| BackgroundAtherosclerosis (AS) and its complications caused by acute cardiovascular disease event have become the primary killer of mankind, its prevention and treatment have been a worldwide public health priorities and challenges. Acute coronary syndrome (ACS) is the most important death cause of coronary atherosclerotic heart disease. ACS is the consequence of rupture or erosion of preexisting atherosclerotic plaque, with subsequent formation of local thrombus leading to critical occlusion of coronary arteries. From the pathophysiological aspect, the main underlying cause of ACS is vulnerable plaque, the plaque which induces thrombus easily or may progress to culprit lesions rapidly.Macrophages are one of the inflammatory cells on local plaques, not only limited to the plaque, but also largely infiltration of the outside vascular bed. Under the ox-LDL and other proinflammatory factors effects, the monocyte-derived macrophages are activated, aggregation, adhesion and migration to the vascular wall through endothelial cells, and secretion cytokines, mitosis-promoting factors, chemokines, metalloproteinases and other proteolytic enzymes, further activating the inflammatory response; with the deterioration of the inflammatory response, macrophages continuous activation trigger cascade reactions that enlarge the inflammatory response. Therefore, the activation of macrophages plays an important role in the pathogenesis of acute coronary syndrome.There are two important potassium channels on macrophages:voltage-dependent potassium channels and Ca2+-dependent potassium channels. The opening of voltage-gated potassium channel Kv1.3caused the membrane hyperpolarization, encouraged the Ca2+influx, then the increasing intracellular Ca2+as a second messenger to activate macrophages. Further studies showed that the voltage-dependent potassium channel Kv was encoded by Kv1.3gene. The voltage-dependent potassium channel Kv1.3is made up of4protein subunits composed by six α-helices, which constitute the single-hole channel through six transmembrane structures. The open of Kv channels are of frequency-dependent inactivation. The channel opens when the membrane potential exceeds-40mv. Therefore, the potassium channels Kv1.3affect macrophage function through the regulation of membrane potential and intracellular signaling pathways.Objective1. To observe the expression of Kv1family in monocyte-derived macrophages;2. To observe the potassium curren in ACS monocyte-derived macrophages;2. To investigate the expression of the voltage-dependent potassium channel Kv1.3in monocyte-derived macrophages.Methods1Separate and culture human monocyte-derived macrophages;2Detection the potassium current density by whole-cell patch clamp in macrophages;3Detection the Kv1.3mRNA expression by RT-PCR;4Detection the Kv1.3protein expression by Western blot.Results1. Data analysis of clinical specimensCompared with the control group, the low density lipoprotein (LDL) cholesterol in the group of acute coronary syndrome were significantly increased (P<0.05). There was no significant difference among the four groups in the age, sex ratio, blood glucose, blood pressure, the proportion of smoking and drug treatment (P>0.05). It was comparable among the four groups. 2. Identification the Kv current in macrophagesThe free Ca2+concentration is about10nmol/L by the EGTA in the electrode solution, reducing the effect of Ca2+-activated potassium channels ultimately. So the main composition of the current is the potassium. And4-AP blocked the current significantly. The above characteristics suggested that the current should be the Kv current.3. The capacitance and Kv current in macrophages among four groupsMembrane capacitance was no significant difference among the four groups (P>0.05). Compared with the control group, the peak current density of Kv in unstable angina group was significantly increased (P<0.01), the acute myocardial infarction group was also significantly increased (P<0.01), the stable angina group was no significant difference (P>0.05).4. The expression of Kv1channel in macrophagesThe mRNA expression of Kv1channels were detected at the fifth day of natural differentiation. The results showed that the Kv1.3mainly expressed in macrophages.5. The mRNA expression of Kv1.3channel in macrophagesCompared with