TRPC5通道在动脉粥样硬化早期斑块发生中的作用及机制研究
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摘要
背景
冠心病已成为严重威胁人类健康的流行病,动脉粥样硬化(atherosclerosis, AS)斑块的发生和发展是其病理生理基础。在斑块的发生和发展过程中,平滑肌细胞的迁移、增殖、表型转换及细胞外基质的产生起到重要作用。因此,研究平滑肌细胞功能在AS斑块发生和发展中的作用对于防治冠心病具有重要意义。
研究发现平滑肌细胞内Ca2+作为第二信使对于平滑肌细胞的功能调节起关键作用。近年来钙库操纵性通道(store-operated channels, SOCs)介导的钙内流在细胞功能及疾病中的作用日益受到重视,特别是经典型瞬时感受器电位通道(classical transient receptor potential channel, TRPC channel)是组成SOCs的主要通道,能被G蛋白偶联磷脂酶C所产生的IP3激活而产生持续的钙内流,它们在平滑肌细胞功能中的作用日益受到重视。近年研究发现人冠状动脉和脑动脉有TRPC通道的表达,TRPC1参与了移植静脉内膜增生。此外,TRPC5在脂质对细胞功能的调节中有重要作用,具有脂质离子亚型受体特性,1-磷酸鞘氨醇作为氧化低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)的成分也能够激活TRPC5。
由于ox-LDL和平滑肌细胞在AS过程中的重要作用,特别是TRPC通道在静脉内膜增生中的作用提示：TRPC通道在AS过程中可能担当重要角色。以往研究仅研究了体外表达系统或细胞系中TRPC5的生理作用,对其在AS发生和发展过程中的作用知之甚少。因此我们提出如下假说：AS早期斑块中TRPC5通道表达增高,阻断TRPC5通道可以通过降低斑块内平滑肌细胞和胶原含量而抑制AS斑块的发展。我们设计了一系列体内和体外实验来证明这一假说。
目的
1.研究AS斑块发生和发展过程中TRPC5通道的表达情况；
2.探讨体内特异性阻断TRPC5通道能否抑制AS早期斑块的发展及其可能机制。
方法
1.AS早期斑块模型建立：8周龄雄性apoE-/-小鼠,全程高脂饮食,行右侧颈总动脉套管(缩窄性硅胶管)术以诱发AS早期斑块。
2.体内阻断TRPC5:随机分为4组：PBS组,尾静脉注射无菌PBS200μl; IgG组：TRPC5通道特异性阻断剂T5E3Ab相同种属来源的IgG100μg,用无菌PBS200μl溶解；TRPC5通道特异性阻断剂T5E3Ab低剂量组：T5E3Ab50μg,用无菌PBS200μl溶解；TRPC5通道特异性阻断剂T5E3Ab高剂量组：T5E3Ab100μg,用无菌PBS200μl溶解。套管术前1天尾静脉给药,每周给药一次,8周末处死,每组20只。
3.动物一般状况的评价：实验8周末测量体重、心率和尾动脉血压,收集小鼠血液,检测小鼠的血细胞、总胆固醇(TC)、甘油三脂(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的水平。
4.病理组织学检测：颈动脉斑块组织学切片分别行苏木素-伊红染色、天狼猩红染色和油红O染色,并进行免疫组织化学染色观察巨噬细胞(MOMA-2)、平滑肌肌动蛋白(a-SM-actin)和TRPC5局部表达。测量斑块面积、胶原、脂质、平滑肌细胞和巨噬细胞免疫组化阳性染色面积,并计算它们占斑块面积的比例。
5.实时定量RT-PCR:检测斑块TRPC通道mRNA表达水平。
6. T5E3Ab对TRPC5通道的特异性阻断作用的验证：培养稳定表达TRPC5通道的HEK293细胞系(TRPC5-HEK293):膜片钳技术检测TRPC5-HEK293细胞的TRPC5通道介导的电流及T5E3Ab的阻断作用；钙离子成像检测TRPC5-HEK293细胞的TRPC5通道介导的细胞内钙离子增加及T5E3Ab的阻断作用。检测含T5E3Ab小鼠血清给对TRPC5通道的抑制作用。
结果
1.T5E3Ab对TRPC5通道的阻断作用：在诱导过表达TRPC5通道的细胞,TRPC5通道的激活剂Gd3+(100μM)可产生细胞内钙离子的增加和通道电流,而T5E3Ab (25μg/ml能够抑制这些反应。而在未诱导表达的细胞,不产生这些变化。与注射对照IgG100μg组小鼠血清比较,含T5E3Ab小鼠血清给药后1天对TRPC5通道抑制率95%(P<0.05),而1周末抑制率为50%(P<0.05,表1),2周末抑制作用消失(P>0.05)。
2.动物一般状况比较：实验8周末,4组ApoE-/-小鼠的体重、血压、血清TC、TG、LDL-C、HDL-C和血细胞成份无显著性差异(P>0.05)。
3.AS早期斑块TRPC通道mRNA表达改变：实验8周末,与未套管侧颈动脉比较,PBS组ApoE-/-小鼠套管处颈动脉TRPC5通道mRNA的表达明显增高(P<0.05),其他亚型TRPC通道无显著差异(P>0.05)。4组ApoE-/-小鼠之间TRPC5通道mRNA表达水平无统计学差异(P>0.05)。
4.AS早期斑块TRPC5通道表达与斑块特征的关系：实验8周末,与未套管侧颈动脉比较,在未干预组(PBS组),ApoE-/-小鼠套管处颈动脉有明显斑块,斑块负荷、脂质含量和TRPC5通道表达含量明显增加(P<0.05)。相关分析表明,PBS组TRPC5通道表达与斑块负荷、脂质含量、平滑肌细胞和胶原含量成正相关(P<0.05),与斑块内巨噬细胞含量无关(P>0.05)。
5.阻断TRPC5通道对AS斑块负荷的影响：实验8周末,与未干预组(PBS组)和IgG (100μg)组比较,TRPC5通道阻断组斑块负荷明显降低(P<0.05),与低剂量组(T5E3Ab50μg)比较,高剂量组(T5E3Ab100μg)斑块负荷进一步降低(P<0.05)。
6.阻断TRPC5通道对AS斑块脂质含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,阻断TRPC5通道可以明显降低脂质含量(P<0.05),虽然高剂量组脂质含量比低剂量组降低,但是组间差别无统计学意义(P>0.05)。
7.阻断TRPC5通道对斑块胶原含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,TRPC5通道阻断组斑块胶原含量明显降低(P<0.05),与低剂量组比较,高剂量组斑块胶原含量进一步降低(P<0.05)。
8.阻断TRPC5通道对AS斑块平滑肌细胞含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,阻断TRPC5通道可以明显降低平滑肌细胞含量(P<0.05),与低剂量组比较,高剂量组斑块平滑肌细胞含量进一步降低(P<0.05)。
9.阻断TRPC5通道对斑块巨噬细胞含量的影响：
实验8周末,斑块内巨噬细胞含量在四组之间差异无统计学意义(P>0.05)。
结论
1.AS早期斑块发生和发展过程中TRPC5通道表达增高,高表达的TRPC5通道与斑块负荷、脂质含量、平滑肌细胞和胶原含量成正相关,与斑块内巨噬细胞含量无关。
2.体内应用TRPC5通道的特异性阻断抗体T5E3Ab可抑制AS早期斑块的发展。
背景
在AS早期斑块的发生和发展过程中平滑肌细胞迁移至内膜层、增殖并吞噬脂质,从而形成平滑肌细胞源性泡沫细胞。因此研究平滑肌细胞功能在AS斑块进展中的作用对于防治AS具有重要意义。
研究发现,细胞内Ca2+不仅在平滑肌细胞收缩和舒张功能中是最关键的调控因素,而且它的持续增加对于平滑肌细胞迁移、增殖及表型转换亦起到关键作用。平滑肌细胞内Ca2+的调控受多种离子通道的影响,近年来研究发现TRPC通道介导多种细胞的持续性钙内流,在平滑肌细胞功能中的作用日益受到重视。
本文的前述研究发现,AS早期斑块发生和发展过程中TRPC5通道表达增高,阻断TRPC5通道可降低斑块内部平滑肌细胞和胶原含量而抑制AS斑块的发展,但其中机制尚不清楚。由于以往研究发现TRPC5通道可作为脂质的细胞表面受体,TRPC通道在平滑肌细胞的迁移中发挥重要作用。因此我们设想阻断TRPC5通道可能通过调控ox-LDL诱导的平滑肌细胞内钙离子增加和钙库依赖的钙离子内流,从而影响平滑肌细胞的增殖和迁移,进而在AS早期斑块发生过程中发挥重要作用。
目的
1.研究TRPC5通道在ox-LDL引起的平滑肌细胞内钙离子和钙库依赖性钙内流中的作用；
2.探讨TRPC5通道在ox-LDL引起的平滑肌细胞增殖和迁移中的作用及机制。
方法
1.不同浓度ox-LDL对平滑肌细胞TRPC5表达影响：体外培养原代人主动脉平滑肌细胞系,根据ox-LDL浓度分为control、ox-LDL25μg/ml、50μg/ml和100μg/ml四组,刺激24h；根据50μg/ml ox-LDL的刺激时间分为control、2h、6h、24h和48h：实时荧光定量RT-PCR检测平滑肌细胞TRPC5通道mRNA表达水平,细胞免疫荧光实验检测平滑肌细胞TRPC5通道蛋白表达水平。
2.钙离子成像实验：研究ox-LDL对平滑肌细胞内钙离子浓度的影响,用thapsigargin (Tg1μM)耗竭细胞内钙库后再复钙研究钙库依赖的钙内流机制,应用TRPC5通道特异性阻断剂T5E3Ab阻断TRPC5通道后证明ox-LDL对平滑肌细胞内钙离子和钙库依赖的钙内流的作用是通过TRPC5通道。
3.膜片钳电生理实验：应用TRPC5通道特异性阻断剂T5E3Ab I阻断TRPC5通道后,研究ox-LDL诱导平滑肌细胞产生的通道电流是TRPC5通道介导的。
4. BrdU细胞增殖实验：应用TRPC5特异性阻断剂T5E3Ab阻断TRPC5通道和EGTA螫合培养基中的钙后,研究TRPC5通道在ox-LDL'(?)|起平滑肌细胞增殖中的作用。
5. Transwell细胞迁移实验：应用TRPC5特异性阻断剂T5E3Ab阻断TRPC5通道和EGTA螫合培养基中的钙后,研究TRPC5通道在ox-LDL引起平滑肌细胞迁移中的作用。
结果
1. ox-LDL对平滑肌细胞TRPC5通道mRNA和蛋白表达的影响：培养人原代主动脉平滑肌细胞系,ox-LDL可以明显增加TRPC5通道mRNA表达水平(P<0.05),具有时间依赖性和浓度依赖性。50μg/ml和100μg/ml刺激间TRPC5通道mRNA改变无统计学差异(P>0.05)。免疫荧光染色结果显示：50μg/ml ox-LDL刺激平滑肌细胞24h明显增加TRPC5阳性细胞的数量即蛋白表达量(P<0.05)。
