摘要
中文摘要Ⅰ补体膜调节蛋白抗动脉粥样硬化作用的实验研究
背景
补体系统的可溶性成分多以前体的形式存在于外周血,当病原入侵或者组织损伤时,可通过经典途径、凝集素激活途径或替代途径被激活。激活的补体系统通过释放白细胞趋化因子C3a和C5a促进炎症反应,并可通过合成膜攻击复合体介导细胞损伤。为了防止自体细胞受到补体系统的攻击,生物体内的多种细胞,尤其是那些接触血液循环的血细胞和内皮细胞,表达了多种结合于细胞膜的补体调节蛋白,其中包括通过GPI结构与细胞膜锚链的CD55和CD59。CD55可以通过促进C3和C5转化酶的降解而调控补体激活,而CD59主要通过抑制膜攻击复合体的形成而发挥作用。
近年的研究表明,动脉粥样硬化(atherosclerosis,AS)是一种发生于动脉血管壁的慢性炎症,天然性与获得性免疫系统的多个成分参与了其发生发展过程。作为天然性免疫系统的一个重要组成部分,补体系统致AS的作用已为许多研究所揭示。各种补体系统的激活产物,调节蛋白和补体受体均可在AS病灶,尤其是易损斑块和已破裂斑块中被探及,并且对人体病变的研究发现,膜攻击复合体的沉积量与病灶进展程度呈现正相关。对补体敲基因动物的研究亦初步揭示了补体致AS的机理:补体激活的中心分子—C3的基因缺陷可以通过上调血甘油三酯的浓度促进AS病灶的发展;C6基因缺陷可以明显减少高胆固醇饮食所诱发的兔主AS,而C5基因敲除却并没有明显地影响ApoE~(-/-)小鼠的斑块大小,意味着补体终末效应物-膜攻击复合体在AS发展的过程中发挥着较为复杂的作用;在补体激活通路的研究方面,替代激活途径的关键因子-Factor B缺陷并没有对AS产生明显的影响,提示另外两条通路可能发挥着更为重要的作用;而经典激活途径的关键因子-Clq基因缺陷却通过影响坏死细胞的清除促进了病变的发展。总之,近来的研究表明,补体系统参与了AS的发病过程,但是其具体的作用机制十分复杂,仍然需要进一步的研究来加以阐明。
作为补体系统的重要组成部分,膜调节蛋白亦被发现存在于AS病灶中,并且研究发现,阿托伐他汀和C反应蛋白均可诱导内皮细胞表达补体膜调节蛋白,从而使之幸免于补体系统的裂解作用。补体膜调节蛋白是否参与了AS病灶的发生和发展过程,具体的作用机制是什么,目前均无研究加以阐明。由于该组蛋白具有明显的补体激活抑制作用,我们提出如下科学假说:补体膜调节蛋白通过抑制补体激活或补体效应物的产生而发挥了抗AS作用。
目的
1.通过分别把CD55~(-/-)和CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得易形成AS斑块的双基因敲除小鼠;
2.通过对双敲小鼠的病变范围、斑块大小和病灶内容物进行分析,研究补体膜调节蛋白-CD55和CD59在AS发生发展中的作用;
3.通过对血脂、血尿、补体激活和T细胞免疫的分析,探讨CD55和CD59抗AS的机制。
方法
1.获取双基因敲除小鼠及高脂喂养
把CD55~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得CD55~(+/+)ApoE~(-/-)杂合子,以杂合子小鼠交配繁殖获得CD55~(-/-)ApoE~(-/-)小鼠和同龄野生型对照-CD55~(+/+)ApoE~(-/-)小鼠。将6周龄的小鼠根据高脂喂养时间和性别分为4组:短期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;短期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;长期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周;长期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周。以与CD55同样的方式,把CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得获得CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠。将6周龄的CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠按照高脂喂养时间和性别分为4组。
各组高脂喂养结束后,小鼠空腹过夜后被处死。自下腔静脉收集血液标本,并冻存。PBS冲洗左侧心腔及动脉腔内的残血后,将心脏分离并包埋于OCT。钝性分离自主动脉弓到腹主动脉分叉处的主动脉节段,进行染色分析。所有的动物实验及相关处理均通过了美国宾夕法尼亚大学动物管理及应用委员会的批准。
2.血液及尿液分析
应用酶法测定血清总胆固醇水平。应用代谢笼连续24小时收集小鼠的尿液,并应用HPLC/MS/MS法分析尿液中脂质代谢产物8,12-iso-iPF2_α-V和肌酐的水平。
3.斑块大小分析
将主动脉纵剖后钉于蜡版,并进行苏丹Ⅳ染色分析。病变范围表示为AS斑块占主动脉表面积的百分比。将小鼠心脏进行6μm连续冰冻切片,收集自出现三个主动脉瓣叶起至瓣尖的切片。对冰冻切片进行油红0染色,照相并应用软件分析。斑块大小表示为斑块占主动脉根面积的百分比。
