平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析
Summary
我们提出了一个标准化的方案来表征晚期小鼠动脉粥样硬化病变的细胞组成, 包括动物解剖、组织包埋、切片、染色和腕头动脉分析的系统方法从动脉粥样硬化平滑肌细胞谱系追踪小鼠。
Abstract
动脉粥样硬化仍然是全世界的主要死因, 尽管有无数临床前研究描述了有希望的治疗目标, 但新的干预措施仍然遥遥无期。这在一定程度上可能是由于依赖临床前预防模型, 调查基因操作或药物治疗对动脉粥样硬化发展的影响, 而不是既定疾病。另外, 由于使用了表面病变分析, 缺乏对病变细胞群的定性, 这些研究的结果往往令人困惑。为了帮助克服这些转化障碍, 我们建议更多地依赖干预模型, 利用免疫荧光染色和共聚焦显微镜对单个细胞水平细胞组成的变化进行研究。为此, 我们描述了一种在小鼠干预模型中测试假定治疗药物的方案, 包括动物解剖、嵌入、切片、染色和臂头动脉病变定量的系统方法。此外, 由于晚期动脉粥样硬化病变中细胞的表型多样性, 我们描述了使用细胞特异性、诱导的谱系追踪小鼠系统的重要性, 以及如何利用这种方法对细胞进行无偏见的表征。动脉粥样硬化病变细胞群。总之, 这些策略可以帮助血管生物学家更准确地模拟治疗干预措施和分析动脉粥样硬化疾病, 并有望转化为更高的成功率的临床试验。
Introduction
动脉粥样硬化是全球大多数冠心病、外周动脉疾病和中风的主要发病和死亡原因。晚期冠状动脉粥样硬化可导致严重并发症, 包括心肌梗塞占世界人口死亡人数的近 16% 1,2。由于其对公众健康的破坏性影响, 已经作出了大量的努力, 以破译机制, 推动动脉粥样硬化的进展, 并制定新的治疗策略。然而, 与其他临床领域 (第一阶段仅为 8.7%) 相比, 心血管疾病临床试验的批准可能性 (loa)是最低的。这在一定程度上可以用动脉粥样硬化对有效药物开发构成的许多障碍来解释, 包括其几乎无处不在的性质、临床上沉默的进展和显著的疾病异质性。此外, 临床前动物研究的次优设计也可以解释临床翻译缺乏成功的原因。具体而言, 我们认为有必要尽可能实施干预研究, 以调查治疗策略的疗效。此外, 有一个迫切需要执行标准化的程序, 病变分析, 包括先进的定性后期动脉粥样硬化病变细胞组成的命运图和表型。
绝大多数动脉粥样硬化研究的重点是模型动脉粥样硬化预防包括药物治疗或基因操作 (敲除或敲入) 健康的幼鼠, 在疾病的开始和进展之前。这些研究发现了大量的基因和信号分子, 它们在动脉粥样硬化的发展中起着作用。然而, 这些目标大多未能转化为人类有效的治疗方法。事实上, 很难推断治疗对健康的幼鼠有影响的老年患者的晚期动脉粥样硬化病变。因此, 在临床前实验管道中实施干预研究可能会更准确地描述一种新治疗方法的相关性和有效性。在采用预防 4、5、6或干预策略7时, 抑制促炎性细胞因子白细胞素-1β (il-1β) 的显著发散效应支持了这一想法。预防和干预研究之间的差异表明, 不同的细胞过程发生在动脉粥样硬化发展的不同阶段, 并突出表明预防研究可能不足以模拟临床情景充分。
美国心脏协会最近发表了一份科学声明, 详细介绍了正确的实验设计、程序标准化、分析和报告动物动脉粥样硬化研究的建议 8。报告强调了该领域使用的主要技术的好处和局限性。例如,面对苏丹主动脉的 iv 染色通常作为第一次读出进行。虽然面对苏丹 iv 染色的脂质沉积是一个合适的方法来评估全球斑块负担, 它是无法区分早期阶段脂肪条纹病变和更先进的晚期病变。因此,对面部染色的解释往往是模糊和肤浅的.使用形态参数血管、病变、腔大小和病变稳定性指标的量化对组织横截面进行仔细分析, 可以更准确地了解实验的效果。
最后, 人类组织病理学研究表明, 细胞组合物是一个更好的预测破裂比病变大小本身, 病变在平滑肌细胞 (smc) 和丰富的巨噬细胞更容易破裂10, 11个。这些观察是基于经典用于细胞鉴定的标记的染色 (即 smc 的 acta2 和巨噬细胞的 lgals3 或 cd68)。然而, 由于包括 smc、内皮细胞和骨髓细胞12在内的多种谱系的可塑性, 这些标记的表达并不严格限于动脉粥样硬化病变中的单个细胞类型。特别是, 在过去十年之前, 几乎不可能在动脉粥样硬化病变中明确识别 smc, 因为这些细胞具有去分化和抑制其谱系特异性标记基因的特性 (这一过程称为表型转换) 在受伤或患病的血管13。由于血统追踪7、14、15、16、17、18、19,20,21,22,23,24. 