The Role And Molecular Mechanism Of K_ATPChannels In Atherosclerosis

BackgroundAtherosclerosis is one of the most serious diseases detrimental to health and vulnerable plaques have been recognized as chief criminal of acute coronary syndrome (ACS) and cerebrovascular disorders. However, the pathogenesis and approach targets of atherosclerotic plaques are still unclear. The main cellular components in plaques include macrophages, smooth muscle cells and endothelial cells, while the decrease of smooth muscle cells and collagen fibers, and the increase of lipids and macrophages happened in vulnerable plaques.Numerous studies have indicated that ion channels are related to vascular functions tightly and play an important role in the progression of atherosclerosis. It has been reported that the calcium channels, potassium channels and chloride channels in endothelial cells regulated the shear stress and inflammation; the disorders of vascular smooth muscle cells, endothelial cells, monocytes/macrophages and platelets induced by transient receptor potential canonical channels (TRPC) participated in the pathogenesis of atherosclerosis. So. the role of ion channels in atherosclerosis should never be neglected. Potassium channels contribute to the membrane potential, preserve the cellular electrical homeostasis and could affect the activities of other ions to regulate the cellular state.ATP-sensitive potassium channels (KATP channels) are heterooctameric complexes composed of Kir6.1or Kir6.2as pore forming subunits, and SUR1, SUR2A or SUR2B as regulatory subunits. KATP channels are sensitive to the change of intracellular ATP as important metabolic sensors, coupling metabolic state with electrical activity of cells. Recently, the role of KATP channels in both of diabetes and cardiovascular diseases has attracted more and more attention. The studies mainly concentrate on the following aspects:the regulation of cardiac functions, under ischemia-reperfusion, myocardial infarction, myocardial remolding, heart failure, ischemic preconditioning and stress; vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors; regulation the effect of endothelium-derived relaxing factor (EDRF), such as nitric oxide (NO), hydrogen sulfide (H2S) and endothelin-1(ET-1). However, the role of KATP channels in atherosclerosis hasn’t been investigated directly.Therefore, we put forward the hypothesis that KATP channels play an important role in the pathogenesis of atherosclerosis and represent the potential therapeutic target of atherosclerosis.Objective1. To establish animal models of progressive atherosclerosis;2. To evaluate the content of smooth muscle cells, macrophages, collagen fibers and lipids in plaques, and the vulnerable index of plaques;3. To investigate the interventional effects of modulators targeting to KATP channels on the progression of atherosclerosis.MethodsWe applied eighty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, with or without intervention of glibenclamide in vivo, followed by a series of experiments.1. Blood biochemical analysis:collect blood samples to detect the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma.2. Tissue harvesting and morphological analysis:the mice were sacrificed0day,1day,4days or14days after transduction according to the assignment, respectively, and the tissue samples were carefully excised for pathological staining and molecular biological experiments. Hematoxylin and eosin (HE)staining was used to show the tissue structure, Oil-red O staining was used to detect the content of lipids, sirius-red staining was used to detect the content of collagen fibers, immunohistochemical staining of p53, SMC-a actin and MOMA-2was used to detect the content of p53protein, smooth muscle cells and macrophages, respectively.3. Calculation of vulnerable index:vulnerable index=(MOMA-2positive staining area/plaque area+lipids staining area/plaque area)/(SMC-a actin positive staining area/plaque area+collagen fibers staining area/plaque area).Results1. The effects of glibenclamide intervention on blood biochemistry and livers in ApoE-/-miceThere were no substantial changes in the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma, and hepatic structures of ApoE-/-mice after intragastric administration of glibenclamide in our experiment (all P>0.05).2. Identification of carotid collar placement and transduction of p53The right carotid arteries proximal to collars showed atherosclerotic plaques by ultrasound. Compared with that of control groups, the staining of p53was significantly increased in Ad5-CMV.p53-treated