IK_(Ca) Channel-Mediated Ca~(2+) Entry and ERK1/2 Activation: Implication in Stiff Substrate-Induced Vascular Smooth Muscle Cell Proliferation
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摘要
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.
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.
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.
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