4.病理学检测
对主动脉窦部冰冻切片行免疫组化染色和天狼星红染色,测量斑块内平滑肌细胞、巨噬细胞、C3、C9和胶原的阳性染色面积,其含量表示为阳性染色面积占斑块总面积的百分比。对切片行CD4和CD8免疫组化染色,计数单位面积内的阳性细胞个数。
5.统计学分析
定量数据均以均数±标准误表示,应用Graphpad公司的Prism3.0进行统计分析。在做分析之前,对所有的数据进行正态分布检验。对于正态分布的数据应用双侧t检验,对非正态分布的数据应用Mann—Whiteney分析。设P<0.05为有统计学意义。
结果
1.CD59的抗AS作用
短期雌性组内,虽然主动脉表面病灶范围并无组间差异(2.07±0.27%vs.1.34±0.21%,P=0.06),但是CD59~(-/-)ApoE~(-/-)小鼠主动脉窦部的斑块明显大于CD59~(+/+)ApoE~(-/-)对照(20.74±1.33%vs.13.12±1.46%;P<0.005)。然而,短期雄性组内,雄性CD59~(-/-)ApoE~(-/-)小鼠与CD59~(+/+)+ApoE~(-/-)对照间未发现显著差异(病变范围:1.35±0.16%vs.1.57±0.19%,P=0.3936;斑块大小:11.23±1.23%vs.12.75±1.41%,P=0.536)。长期雌性组内,CD59基因缺陷明显地增加了雌性小鼠的病变覆盖范围(17.13±1.14%vs.9.72±1.14%;P<0.001),但是却未对主动脉窦部的斑块大小产生明显的影响(32.67±1.58%vs.34.44±2.52%;P=0.315)。与短期雄性组的结果相似,在长期雄性组内未发现显著差异(病变范围:12.88±1.49%vs.10.98±0.95%,P=0.779;斑块大小:26.84±1.97%vs.29.46±2.3%,P=0.408)。CD59基因缺陷导致了病变覆盖范围的扩大或局部斑块的增大,提示CD59具有明显的抗AS作用。然而,无论是在短期还是长期雄性组内,均未发现显著的差异,提示CD59的抗AS作用存在明显的性别差异。
2 CD55缺陷与AS
与CD59基因缺陷的结果不同,无论在雄性还是雌性组,CD55基因敲除均未引起明显的病变范围或斑块大小的改变。在短期雌性组,雌性CD55~(-/-)ApoE~(-/-)和CD55~(+/+)ApoE~(-/-)小鼠的病变范围(0.80±0.17%vs 1.37±0.22%;P=0.07)和斑块大小(21.45±1.33%vs 23.58±2.1096;P=0.337)无显著差异:同样的结果也见于短期雄性组(病变范围:1.14±0.17%vs 0.80±0.14%,P=0.207:斑块大小:15.68±1.58%vs 16.89±1.65%,P=0.921)。在长期雌性组,CD55~(-/-)ApoE~(-/-)与CD55~(+/+)ApoE~(-/-)小鼠的病变范围(6.39±0.59%vs 5.81±0.51%:P=0.303)和斑块大小(29.81±2.78%vs 29.11±3.25%;P=0.918)无显著差异;长期雄性组内,也未发现明显的差异(病变范围:6.09±0.83%vs 6.16±0.69%,P=0.95;斑块大小:25.25±3.02%vs 30.94±3.22%;P=0.281)。
3 CD59缺陷对斑块构成的影响
通过对短期雌性组的病理学分析,我们研究了CD59基因缺陷对斑块内各种成份的影响。CD68染色显示,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠巨噬细胞的分布和相对含量较为一致(18.74±2.79%vs.18.66±2.41%;P=0.983),提示CD59并没有参与巨噬细胞的粘附、浸润或增殖过程,仅仅通过其他途径促进了斑块的发展,而巨噬细胞呈现等比例的增加;α-SMC actin染色显示,CD59~(-/-)ApoE~(-/-)小鼠的平滑肌细胞显著少于对照组(3.69±0.51%vs.6.42±0.86%;P<0.05);苦味酸-天狼星红染色发现,CD59基因缺陷小鼠的胶原含量较对照小鼠明显增加(8.14±0.33%vs.6.07±0.82%:P<0.05)。
2.4 CD59抗AS作用的机制
