Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,ther...Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,there are few reports about its short term effect on MSC proliferation.In the present study,effects of short-term mechanical stretch on the proliferation and osteogenic differentiation of mesenchymal stem cell proliferation were investigated.In addition,the stretchinfluenced expression of transient receptor potential cation channel,subfamily C,member 1(TRPC1)was also investigated due to its mechanosensitivity and positive correlation with MSC proliferation.Methods MSCs,harvested from rat bone marrow,were seeded on collagen l-coated silicone chamber and exposed to mechanical stretch with various magnitude(0%,5%,10%and 15%)or various duration(2 h,6 h,12 h and 24 h).Cell proliferation was examined by cell counting kit-8(CCK-8)assay and cell cycle analysis.The gene and protein expression of two makers for osteogenic differentiation,collagen I and Cbfα1,and TRPC1 were determined by RT-PCR and western blotting,respectively.BMSC were harvested,and total RNA was isolated with Trizol reagent.A 2μg portion of total RNA was synthesized to cDNA according to the manufacturer s instructions.cDNA was used as a template for each PCR amplification.BMSC were solubilized in RIPA lysis buffer on ice for 30 min.Phenylmethane sulfonyl fluoride(PMSF)was added to avoid proteolysis.Equal portions of the cell lysates were separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with primary antibodies to TRPC1 and GAPDH at4℃overnight to identify the specific proteins.The PVDF membranes were washed with TBST three times and incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody.Immunoreactive bands were visualized using an enhanced chemiluminescent(ECL)system.Results The OD value for the three stretch cases(5%-15%)was increased^1.4-fold compared with that for control(0%).There is no significantly difference among the three stretch cases.The percentage of cells for three stretch cases were more in the S phase but less in the G0/G1 phase compared to those for control.The cell cycle distribution still had no significant difference among the three stretch cases.In addition,the stretch application for 24 h didn’t affect the gene or protein level for collagen I and Cbfα1 compared with those of control.Application of 10%stretch for 2 h didn’t affect TRPC1 gene or protein expression,but that application for 6-24 h significantly up-regulated TRPCl gene and protein level.That increase exhibited a stretch duration-independent manner.Conclusions Short-term mechanical stretch promoted MSC proliferation in a magnitude-independent manner,whereas had no effect on its oesteogenic differentiation.Paralleled to which,TRPC1 was up-regulated by stretch,implying that TRPCl may be implicated in that proliferation courses.Future work is still needed to confirm whether TRPC1 participates in that stretch-induced MSC proliferation using RPC1 blockade or knockout.展开更多
Objective:Arterial stiffening occurs in the progression of natural aging and cardiovascular diseases.Vascular smooth muscle cells(VSMCs),the major components of vascular walls,which largely contribute to the pathophys...Objective:Arterial stiffening occurs in the progression of natural aging and cardiovascular diseases.Vascular smooth muscle cells(VSMCs),the major components of vascular walls,which largely contribute to the pathophysiological states of blood vessels,are influenced by environmental cues of blood vessels reciprocally as well.Consistently,the increased proliferation of VSMCs has been reported to be observed in stiffening blood vessel and on rigid substrates,the underlying mechanism of which remains not yet fully clarified.Our previous work has demonstrated that Ca2+-activated K+(IKCa)channel participates in the stiff substrate-induced vascular smooth muscle cell(VSMC)proliferation.In the present work,from the standpoint of calcium entry and extracellular regulated protein kinases 1 and 2(ERK 1/2)activation,we further investigated the underlying mechanisms by which IKCa channels functions in the process mentioned above.Methods Soft(0.21 MPa)and stiff(1.72 MPa)PDMS substrates where VSMCs were seeded after coated with fibronectin(FN),were fabricated through the blending of sylgard 184 gel and sylgard 527 gel.After that,intracellular calcium level of VSMCs was compared with or without the treatment of IKCa specific blocker,TRAM-34,using a calcium-sensitive dye,fluo 4-AM.1mM Ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid(EGTA)was added into the culture media for the removal of the extracellular calcium ions as well as cell counting kit-8(CCK-8)assay was applied,which is to explore the role that calcium ion entry played in proliferation process.The activation level of ERK 1/2 was described by the expression level of phospho-ERK 1/2 using western blotting with or without TRAM-34 treatment.The role of the activation of ERK1/2 in VSMC proliferation was examined by CCK-8 assay with or without the treatment of PD98095,an ERK1/2 inhibitor.Results Compared with soft substrate,stiff substrate caused an increase of intracellular calcium level,which was attenuated by IKCa blockade.In addition,compared with soft substrate,stiff substrate also caused an activation of ERK1/2,which was significantly suppressed by IKCa blockade.Furthermore,extracellular calcium ion reduction by adding EGTA significantly inhibited the stiff substrate-induced VSMC proliferation,which whereas had no effect on VSMC proliferation on soft substrate.Finally,ERK1/2 inhibition had similar inhibitory effect on stiff substrate-induced proliferation.Conclusions Stiff substrate causes an IKCa channel-mediated calcium entry and ERK1/2 activation,both of which play important roles in stiff substrate-induced VSMC proliferation.Combined the previous results that IKCa channel participated in stiff substrate-induced VSMC proliferation,our present work suggests that IKCa channel functioned in the proliferation process through mediating calcium entry and subsequent ERK1/2 activations.These findings provide a new insight into how substrate stiffness regulates VSMC proliferation,and additional considerations for vascular tissue engineering and vascular disease treatment.展开更多