2. TRPC5通道在ox-LDL调控的平滑肌细胞内钙离子及钙库依赖的钙内流中的作用：与对照组比较,50Mg/ml ox-LDL作用30min明显增加平滑肌细胞内钙离子浓度(P<0.05)。在无钙细胞外液中加入Tg(1μM)30min以耗竭细胞内钙库,然后再复钙(细胞外液2mM Ca2+),能够引起细胞内钙离子的持续增加,这种现象是钙库依赖的钙内流。在50μg/ml ox-LDL预刺激30min的平滑肌细胞,钙库依赖的钙内流明显增强(P<0.05)。TRPC5通道阻断剂’T5E3Ab (50μg/ml)可以抑制ox-LDL引起的细胞内钙离子增加和钙库依赖的钙内流(P<0.05)。
3. TRPC5通道在ox-LDL诱导的通道电流中的作用：Tg(1μM)在正常平滑肌细胞不引起明显的电流,而在50μg/ml ox-LDL预刺激30min的平滑肌细胞可以引起明显的通道电流(P<0.05),TRPC5通道阻断性抗体T5E3Ab (50μg/ml)可以抑制这种通道电流(P<0.05)。
4. TRPC5通道在ox-LDL诱导的平滑肌细胞增殖中的作用：50μg/ml ox-LDL明显增加平滑肌细胞的增殖(P<0.05),TRPC5特异性阻断剂T5E3Ab (50μg/ml)和钙螯合剂EGTA (10mmM)可以明显降低ox-LDL引起的平滑肌细胞增殖(P<0.05)。
5. TRPC5通道在ox-LDL诱导的平滑肌细胞迁移中的作用：50μg/ml ox-LDL明显增加平滑肌细胞的迁移(P<0.05),TRPC5特异性阻断剂T5E3Ab (50μg/ml)和钙螯合剂EGTA (10mM)可以明显降低ox-LDL引起的平滑肌细胞迁移(P<0.05)。
结论
1. ox-LDL可上调血管平滑肌细胞TRPC5通道表达；
2. TRPC5通道介导了ox-LDL引起的平滑肌细胞内钙离子增加和钙库依赖的钙内流机制；
3.特异性阻断TRPC5通道可明显降低ox-LDL引起的平滑肌细胞增殖和迁移,这可能是TRPC5通道影响AS早期斑块发生和发展的主要机制。
背景
本文前述的研究发现,AS早期斑块发生和发展过程中TRPC5通道表达增高,阻断TRPC5通道可以降低斑块内部平滑肌细胞和胶原含量而抑制AS斑块的发展,进一步体外实验研究发现ox-LDL可上调血管平滑肌细胞TRPC5通道,而TRPC5介导了ox-LDL引起的平滑肌细胞内钙离子增加和钙库依赖的钙内流机制。
由于ox-LDL是AS非常重要的致病因素,因而进一步深入研究ox-LDL对TRPC5通道功能的影响及其机制对于揭示TRPC5通道在AS中的作用具有重要意义。他汀类药物是治疗AS的最重要药物,以往研究发现他汀具有调脂以外的多效性作用(pleiotropic effects),但他汀是否影响TRPC5通道功能尚未见报道。
以往研究发现细胞内活性氧(reactive oxygen species, ROS)对于离子通道的功能有明显影响,最近报道TRPC5通道对抗氧化剂亦非常敏感。许多证据已经证明,ox-LDL可以引起细胞内ROS增加,而他汀类药物可降低细胞内ROS,因此我们推测ox-LDL和他汀可通过影响细胞内ROS而影响TRPC5通道功能,如果此假说得到验证,则不仅可揭示TRPC5通道的新调控机制,也将进一步证明他汀多效性作用的新靶点。
目的
1.探讨ox-LDL对于稳定表达的TRPC5通道功能的作用；
2.探讨辛伐他汀对于稳定表达的TRPC5通道功能的作用：
3.揭示ox-LDL和辛伐他汀调控TRPC5通道功能的可能机制。
方法
1.稳定表达TRPC5通道的HEK293细胞系(HEK-TRPC5)的培养：常规方法培养HEK-TRPC5细胞,其携带四环素(tetracycline, Te)转录抑制子,需要用1μg/ml Te刺激24h来诱导TRPC5通道的过表达。
2.钙离子成像实验：用thapsigargin (Tg1μM)耗竭细胞内钙库后再复钙研究钙库依赖的钙内流机制；记录不同浓度ox-LDL(25μg/ml、50μg/ml和100μg/ml)和辛伐他汀(10μM、20μM和50μM)对TRPC5通道介导的钙离子内流和钙库依赖的钙内流的作用。
3.膜片钳电生理实验：记录不同浓度ox-LDL和辛伐他汀对稳定表达的TRPC5通道电流的作用。
4.细胞内活性氧荧光探针实验：10μM的H2DCFDA荧光探针标记细胞内ROS,检测ox-LDL和辛伐他汀对稳定表达的TRPC5通道的HEK293细胞内ROS的影响。
结果
1.稳定表达的TRPC5通道功能研究：在Te诱导的HEK-TRPC5细胞,钙离子成像显示TRPC5通道激活剂Gd3+(100μM)可以诱导细胞内钙离子的增加(P<0.05),膜片钳记录显示TRPC5通道激活剂Gd3+(100μM)可以诱导TRPC5通道电流(P<0.05),在其电流电压曲线上显示明显手指结构,TRPC5通道特异阻断剂T5E3Ab可以抑制这些反应(P<0.05)。而在Te未诱导的细胞,不产生这些变化(P<0.05)。
2.稳定表达的TRPC5通道介导的钙库依赖性钙内流：在无钙细胞外液中加入Tg1μM30min以耗竭细胞内钙库,然后再复钙(细胞外液2mM Ca2+),能够引起细胞内钙离子的持续增加,这种现象是钙库依赖的钙内流。与未诱导的细胞比较,Te诱导的HEK-TRPC5细胞能产生非常明显的钙库依赖的钙内流(P<0.05)。
3.ox-LDL对TRPC5通道功能的影响：不同浓度ox-LDL (25μg/ml、50μg/ml和100μg/ml)可以增加TRPC5通道介导的细胞内钙离子内流和钙库依赖性钙内流的作用(P<0.05):ox-LDL也可以激活TRPC5通道介导的通道电流(P<0.05)；TRPC5通道特异性阻断剂T5E3Ab(50μg/ml)可以抑制ox-LDL引起的细胞内钙离子增加和通道电流(P<0.05)。
4.辛伐他汀对TRPC5通道功能的影响：不同浓度辛伐他汀(10μM、20μM和50μM)可以抑制TRPC5通道介导的细胞内钙离子内流和钙库依赖性钙内流(P<0.05)；不同浓度辛伐他汀也可以抑制TRPC5通道介导的通道电流(P<0.05)。
5. ox-LDL与辛伐他汀对TRPC5通道功能调节的机制：抗氧化剂(Tiron1mM和DPI10μM)可以抑制TRPC5通道激活剂Gd3+(100μM诱导细胞内钙离子的增加和TRPC5通道电流(P<0.05)。抗氧化剂可以阻断ox-LDL诱导的TRPC5通道的激活(P<0.05)；外源性H2O2(100μM可以激活TRPC5通道并逆转辛伐他汀对TRPC5通道的阻断作用(P<0.05): ox-LDL增强细胞内ROS,可以被抗氧化剂降低(P<0.05)：辛伐他汀可以降低细胞内ROS,可以被外源性H2O2逆转(P<0.05)。
结论
1.ox-LDL可以激活稳定表达的TRPC5通道引起细胞内钙离子增加和TRPC5
通道电流；
2.辛伐他汀可以抑制稳定表达的TRPC5通道引起细胞内钙离子增加和TRPC5通道电流；
3.ox-LDL和他汀调控TRPC5通道功能的机制是通过影响细胞内ROS。
Backgrounds
Coronary heart disease (CHD) has become a major cause of mortality and morbidities worldwide. Atherosclerosis (AS) is the key pathophysiology basis of CHD. Smooth muscle cells (SMCs) play a key role in the formation and development of AS plaque through migration, proliferation, phenotype transformation and production of extracellular matrix. Therefore, it is very important for prevention and treatment of CHD to study the mechanism of function changes about SMCs in the AS.
Previous studies found that intracellular Ca2+was a key second messenger and play an important role in regulating functions of SMCs. In recent years, calcium influx conducted by store-operated channels (SOCs) have been paid more and more attention in the cell function and diseases. Classical transient receptor potential (TRPC) channel is the main subtype of SOCs and can be activated by the activation of IP3leading to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.
Expression of TRPC channels was found in human coronary artery and cerebral artery; TRPC participates in the vein graft intimal hyperplasia. At the same time, studies have found that TRPC5play an important role in regulating cell function induced by lipids and is considered as a lipid receptor. Sphingosine1-phosphate (SIP) as one component of ox-LDL can activate TRPC5channel.
As we known. both ox-LDL and SMCs are important effectors in the development of AS. especially TRPC channels participated in the vein intimal hyperplasia. we can conclude that TRPC channels may play an important role in AS. However, previous studies only investigated TRPC5in vitro expression system or its physiological effect, it is unclear whether TRPC5participate in the occurrence and development of AS plaque. Therefore, we hypothesized:TRPC5channel was upregulated in AS early plaque; blocking TRPC5channel could inhibit the development of AS early plaques by reducing numbers of SMCs and collagen content of plaques. We designed a series of in vivo and in vitro experiments to prove this hypothesis.
