Background&Objective Knee osteoarthritis(OA)is a degenerative disease,which not only induces superficial cartilage defects and full-thickness cartilage defects,but also exacerbates the microenvironment of the knee...Background&Objective Knee osteoarthritis(OA)is a degenerative disease,which not only induces superficial cartilage defects and full-thickness cartilage defects,but also exacerbates the microenvironment of the knee joint and affects the mechano-chemical responses of the organ.As a growth/repair factor,mechanical growth factor(MGF)has the function of preventing OA,promoting cartilage regeneration and repairing damaged ligaments.activating transcription factor 2(ATF-2),a transcription factor,has the property of binding to cytokines,which makes it involved in the transcriptional regulation of various pathways in response to cellular stress,inflammatory cytokine and growth factors.At present,little is known about the effect of MGF on human osteoarthritis ligament fibroblasts(OA-LFs),and whether the approach can promote OA-LFs timely response to the mechanical injury and initiate signaling pathway for cell survival.Therefore,the purpose of this study is to investigate whether MGF promotes mechanical response to ligament fibroblasts in osteoarthritis knee cavity via ATF-2.Methods OA-LFs were seeded onto six-cell BioFlex plates and suffered from 12%static mechanical stretch[60 cycles/minute(1 Hz)]for 12 hours to mimic mechanical force mediated ligament injury.Meanwhile,OA-LFs were treated with MGF before and during mechanical stretch.Intracellular reactive oxygen species(ROS)and GRP78 mRNA expression were investigated to detect the cellular stress response of OA-LFs.The scratch test was performed to detect the migration ability of cells,gelatin zymography was used to examine the effect of MGF on the activity of matrix metalloproteinase 2(MMP-2)in OA-LFs,and cell deformation was detected by phalloidin-FITC staining after stretching.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to screen the messenger RNA(mRNA)expression of ATF family members after OALFs treatment with MGF.Western blotting further proved that MGF is capable to activate the p-ATF-2.Results OA delays LFs response to mechanical injury,while MGF pretreatment can promote cells timely feedback the mechanically stimuli by inducing cellular stress.MGF treatment can alleviate the decline in cell migration ability caused by mechanical injury and further promote cell migration.In addition,MGF can reduce the activity of MM P-5 and alleviate the stretch-induced deformation of OA-LFs.Furthermore,the mRNA expression of ATF-2 up-regulated in a dose-dependent manner upon MGF treatment compared with control,while the expression of ATF-5 gene was down-regulated in a dose-dependent.Protein levels showed that the expression of p-ATF-2 increased with increasing MGF concentration.Conclusions Our study shows that MGF pretreatment of OA-LFs can respond quickly to mechanical damage and accelerate the ligament injury repair by promoting cell migration,decreasing the MMP-2 activity,and remitting the cell deformation.Therefore,MGF has potential as a therapeutic for OA patients.展开更多
Introduction Primary cilium is a non-motile microstructure,protruding from cell surface of most mammalian cells.It was previously thought to be vestigial.However,recent studies indicate that it may serve as one of the...Introduction Primary cilium is a non-motile microstructure,protruding from cell surface of most mammalian cells.It was previously thought to be vestigial.However,recent studies indicate that it may serve as one of the most vital mechanosensors for many types of cells such as epithelial and endothelial cells and osteocytes.Protruding from the apical membrane,the primary cilium can directly sense subtle variation of mechanical forces exerted on the cell and then transduce the mechanical cues into biochemical signals into the cell,although the mechanism remain elusive.Vascular endothelial cells(ECs)lining the inner wall of our blood vessels are continuously exposed to the blood flow.In order to maintain proper functions for the cardiovascular system,ECs should have a variety of mechano-sensors and transducers to sense the blood flow change and adjust the vessel size and transport across the vessel wall accordingly.Among more than a dozen recognized EC mechano-sensors,the primary cilium has drawn more and more attention recently.Primary cilium on endothelial cells is essential for the homeostasis of vessels.It is reported to be prevalent in areas of disturbed flow where atherosclerosis and intracranial aneurysm usually occur.Deficiencies of primary cilia may promote atherosclerosis,endothelial-to-mesenchymal transition(EndoMT)and loss of direction orientation,to name a few.Therefore understanding why the primary cilia are necessary to maintain the homeostasis of blood vessels and how will help us develop better treatment strategies for the common cardiovascular diseases.Dimension and structure of primary cilium Primary cilium is reported to be shorter than 8 in length and about 0.2 in diameter.The length of primary cilium varies in different cell types and under different conditions.The major structural components of the primary cilium include basal body,ciliary axoneme(consisting of nine doublet microtubules),ciliary membrane,transition zone,basal feet,and striated rootlets.Each part of the primary cilium is essential and has specific function.Current methods investigating the EC primary cilium as a mechano-sensor:Immunostaining