control group, the mRNA expression of Kvl.3in unstable angina and acute myocardial infarction groups were significantly increased (P<0.05); stable angina group was no significant change (P>0.05).6. The protein expression of Kv1.3channel in macrophagesCompared with control group, the protein expression of Kv1.3channel in unstable angina and acute myocardial infarction groups were significantly increased (P<0.05); stable angina group was no significant difference (P>0.05).Conclusions1. The Kv current density was significantly increased in macrophages of acute coronary syndrome;2. The expression of Kv1.3is priority in the voltage-dependent outward Kvl channel of monocyte-derived macrophages;3. The mRNA and protein expression of Kv1.3channel were increased in monocyte derived macrophages of acute coronary syndrome. BackgroundMonocytes/macrophages play an important role in process of atherosclerosis (AS). Monocytes attached to the wall, transformation to macrophages, modification of ox-LDL, cell migration, uptake the ox-LDL, phagocytosis, foaming and the generation of plaque matrix components depends on the direct involvement and effect of monocytes/macrophages. Activated macrophages infiltrate the vascular wall, damage vascular endothelial cells, secrete a variety of metalloproteinases including collagenase to degrade the extracellular matrix and weaken the fibrous cap to make it thinner, that change the stable plaques into vulnerable plaques. So that some plaques were unstable and easy to rupture, causing thrombosis formation.Ox-LDL is the main activation in macrophages. There were a large number of ox-LDL accumulation in the vulnerable plaque at the early and late state of AS. Previous vitro studies showed that ox-LDL has the characteristics of AS generation, chemotaxis for monocyte-macrophage, inducts the expression of monocyte chemoattractant protein-1, and devolves the expression of vascular endothelial cell adhesion molecule-1, intercellular adhesion molecule-1and P-selectin etc; ox-LDL can also influence the macrophage gene expression, then can affect the macrophage inflammatory response and atherosclerosis process.The voltage-dependent potassium channel Kv is the critical channel on the macrophage cell membrane the resting macrophage. Kv channels have two basic characteristics:(1) voltage dependentd;(2) The high degree of selectivity for K+. With the development of molecular biology, Kvl.3subtype has been identified the main outward delayed potassium channels in macrophages. Kv channels can indirectly influence the Ca2+signal by regulating the macrophage resting membrane potential, then play an important role in the cytokine secretion and cell proliferation.Objective1. To investigate the expression of Kv1.3induced by ox-LDL in macrophages;2. To explore the role of Kv1.3on potassium current in macrophages induced by ox-LDL;3. To investigate the effect of Kv1.3on migration and secretion in macrophages induced by ox-LDL.MethodsWe took primary human monocyte-derived macrophages as the object of this research, then quantitative real time PCR, western blot, ELISA and Transwell techniques were used to observe respectively:1. Macrophages were treated with various concentrations of ox-LDL (0,5,10,20,40and80μg/ml) for various times (0,4,8,12,24and48h), then detect Kv1.3levels;2. After overexpressing or silencing Kv1.3expression, the change of potassium current in macrophages were measured;3. After overexpressing or silencing Kv1.3expression, the change of migration in macrophages were measured;4. After overexpressing or silencing Kv1.3expression, the change of TNF-α secretion in macrophages were measured.Results1. Kv1.3expression in macrophages induced by ox-LDLMacrophages were treated with various concentrations of ox-LDL (0,5,10,20,40and80μg/ml) for24h, then it showed that ox-LDL can effectively stimulate the Kv1.3mRNA upregulation in macrophages, which expresses as dose-depended. The Kv1.3mRNA expression significantly increase at the concentration of40μg/ml ox-LDL, and80μg/ml ox-LDL was strongest.Macrophages were treated with1μg/ml ox-LDL for various times (0,4,8,12,24and48h), then it showed that Kv1.3mRNA expression increased with increasing treatment time. The Kv1.3mRNA expression significantly increased at24h, and expression was strongest at48h. It was further confirmed