血清学分析显示,短期雌性组内,CD59~(-/-)ApoE~(-/-)小鼠的血清总胆固醇显著高于CD59~(+/+)ApoE~(-/-)小鼠(1404±43.74vs.1208±57.78mg/dL;P<0.05),提示CD59可能通过降低血清总胆固醇发挥抗AS作用。通过对尿液中氧化应激标志物的质谱分析,发现在雌性CD59~(-/-)ApoE~(-/-)小鼠和CD59~(+/+)ApoE~(-/-)对照间无显著差异(短期雌性组:2.98±1.01 vs.3.12±0.96 ng/mg肌酐,P=0.92;长期雌性组:1.65±0.56 vs.1.59±0.38 ng/mg肌酐,P=0.92),因而CD59基因缺陷并未对该系统产生显著的影响。短期雌性组的C3免疫组化染色发现,CD59~(-/-)ApoE~(-/-)小鼠与对照小鼠间斑块内的C3沉积无显著差异(28.65±1.69%vs.25.33±3.07%,P=0.37),提示CD59基因缺陷并没有影响局部的补体激活;而C9染色发现,CD59~(-/-)ApoE~(-/-)小鼠斑块内的C9沉积明显多于对照组(17.32±1.52%vs.8.74±3.63%,p<0.05),提示CD59基因缺陷时,局部的补体膜攻击复合体形成明显增加。为了明确CD59是否通过影响T细胞而发挥抗AS作用,对短期雌性组的小鼠切片做了CD4和CD8染色分析。结果发现,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠斑块内的T细胞含量无差异(CD4~+T细胞:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66:CD8~+T细胞:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40)。
结论
1 CD59可能通过降低血清总胆固醇和减少补体效应产物-膜攻击复合体的生成而发挥抗AS的作用。该作用与氧化应激和GPI介导的T细胞激活无明显的相关性。CD59的抗AS作用仅见于雌性,具有明显的性别差异。
2 CD59并没有参与斑块内巨噬细胞的粘附、迁移和增殖过程,该因子缺陷时通过影响其他方面导致了等幅度的巨噬细胞浸润;CD59通过抑制膜攻击复合体的形成保护血管壁平滑肌细胞免受补体系统的攻击。
3 CD55基因缺陷并没有引起明显的斑块大小改变,提示在动脉壁保护的过程中,其他的C3转化酶抑制剂如Factor H和Crry可能发挥着更重要的作用。在CD55基因缺陷的情况下,C3转化酶的功能仍然得到了有效的抑制,补体系统并未被有效地激活。
4主动脉表面的病变范围和窦部的斑块大小存在失偶联的现象,很可能是由于根部的空间有限,在ApoE缺陷较强的致AS背景下,其他基因很难引起明显的斑块大小改变。主动脉表面具有广阔的空间,可以作为更恒定的评价指标。
中文摘要Ⅱ补体过敏毒素调节Toll样受体致炎作用的体外研究
背景
天然免疫系统是机体抵御病原微生物感染的第一道防线。Toll样受体(tolllike receptors,TLRs)和补体是天然免疫系统的两个重要组成部分,通过迅速诱发炎症反应和激活继发的获得性免疫而在应对致病原的过程中发挥着重要的作用。
哺乳动物的TLRs,是果蝇Toll样蛋白的同源体,属于病原模式识别受体家族,可以通过识别病原或坏死细胞的相关分子活化天然免疫系统,同时也可以通过向抗原递呈细胞提供信号而启动获得性免疫系统,在细胞吞噬的调节、炎症反应的激活等方面发挥着重要的作用。补体是由30余种可溶性蛋白和膜结合性蛋白组成的多分子系统。其可溶性成分多以无活性的前体形式存在于血液循环中,需要时,在激活物如抗原-抗体复合物等的作用下,被依次激活,形成膜攻击复合体,发挥溶解、破坏细菌、病毒等致病微生物的作用。除膜攻击复合体外,补体系统激活时尚可以通过产生过敏毒素-C3a和C5a,参与机体的抗感染免疫,扩大体液免疫效应,调节免疫应答。
研究发现,许多TLRs的天然激活物如革兰阴性菌来源的脂多糖和来源于酵母细胞壁的酵母多糖也可以激活补体系统,提示在这两系统间可能存在一定的相互联系。探讨补体和TLRs之间是否存在相互作用及其具体的作用机制,对于深入理解炎症和机体的免疫反应具有极其重要的意义。日前,我们的一项研究发现,LPS等TLRs激活剂在CD55~(-/-)小鼠诱发的炎症反应明显强于对照小鼠,提示补体上调了TLRs的致炎作用。后继的机制研究发现,该作用是由补体过敏毒素-C3a和C5a所介导的。体内实验中,补体与TLRs的协同作用发生迅速,TLRs激活剂注入体内后3小时即达到顶峰,提示该作用很可能是由与循环系统密切接触的器官或者组织所介导的。内皮细胞构成了机体内最大的“内分泌器官”,并且与循环系统直接接触,故而我们提出如下科学假说:内皮细胞介导了补体对TLRs致炎作用的调节。在本研究中,我们应用人脐静脉内皮细胞(HUVEC)和小鼠心脏内皮细胞(H5V)对内皮系统在这一过程中的作用进行了探讨。
目的
研究内皮细胞是否介导了补体C3a和C5a对TLRs致炎作用的调控,并探讨其具体的作用机制。
方法
1细胞及其培养
人脐静脉内皮细胞,培养于含10%胎牛血清、0.05 mg/ml内皮细胞生长因子、2mML—谷氨酰胺、0.1mg/ml肝素和100 U混合抗生素的以M199为主体的培养基中。H5V为来源于小鼠心脏的内皮细胞系,培养于含10%胎牛血清的DMEM培养基内。内皮细胞贴壁生长于以0.1%明胶预先包被的培养皿中,37℃、5%CO_2的条件下,2-3天传代一次。其中第5-7代的HUVEC用于实验。
2细胞毒性实验
以单剂细胞增殖分析法研究C3a、C5a、Pam3CKS4、Poly I:C、LPS和CPG对内皮细胞的毒性作用,以选出可用的浓度范围。
3内皮细胞C5a受体的测定