Objectives
1. To study expression changes of TRPC channel in AS:
2. To investigate whether blocking TRPC5channels in vivo can inhibit development of AS early plaque and the underline mechanism.
Methods
1. Animal model of AS early plaque:ApoE-/-mice (male.8week old) were fed with high fat diet and underwent surgery with a perivascular collar around the right carotid artery to induce AS plaques.
2. Blocking TRPC5channels in vivo:Mice were administrated200μl of different reagents the day before surgery, and were randomly divided into4groups (20per group):PBS group. IgG group(100μg). TRPC5T5E3Ab low-dose group(50μg) and TRPC5T5E3Ab high-dose group(100μg). Mice were given with drugs once a week, and were euthanized at the end of8weeks after surgery.
3. General information of mice:At the end of8weeks, body weight, heart rate (HR) and blood pressure were measured by a noninvasive tail-cuff system. Serum levels of total cholesterol (TC). triglyceride (TG). low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured using an ELISA.
4. Histological and immunohistochemical analysis:Serial transverse cryosections of carotid artery plaques were stained with hematoxylin and eosin, picosirius red. And oil-red O staining. Corresponding sections were used for immunohistochemical analysis with the following antibodies:anti-mouse monocyte/macrophage monoclonal antibodies(MOMA-2). anti-alpha smooth muscle actin antibodies(a-SM-actin).. anti-mouse TRPC5channel antibody. The plaque area, lipid area, collagen area and the intensive optical density (IOD) of immunostaining positive area for smooth muscle and macrophage were measured with an automated image analysis system and calculated as the proportion of plaque area.
5. Real-time quantitative RT-PCR reaction was used to detect mRNA expression level of TRPC channels.
6. Verification of the specificity of T5E3Ab blocking effect on TRPC5channel: TRPC5channel was stable expressed in HEK293cell line (TRPC5-HEK293cells), patch clamp technique was used to detect blocking effects of T5E3Ab on TRPC5channel current, calcium imaging was used to detect blocking effects of T5E3Ab on intracellular calcium increasing mediated by TRPC5channel.
Results
1. Verification of blocking effects of T5E3Ab on TRPC5channel:TRPC5channel currents were recorded in TRPC5-HEK293cells, patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents and intracellular calcium increase, which could be blocked by T5E3Ab.
2. Comparing general data of mice:At the end of8weeks after surgery, there was no significant difference about body weight, SBP, DBP or serum levels of TC, TG, LDL-C, HDL-C and blood cell components among the4groups of ApoE-/-mice (P>0.05).
3. Expression of TRPC channels in AS early plaques:At the end of8weeks after surgery, TRPC5channel mRNA expressions of plaque tissues were higher than the artery without perivascular collar (P<0.05). There were no significant difference about other types of TRPC channel mRNA expressiuon and about TRPC5channel mRNA among the4groups of ApoE-/-mice (P>0.05).
4. The relationship between TRPC5mRNA expression and characteristics of AS early plaque:At the end of8weeks after surgery, compared with the artery without perivascular collar in PBS group, plaques were obvious with more lipid content and TRPC5expression (P<0.05). The correlation analysis showed that TRPC5channel expression was positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content (P<0.05). with no correlation with macrophage content of plaque (P>0.05).
5. Effect of blocking TRPC5channels on the AS plaque burden:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque burden(P<0.05). Compared with low dose group (T5E3Ab50μg). plaque burden was further reduced in high dose group (T5E3Ab100μg,.P<0.05).6. Effect of blocking TRPC5channels on lipid content of AS plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque lipid content (?<0.05). Compared with low dose group (T5E3Ab50μg). lipid content was further reduced in high dose group (T5E3Ab100ug) without statistically significance(P 7. Effect of blocking TRPC5channels on collagen content of plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased collagen content of plaques (P<0.05), Compared with low dose group (T5E3Ab50μg). collagen content of plaques was further reduced in high dose group (T5E3Ab100μg, P<0.05).