and imaging techniques have been used to investigate the molecular mechanisms by which EC primary cilium serves as a mechano-sensor and transducer.It has been found that various proteins locate on the primary cilium,working together to maintain the function of primary cilium.Some proteins function as ion-channels,mediating Ca2+entry into the primary cilium.Some are involved in the cascade signal pathway.Others are related to the assembly and maintenance of primary cilium.Briefly,the flow induces the deflection of the EC primary cilium,which triggers calcium increase via opening of the PC2 cation channel that is responsible for calcium ion influx.This PC2 cation channel is localized to the primary cilium and is assumed to be stretch-activated.The resulting change in the intracellular calcium concentration then regulates numerous molecular activities inside the cell that contribute to vessel homeostasis.In addition to triggering calcium release,another mechanism has also been found in blood-pressure maintenance in the vasculature,where the vessel diameter is regulated by endothelial primary cilia through adjusting nitric oxide production.So far,little is known about the mechanical mechanism behind this deflection-triggered o-pening of signaling pathways.For example,what is the flow induced bending behavior and force distribution? What is the threshold value of stretch/defection for activating a corresponding signaling pathway? These all remain to be answered.In combination of image data and experiments,several computational models have been established to answer these questions.However,the current models are not able to include the complex structure of primary cilium and the model predictions are limited.Future studies With the development of super high resolution optical microscopy,more detailed images for the structural(molecular)components of EC primary cilia will be revealed,especially when the ECs are alive and the forces are known.Combining these experimental observations with more sophisticated mathematical models will elucidate the mechano-sensing mechanism of EC primary cilia,as the force and stress distribution on cilium along with other mechanical properties are still beyond the capability of experimental approaches due to the scales of the quantities involved.By using numerical approaches,much more detailed dynamic information can be obtained.展开更多
基金supported by the National Natural Science Foundation of China ( 11532004,31270990, 31600762)Innovation and Attracting Talents Program for College and University( “111”Project) ( B06023)
文摘Background&Objective Knee osteoarthritis(OA)is a degenerative disease,which not only induces superficial cartilage defects and full-thickness cartilage defects,but also exacerbates the microenvironment of the knee joint and affects the mechano-chemical responses of the organ.As a growth/repair factor,mechanical growth factor(MGF)has the function of preventing OA,promoting cartilage regeneration and repairing damaged ligaments.activating transcription factor 2(ATF-2),a transcription factor,has the property of binding to cytokines,which makes it involved in the transcriptional regulation of various pathways in response to cellular stress,inflammatory cytokine and growth factors.At present,little is known about the effect of MGF on human osteoarthritis ligament fibroblasts(OA-LFs),and whether the approach can promote OA-LFs timely response to the mechanical injury and initiate signaling pathway for cell survival.Therefore,the purpose of this study is to investigate whether MGF promotes mechanical response to ligament fibroblasts in osteoarthritis knee cavity via ATF-2.Methods OA-LFs were seeded onto six-cell BioFlex plates and suffered from 12%static mechanical stretch[60 cycles/minute(1 Hz)]for 12 hours to mimic mechanical force mediated ligament injury.Meanwhile,OA-LFs were treated with MGF before and during mechanical stretch.Intracellular reactive oxygen species(ROS)and GRP78 mRNA expression were investigated to detect the cellular stress response of OA-LFs.The scratch test was performed to detect the migration ability of cells,gelatin zymography was used to examine the effect of MGF on the activity of matrix metalloproteinase 2(MMP-2)in OA-LFs,and cell deformation was detected by phalloidin-FITC staining after stretching.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to screen the messenger RNA(mRNA)expression of ATF family members after OALFs treatment with MGF.Western blotting further proved that MGF is capable to activate the p-ATF-2.Results OA delays LFs response to mechanical injury,while MGF pretreatment can promote cells timely feedback the mechanically stimuli by inducing cellular stress.MGF treatment can alleviate the decline in cell migration ability caused by mechanical injury and further promote cell migration.In addition,MGF can reduce the activity of MM P-5 and alleviate the stretch-induced deformation of OA-LFs.Furthermore,the mRNA expression of ATF-2 up-regulated in a dose-dependent manner upon MGF treatment compared with control,while the expression of ATF-5 gene was down-regulated in a dose-dependent.Protein levels showed that the expression of p-ATF-2 increased with increasing MGF concentration.Conclusions Our study shows that MGF pretreatment of OA-LFs can respond quickly to mechanical damage and accelerate the ligament injury repair by promoting cell migration,decreasing the MMP-2 activity,and remitting the cell deformation.Therefore,MGF has potential as a therapeutic for OA patients.