以流式细胞仪分析HUVEC和H5V表面的C5a受体的表达情况,以确认内皮细胞可表达C5aR。
4干预实验
以1×10~5个/孔的浓度将细胞接种于24孔板,培养24小时后细胞大约融合至80%。在分析浓度-效应时,将各TLRs激活剂以培养基梯度稀释成5个浓度,刺激24小时;在分析时间-效应时,以选定的浓度稀释TLRs激活剂,分别刺激内皮细胞3、6、9、12、15和24个小时。刺激实验结束后,收集培养液用于IL-6或IL-8的ELISA分析,以选出合适的作用浓度和时间。过敏毒素和TLRs激活剂联合作用时,用不同浓度的C3a和C5a合并选定浓度的TLRs激活剂刺激80%融合的单层内皮细胞24小时。取培养液用于IL-6、IL-8或chemokines KC的ELISA分析,研究过敏毒素是否上调了TLRs的致炎作用。
5酶联免疫吸附实验(enzyme linked immunoad sorbent assay,ELISA)按照生产厂家提供的实验步骤,对稀释过的培养液进行IL-6、IL-8或chemokine KC的ELISA分析。
结果
1细胞毒性实验
应用单剂细胞增殖分析试剂盒,检测了补体过敏毒素和TLRs对H5V的细胞毒性作用。结果显示,C3a在0-100nM,C5a在0-200nM,Pam3CKS4在0-10000 ng/ml,PolyI:C在0-50μg/ml,LPS在0-1000 ng/ml,CPG在0-160ng/ml为安全的浓度区域,并未引起明显的细胞坏死。
2 C5a受体表达
应用流式分析,发现在HUVEC和H5V表面有相当数量的C5aR表达,而同时染色的阴性对照-HEK细胞上几乎没有荧光信号。
3 C5a和C3a对TLR4致炎作用的影响
LPS浓度-效应的分析发现,10ng/ml LPS即可以明显地增加H5V细胞的IL-6表达,随后IL-6的生成随着LPS浓度的增高而增加,当LPS浓度为500ng/ml时达到顶峰;时间-效应的分析发现,100ng/ml LPS作用3小时即可以诱导H5V细胞IL-6的释放增加,其效应随着作用时间的延长而增加,在刺激24小时时达到顶峰。
根据上述实验结果,我们选用10 ng/ml和100 ng/ml作为LPS的作用浓度,与C3a和C5a联合作用于内皮细胞。刺激24小时后,100 ng/ml的LPS明显地增加了IL-6的生成,其作用约为10 ng/ml组的2.7倍。但无论是合并10 ng/ml还是100 ng/ml的LPS,50nM C3a或C5a均未显著地增加LPS的诱导IL-6或chemokine KC的生成。应用不同浓度的C5a与100 ng/ml LPS同时刺激H5V,结果在其他浓度时,并未发现明显的上调作用。应用HUVEC重复了上述实验,也只获得了类似的结果。
4 C3a和C5a对其他补体激活物致炎作用的影响
100ng/ml Pam3CSK 4即可引起明显的IL-6表达增加,其刺激作用在1000ng/ml时达到最高;IL-6的生成亦随着Pam3CSK 4作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,100ng/ml Pam3CSK 4明显增加了内皮细胞的IL-6生成,但50hM C3a或50nM C5a均未对Pam3CSK4的致炎作用产生明显影响。
1μg/ml PolyI:C即可引起明显IL-6表达增加,其刺激作用在50μg/ml时达到最高:IL-6的生成随着作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,1μg/mlPolyI:C明显增加了内皮细胞的IL-6表达,但50hM C3a或50nM C5a均未对PolyI:C的致炎作用产生明显影响。
分析CPG的浓度-效应时,先后分析了5个浓度:200ng/ml、2μg/ml、20μg/ml、100μg/ml和200μg/ml,均没有引起明显的IL-6表达增加;时间-效应分析发现,20μg/mlCPG刺激24小时后,未引起明显的IL-6表达增加。合并50nM C3a或C5a时,20μg/mlCPG对内皮细胞刺激24小时,未发现明显的IL-6和chemokine KC表达增加。
应用HUVEC替代H5V后,发现了Pam3CSK4和Poly I:C可以明显地诱导IL-8表达,但并未发现C3a和C5a可以上调TLRs激活物的致炎作用。
结论
1 Pam3CSK4、Poly I:C和LPS可以激活相应的TLRs,引起明显的炎性细胞因子释放。心脏内皮细胞也具有与人脐静脉内皮细胞相似的反应性,可以在Pam3CSK4、Poly I:C或LPS的刺激下,产生大量的炎性因子。
2无论是HUVEC还是H5V,过敏毒素-C3a或C5a的联合刺激均未显著地增加TLRs的致炎作用,提示内皮细胞并未参与补体上调TLRs致炎作用这一过程。
Background