8. Effect of blocking TRPC5channels on smooth muscle cell content of plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased smooth muscle cell content of plaques (P<0.05). Compared with low dose group (T5E3Ab50μg). smooth muscle cell content of plaques was further reduced in high dose group (T5E3Ab100μg. p<0.05).
9. Effect of blocking TRPC5channels on macrophage content of plaques:At the end of8weeks after surgery, there were no significant difference about macrophage content of plaques among the4groups of ApoE-/-mice(P>0.05).
Conclusions
1. TRPC5channel is upregulated in AS early plaque development. upregulated TRPC5channel is positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content with no relation to plaque macrophage content.
2. Blocking TRPC5channel with specific blocking antibody T5E3Ab can inhibit AS early plaque development.
Backgrounds
In the development of AS early plaque, smooth muscle cells (SMCs) migrate to intima. proliferate, and phagocytize lipid resulting in formation of foam cells. Therefore it is very important for prevention and treatment of CHD to study the mechanism of functional regulation about SMCs in the AS.
In SMCs. intracellular Ca2+not only participate in the regulation of contraction and relaxation, but also regulates various cellular functions, such as proliferation. secretion, and differentiation. Regulation of SMCs intracellular Ca2+is affected by a variety of ion channels. In recent years, classical transient receptor potential (TRPC) channel was found to lead to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.
Our above study found that TRPC5channel is upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. However, the underline mechanism is unclear. Previous studies have found that TRPC5channel can be used as a lipid receptor and that TRPC channels play an important role in the migration of SMCs. Therefore we assume that the underline mechanism of TRPC5channel in inhibiting AS plaque development:TRPC5channel participate in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx, thus affect the proliferation and migration of smooth muscle cells.
Objectives
1. To investigate the role of TRPC5channels in the ox-LDL induced smooth muscle intracellular calcium increase and store-operated calcium influx;
2. To investigate the role and underline mechanism of TRPC5channels in the proliferation and migration of smooth muscle cells induced by ox-LDL
Methods
1. Culture primary human aortic smooth muscle cells, stimulate SMCs with different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) for24h, and treat SMCs with50μg/ml ox-LDL for different times (2h、6h、24h and48h). Real-time fluorescence quantitative RT-PCR was used to detect mRNA expression of TRPC5channel.
2. Calcium imaging experiments:SMCs were cultured on13mm diameter coverslips and treated with50μg/ml ox-LDL for24h. Thapsigargin (Tg) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, which is called store-operated calcium influx. With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx.
3. Patch-clamp electrophysiology experiments:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced calcium channel currents.
4. BrdU cell proliferation assay:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell proliferation.
5. Transwell cell migration experiment:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell migration.
Results
1. Effect of ox-LDL on the expression of TRPC5channel mRNA expression in SMCs:ox-LDL could significantly increase the expression of TRPC5channel mRNA with time-and concentration-dependence (P<0.05).
2. The role of TRPC5channel in ox-LDL induced intracellular calcium increasing and store-operated calcium influx of SMCs:Compared with control.50μg/ml ox-LDL induced intracellular calcium increasing (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05). Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. When pretreated with50μg/ml ox-LDL for30min. store-operated calcium influx was higher than the control (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).
3. The role of TRPC5channel in ox-LDL induced channel currents of SMCs: Compared with control.50μg/ml ox-LDL induced channel currents(P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).
4. The role of TRPC5channel in ox-LDL induced proliferation of SMCs: Compared with control,50μg/ml ox-LDL induced more proliferation of SMCs (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA (P<0.05).
5. The role of TRPC5channel in ox-LDL induced migration of SMCs: Compared with control.50μg/ml ox-LDL induced more migration of SMCs (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA(P<0.05).
Conclusions
1. TRPC5channel is unregulated by ox-LDL in artery smooth muscle cells;
2. TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx:
3. Blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells induced by ox-LDL
Background
Our above study found that TRPC5channel was upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. In vitro experiments, we revealed that TRPC5channel was unregulated by ox-LDL in artery smooth muscle cells, TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx and that blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells.
Because ox-LDL is a key factor in the pathogenesis of AS, it is very important for revealing the special role of TRPC5channel in AS to study the regulatory effect and mechanism of ox-LDL on TRPC5channel. On the other hand, statins are one of the most important drugs for treatment of AS, previous studies have shown that statins have pleiotropic effects beyond their lowering lipid. However, it has not been reported whether statins can affect the TRPC5channel function.
Meanwhile, studies found that intracellular reactive oxygen species (ROS) play an important role in the regulation of ion channels. Recent study has found that TRPC5is sensitive to antioxidant. A lot of studies have also proved that ox-LDL increase intracellular ROS and that statins reduce intracellular ROS. So we hypothesized that ox-LDL and statins regulate TRPC5channel function via affecting intracellular ROS. If this hypothesis is confirmed, we not only reveal a new regulation mechanism to regulating the TRPC5channel, but also further prove that the new target of statins pleiotropic effects.
Objectives
1. To study the effect of ox-LDL on stably expressed TRPC5channel:
2. To investigate the effect of simvastatin on stably expressed TRPC5;
3. To explore the possible mechanism of regulating ox-LDL and simvastatin effect on TRPC5channel function.
Methods
1. HEK293cell line with stable expression of TRPC5channel (HEK-TRPC5): cultured HEK-TRPC5cells in standard methods. Because the cell carries the tetracycline (Te) transcriptional repressor. TRPC5channel overexpression was induced by1μg/ml Te for24h.
2. Calcium imaging experiments:Thapsigargin (Tg1μM) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution. which is called store-operated calcium influx. To study effects of different concentrations of ox-LDL (25μg/ml.50μg/ml.100μ g/ml) and simvastatin (10μM,20μM,50μM) on TRPC5channel-mediated calcium increasing and store-operated calcium influx.
3. Patch-clamp electrophysiology experiments:we explore effects of different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) and simvastatin (10μ M，20μM,50μM) on TRPC5channel currents.
4. Intracellular ROS fluorescence probe experiments were used to detect effects of ox-LDL and simvastatin on the intracellular ROS in TRPC5channel stable expressed HEK293cells.
Results
1. Study of TRPC5channel function in HEK-TRPC5cells: TRPC5channel overexpression was induced by1μg/ml Te. In Te induced (Te+)cells. patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents (P<0.05) and intracellular calcium increase (P<0.05), which could be blocked by T5E3Ab(P<0.05). In non-induced (Te-) cells, there was no response to Gd33+(100μM).
2. Store-operated calcium influx in HEK-TRPC5cells:Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. Compared with Te-cells, store-operated calcium influx was higher in Te+cells (P<0.05).
3. Effect of ox-LDL on stably expressed TRPC5channel:Different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents, which could be inhibited by specific TRPC5channel blocker T5E3Ab (P<0.05).
4. Effect of simvastatin on stably expressed TRPC5channel:Different concentrations of simvastatin (10μM,20μM,50μM) could inhibit TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents (P<0.05).
5. The possible mechanism of regulating TRPC5channel function by ox-LDL and simvastatin:Antioxidants (tiron,1mM and DPI,10μM) could inhibit TRPC5channel activator Gd3+induced intracellular calcium increase and TRPC5channel currents (P<0.05). Antioxidants could also block the activation of TRPC5channel induced by ox-LDL (P<0.05). Exogenous H2O2(100μM) could activate TRPC5channel and overcome the inhibiting effect of simvastatin on TRPC5channel (P<0.05). ox-LDL enhanced intracellular ROS which could be reduced by antioxidants (P<0.05):simvastatin could reduce the intracellular ROS which could be reversed by exogenous H2O2(P<0.05).
Conclusions
1. ox-LDL increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents.
2. Simvastatin could inhibit TRPC5channel mediated intracellular calcium increase. store-operated calcium influx and TRPC5channel currents.
3. ox-LDL and simvastatin regulated TRPC5channel function via affecting intracellular ROS.