基金supported by grants ( 11421202,11572029) from National Natural Science Foundation of China
文摘Introduction Primary cilium is a non-motile microstructure,protruding from cell surface of most mammalian cells.It was previously thought to be vestigial.However,recent studies indicate that it may serve as one of the most vital mechanosensors for many types of cells such as epithelial and endothelial cells and osteocytes.Protruding from the apical membrane,the primary cilium can directly sense subtle variation of mechanical forces exerted on the cell and then transduce the mechanical cues into biochemical signals into the cell,although the mechanism remain elusive.Vascular endothelial cells(ECs)lining the inner wall of our blood vessels are continuously exposed to the blood flow.In order to maintain proper functions for the cardiovascular system,ECs should have a variety of mechano-sensors and transducers to sense the blood flow change and adjust the vessel size and transport across the vessel wall accordingly.Among more than a dozen recognized EC mechano-sensors,the primary cilium has drawn more and more attention recently.Primary cilium on endothelial cells is essential for the homeostasis of vessels.It is reported to be prevalent in areas of disturbed flow where atherosclerosis and intracranial aneurysm usually occur.Deficiencies of primary cilia may promote atherosclerosis,endothelial-to-mesenchymal transition(EndoMT)and loss of direction orientation,to name a few.Therefore understanding why the primary cilia are necessary to maintain the homeostasis of blood vessels and how will help us develop better treatment strategies for the common cardiovascular diseases.Dimension and structure of primary cilium Primary cilium is reported to be shorter than 8 in length and about 0.2 in diameter.The length of primary cilium varies in different cell types and under different conditions.The major structural components of the primary cilium include basal body,ciliary axoneme(consisting of nine doublet microtubules),ciliary membrane,transition zone,basal feet,and striated rootlets.Each part of the primary cilium is essential and has specific function.Current methods investigating the EC primary cilium as a mechano-sensor:Immunostaining and imaging techniques have been used to investigate the molecular mechanisms by which EC primary cilium serves as a mechano-sensor and transducer.It has been found that various proteins locate on the primary cilium,working together to maintain the function of primary cilium.Some proteins function as ion-channels,mediating Ca2+entry into the primary cilium.Some are involved in the cascade signal pathway.Others are related to the assembly and maintenance of primary cilium.Briefly,the flow induces the deflection of the EC primary cilium,which triggers calcium increase via opening of the PC2 cation channel that is responsible for calcium ion influx.This PC2 cation channel is localized to the primary cilium and is assumed to be stretch-activated.The resulting change in the intracellular calcium concentration then regulates numerous molecular activities inside the cell that contribute to vessel homeostasis.In addition to triggering calcium release,another mechanism has also been found in blood-pressure maintenance in the vasculature,where the vessel diameter is regulated by endothelial primary cilia through adjusting nitric oxide production.So far,little is known about the mechanical mechanism behind this deflection-triggered o-pening of signaling pathways.For example,what is the flow induced bending behavior and force distribution? What is the threshold value of stretch/defection for activating a corresponding signaling pathway? These all remain to be answered.In combination of image data and experiments,several computational models have been established to answer these questions.However,the current models are not able to include the complex structure of primary cilium and the model predictions are limited.Future studies With the development of super high resolution optical microscopy,more detailed images for the structural(molecular)components of EC primary cilia will be revealed,especially when the ECs are alive and the forces are known.Combining these experimental observations with more sophisticated mathematical models will elucidate the mechano-sensing mechanism of EC primary cilia,as the force and stress distribution on cilium along with other mechanical properties are still beyond the capability of experimental approaches due to the scales of the quantities involved.By using numerical approaches,much more detailed dynamic information can be obtained.