The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury via three different pathways,the classical pathway,the lectin pathway and the alternative pathway. Activated complement system promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).To prevent complement-mediated autologous tissue injury,many kinds of host cells,particularly those in the vascular space such as blood cells and endothelial cells,express several membrane-bound complement regulatory proteins.DAF and CD59 are two of them that link to the cell membrane via a glycosylphosphatidylinositol(GPI) anchor.DAF regulates complement activation by promoting the decay of C3 and C5 convertases,whereas CD59 inhibits the formation of the MAC.
Recent studies have indicated that atherosclerosis is a chronic inflammatory condition in which many components of innate and adaptive immune systems are involved.As an important part of innate immunity,complement system has been implicated in the pathogenesis of atherosclerosis by several studies.For example,various complement activation products,regulatory proteins,and complement receptors have been detected in human atherosclerotic lesions,particularly in vulnerable and ruptured plaques,and deposition of C5b-9 has been shown to correlate with the disease state. Experimental studies with complement deficient animals have also been performed to elucidate the role of the complement system in atherogenesis.A cholesterol-rich diet was shown to induce less atherosclerosis in rabbits deficient in C6 than in controls with a fully functional complement system.Contrasting results were obtained, however,in C5 deficient ApoE~(-/-) mice,which showed a similar extent of atherosclerotic lesions compared to complement-competent control mice.In other studies,C3 deficiency was found to increase lipid-positive lesions in the mouse aorta, as well as altering the plasma lipid profile.On the other hand,deficiency of factor B affected neither lipid levels nor lesion size.More recently,Clq deficiency was found to cause larger atherosclerotic lesions in low-density lipoprotein receptor knockout mice,possibly reflecting a role of Clq in the disposal of dying cells.Together,these results suggested a variable and complex role of complement components in atherosclerosis.