冠心病已成为严重威胁人类健康的流行病,动脉粥样硬化(atherosclerosis, AS)斑块的发生和发展是其病理生理基础。在斑块的发生和发展过程中,平滑肌细胞的迁移、增殖、表型转换及细胞外基质的产生起到重要作用。因此,研究平滑肌细胞功能在AS斑块发生和发展中的作用对于防治冠心病具有重要意义。
研究发现平滑肌细胞内Ca2+作为第二信使对于平滑肌细胞的功能调节起关键作用。近年来钙库操纵性通道(store-operated channels, SOCs)介导的钙内流在细胞功能及疾病中的作用日益受到重视,特别是经典型瞬时感受器电位通道(classical transient receptor potential channel, TRPC channel)是组成SOCs的主要通道,能被G蛋白偶联磷脂酶C所产生的IP3激活而产生持续的钙内流,它们在平滑肌细胞功能中的作用日益受到重视。近年研究发现人冠状动脉和脑动脉有TRPC通道的表达,TRPC1参与了移植静脉内膜增生。此外,TRPC5在脂质对细胞功能的调节中有重要作用,具有脂质离子亚型受体特性,1-磷酸鞘氨醇作为氧化低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)的成分也能够激活TRPC5。
由于ox-LDL和平滑肌细胞在AS过程中的重要作用,特别是TRPC通道在静脉内膜增生中的作用提示：TRPC通道在AS过程中可能担当重要角色。以往研究仅研究了体外表达系统或细胞系中TRPC5的生理作用,对其在AS发生和发展过程中的作用知之甚少。因此我们提出如下假说：AS早期斑块中TRPC5通道表达增高,阻断TRPC5通道可以通过降低斑块内平滑肌细胞和胶原含量而抑制AS斑块的发展。我们设计了一系列体内和体外实验来证明这一假说。
目的
1.研究AS斑块发生和发展过程中TRPC5通道的表达情况；
2.探讨体内特异性阻断TRPC5通道能否抑制AS早期斑块的发展及其可能机制。
方法
1.AS早期斑块模型建立：8周龄雄性apoE-/-小鼠,全程高脂饮食,行右侧颈总动脉套管(缩窄性硅胶管)术以诱发AS早期斑块。
2.体内阻断TRPC5:随机分为4组：PBS组,尾静脉注射无菌PBS200μl; IgG组：TRPC5通道特异性阻断剂T5E3Ab相同种属来源的IgG100μg,用无菌PBS200μl溶解；TRPC5通道特异性阻断剂T5E3Ab低剂量组：T5E3Ab50μg,用无菌PBS200μl溶解；TRPC5通道特异性阻断剂T5E3Ab高剂量组：T5E3Ab100μg,用无菌PBS200μl溶解。套管术前1天尾静脉给药,每周给药一次,8周末处死,每组20只。
3.动物一般状况的评价：实验8周末测量体重、心率和尾动脉血压,收集小鼠血液,检测小鼠的血细胞、总胆固醇(TC)、甘油三脂(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的水平。
4.病理组织学检测：颈动脉斑块组织学切片分别行苏木素-伊红染色、天狼猩红染色和油红O染色,并进行免疫组织化学染色观察巨噬细胞(MOMA-2)、平滑肌肌动蛋白(a-SM-actin)和TRPC5局部表达。测量斑块面积、胶原、脂质、平滑肌细胞和巨噬细胞免疫组化阳性染色面积,并计算它们占斑块面积的比例。
5.实时定量RT-PCR:检测斑块TRPC通道mRNA表达水平。
6. T5E3Ab对TRPC5通道的特异性阻断作用的验证：培养稳定表达TRPC5通道的HEK293细胞系(TRPC5-HEK293):膜片钳技术检测TRPC5-HEK293细胞的TRPC5通道介导的电流及T5E3Ab的阻断作用；钙离子成像检测TRPC5-HEK293细胞的TRPC5通道介导的细胞内钙离子增加及T5E3Ab的阻断作用。检测含T5E3Ab小鼠血清给对TRPC5通道的抑制作用。
结果
1.T5E3Ab对TRPC5通道的阻断作用：在诱导过表达TRPC5通道的细胞,TRPC5通道的激活剂Gd3+(100μM)可产生细胞内钙离子的增加和通道电流,而T5E3Ab (25μg/ml能够抑制这些反应。而在未诱导表达的细胞,不产生这些变化。与注射对照IgG100μg组小鼠血清比较,含T5E3Ab小鼠血清给药后1天对TRPC5通道抑制率95%(P<0.05),而1周末抑制率为50%(P<0.05,表1),2周末抑制作用消失(P>0.05)。
2.动物一般状况比较：实验8周末,4组ApoE-/-小鼠的体重、血压、血清TC、TG、LDL-C、HDL-C和血细胞成份无显著性差异(P>0.05)。
3.AS早期斑块TRPC通道mRNA表达改变：实验8周末,与未套管侧颈动脉比较,PBS组ApoE-/-小鼠套管处颈动脉TRPC5通道mRNA的表达明显增高(P<0.05),其他亚型TRPC通道无显著差异(P>0.05)。4组ApoE-/-小鼠之间TRPC5通道mRNA表达水平无统计学差异(P>0.05)。
4.AS早期斑块TRPC5通道表达与斑块特征的关系：实验8周末,与未套管侧颈动脉比较,在未干预组(PBS组),ApoE-/-小鼠套管处颈动脉有明显斑块,斑块负荷、脂质含量和TRPC5通道表达含量明显增加(P<0.05)。相关分析表明,PBS组TRPC5通道表达与斑块负荷、脂质含量、平滑肌细胞和胶原含量成正相关(P<0.05),与斑块内巨噬细胞含量无关(P>0.05)。
5.阻断TRPC5通道对AS斑块负荷的影响：实验8周末,与未干预组(PBS组)和IgG (100μg)组比较,TRPC5通道阻断组斑块负荷明显降低(P<0.05),与低剂量组(T5E3Ab50μg)比较,高剂量组(T5E3Ab100μg)斑块负荷进一步降低(P<0.05)。
6.阻断TRPC5通道对AS斑块脂质含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,阻断TRPC5通道可以明显降低脂质含量(P<0.05),虽然高剂量组脂质含量比低剂量组降低,但是组间差别无统计学意义(P>0.05)。
7.阻断TRPC5通道对斑块胶原含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,TRPC5通道阻断组斑块胶原含量明显降低(P<0.05),与低剂量组比较,高剂量组斑块胶原含量进一步降低(P<0.05)。
8.阻断TRPC5通道对AS斑块平滑肌细胞含量的影响：实验8周末,与未干预组(PBS组)和IgG组比较,阻断TRPC5通道可以明显降低平滑肌细胞含量(P<0.05),与低剂量组比较,高剂量组斑块平滑肌细胞含量进一步降低(P<0.05)。
9.阻断TRPC5通道对斑块巨噬细胞含量的影响：
实验8周末,斑块内巨噬细胞含量在四组之间差异无统计学意义(P>0.05)。
结论
1.AS早期斑块发生和发展过程中TRPC5通道表达增高,高表达的TRPC5通道与斑块负荷、脂质含量、平滑肌细胞和胶原含量成正相关,与斑块内巨噬细胞含量无关。
2.体内应用TRPC5通道的特异性阻断抗体T5E3Ab可抑制AS早期斑块的发展。
背景
在AS早期斑块的发生和发展过程中平滑肌细胞迁移至内膜层、增殖并吞噬脂质,从而形成平滑肌细胞源性泡沫细胞。因此研究平滑肌细胞功能在AS斑块进展中的作用对于防治AS具有重要意义。
研究发现,细胞内Ca2+不仅在平滑肌细胞收缩和舒张功能中是最关键的调控因素,而且它的持续增加对于平滑肌细胞迁移、增殖及表型转换亦起到关键作用。平滑肌细胞内Ca2+的调控受多种离子通道的影响,近年来研究发现TRPC通道介导多种细胞的持续性钙内流,在平滑肌细胞功能中的作用日益受到重视。
本文的前述研究发现,AS早期斑块发生和发展过程中TRPC5通道表达增高,阻断TRPC5通道可降低斑块内部平滑肌细胞和胶原含量而抑制AS斑块的发展,但其中机制尚不清楚。由于以往研究发现TRPC5通道可作为脂质的细胞表面受体,TRPC通道在平滑肌细胞的迁移中发挥重要作用。因此我们设想阻断TRPC5通道可能通过调控ox-LDL诱导的平滑肌细胞内钙离子增加和钙库依赖的钙离子内流,从而影响平滑肌细胞的增殖和迁移,进而在AS早期斑块发生过程中发挥重要作用。
目的
1.研究TRPC5通道在ox-LDL引起的平滑肌细胞内钙离子和钙库依赖性钙内流中的作用；
2.探讨TRPC5通道在ox-LDL引起的平滑肌细胞增殖和迁移中的作用及机制。
方法
1.不同浓度ox-LDL对平滑肌细胞TRPC5表达影响：体外培养原代人主动脉平滑肌细胞系,根据ox-LDL浓度分为control、ox-LDL25μg/ml、50μg/ml和100μg/ml四组,刺激24h；根据50μg/ml ox-LDL的刺激时间分为control、2h、6h、24h和48h：实时荧光定量RT-PCR检测平滑肌细胞TRPC5通道mRNA表达水平,细胞免疫荧光实验检测平滑肌细胞TRPC5通道蛋白表达水平。
2.钙离子成像实验：研究ox-LDL对平滑肌细胞内钙离子浓度的影响,用thapsigargin (Tg1μM)耗竭细胞内钙库后再复钙研究钙库依赖的钙内流机制,应用TRPC5通道特异性阻断剂T5E3Ab阻断TRPC5通道后证明ox-LDL对平滑肌细胞内钙离子和钙库依赖的钙内流的作用是通过TRPC5通道。
3.膜片钳电生理实验：应用TRPC5通道特异性阻断剂T5E3Ab I阻断TRPC5通道后,研究ox-LDL诱导平滑肌细胞产生的通道电流是TRPC5通道介导的。
4. BrdU细胞增殖实验：应用TRPC5特异性阻断剂T5E3Ab阻断TRPC5通道和EGTA螫合培养基中的钙后,研究TRPC5通道在ox-LDL'(?)|起平滑肌细胞增殖中的作用。
5. Transwell细胞迁移实验：应用TRPC5特异性阻断剂T5E3Ab阻断TRPC5通道和EGTA螫合培养基中的钙后,研究TRPC5通道在ox-LDL引起平滑肌细胞迁移中的作用。
结果
1. ox-LDL对平滑肌细胞TRPC5通道mRNA和蛋白表达的影响：培养人原代主动脉平滑肌细胞系,ox-LDL可以明显增加TRPC5通道mRNA表达水平(P<0.05),具有时间依赖性和浓度依赖性。50μg/ml和100μg/ml刺激间TRPC5通道mRNA改变无统计学差异(P>0.05)。免疫荧光染色结果显示：50μg/ml ox-LDL刺激平滑肌细胞24h明显增加TRPC5阳性细胞的数量即蛋白表达量(P<0.05)。
2. TRPC5通道在ox-LDL调控的平滑肌细胞内钙离子及钙库依赖的钙内流中的作用：与对照组比较,50Mg/ml ox-LDL作用30min明显增加平滑肌细胞内钙离子浓度(P<0.05)。在无钙细胞外液中加入Tg(1μM)30min以耗竭细胞内钙库,然后再复钙(细胞外液2mM Ca2+),能够引起细胞内钙离子的持续增加,这种现象是钙库依赖的钙内流。在50μg/ml ox-LDL预刺激30min的平滑肌细胞,钙库依赖的钙内流明显增强(P<0.05)。TRPC5通道阻断剂’T5E3Ab (50μg/ml)可以抑制ox-LDL引起的细胞内钙离子增加和钙库依赖的钙内流(P<0.05)。
3. TRPC5通道在ox-LDL诱导的通道电流中的作用：Tg(1μM)在正常平滑肌细胞不引起明显的电流,而在50μg/ml ox-LDL预刺激30min的平滑肌细胞可以引起明显的通道电流(P<0.05),TRPC5通道阻断性抗体T5E3Ab (50μg/ml)可以抑制这种通道电流(P<0.05)。