groups, including1,4,14-day subgroups, and the difference increased over time (all P<0.05).3. The staining of smooth muscle cells and collagen fibers in atherosclerotic plaquesCompared to Ad5-CMV.lacZ-treated groups, the content of smooth muscle cells and collagen fibers in atherosclerotic plaques was decreased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).4. The staining of macrophages and lipids in atherosclerotic plaquesCompared to Ad5-CMV.lacZ-treated groups, the content of macrophages and lipids in atherosclerotic plaques was increased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).5. Glibenclamide intervention stabilizing atherosclerotic plaques in vivoIn Ad5-CMV.lacZ-treated-14days groups, the content of macrophages and lipids decreased obviously (both P<0.05), and the content of collagen fibers increased (P<0.05), while the content of smooth muscle cells was not different significantly after intervention of glibenclamide for14days in vivo.6. Calculation of vulnerable indexThe vulnerability index (VI), which was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions to estimate the vulnerable possibility of plaques, was higher in p53-treated groups than that in control groups after4days and14days and the gap was widened over time (both P<0.05).Conclusions1. Transduction of Ad5-CMV.p53induced increased vulnerable index of plaques in ApoE-/-mice with high fat diet and collar placement;2. Glibenclamide intervention didn’t affect the blood biochemistry and structure of livers obviously in ApoE-/-mice transfected with Ad5-CMV.p53;3. Glibenclamide ameliorated the progression of atherosclerosis induced by Ad5-CMV.p53transduction in vivo. BackgroundAtherosclerosis (AS) is related to membrane ion channels closely. K+current is the primary component of membrane potential and vitally important to cellular functions. ATP sensitive potassium channels (KAtp) could regulate the concentration of K+and preserve membrane potential to modulate activities of other ions and the whole cell, and couple cellular metabolism with membrane potential. When the intracellular ADP/ATP is raised due to ischemia, hypoxia, metabolic acidosis and so on, KATP channels will open, the cell hyperpolarize, voltage-dependent calcium channels (VDCCs) close, calcium influx decrease and intracellular Ca2+reduce to mediate cardiac protection.KATP channel is a hetero-octamer complex of four pore forming subunits, inwardly rectifying potassium channels Kir6.X, and four regulatory subunits according to intracellular ATP concentration, sulfonylurea receptors (SURs), member of ATP binding cassette (ABC) superfamily. Kir6.X, including Kir6.1and Kir6.2, links with SURs, including SUR1, SUR2A and SUR2B, to constitute functional KATP channels. KATP channels exist in various tissues and carry an important cellular function with tissue specificity. In cells expressing Kir6.1and Kir6.2at the same time, the subunits often comprise heterogeneous polymers in a different proportion. Accordingly, KATP channels in different tissues exhibit different physiologic characteristics and response with specific reagents. For example, SUR2A/Kir6.2 subtype mainly expresses in myocardial cells and shows cardiac protection under conditions of ischemia, hypoxia and other stress; SUR1/Kir6.2subtype mainly expresses in pancreatic P cells to regulate secretion of insulin; SUR2B/Kir6.1mainly expresses in vascular smooth muscle cells and endothelial dcells and participate in vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors. KATP channels are important metabolic sensors, while AS and inflammation are associated with metabolism closely.Therefore, we put forward the hypothesis that KATP channels express in vascular smooth muscle cells, endothelial cells and macrophages of atherosclerotic plaques, and should be correlated with vulnerability of plaques.Objective1. To detect the expression and distribution of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in atherosclerotic plaques;2. To make correlation analysis between content of KATP subunits and vulnerability of plaques;3. To evaluate the effect of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages on progression of atherosclerosis.MethodsWe applied fifty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, respectively, followed by a series of experiments.1. Morphological staining:Hematoxylin and eosin (HE) staining was used to show the tissue structure, sirius-red staining was used to detect the content of collagen fibers and Oil-red O staining was used to detect the content of lipids;2. SMC-a actin and MOMA-2staining:Immunohistochemical