In contrast to the above studies on individual complement components in atherogenesis,there have been few studies testing the role of complement regulatory proteins in the development of this disease.Previous studies have detected the presence of complement regulators in atherosclerotic lesions and recently,both statins and C-reactive protein,the latter a marker of chronic inflammation and strong predictor of vascular diseases,have been shown to induce the expression of endothelial complement regulators.However,it is still not clear whether complement membrane-bound regulatory proteins are involved in the atherosclerosis.In this study, our hypothesis is that complement membrane-bound regulatory proteins protect agaist atherosclerosis through inhibiting the activation of complement system or the production of complement effectors.
Aims
1.To produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice by crossing CD55~(-/-)and CD59 ~(-/-) mice with ApoE~(-/-) mice respectively.
2.To illustrate the anti-atherosclerotic role of membrane-bound regulatory proteins, by analyzing the lesion size and lesion contents of double knockout mice after a high-fat diet.
3.To reveal the mechanism of the anti-atherosclerotic role of membrane-bound regulatory proteins.
Methods
1.Mice and diets
CD55 knockout mice and CD59 knockout mice were crossed with ApoE~(-/-) mice respectively to produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice.CD55~(+/+) ApoE~(-/-) mice and CD59~(+/+) ApoE~(-/-) mice were used as controls.Starting from 6 weeks of age,they were fed a high fat diet.At 14 or 22 weeks of age,they were fasted overnight and killed by CO2 asphyxiation.Blood was collected for plasma lipid analysis by vena cava nicking and then stored in -80℃refrigerators.The aorta was perfused with cold PBS by inserting a cannula into the left ventricle.The hearts were dissected and embedded in the OCT.Perfused aortas were dissected from aortic arch to iliac bifurcation.All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania.
2.Serum and urine sample analysis
Plasma samples were collected after 12 hours of fasting and stored at -80℃.Total cholesterol was measured.Twenty-four hour urine samples were collected in metabolic cages.The nonenzymatic lipid peroxidation product,8,12-iso-iPF2α-Ⅵ, was measured by HPLC/MS/MS.Metabolite levels were corrected for urinary creatinine.
3.Processing and analysis of the aorta
Aortas were pinned on wax plates and stained with SudanⅣ.The extent of atherosclerosis was expressed as the percentage of the aorta surface covered by positive staining.Serial 6-μm-thick cryostat sections were prepared from the origin of the aortic valve cusps and cross-sectional analysis for atherosclerotic lesion was performed every 60μm over 360μm by staining with oil-red O.The extent of atherosclerosis was expressed as the percentage of aortic root covered by lesions. Images were captured digitally with a video camera and analyzed by computerized image analysis.
4.Histological analysis of aortic lesions
The aortic root sections were stained for smooth muscle cells,macrophages,collagen, C3,C9,CD4 and CD8.The ratio of positive area or cells to aortic root area was recorded.
5.Statistical Analysis
Statistical analyses were performed with GraphPad software(Prism 3.0).Data were analyzed by two-tailed Student t-test(for data with normal distribution) or Mann-Whitney test(for data with non-parametric distribution).Results are considered to be significant at values of P<0.05 and are presented as mean±SEM.
Results
1.CD59 protects against atherosclerosis
After 8 weeks of HFD,we found that female CD59~(-/-) ApoE~(-/-) mice developed significantly larger atherosclerotic lesions in their aortas,as shown by both en face (2.07±0.27%vs.1.34±0.21%,P=0.06) and aortic root section analysis(20.74±1.33% vs.13.12±1.46%,P<0.005).Interestingly,the increased sensitivity to atherosclerosis development in CD59~(-/-) ApoE~(-/-) mice appeared to be gender-biased as we observed no significant difference between male CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either en face(1.35±0.16%vs.1.57±0.19%,P=0.393) or aortic root section analysis (11.23±1.23%vs.12.75±1.41%,P=0.536).
After 16 weeks of HFD,a more pronounced difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice was observed by en face staining (17.13±1.14%vs.9.72±1.14%,P<0.001).However,a difference could not be detected between the two groups mice by aortic root section analysis at this advanced stage of lesion development(32.67±1.58%vs.34.44±2.52%,P=0.315).Similar to the finding at 8 weeks of HFD feeding,we detected no significant difference at 16 weeks between male CD59~(+/+) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either the en face (12.88±1.49%vs.10.98±0.95%,P=0.779) or aortic root section analysis (26.845±1.97%vs.29.46±2.3%,P=0.408).