4. TRPC5通道在ox-LDL诱导的平滑肌细胞增殖中的作用：50μg/ml ox-LDL明显增加平滑肌细胞的增殖(P<0.05),TRPC5特异性阻断剂T5E3Ab (50μg/ml)和钙螯合剂EGTA (10mmM)可以明显降低ox-LDL引起的平滑肌细胞增殖(P<0.05)。
5. TRPC5通道在ox-LDL诱导的平滑肌细胞迁移中的作用：50μg/ml ox-LDL明显增加平滑肌细胞的迁移(P<0.05),TRPC5特异性阻断剂T5E3Ab (50μg/ml)和钙螯合剂EGTA (10mM)可以明显降低ox-LDL引起的平滑肌细胞迁移(P<0.05)。
结论
1. ox-LDL可上调血管平滑肌细胞TRPC5通道表达；
2. TRPC5通道介导了ox-LDL引起的平滑肌细胞内钙离子增加和钙库依赖的钙内流机制；
3.特异性阻断TRPC5通道可明显降低ox-LDL引起的平滑肌细胞增殖和迁移,这可能是TRPC5通道影响AS早期斑块发生和发展的主要机制。
背景
本文前述的研究发现,AS早期斑块发生和发展过程中TRPC5通道表达增高,阻断TRPC5通道可以降低斑块内部平滑肌细胞和胶原含量而抑制AS斑块的发展,进一步体外实验研究发现ox-LDL可上调血管平滑肌细胞TRPC5通道,而TRPC5介导了ox-LDL引起的平滑肌细胞内钙离子增加和钙库依赖的钙内流机制。
由于ox-LDL是AS非常重要的致病因素,因而进一步深入研究ox-LDL对TRPC5通道功能的影响及其机制对于揭示TRPC5通道在AS中的作用具有重要意义。他汀类药物是治疗AS的最重要药物,以往研究发现他汀具有调脂以外的多效性作用(pleiotropic effects),但他汀是否影响TRPC5通道功能尚未见报道。
以往研究发现细胞内活性氧(reactive oxygen species, ROS)对于离子通道的功能有明显影响,最近报道TRPC5通道对抗氧化剂亦非常敏感。许多证据已经证明,ox-LDL可以引起细胞内ROS增加,而他汀类药物可降低细胞内ROS,因此我们推测ox-LDL和他汀可通过影响细胞内ROS而影响TRPC5通道功能,如果此假说得到验证,则不仅可揭示TRPC5通道的新调控机制,也将进一步证明他汀多效性作用的新靶点。
目的
1.探讨ox-LDL对于稳定表达的TRPC5通道功能的作用；
2.探讨辛伐他汀对于稳定表达的TRPC5通道功能的作用：
3.揭示ox-LDL和辛伐他汀调控TRPC5通道功能的可能机制。
方法
1.稳定表达TRPC5通道的HEK293细胞系(HEK-TRPC5)的培养：常规方法培养HEK-TRPC5细胞,其携带四环素(tetracycline, Te)转录抑制子,需要用1μg/ml Te刺激24h来诱导TRPC5通道的过表达。
2.钙离子成像实验：用thapsigargin (Tg1μM)耗竭细胞内钙库后再复钙研究钙库依赖的钙内流机制；记录不同浓度ox-LDL(25μg/ml、50μg/ml和100μg/ml)和辛伐他汀(10μM、20μM和50μM)对TRPC5通道介导的钙离子内流和钙库依赖的钙内流的作用。
3.膜片钳电生理实验：记录不同浓度ox-LDL和辛伐他汀对稳定表达的TRPC5通道电流的作用。
4.细胞内活性氧荧光探针实验：10μM的H2DCFDA荧光探针标记细胞内ROS,检测ox-LDL和辛伐他汀对稳定表达的TRPC5通道的HEK293细胞内ROS的影响。
结果
1.稳定表达的TRPC5通道功能研究：在Te诱导的HEK-TRPC5细胞,钙离子成像显示TRPC5通道激活剂Gd3+(100μM)可以诱导细胞内钙离子的增加(P<0.05),膜片钳记录显示TRPC5通道激活剂Gd3+(100μM)可以诱导TRPC5通道电流(P<0.05),在其电流电压曲线上显示明显手指结构,TRPC5通道特异阻断剂T5E3Ab可以抑制这些反应(P<0.05)。而在Te未诱导的细胞,不产生这些变化(P<0.05)。
2.稳定表达的TRPC5通道介导的钙库依赖性钙内流：在无钙细胞外液中加入Tg1μM30min以耗竭细胞内钙库,然后再复钙(细胞外液2mM Ca2+),能够引起细胞内钙离子的持续增加,这种现象是钙库依赖的钙内流。与未诱导的细胞比较,Te诱导的HEK-TRPC5细胞能产生非常明显的钙库依赖的钙内流(P<0.05)。
3.ox-LDL对TRPC5通道功能的影响：不同浓度ox-LDL (25μg/ml、50μg/ml和100μg/ml)可以增加TRPC5通道介导的细胞内钙离子内流和钙库依赖性钙内流的作用(P<0.05):ox-LDL也可以激活TRPC5通道介导的通道电流(P<0.05)；TRPC5通道特异性阻断剂T5E3Ab(50μg/ml)可以抑制ox-LDL引起的细胞内钙离子增加和通道电流(P<0.05)。
4.辛伐他汀对TRPC5通道功能的影响：不同浓度辛伐他汀(10μM、20μM和50μM)可以抑制TRPC5通道介导的细胞内钙离子内流和钙库依赖性钙内流(P<0.05)；不同浓度辛伐他汀也可以抑制TRPC5通道介导的通道电流(P<0.05)。
5. ox-LDL与辛伐他汀对TRPC5通道功能调节的机制：抗氧化剂(Tiron1mM和DPI10μM)可以抑制TRPC5通道激活剂Gd3+(100μM诱导细胞内钙离子的增加和TRPC5通道电流(P<0.05)。抗氧化剂可以阻断ox-LDL诱导的TRPC5通道的激活(P<0.05)；外源性H2O2(100μM可以激活TRPC5通道并逆转辛伐他汀对TRPC5通道的阻断作用(P<0.05): ox-LDL增强细胞内ROS,可以被抗氧化剂降低(P<0.05)：辛伐他汀可以降低细胞内ROS,可以被外源性H2O2逆转(P<0.05)。
结论
1.ox-LDL可以激活稳定表达的TRPC5通道引起细胞内钙离子增加和TRPC5
通道电流；
2.辛伐他汀可以抑制稳定表达的TRPC5通道引起细胞内钙离子增加和TRPC5通道电流；
3.ox-LDL和他汀调控TRPC5通道功能的机制是通过影响细胞内ROS。
Backgrounds
Coronary heart disease (CHD) has become a major cause of mortality and morbidities worldwide. Atherosclerosis (AS) is the key pathophysiology basis of CHD. Smooth muscle cells (SMCs) play a key role in the formation and development of AS plaque through migration, proliferation, phenotype transformation and production of extracellular matrix. Therefore, it is very important for prevention and treatment of CHD to study the mechanism of function changes about SMCs in the AS.
Previous studies found that intracellular Ca2+was a key second messenger and play an important role in regulating functions of SMCs. In recent years, calcium influx conducted by store-operated channels (SOCs) have been paid more and more attention in the cell function and diseases. Classical transient receptor potential (TRPC) channel is the main subtype of SOCs and can be activated by the activation of IP3leading to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.
Expression of TRPC channels was found in human coronary artery and cerebral artery; TRPC participates in the vein graft intimal hyperplasia. At the same time, studies have found that TRPC5play an important role in regulating cell function induced by lipids and is considered as a lipid receptor. Sphingosine1-phosphate (SIP) as one component of ox-LDL can activate TRPC5channel.
As we known. both ox-LDL and SMCs are important effectors in the development of AS. especially TRPC channels participated in the vein intimal hyperplasia. we can conclude that TRPC channels may play an important role in AS. However, previous studies only investigated TRPC5in vitro expression system or its physiological effect, it is unclear whether TRPC5participate in the occurrence and development of AS plaque. Therefore, we hypothesized:TRPC5channel was upregulated in AS early plaque; blocking TRPC5channel could inhibit the development of AS early plaques by reducing numbers of SMCs and collagen content of plaques. We designed a series of in vivo and in vitro experiments to prove this hypothesis.
Objectives
1. To study expression changes of TRPC channel in AS:
2. To investigate whether blocking TRPC5channels in vivo can inhibit development of AS early plaque and the underline mechanism.
Methods
1. Animal model of AS early plaque:ApoE-/-mice (male.8week old) were fed with high fat diet and underwent surgery with a perivascular collar around the right carotid artery to induce AS plaques.