staining of SMC-a actin and MOMA-2was used to detect the content of smooth muscle cells and macrophages, respectively;3. KATP Subunits staining:Immunohistochemical staining of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in AS plaques to investigate the expression and distribution of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages;4. Correlation analysis:The content of smooth muscle cells, collagens, macrophages and lipids in plaques was evaluated, the vulnerable index was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions, and then, we anlysed the correlation between AS components, vulnerable index and KATP Subunits;5. Immunofluorescence:Immunofluorescence double staining was used to detect the co-localization of atherosclerotic cells and KATP Subunits;6. RT-PCR quantitative analysis of KATP Subunits:The mRNA expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in carotid arteries was detected by RT-PCR.Results1. The staining of KATP Subunits in atherosclerotic plaquesPositive staining of SUR2A and Kir6.2subunits mainly accumulated in plaque shoulder and fatty streak with more macrophages, and quantitative analysis showed similar expressional tendency:SUR2A and Kir6.2expression was detected mostly in p53-treated4-day group and was higher than that of lacZ treated-control groups after1day and4days treatment (P<0.05). Positive staining of SUR1subunit mainly accumulated in plaque shoulder and fatty streak, as well as endothelial cells, and quantitative analysis showed that the staining was most markedly in p53-treated4-day group and was higher than that of control group (P<0.05). Positive staining of Kir6.1subunit distributed in plaque shoulder and fatty streak, as well as endothelial cells and smooth muscle cells, and quantitative analysis showed that the staining was higher in p53treated groups after1day and4days compared with that of lacZ treated groups (P<0.05). Positive staining of SUR2B subunit mainly accumulated in vascular smooth muscle cells and endothelial cells, and quantitative analysis showed that the staining was decreased in p53-treated4-day group compared with that of lacZ treated group (P<0.05).2. The mRNA expression of KATP subunits in carotid arteriesAt mRNA level, the expression of SUR2A, Kir6.2and SUR1subunits was higher in p53-treated group than that of control group after4-day treatment (P<0.05); the expression of Kir6.1subunit was higher in p53-treated group than that of control group after1-day treatment (P<0.05); and the largest value of SUR2B transtripts was found in blank control group (P<0.05).3. The correlation analysis between KATP subunits and plaque burdenThe expression of SUR2A and Kir6.2subunits in plaques was positively correlated with vulnerable index and the content of macrophages in plaques, and negatively correlated with the content of smooth muscle cells in plaques (all P<0.05).4. Co-localization of MOMA-2with SUR2A and Kir6.2subunits in plaquesThe result of immunofluorescence double staining showed that MOMA-2and SUR2A, as well as MOMA-2and Kir6.2, co-located at macrophages attached to vascular walls and within plaques.Conclusions1. KATP subunits expressed in smooth muscle cells, endothelial cells and macrophages of AS plaques;2. SUR2A and Kir6.2subunits were associated with plaque burden closely;3. The subtype of SUR2A/Kir6.2mainly located in macrophages of atherosclerotic plaques. BackgroundAtherosclerotic plaques consist of macrophages, smooth muscle cells and endothelial cells. Differentiating from vascular smooth muscle cells and endothelial cells, macrophages derive from circular monocytes and contribute to immune inflammatory response. The activation of monocytes/macrophages is considered as the initiation of atherosclerosis and works through the whole process of atherosclerosis with accumulating, adhesion, migration and producing inflammatory cytokines.The previous studies have showed that ion channels expressed in monocytes/macrophages played an important role in atherosclerosis. Dysfunction of monocytes/macrophages mediated by TRPC (transient receptor potential canonical) channels was associated with the development of atherosclerosis; the changes of voltage-dependent inwardly rectifying potassium channels (Kir) and delayed outwardly rectifying potassium channels induced by inflammatory cytokines promoted the activation and differentiation of monocytes; disordered volume-regulated Cl channels promoted foam cell formation from macrophages in atherosclerosis; moreover, MCP-1and LPC regulated the chloride channels and calcium activated potassium