The environment-sensing aryl-hydrocarbon receptor inhibits the chondrogenic fate of modulated smooth muscle cells in atherosclerotic lesions

Aryl-hydrocarbon receptor protects against endochondral ossification of modulated smooth muscle cells in atherosclerosis Introduction: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. Methods: We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. Results: Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term chondromyocytes (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. Conclusion: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues. Overall design: We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.

芳基烃受体（Aryl-hydrocarbon receptor, AHR）可抑制动脉粥样硬化中调节型平滑肌细胞的软骨内化骨
引言：平滑肌细胞（smooth muscle cells, SMC）在动脉粥样硬化进程中发挥关键作用。芳基烃受体（AHR）是一类环境感知型转录因子，参与血管发育，并与冠状动脉粥样硬化性心脏病（coronary artery disease, CAD）的发病风险相关。我们提出假说：AHR可通过调控SMC的表型转化影响动脉粥样硬化进程。
方法：我们在人冠状动脉平滑肌细胞（human coronary artery SMC, HCASMC）中联合运用RNA测序（RNA-Seq）、染色质免疫共沉淀测序（ChIP-Seq）、转座酶可及性测序（ATAC-Seq）及体外实验，并结合动脉粥样硬化SMC特异性谱系示踪Ahr敲除小鼠模型中的单细胞RNA测序（scRNA-Seq）、组织学分析及RNAscope技术，以深入解析AHR在血管疾病中的作用机制。
结果：培养的HCASMC中的基因组学研究结合功能实验表明，AHR可调控HCASMC的表型，并抑制这些细胞的骨化进程。小鼠主动脉窦的谱系示踪与活性示踪研究显示，Ahr通路在动脉粥样硬化病变帽部的调节型SMC中处于激活状态。进一步针对SMC特异性Ahr敲除小鼠的scRNA-Seq研究发现，表达软骨细胞标志物（如Col2a1与Alpl）的调节型SMC比例显著升高，这些细胞定位于病变新内膜中。我们将这类细胞命名为“软骨肌细胞（chondromyocytes, CMC）”，其在人类冠状动脉的病变新内膜中也可被检测到。组织学分析显示，与野生型小鼠相比，Ahr敲除小鼠的病变体积更大、谱系示踪标记的SMC在病变中的浸润比例更高，但病变帽部的谱系示踪SMC数量减少，且病变中的碱性磷酸酶活性升高。基于上述实验数据及人类遗传学分析的推论，我们认为AHR具有保护作用。
结论：综上，我们认为AHR可维持病变帽部的结构完整性，并抑制动脉粥样硬化组织中SMC向CMC的疾病相关性转化。
整体实验设计：我们提出假说“AHR可通过调控SMC的表型转化影响动脉粥样硬化进程”。我们在人冠状动脉平滑肌细胞（HCASMC）中联合运用RNA-Seq、ChIP-Seq、ATAC-Seq及体外实验，并结合动脉粥样硬化SMC特异性谱系示踪Ahr敲除小鼠模型中的scRNA-Seq、组织学分析及RNAscope技术，以深入解析AHR在血管疾病中的作用机制。