2. Blocking TRPC5channels in vivo:Mice were administrated200μl of different reagents the day before surgery, and were randomly divided into4groups (20per group):PBS group. IgG group(100μg). TRPC5T5E3Ab low-dose group(50μg) and TRPC5T5E3Ab high-dose group(100μg). Mice were given with drugs once a week, and were euthanized at the end of8weeks after surgery.
3. General information of mice:At the end of8weeks, body weight, heart rate (HR) and blood pressure were measured by a noninvasive tail-cuff system. Serum levels of total cholesterol (TC). triglyceride (TG). low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured using an ELISA.
4. Histological and immunohistochemical analysis:Serial transverse cryosections of carotid artery plaques were stained with hematoxylin and eosin, picosirius red. And oil-red O staining. Corresponding sections were used for immunohistochemical analysis with the following antibodies:anti-mouse monocyte/macrophage monoclonal antibodies(MOMA-2). anti-alpha smooth muscle actin antibodies(a-SM-actin).. anti-mouse TRPC5channel antibody. The plaque area, lipid area, collagen area and the intensive optical density (IOD) of immunostaining positive area for smooth muscle and macrophage were measured with an automated image analysis system and calculated as the proportion of plaque area.
5. Real-time quantitative RT-PCR reaction was used to detect mRNA expression level of TRPC channels.
6. Verification of the specificity of T5E3Ab blocking effect on TRPC5channel: TRPC5channel was stable expressed in HEK293cell line (TRPC5-HEK293cells), patch clamp technique was used to detect blocking effects of T5E3Ab on TRPC5channel current, calcium imaging was used to detect blocking effects of T5E3Ab on intracellular calcium increasing mediated by TRPC5channel.
Results
1. Verification of blocking effects of T5E3Ab on TRPC5channel:TRPC5channel currents were recorded in TRPC5-HEK293cells, patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents and intracellular calcium increase, which could be blocked by T5E3Ab.
2. Comparing general data of mice:At the end of8weeks after surgery, there was no significant difference about body weight, SBP, DBP or serum levels of TC, TG, LDL-C, HDL-C and blood cell components among the4groups of ApoE-/-mice (P>0.05).
3. Expression of TRPC channels in AS early plaques:At the end of8weeks after surgery, TRPC5channel mRNA expressions of plaque tissues were higher than the artery without perivascular collar (P<0.05). There were no significant difference about other types of TRPC channel mRNA expressiuon and about TRPC5channel mRNA among the4groups of ApoE-/-mice (P>0.05).
4. The relationship between TRPC5mRNA expression and characteristics of AS early plaque:At the end of8weeks after surgery, compared with the artery without perivascular collar in PBS group, plaques were obvious with more lipid content and TRPC5expression (P<0.05). The correlation analysis showed that TRPC5channel expression was positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content (P<0.05). with no correlation with macrophage content of plaque (P>0.05).
5. Effect of blocking TRPC5channels on the AS plaque burden:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque burden(P<0.05). Compared with low dose group (T5E3Ab50μg). plaque burden was further reduced in high dose group (T5E3Ab100μg,.P<0.05).6. Effect of blocking TRPC5channels on lipid content of AS plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque lipid content (?<0.05). Compared with low dose group (T5E3Ab50μg). lipid content was further reduced in high dose group (T5E3Ab100ug) without statistically significance(P
8. Effect of blocking TRPC5channels on smooth muscle cell content of plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased smooth muscle cell content of plaques (P<0.05). Compared with low dose group (T5E3Ab50μg). smooth muscle cell content of plaques was further reduced in high dose group (T5E3Ab100μg. p<0.05).
9. Effect of blocking TRPC5channels on macrophage content of plaques:At the end of8weeks after surgery, there were no significant difference about macrophage content of plaques among the4groups of ApoE-/-mice(P>0.05).
Conclusions
1. TRPC5channel is upregulated in AS early plaque development. upregulated TRPC5channel is positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content with no relation to plaque macrophage content.
2. Blocking TRPC5channel with specific blocking antibody T5E3Ab can inhibit AS early plaque development.
Backgrounds
In the development of AS early plaque, smooth muscle cells (SMCs) migrate to intima. proliferate, and phagocytize lipid resulting in formation of foam cells. Therefore it is very important for prevention and treatment of CHD to study the mechanism of functional regulation about SMCs in the AS.