that Kvl.3protein expression increased significantly treated with40μg/ml ox-LDL for24h by western blot.2. Effect of ox-LDL on survival rate of macrophagesMacrophages were treated with various concentrations of ox-LDL (0,5,10,20,40and80μg/ml) for24h, MTT showed that survival rate of macrophages was significantly decreased treated with80μg/ml ox-LDL.Macrophages were treated with1μg/ml ox-LDL for various times (0,4,8,12,24and48h), it showed that survival significantly decreased treated with ox-LDL for48h in macrophages. Therefore, we choose40μg/ml ox-LDL treated for24h as the processing condition.3. Effect of Kvl.3on potassium current in macrophages induced by ox-LDLCompared with control cells, current density of Kv1.3-overexpressing macrophages was increased (P<0.01); with ox-LDL treatment, Kv1.3-overexpressing macrophages was further increased (P<0.01) compared with controls. Factorial analyses demonstrated no significant interaction between overexpression of Kv1.3and stimulation with ox-LDL on macrophage K+current density.Kv1.3-silenced macrophages showed decreased K+current density compared with controls (P<0.01); Kv1.3-silenced cells treated with ox-LDL showed increased K+current density (P<0.01) compared with controls. Silenced Kv1.3and ox-LDL stimulation showed significant interaction for macrophage K+current density (P<0.01),which suggests that Kv1.3knockdown in part resisted macrophage K+current density by ox-LDL.4. Effect of Kvl.3on migration in macrophages induced by ox-LDLCompared with control cells, the migration rate of Kvl.3-overexpressing macrophages was increased (P<0.01); with ox-LDL treatment, Kvl.3-overexpressing macrophages was further increased (P<0.01) compared with controls. Factorial analyses revealed no significant interaction between overexpression of Kv1.3and stimulation with ox-LDL on macrophage migration rate. Kv1.3-silenced macrophages showed decreased migration rate compared with controls (P<0.01); Kv1.3-silenced cells treated with ox-LDL showed increased K+current density (P<0.01) compared with controls. Silenced Kvl.3and ox-LDL stimulation showed significant interaction for macrophage migration rate(P=0.039), which suggests that Kv1.3knockdown in part resisted macrophage migration by ox-LDL,5. Effect of Kvl.3on secretion in macrophages induced by ox-LDLCompared with control cells, the TNF-a secretion of Kvl.3-overexpressing macrophages was increased (P<0.01); with ox-LDL treatment, Kvl.3-overexpressing macrophages was further increased (P<0.01) compared with controls. Factorial analyses reveled no significant interaction between overexpression of Kvl.3and stimulation with ox-LDL on macrophage migration rate.Kv1.3-silenced macrophages showed decreased TNF-a secretion compared with controls (P<0.01); Kv1.3-silenced cells treated with ox-LDL showed increased TNF-a secretion (P<0.01) compared with controls. Silenced Kvl.3and ox-LDL stimulation showed significant interaction for macrophage TNF-a secretion(P<0.01),which suggests that Kv1.3knockdown in part resisted macrophage TNF-a secretion by ox-LDL.Conclusions1. Kv1.3expression was upregulated in macrophages in a dose-and time-dependent manner on stimulation with ox-LDL.2. Kv1.3in part mediates the increase of potassium current density in macrophage induced by ox-LDL;3. Kv1.3in part mediates the migration and secretion in macrophage induced by ox-LDL. BackgroundThe resting membrane potential is mainly dependent on the K+concentration of intracellular and extracellular solution gradient, which play a joint role in the repolarization after membrane potential depolarization. The potassium channels and other transport proteins maintain the stability of the membrane potential. Recently studies found that voltage-dependent outward delayed potassium channel in macrophages is one of the membrane protein involved in cellular immune response. The potassium currents play an important role in macrophages secretion, even as an important signaling molecule, regulating the macrophages inflammation.Voltage-dependent potassium channel regulate the Ca2+by controlling the membrane potential. The rest membrane potential is negative, promoting the extracellular Ca2+to the cells; when the Kv channels open, the outflow of K+lead to the hyperpolarization, promoting Ca2+influx, Ca2+of intracellular increased; when