Background:We previously confirmed that mechanical stimulation is an important factor in the repair of tendon-bone insertion(TBI)injuries and that mechanoreceptors such as transient receptor potential ion-channel subf...Background:We previously confirmed that mechanical stimulation is an important factor in the repair of tendon-bone insertion(TBI)injuries and that mechanoreceptors such as transient receptor potential ion-channel subfamily V member 4(TRPV4;also known as transient receptor potential vanilloid 4)are key to transforming mechanical stimulation into intracellular biochemical signals.This study aims to elucidate the mechanism of mechanical stimulation regulating TRPV4.Methods:Immunohistochemical staining and western blotting were used to evaluate cartilage repair at the TBI after injury.The RNA expression and protein expression of mechanoreceptors and key pathway molecules regulating cartilage proliferation were analyzed.TBI samples were collected for transcriptome sequencing to detect gene expression.Calcium-ion imaging and flow cytometry were used to evaluate the function of TPRV4 and cellular communication network factor 2(CCN2)after the administration of siRNA,recombinant adenovirus and agonists.Results:We found that treadmill training improved the quality of TBI healing and enhanced fibrochondrocyte proliferation.The transcriptome sequencing results suggested that the elevated expression of the mechanistically stimulated regulator CCN2 and the exogenous administration of recombinant human CCN2 significantly promoted TRPV4 protein expression and fibrochondrocyte proliferation.In vitro,under mechanical stimulation conditions,small interfering RNA(siRNA)-CCN2 not only inhibited the proliferation of primary fibrochondrocytes but also suppressed TRPV4 protein expression and activity.Subsequently,primary fibrochondrocytes were treated with the TRPV4 agonist GSK1016790A and the recombinant adenovirus TRPV4(Ad-TRPV4),and GSK1016790A partially reversed the inhibitory effect of siRNA-CCN2.The phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway participated in the above process.Conclusions:Mechanical stimulation promoted fibrochondrocyte proliferation and TBI healing by activating TRPV4 channels and the PI3K/AKT signaling pathway,and CCN2 may be a key regulatory protein in maintaining TRPV4 activation.展开更多
Background Connective tissue growth factor （CTGF） is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an importan...Background Connective tissue growth factor （CTGF） is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an important role in extracellular matrix production by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. We herein evaluated whether CTGF was required for extracellular matrix synthesis of meniscal fibrochondrocytes and/or angiogenesis during the repair of meniscal tears. Methods Meniscal fibrochondrocytes were isolated from the inner-I/2 of rabbit meniscus by trypsin collagenase treatment and further treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analyzing CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. Histological sections prepared from a 3-mm portion of a longitudinal tearing defect in the middle of the rabbit meniscus were subjected to fluorescence-immunohistochemistry analysis at 1, 4 and 10 weeks following surgical treatment with 1.5 IJg of CTGF/fibrin-glue composites. Results Quantitative RT-PCR assay showed that types I and II collagen and vascular endothelial growth factor mRNA expression in the 100 ng/ml CTGF group were remarkably enhanced as compared to levels in the no-dose group at 14 days （（2.38±0.63） fold, （2.96±0.87） fold, （2.14±0.56） fold, respectively）. Likewise, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, types Ⅰand Ⅱcollagen, as well as the capillaries, completely filled the defect by 10 weeks, postoperatively. In contrast, only soft tissue repair occurred when PBS-fibrin glue was implanted. Conclusions These findings suggest that CTGF can significantly promote extracellular matrix deposition （types Ⅰ and Ⅱcollagen） within the meniscal avascular zone; CTGF can greatly heighten the expression of vascular endothelial growth factor activity simultaneously in vivo, further enhancing the repair of meniscal tears in the avascular zone.展开更多
基金supported by the National Natural Science Foundation of China(NSFC,Nos.82130071 and 82072516).
文摘Background:We previously confirmed that mechanical stimulation is an important factor in the repair of tendon-bone insertion(TBI)injuries and that mechanoreceptors such as transient receptor potential ion-channel subfamily V member 4(TRPV4;also known as transient receptor potential vanilloid 4)are key to transforming mechanical stimulation into intracellular biochemical signals.This study aims to elucidate the mechanism of mechanical stimulation regulating TRPV4.Methods:Immunohistochemical staining and western blotting were used to evaluate cartilage repair at the TBI after injury.The RNA expression and protein expression of mechanoreceptors and key pathway molecules regulating cartilage proliferation were analyzed.TBI samples were collected for transcriptome sequencing to detect gene expression.Calcium-ion imaging and flow cytometry were used to evaluate the function of TPRV4 and cellular communication network factor 2(CCN2)after the administration of siRNA,recombinant adenovirus and agonists.Results:We found that treadmill training improved the quality of TBI healing and enhanced fibrochondrocyte proliferation.The transcriptome sequencing results suggested that the elevated expression of the mechanistically stimulated regulator CCN2 and the exogenous administration of recombinant human CCN2 significantly promoted TRPV4 protein expression and fibrochondrocyte proliferation.In vitro,under mechanical stimulation conditions,small interfering RNA(siRNA)-CCN2 not only inhibited the proliferation of primary fibrochondrocytes but also suppressed TRPV4 protein expression and activity.Subsequently,primary fibrochondrocytes were treated with the TRPV4 agonist GSK1016790A and the recombinant adenovirus TRPV4(Ad-TRPV4),and GSK1016790A partially reversed the inhibitory effect of siRNA-CCN2.The phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway participated in the above process.Conclusions:Mechanical stimulation promoted fibrochondrocyte proliferation and TBI healing by activating TRPV4 channels and the PI3K/AKT signaling pathway,and CCN2 may be a key regulatory protein in maintaining TRPV4 activation.
文摘Background Connective tissue growth factor （CTGF） is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an important role in extracellular matrix production by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. We herein evaluated whether CTGF was required for extracellular matrix synthesis of meniscal fibrochondrocytes and/or angiogenesis during the repair of meniscal tears. Methods Meniscal fibrochondrocytes were isolated from the inner-I/2 of rabbit meniscus by trypsin collagenase treatment and further treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analyzing CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. Histological sections prepared from a 3-mm portion of a longitudinal tearing defect in the middle of the rabbit meniscus were subjected to fluorescence-immunohistochemistry analysis at 1, 4 and 10 weeks following surgical treatment with 1.5 IJg of CTGF/fibrin-glue composites. Results Quantitative RT-PCR assay showed that types I and II collagen and vascular endothelial growth factor mRNA expression in the 100 ng/ml CTGF group were remarkably enhanced as compared to levels in the no-dose group at 14 days （（2.38±0.63） fold, （2.96±0.87） fold, （2.14±0.56） fold, respectively）. Likewise, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, types Ⅰand Ⅱcollagen, as well as the capillaries, completely filled the defect by 10 weeks, postoperatively. In contrast, only soft tissue repair occurred when PBS-fibrin glue was implanted. Conclusions These findings suggest that CTGF can significantly promote extracellular matrix deposition （types Ⅰ and Ⅱcollagen） within the meniscal avascular zone; CTGF can greatly heighten the expression of vascular endothelial growth factor activity simultaneously in vivo, further enhancing the repair of meniscal tears in the avascular zone.