channels to accelerate the migration of monocytes. The migration of monocytes initiates the process of atherosclerosis and the formation of considerable foam cells designates the vulnerable plaques. Whereas, KATP channels in monocytes/macrophages have drawn relatively a little attention.KATP channels are octamers of four pore forming subunits-inwardly rectifying potassium channel Kir6.X and four regulatory subunits-a member of ATP binding cassette (ABC) superfamily called sulfonylurea receptor (SUR). The former contains Kir6.1and Kir6.2subtypes, and the latter contains SUR1, SUR2A and SUR2B subtypes. They comprise various subtypes of KATP channels in different tissues with distinct physiologic and pathological properties. It has been reported that the blocker of KATP channels, glibenclamide, could inhibit the release of proinflammatory cytokines induced by Lipopolysaccharide (LPS) in condition of hypoxia and acidosis. The inflammation mediated by monocytes/macrophages play an important role in initiation and progression of atherosclerotic lesions. LPS, a typical bacterial pathogens known as most powerful activators of macrophages, could stimulate monocytes/macrophages to produce proinflammatory cytokines such as TNF-a by bounding with toll like receptors (TLRs) through two major signal transduction pathways:nuclear factor κB (NF-κB) proteins and mitogen-activated protein kinases (MAPKs), the latter including extracellular signal related kinase (ERK1/2), p38MAPK (p38) and c-Jun N-terminal kinase (JNK) pathways.Therefore, we put forward the hypothesis that KATP channels in monocytes/macrophages play an important role in inflammation to mediate the progression of atherosclerotic lesions and they work through NF-κB/MAPKs signal transduction pathways to regulate the inflammatory response.Objective1. To detect the expression of KATP subunits and TNF-a induced by LPS in macrophages;2. To explore the effect of KATP channels on the expression of TNF-a induced by LPS in macrophages;3. To investigate the the effect of KATP channels on NF-κB and MAPKs pathways induced by LPS in macrophages. MethodsWe used the cell line of mice monocytes/macrophages, RAW264.7cells, as the object of this study, pretreated with the opener or blocker of KATP channels, pinacidil or glibenclamide, followed by stimulation of LPS. And then we applied real time RT-PCR and western blotting to detect the role and mechanisms of KATP channels in macrophages.1. RAW264.7cells were treated with LPS (1μg/ml) for0,2,4,8or24h to detect the expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subunits, as well as TNF-a.2. RAW264.7cells were pretreated with the opener or blocker of KATP channels, pinacidil (10μM) or glibenclamide (10μM), to detect the expression of TNF-a induced by LPS and the phosphorylation of NF-κB and MAPKs signal transduction proteins at various time (15min,30min,1h, and24h).3. After intervention with inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), the expression of KATP subunits and TNF-a inducted by LPS in macrophages was investigated.Results1. LPS induced the expression of KATP subunits and TNF-a in RAW264.7cellsCompared with control group, the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits, as well as TNF-a, was increased in LPS (1μg/ml) treated RAW264.7cells for4h (all P<0.05). However, the expression of SUR2B subunit was not detected.2. The regulation of KATP channels in expression of TNF-a and phosphorylation of NF-κB and MAPKs signal proteinsThe blocker of KATP channels, glibenclamide (10κM). could reduce the expression of TNF-a induced by LPS in RAW264.7cells for4h. while the opener pinacidil (10λM) raised the expression slightly (both P<0.05). Moreover, glibenclamide decreased the phosphorylation of p65and ERK1/2induced by LPS for15min (both P<0.05), while the effect of pinacidil was not obvious (P>0.05).3. KATP channels located at upstream of NF-κB activated by LPS in RAW264.7 cellsThe inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), decreased the expression the TNF-a induced by LPS in RAW264.7cells obviously (P<0.05), and glibenclamide could enhance the effect of NF-κB inhibitors, while pinacidil antagonized their effect (P<0.05).PDTC (100μM) and DMFR (100μM) increased the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits induced by LPS in RAW264.7cells significantly (P<0.05).Conclusions1. LPS increased the expression of KATP channels in macrophages;2. KATP channels in macrophages participated in inflammatory response induced by LPS;3. KATP channels in macrophages regulated inflammation via NF-κB and ERK1/2pathways.