In SMCs. intracellular Ca2+not only participate in the regulation of contraction and relaxation, but also regulates various cellular functions, such as proliferation. secretion, and differentiation. Regulation of SMCs intracellular Ca2+is affected by a variety of ion channels. In recent years, classical transient receptor potential (TRPC) channel was found to lead to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.
Our above study found that TRPC5channel is upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. However, the underline mechanism is unclear. Previous studies have found that TRPC5channel can be used as a lipid receptor and that TRPC channels play an important role in the migration of SMCs. Therefore we assume that the underline mechanism of TRPC5channel in inhibiting AS plaque development:TRPC5channel participate in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx, thus affect the proliferation and migration of smooth muscle cells.
Objectives
1. To investigate the role of TRPC5channels in the ox-LDL induced smooth muscle intracellular calcium increase and store-operated calcium influx;
2. To investigate the role and underline mechanism of TRPC5channels in the proliferation and migration of smooth muscle cells induced by ox-LDL
Methods
1. Culture primary human aortic smooth muscle cells, stimulate SMCs with different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) for24h, and treat SMCs with50μg/ml ox-LDL for different times (2h、6h、24h and48h). Real-time fluorescence quantitative RT-PCR was used to detect mRNA expression of TRPC5channel.
2. Calcium imaging experiments:SMCs were cultured on13mm diameter coverslips and treated with50μg/ml ox-LDL for24h. Thapsigargin (Tg) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, which is called store-operated calcium influx. With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx.
3. Patch-clamp electrophysiology experiments:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced calcium channel currents.
4. BrdU cell proliferation assay:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell proliferation.
5. Transwell cell migration experiment:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell migration.
Results
1. Effect of ox-LDL on the expression of TRPC5channel mRNA expression in SMCs:ox-LDL could significantly increase the expression of TRPC5channel mRNA with time-and concentration-dependence (P<0.05).
2. The role of TRPC5channel in ox-LDL induced intracellular calcium increasing and store-operated calcium influx of SMCs:Compared with control.50μg/ml ox-LDL induced intracellular calcium increasing (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05). Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. When pretreated with50μg/ml ox-LDL for30min. store-operated calcium influx was higher than the control (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).
3. The role of TRPC5channel in ox-LDL induced channel currents of SMCs: Compared with control.50μg/ml ox-LDL induced channel currents(P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).
4. The role of TRPC5channel in ox-LDL induced proliferation of SMCs: Compared with control,50μg/ml ox-LDL induced more proliferation of SMCs (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA (P<0.05).
5. The role of TRPC5channel in ox-LDL induced migration of SMCs: Compared with control.50μg/ml ox-LDL induced more migration of SMCs (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA(P<0.05).
Conclusions
1. TRPC5channel is unregulated by ox-LDL in artery smooth muscle cells;
2. TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx:
3. Blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells induced by ox-LDL
Background
Our above study found that TRPC5channel was upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. In vitro experiments, we revealed that TRPC5channel was unregulated by ox-LDL in artery smooth muscle cells, TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx and that blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells.
Because ox-LDL is a key factor in the pathogenesis of AS, it is very important for revealing the special role of TRPC5channel in AS to study the regulatory effect and mechanism of ox-LDL on TRPC5channel. On the other hand, statins are one of the most important drugs for treatment of AS, previous studies have shown that statins have pleiotropic effects beyond their lowering lipid. However, it has not been reported whether statins can affect the TRPC5channel function.
Meanwhile, studies found that intracellular reactive oxygen species (ROS) play an important role in the regulation of ion channels. Recent study has found that TRPC5is sensitive to antioxidant. A lot of studies have also proved that ox-LDL increase intracellular ROS and that statins reduce intracellular ROS. So we hypothesized that ox-LDL and statins regulate TRPC5channel function via affecting intracellular ROS. If this hypothesis is confirmed, we not only reveal a new regulation mechanism to regulating the TRPC5channel, but also further prove that the new target of statins pleiotropic effects.
Objectives
1. To study the effect of ox-LDL on stably expressed TRPC5channel:
2. To investigate the effect of simvastatin on stably expressed TRPC5;
3. To explore the possible mechanism of regulating ox-LDL and simvastatin effect on TRPC5channel function.
Methods
1. HEK293cell line with stable expression of TRPC5channel (HEK-TRPC5): cultured HEK-TRPC5cells in standard methods. Because the cell carries the tetracycline (Te) transcriptional repressor. TRPC5channel overexpression was induced by1μg/ml Te for24h.
2. Calcium imaging experiments:Thapsigargin (Tg1μM) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution. which is called store-operated calcium influx. To study effects of different concentrations of ox-LDL (25μg/ml.50μg/ml.100μ g/ml) and simvastatin (10μM,20μM,50μM) on TRPC5channel-mediated calcium increasing and store-operated calcium influx.
3. Patch-clamp electrophysiology experiments:we explore effects of different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) and simvastatin (10μ M，20μM,50μM) on TRPC5channel currents.
4. Intracellular ROS fluorescence probe experiments were used to detect effects of ox-LDL and simvastatin on the intracellular ROS in TRPC5channel stable expressed HEK293cells.
Results
1. Study of TRPC5channel function in HEK-TRPC5cells: TRPC5channel overexpression was induced by1μg/ml Te. In Te induced (Te+)cells. patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents (P<0.05) and intracellular calcium increase (P<0.05), which could be blocked by T5E3Ab(P<0.05). In non-induced (Te-) cells, there was no response to Gd33+(100μM).
2. Store-operated calcium influx in HEK-TRPC5cells:Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. Compared with Te-cells, store-operated calcium influx was higher in Te+cells (P<0.05).
3. Effect of ox-LDL on stably expressed TRPC5channel:Different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents, which could be inhibited by specific TRPC5channel blocker T5E3Ab (P<0.05).
4. Effect of simvastatin on stably expressed TRPC5channel:Different concentrations of simvastatin (10μM,20μM,50μM) could inhibit TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents (P<0.05).
5. The possible mechanism of regulating TRPC5channel function by ox-LDL and simvastatin:Antioxidants (tiron,1mM and DPI,10μM) could inhibit TRPC5channel activator Gd3+induced intracellular calcium increase and TRPC5channel currents (P<0.05). Antioxidants could also block the activation of TRPC5channel induced by ox-LDL (P<0.05). Exogenous H2O2(100μM) could activate TRPC5channel and overcome the inhibiting effect of simvastatin on TRPC5channel (P<0.05). ox-LDL enhanced intracellular ROS which could be reduced by antioxidants (P<0.05):simvastatin could reduce the intracellular ROS which could be reversed by exogenous H2O2(P<0.05).
Conclusions
1. ox-LDL increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents.
2. Simvastatin could inhibit TRPC5channel mediated intracellular calcium increase. store-operated calcium influx and TRPC5channel currents.
3. ox-LDL and simvastatin regulated TRPC5channel function via affecting intracellular ROS.
引文
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1. Berridge MJ. Smooth muscle cell calcium activation mechanisms. J Physiol.2008 Nov 1;586(Pt21):5047-61.
2. Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med Indones.2007 Apr-Jun:39(2):86-93.
3. Schachter M. Vascular smooth muscle cell migration, atherosclerosis、and calcium channel blockers. Int J Cardiol.1997 Dec 31:62 Suppl 2:S85-90.
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12. Clapham DE. TRP channels as cellular sensors. Nature.2003.426(6966):517-524.
13. Minke B. TRP channels and Ca2+ signaling. Cell Calcium.2006.40(3):261-275.
14. Nilius B. Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev.2007 Jan;87(1):165-217.
15. Jordt SE, Ehrlich BE. TRP channels in disease. Subcell Biochem. 2007:45:253-71.
16. Zhu Z. Luo Z. Ma S. Liu D. TRP channels and their implications in metabolic diseases. Pflugers Arch.2011 Feb:461(2):211-23.
17. Yip H,Chan WY, Leung PC, Kwan HY, Liu C. Huang Y. Michel V, Yew DT. Yao X, Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries. Histochem Cell Biol,2004,122(6):553-561.
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