基金supported by the National Natural Science Foundation of China ( 11872010)
文摘Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,there are few reports about its short term effect on MSC proliferation.In the present study,effects of short-term mechanical stretch on the proliferation and osteogenic differentiation of mesenchymal stem cell proliferation were investigated.In addition,the stretchinfluenced expression of transient receptor potential cation channel,subfamily C,member 1(TRPC1)was also investigated due to its mechanosensitivity and positive correlation with MSC proliferation.Methods MSCs,harvested from rat bone marrow,were seeded on collagen l-coated silicone chamber and exposed to mechanical stretch with various magnitude(0%,5%,10%and 15%)or various duration(2 h,6 h,12 h and 24 h).Cell proliferation was examined by cell counting kit-8(CCK-8)assay and cell cycle analysis.The gene and protein expression of two makers for osteogenic differentiation,collagen I and Cbfα1,and TRPC1 were determined by RT-PCR and western blotting,respectively.BMSC were harvested,and total RNA was isolated with Trizol reagent.A 2μg portion of total RNA was synthesized to cDNA according to the manufacturer s instructions.cDNA was used as a template for each PCR amplification.BMSC were solubilized in RIPA lysis buffer on ice for 30 min.Phenylmethane sulfonyl fluoride(PMSF)was added to avoid proteolysis.Equal portions of the cell lysates were separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with primary antibodies to TRPC1 and GAPDH at4℃overnight to identify the specific proteins.The PVDF membranes were washed with TBST three times and incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody.Immunoreactive bands were visualized using an enhanced chemiluminescent(ECL)system.Results The OD value for the three stretch cases(5%-15%)was increased^1.4-fold compared with that for control(0%).There is no significantly difference among the three stretch cases.The percentage of cells for three stretch cases were more in the S phase but less in the G0/G1 phase compared to those for control.The cell cycle distribution still had no significant difference among the three stretch cases.In addition,the stretch application for 24 h didn’t affect the gene or protein level for collagen I and Cbfα1 compared with those of control.Application of 10%stretch for 2 h didn’t affect TRPC1 gene or protein expression,but that application for 6-24 h significantly up-regulated TRPCl gene and protein level.That increase exhibited a stretch duration-independent manner.Conclusions Short-term mechanical stretch promoted MSC proliferation in a magnitude-independent manner,whereas had no effect on its oesteogenic differentiation.Paralleled to which,TRPC1 was up-regulated by stretch,implying that TRPCl may be implicated in that proliferation courses.Future work is still needed to confirm whether TRPC1 participates in that stretch-induced MSC proliferation using RPC1 blockade or knockout.
基金supported by the National Natural Science Foundation of China ( 11872010)
文摘Objective:Arterial stiffening occurs in the progression of natural aging and cardiovascular diseases.Vascular smooth muscle cells(VSMCs),the major components of vascular walls,which largely contribute to the pathophysiological states of blood vessels,are influenced by environmental cues of blood vessels reciprocally as well.Consistently,the increased proliferation of VSMCs has been reported to be observed in stiffening blood vessel and on rigid substrates,the underlying mechanism of which remains not yet fully clarified.Our previous work has demonstrated that Ca2+-activated K+(IKCa)channel participates in the stiff substrate-induced vascular smooth muscle cell(VSMC)proliferation.In the present work,from the standpoint of calcium entry and extracellular regulated protein kinases 1 and 2(ERK 1/2)activation,we further investigated the underlying mechanisms by which IKCa channels functions in the process mentioned above.Methods Soft(0.21 MPa)and stiff(1.72 MPa)PDMS substrates where VSMCs were seeded after coated with fibronectin(FN),were fabricated through the blending of sylgard 184 gel and sylgard 527 gel.After that,intracellular calcium level of VSMCs was compared with or without the treatment of IKCa specific blocker,TRAM-34,using a calcium-sensitive dye,fluo 4-AM.1mM Ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid(EGTA)was added into the culture media for the removal of the extracellular calcium ions as well as cell counting kit-8(CCK-8)assay was applied,which is to explore the role that calcium ion entry played in proliferation process.The activation level of ERK 1/2 was described by the expression level of phospho-ERK 1/2 using western blotting with or without TRAM-34 treatment.The role of the activation of ERK1/2 in VSMC proliferation was examined by CCK-8 assay with or without the treatment of PD98095,an ERK1/2 inhibitor.Results Compared with soft substrate,stiff substrate caused an increase of intracellular calcium level,which was attenuated by IKCa blockade.In addition,compared with soft substrate,stiff substrate also caused an activation of ERK1/2,which was significantly suppressed by IKCa blockade.Furthermore,extracellular calcium ion reduction by adding EGTA significantly inhibited the stiff substrate-induced VSMC proliferation,which whereas had no effect on VSMC proliferation on soft substrate.Finally,ERK1/2 inhibition had similar inhibitory effect on stiff substrate-induced proliferation.Conclusions Stiff substrate causes an IKCa channel-mediated calcium entry and ERK1/2 activation,both of which play important roles in stiff substrate-induced VSMC proliferation.Combined the previous results that IKCa channel participated in stiff substrate-induced VSMC proliferation,our present work suggests that IKCa channel functioned in the proliferation process through mediating calcium entry and subsequent ERK1/2 activations.These findings provide a new insight into how substrate stiffness regulates VSMC proliferation,and additional considerations for vascular tissue engineering and vascular disease treatment.