它包括永久标记 smc 及其后代, 以跟踪其在动脉粥样硬化进展过程中的命运和表型演变, 使用 smc 特定启动子驱动的 cre 重组酶的表达组合 (即, myh117、15、17、18、19、20、21、22、23,24, acta225、26和sm2α14、16) 和记者的激活 (例如, 荧光蛋白、β-半乳糖苷酶) [在 btzon 和 majesky 中评论201827]。在最早的一项研究中, speer 等人在胚胎发生环境之外进行了研究, 其中一项研究提供了证据, 证明 smc 可以在胚胎发生过程中调节其表型并转化为软骨细胞, 方法是sm22α cre r26r lacz 谱系追踪模型。尽管这些研究开创了 smc 谱系追踪的先河, 但他们在一定程度上模棱两可, 因为任何在疾病发生时表达 sm2α的给定非 smc 都会被记者标记。通过开发和使用允许对细胞标记进行时间控制的他莫西芬诱导 cre ertlexp, 绕过了这一限制。细胞标记仅在他莫西芬分娩过程中发生, 并将仅限于在他莫西芬接触时表达细胞类型特特的细胞, 从而避免追踪激活 cre 的替代细胞类型。疾病进展的设置。对于动脉粥样硬化中 smc 的谱系追踪, 与荧光记者相关的他莫西芬诱导myh11-cre/ert2转基因 (eyfp7、15、17、18),21、mtmg19、25、五彩食品20、22、23为克隆扩频研究已证明了 smc 标签的显著效率和特异性, 并具有显著的应用效率和特异性。在最近的研究中, smc 种群被用于命运图。重要的是, 这些研究表明: 1) 在晚期动脉粥样硬化病变中, 80% 的 smc 不能表达免疫组织学分析中使用的任何常规 smc 标记物 (acta2, myh11), 因此在没有谱系追踪的情况下, 会被误认错误。17岁;2) 包括巨噬细胞标记或间充质干细胞标记 16、17、19的替代细胞类型 smc 表达标记的子集;smc 通过寡克隆扩张和 smc 克隆体保留可塑性, 过渡到表型不同的 20,23。总之, 现在可以清楚地看到, 平滑肌细胞在动脉粥样硬化病变中具有显著的表型多样性, 并可根据其表型转变的性质对病变发病机制产生有益或有害的作用。这些发现是针对 smc 动脉粥样硬化促进表型转变在晚期动脉粥样硬化中的一个显著的新治疗途径。
在此, 我们提出了一个标准化的程序, 分析晚期小鼠动脉粥样硬化病变, 包括动物解剖, 嵌入, 切片, 染色, 和定量的腕头动脉病变。为了确定白细胞介素-1β抑制 smc 的影响, 我们使用 smc 系追踪 apoe-/-小鼠喂养了18个月的西方饮食, 然后接受每周注射抗 il1 β抗体或同位素匹配的 igg 控制。
Protocol
Representative Results
Discussion
尽管在动脉粥样硬化的研究方面进行了几十年的研究和技术进步, 但该领域将科学发现转化为临床疗法的历史令人失望,34, 35.这种现象的部分原因可能是动物模型、实验设计和病变分析的差异。在此, 我们描述了一个实验管道, 我们用来分析细胞组成的晚期动脉粥样硬化病变使用谱系追踪小鼠7。这种方法可以对细胞命运图和表型进行细?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢匹兹堡大学生物成像中心 (由 nih 1S10OD019973-01 提供的支持) 的协助。这项工作得到了美国心脏协会科学发展赠款15sdg2580021 的支持, d. g. r. a. b. 得到了国家卫生研究院 f30 hl136188 赠款的支持。
Materials
| 16% Paraformaldehyde aqueous solution | Electron Microscopy Sciences | RT 15710 | Tissue perfusion and fixation |
| 23G butterfly needle | Fisher | BD367342 | |
| 25G needle | Fisher | 14-821-13D | |
| A1 Confocal microscope | Nikon | Confocal microscope | |
| ACTA2-FITC antibody (mouse) | Sigma Aldrich | F3777 | Primary Antibody |
| Alexa-647 anti goat | Invitrogen | A-21447 | Secondary antibody |
| Antigen Unmasking solution, Citric acid based | Vector Labs | H-3300 | Antigen retrieval solution |
