox/flox;Vav-Cre(refer to Atg7-/-)in which Atg 7 had been genetically deleted in the hematopoietic system for phenotypic analysis.Methods: Atg7 knockout was verified by western blotting.Micro-CT was performed for bone mineral density and spatial structure parameter,including bone mineral density,trabecular number,bone volume/tissue volume,trabecular thickness and trabecular space.Trabecular microstructure was observed by scanning electron microscopy analysis.Calcein double labeling was stained for bone mineral apposition rate.Phalloidin was stained for immunofluorescence.Tibia was collected for H&E stain and Masson stain.Three-point bending test was performed for bone biomechanical properties.Whole body skeleton from neonatal mice was stained for Alizarin red/Alcian blue.Osteocyte was examined by phalloidin staining.Immunofluorescence of tibia frozen section was stained with mitochondria marker.DNA damage was measured by ?-H2 AX stain.ROS level of bone tissue was tested by flow cytometry.RNA expression of Sp7,Runx2,Bmp2,Bmp6,Ctsk and Trap5 were measured by RT-PCR.Results: Deletion of Atg7 gene impaired hematopoietic autophagy,shown by the missing band of ATG7 protein and lacking of lipidation of LC3-I to LC3-II.However,autophagy protein expression was not changed in Atg7-/-bone tissue.Micro-CT revealed severe bone loss and decreased bone mineral density,trabecular number,bone volume/tissue volume and trabecular thickness in Atg7-/-femur(p<0.05).Scaning electron microscopy indicated trabecular microstructure undermined.Calcein double labeling revealed a reduced rate of bone formation(p<0.05).Immunofluorescence assay revealed abnormal bone marrow microstructure in Atg7-/-mice.Three point bending test indicated deteriorated bone quality in Atg7-/-mice,with reduced load,stress and stiffness(p<0.05).H&E and Masson staining showed broken collagenous fiber and elastic fibers in Atg7-/-mice.There was no virtually distinguishable different for Alizarin red/Alcian blue stain.Phalloidin stain revealed decreased osteocytes and attenuated formation of osteocyte network in Atg7-/-mice.Mitotracker Deep Red staining showed higher mitochondrial mass in Atg7-/-mice;Flow cytometry of bone tissue also showed increased ROS level(p<0.05).?-H2 AX stain showed increased DNA damage in Atg7-/-mice.Runx2,Bmp2,Bmp6,Ctsk and Trap5 expression were inhibited in Atg7-/-mice bone tissue(p<0.05).Conclusion: Genetic disruption of autophagy in the hematopoietic system causes bone loss in mice.Hematopoietic autophagy deficiency impairs osteocyte homeostasis,which results from enhanced oxidative stress.Part IV Transcriptomic and proteomic analysis on the connection between autophagy defect and bone loss Objective: Based on the observation on hematopoietic autophagy dysfunction leading to bone loss,the objective is to explore functional connection between hematopoietic autophagy and bone homeostasis.Methods: Collected from Atg7-/-tibia,hematopoietic stem and progenitor cells were analyzed by RNA sequencing.Proteomic analysis was performed in bone tissue without bone marrow.Osteogenesis proteins were analyzed by KEGG and STRING.Proteomic results were verified by immunohistochemistry.Results: Transcriptomic profiling showed down-regulation of osteocyte differentiation and calcium metabolism and enhanced iron activity.Proteomic biological process analysis revealed reduction in collagen fibril organization,endochondral bone morphogenesis and bone morphogenesis in Atg7-/-mice;KEGG and STRING analysis revealed inhibition of extracellular matrix and collagen expression.Immunohistochemistry showed less expression of collagen 1 in Atg7-/-tibia.Conclusion: Osteogenesis was inhibited by extracellular matrix collagen 1 in hematopoietic autophagy defect mice.Part V Identification of direct link between hematopoietic autophagy defect and bone loss Objective: Given that hematopoietic autophagy defect leads to bone loss,and the bone tissue type H vessel(CD31hiEMCNhi)connects both hematopoietic system and bonehomeostasis,the type H vessel will be examined in hematopoietic Atg7 deleted mice.Methods: Vegf gene family expression was measured by RT-PCR in Atg7 knockout mice.Tibia frozen section was stained with type H vessel(CD31hi EMCNhi)in Atg7 knockout mice.Results: RT-PCR showed Vegfa was down-regulated in Atg7-/-mice(p<0.05).There was no significant difference for Vegfb or Vegfc.Bone tissue type H vessel was inhibited in Atg7-/-mice.Conclusion: Hematopoietic autophagy defect causes disruption of type H vessel via inhibition of Vegfa,resulting in bone loss.