Kv channels blocked, the reduced outflow of K+lead to the membrane depolarization, inhibiting of Ca2+influx, Ca2+of intracellular decreased. Potassium channels were involed in the macrophage chemotaxis and secretion through the regulation of Ca2+.It is a complex network system for regulation the signal of macrophage inflammation. Various signaling molecules such as oxidized low-density lipoprotein (ox-LDL), nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) are involved in the regulation of macrophage inflammatory activation. The open of voltage-dependent potassium channel result in the membrane hyperpolarization, Ca2+influx increased, thn activating the MAPK and the NF-κB. signaling pathway. The activation of MAPKs and NF-κB signaling pathways can lead to macrophage activation, and thus play an important role in the development of vulnerable plaque."Therefore, this study proposed the following hypothesis:voltage-dependent potassium channel Kv1.3mediated macrophage calcium influx and involved in ox-LDL-induced macrophage activation by regulating Ca2+-MAPKs-NF-κB signal transduction pathways, thereby play an important role in the development of vulnerable plaque.Objective1. To observe the effect of ox-LDL on the phosphorylation MAPKs and NF-κB;2. To explore Kv1.3on the regulation of Ca2+on ox-LDL induced macrophages;3. To explore Kv1.3on the MAPKs and NF-κB signal transduction pathways on ox-LDL induced macrophages.MethodsWe took primary human monocyte-derived macrophages as the object of this research, then Patch clamp technique and western blot analysis were used to observe respectively:1. After overexpressing or silencing Kv1.3expression respectively, the Ca2+fluorescence intensity changes were observed in macrophages;2. After overexpressing or silencing Kv1.3expression respectively, the change of MAPks ERK1/2and NF-κB levels in macrophages were mesured.Results1. Effects of Kv1.3on the Ca2+fluorescence intensityCompared with control cells, the Ca2+fluorescence intensity of Kv1.3overexpression was increased (P<0.01); compared with ox-LDL treatment lonely, the Ca2+fluorescence intensity in both overexpression of Kv1.3and ox-LDL treatment was further increased (P<0.01).Kv1.3-silenced macrophages showed decreased Ca2+fluorescence intensity treatment compared with controls(P<0.01); Kv1.3-silenced cells treated with ox-LDL showed reduced Ca2+fluorescence intensity compared with ox-LDL treatment lonely(P<0.01). 2. Effects of ox-LDL on MAPKs in macrophagesTreatment macrophages with40μg/mL ox-LDL for Oh,24h,48h, the results show, that ox-LDL can stimulate ERK1/2phosphorylation in macrophages, the phosphorylation level of ERK1/2were significantly increased in time-dependent manner; total ERK1/2had no change significantly.3. Effects of Kv1.3on MAPKs in macrophagesCompared with control cells, the p-ERK1/2levels of Kv1.3-overexpressing macrophages was increased (P<0.01); with ox-LDL treatment, the p-ERK1/2levels of Kv1.3-overexpressing macrophages was further increased (P<0.01) compared with ox-LDL treatment lonely.Kv1.3-silenced macrophages showed decreased p-ERK1/2levels treatment compared with controls(P<0.01); Kv1.3-silenced cells treated with ox-LDL showed reduced p-ERK1/2levels compared with ox-LDL treatment lonely(P<0.01).4. Effects of ox-LDL on NF-κB in macrophagesTreatment macrophages with40μg/mL ox-LDL for16h,24h,48h, results showed that the p-p65at16h increased (P<0.01), the p-p65at24h also increased (P<0.01), the p-p65at48h decreased (P<0.01).5. Effects of Kv1.3on NF-κB in macrophagesCompared with control cells, the p-p65levels of Kv1.3-overexpressing macrophages was increased (P<0.01); with ox-LDL treatment, the p-p65levels of Kv1.3-overexpressing macrophages was further increased (P<0.01) compared with ox-LDL treatment lonely.Kv1.3-silenced macrophages showed decreased p-p65levels treatment compared with controls(P<0.01); Kv1.3-silenced cells treated with ox-LDL showed reduced p-p65levels compared with ox-LDL treatment lonely(P<0.01).Conclusions1. Voltage-dependent potassium channel Kv1.3influenced the intracellular calcium by regulating the membrane potential;2. Voltage-dependent potassium channel Kv1.3was involeved in Ca2+-MAPKs-NF-KB signal transduction pathways to regulate the activation of macrophages.
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