2.CD55 has no influence on the development of atherosclerosis
In contrast to the effect of CD59 deficiency on atherosclerosis in female ApoE~(-/-) mice, we observed no significant difference in either gender between CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice,regardless of the length of HFD feeding.En face analysis showed that the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD feeding were 0.80±0.17%vs 1.37±0.22%(P=0.07), and 6.39±0.59%vs 5.81±0.51%,(P=0.303),and that for male mice were 1.14±0.17% vs 0.805±0.14%(P=0.207),and 6.09±0.83%vs 6.16±0.69%(P=0.693),respectively. Aortic root section analysis showed the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD to be 21.45±1.33%vs 23.58±2.10%(P=0.337),and 29.81±2.78%vs 29.11±3.25%(P=0.918),and that for male mice were 15.68±1.58%vs 16.89±1.65%(P=0.921),and 25.25±3.02%vs 30.94±3.22%(P=0.281),respectively.
3.The influence of CD59 knockout on the components of atherosclerotic lesions We next performed histological studies in female CD59-deficient and-sufficient mice to evaluate the complexity of the atherosclerotic lesions.Using CD68 as a marker,we measured the lesion areas that were positive for macrophage infiltration.No significant difference was observed between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice in macrophage distribution and content(18.74±2.79%vs. 18.66±2.41%,P=0.983).On the other hand,after 8 weeks of HFD feeding,we detected more collagen(8.14±0.33%vs.6.07±0.82%,P<0.05) and less smooth muscle cell staining(3.69±0.51%vs.6.42±0.86%,P<0.05) in the lesions of CD59-deficient mice compared with their littermate controls.However,the difference in smooth muscle cell content was not observed at 16 weeks of HFD feeding (2.25±0.24%vs.2.28±0.54%,P=0.97).
4.The anti-atheroslerotic mechanisms of CD59 Plasma lipid analysis showed a moderate increase in total plasma cholesterol levels in female CD59~(-/-) ApoE~(-/-) mice compared with their CD59~(+/+) ApoE~(-/-) littermates,but the difference reached statistical significance only at 8 weeks of HFD feeding (1404±43.74 vs.1208±57.78 mg/dL,P<0.05 ).We observed no significant difference in 8,12-iso-iPF2α-Ⅵbetween female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice either at 8 weeks or 16 weeks of HFD feeding(8 weeks:2.98±1.01 vs.3.12±0.96 ng/mg creatinine,P=0.92;16 weeks:1.59±0.38 vs.1.65±0.56 ng/mg creatinine,P=0.92). Staining of aortic root sections with anti-C3 and anti-C9 antibodies showed a similar C3 deposition(28.65±1.69%vs.25.33±3.07%,P=0.371) in female CD59~(-/-) ApoE~(-/-) mice and controls,but significantly increased C9 deposition was found in the sections of female CD59~(-/-) ApoE~(-/-) mice than those of the controls(17.32±1.52%vs.8.74±3.63%,P<0.05) after 8 weeks of HFD feeding.This result suggested that CD59 deficiency led to more deposition of the membrane attack complex within the atherosclerotic lesions.We also examined CD4+ and CDS+ T cell infiltration into the lesions but observed no significant difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice(for CD4+ T cells:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66; for CDS+ T cells:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40).
Conclusions
1.CD59 offers protection against atherosclerosis in the context of ApoE deficiency, most likely through lowering total cholesterol and inhibiting the formation of membrane attack complex.Its anti-atherosclerotic role is independent of oxidative stress and GPI-mediated T cell activation,and gender-biased,which maybe related to the different level of sex hormones.
2.CD59 is not involved in the adhesion,migration and proliferation of macrophages; CD59 protect smooth muscle cells against complement attack by inhibiting the formation of MAC
3.CD55 shows no obvious influence on atherosclerosis.This maybe because that other complement regulators such as factor H or Crry have compensated for the lack of CD55.Even under the deficiency of CD55,C3 convertase can be inhibited effectively.
4.There is a loss of correlation between en face and aortic root analysis.The aortic roots may be particularly sensitive to lesion development caused by ApoE-deficiency, and any additive effect brought upon by other genetic alterations could be difficult to be detected,especially after prolonged HFD feeding.Thus,en face analysis appears to be a more stable method than aortic root analysis.