| Chow Diet | Harlan Teklad | TD.7012 | |
| Coverslip | Fisher | 12-544-14 | Any 50 x 24 mm cover glass |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | Nucleus fluorescent counterstaining |
| Donkey Alexa-488 anti-rabbit | Invitrogen | A-21206 | Secondary antibody |
| Donkey Alexa-555 anti-rat | Abcam | ab150154 | Secondary antibody |
| DPBS 10X without Calcium and Magnesium | Gibco | 14200166 | PBS for solution dilutions and washes. Dilute to 1x in deionized water |
| Embedding cassette | Fisher | 15-182-701D | |
| ETDA vacuum tube | Fisher | 02-685-2B | |
| Ethanol 200 proof | Decon | 2701 | |
| Foam pad | Fisher | 22-222-012 | |
| Gelatin from cold water fish skin | Sigma Aldrich | G7765 | |
| GFP antibody (goat) | abcam | ab6673 | Primary antibody |
| goat IgG control | Vector Labs | I-5000 | IgG control |
| High Fat Diet | Harlan Teklad | TD.88137 | |
| ImageJ | NIH | Computer program https://imagej.nih.gov/ij/ | |
| LGALS3 antibody (rat) | Cedarlane | CL8942AP | Primary antibody |
| LSM700 confocal microscope | Zeiss | Confocal microscope | |
| Microscope Slides, Superfrost Plus | Fisher | 12-550-15 | |
| Microtome blades | Fisher | 30-538-35 | |
| Mouse IgG control | Vector Labs | I-2000 | IgG control |
| NIS element imaging software | Nikon | Imaging software for z-stack image acquisition | |
| Normal Horse serum | Sigma Aldrich | H1270 | |
| Pap Pen | Fisher | 50-550-221 | |
| Peanut oil | Sigma | P2144 | |
| Prolong gold Antifade mountant | Invitrogen | P36930 | Mounting medium |
| Rabbit IgG control | Vector Labs | I-1000 | IgG control |
| Rat IgG control | Vector Labs | I-4000 | IgG control |
| RUNX2 antibody (rabbit) | Abcam | ab192256 | Primary Antibody |
| Syringe | BD | 309628 | 1 ml syringe |
| Tamoxifen | Sigma | T5648 | |
| Xylene | Fisher | X55K-4 | |
| Zen imaging software | Zeiss | Imaging software for z-stack image acquisition |
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