Background
Innate immunity is the first line of host defense against pathogens.The Toll-like receptors(TLRs) and complement are 2 critical components of the innate immune system.They play an essential role in host defense by eliciting rapid inflammatory reactions and orchestrating adaptive immune responses to microbial infection. The mammalian TLRs,one kind of the pattern recognition receptors,are horologes of Toll proteins from drosophila.Through recognizing the invading microbes and dead cells,they can activate the innate immunity,and initiate the adaptive immunity by interacting with the antigen-presenting cells.They play critical roles in the regulation of phagocytosis,signal transduetion and cell apoptosis.The complement system consists of soluble proteins and membrane-bound proteins.The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury.Activated complement promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).
Many common PAMPs,such as lipopolysaccharide(LPS) from gram-negative bacteria and zymosan,an insoluble carbohydrate from the yeast cell wall,act both as TLR ligands and activators of complement.Whether and how the TLR and the complement systems,when coactivated in vivo,interact with each other and how potential cross talks between the 2 systems might impact the inflammatory and adaptive immune responses of the host has not been well studied.Recently,one of our studies showed that TLRs agonist induced significant inflammatory response in CD55 deficient mice,indicating that complement system upregulates the TLR-mediated inflammatory response.Subsequent analysis showed that this function was mediated by the anaphylatoxins.In vivo study showed that complement upregulated the cytokines production 3 hours after LPS challenge.This means that the function is most likely mediated by the organs or tissues which have direct contaction with circulation system.Endothelial system is the largest endocrinal organ and has direct contaction with blood.This led to our hypothesis that endothelial cells mediate the upregulation of TLRs-mediated inflammatory response by complement.
Aim
To elucidate whether and how endothelial cedis are involved in the upregulation of the TLR-mediated inflammatory response by complement.
Methods
1 Cells culture
HUVEC were cultured in Medium 199 containing 10%fetal bovine serum,0.05 mg/ml endothelial cell growth supplement,2mML-glutamine,0.1mg/ml heparin,and 100 U penieillinstreptomycin.H5V,an endothelial cell line derived from mouse heart, was cultured in DMEM supplemented by 10%fetal bovine serum.Endothelial cells were cultured at 37℃in a humidified atmosphere containing.5%CO2 and passaged every two or three days at a split ratio of 1:3.Passages 5-7 of HUVEC were used for experiments.
2 Cell toxicity testing
The cell toxicity of C3a,C5a,LPS,Poly I:C,Pam3CKS4 and CPG to endothelial cells was conducted with promega's one solution cell proliferation assay.This test was performed to find the dose range for the stimulation of endothelial cells.
3 C5aR expressions in endothelial cells
C5aR expression in HUVEC or H5V was measured by flow cytometry
4 Treatment of HUVEC
Endothelial cells were seed in 24-well plate at 1×105 per well.After 24 hours,the cells were 80%confluent and were stimulated by various concentration of TLRs agonists or for different time to find the way for the joint stimulation. For the joint stimulation,80%confluent endothelial cells were stimulated by anaphylatoxins and different TLRs agonists for 24 hours.
5 ELISA analyses
ELISA for IL-6,IL-8 and chemokine KC was conducted according to the instructions from companies.
Results
1 The following dose ranges were found with no obvious cell toxicity to endothelial cells:0-100nM for C3a,0-200nM for C5a,0-10000 ng/ml for Pam3CKS4,0-50μg/m for Poly I:C,0-1000 ng/ml for LPS and 0-160ng/ml for CPG.
2 A number of C5aR were found in HUVEC and H5V,but not in the negative controls.
3 10 ng/ml LPS significantly induced IL-6 expression.More IL-6 was produced along with the increase of LPS concentration until 500ng/ml.Time course analysis showed that significant IL-6 production was detected after 3 hours'LPS stimulation and peaked after 24 hours.According to the concentration and time course analysis,we chose 10 ng/ml and 100 ng/ml LPS for the joint stimulation.Either 50nM C3a or C5a didn't show any influence on the IL-6 or Chemokine KC production induced by LPS. Then this conclusion was confirmed by using different level of C5a.
4 In the same way,we analyzed the influence of C3a and C5a on other TLR-mediated inflammatory response.50nM C3a or C5a showed no influence on IL-6,IL-8 or chemokine KC production induced by Pam3CSK4,Poly I:C and CPG.
Conclusions
1 Pam3CSK4,Poly I:C and LPS but not CPG can activate toll like receptors on the membrane of endothelial cells.Similar to HUVEC,heart endothelial cells can produce a lot of cytokines under the stimulation of Pam3CSK4,Poly I:C and LPS.
2 Endothelial cells are not involved in the regulation of TLR-mediated inflammatory response by complement
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