Endothelial Cell to Mesenchymal Cell Transitions Play a Critical Biological Sex- and Aging-Dependent Role in Formation and Maintenance of the Acta2+ Atherosclerotic Lesion Protective Fibrous Cap

由不稳定的动脉粥样硬化斑块破裂或侵蚀引起的动脉粥样硬化血栓形成是动脉粥样硬化的主要原因。 全世界的死亡然而，调节晚期动脉粥样硬化病变稳定性的机制仍然存在 不太了解。我们实验室最近的研究（2021 Nature Metabolism）显示，平滑肌细胞（SMC） 在ApoE-/-小鼠中，血小板衍生生长因子受体β（PDGFR β）的条件性敲除（KO）与 SMC几乎完全不能进入病变或纤维帽。尽管几乎完成了 SMC PDGFR基因敲除小鼠的病变中没有SMC，令人惊讶的是，我们没有观察到病变大小或指数的变化 包括ACTA 2+纤维帽的厚度，在18周的西方饮食（WD）之后。 进一步的研究表明：1）与Acta 2+纤维帽细胞几乎完全来自 SMC，我们发现它们仅占晚期ApoE-/-头臂型（BCA）或人类ACTA 2+细胞的60-75%。 冠状动脉（CA）病变，其余来自内皮细胞（EC）-间充质-肌成纤维细胞 转化（EndoMT-MFT，15-20%）和巨噬细胞向肌成纤维细胞转化（MMFT，10-15%）; 2）SMC丢失 用SMC PDGFRmRNA KO或骨髓移植（BMT）对病变进行投资， EndoMT-MFT和MMFT增加; 3）EndoMT-MFT增加，当 WD喂养延长至26周，表明纤维帽细胞维持 病变稳定性取决于其来源; 4）EC对ACTA 2+纤维帽的贡献随着病变的增加而增加 通过给小鼠喂食18周WD，随后喂食12周普通饲料进行消退;以及5）雌性但非雄性小鼠 显示IL 1 R1依赖性EndoMT。本文的研究将检验EC对ACTA 2+的贡献 纤维帽随着致死性辐照-BMT、SMC PDGFR mRNA KO、衰老、女性和/或斑块消退而增加 导致：1）EC增殖和复制性衰老增加; 2）EC完整性丧失和/或抗- 血栓性质;和3）增加的对斑块侵蚀或破裂的敏感性。目标1将决定是否标记 致死性辐照后EndoMT-MFT对ACTA 2+纤维帽的贡献增加-BMT、SMC PDGFR基因敲除或斑块消退与增殖增加、克隆扩增、复制性 衰老和/或EC功能障碍。目标2将确定我们的SMC-或EC-谱系追踪ApoE-/-的治疗是否 或我们的新型Myh 11-CreERT 2/Rosa-eYFP/SR-BI BSCT/BSCT/LDLR-（BSCT）小鼠，其具有晚期BCA和CA病变， 衰老清除药物与斑块稳定性指数的有益变化、生存率的提高、MI的减少或 卒中，降低对EndoMT-MFT维持ACTA 2+纤维帽的依赖性，和/或改善EC 完整性和/或功能。所有目的均包括对稳定与高风险颈动脉内膜切除术的人体验证研究 样本，并评估晚期病变发病机制的性别依赖性决定因素。拟议的研究 将提供关于调节细胞和细胞外基质（ECM）组成的机制的新见解 的纤维帽，并可能导致开发新的治疗方法，以提高斑块的稳定性。

Atherothrombosis, resulting from rupture or erosion of unstable atherosclerotic plaques, is the leading cause of death worldwide. However, the mechanisms that regulate the stability of late stage atherosclerotic lesions remain poorly understood. Recent studies from our lab (2021 Nature Metabolism) showed that smooth muscle cell (SMC) conditional knockout (KO) of the platelet-derived growth factor receptor- (PDGFR) in ApoE-/- mice was associated with nearly complete failure of SMC to invest into lesions or the fibrous cap. However, despite nearly complete absence of SMC in lesions of SMC PDGFR KO mice, surprisingly we observed no changes in lesion size or indices of plaque stability, including the thickness of the ACTA2+ fibrous cap, following 18 weeks of Western diet (WD). Further studies showed that: 1) contrary to dogma that Acta2+ fibrous cap cells are derived almost exclusively from SMC, we found they account for only 60-75% of ACTA2+ cells in advanced ApoE-/- brachiocephalic (BCA) or human coronary artery (CA) lesions with the remainder coming from endothelial cell (EC)-to-mesenchymal-to-myofibroblast transitions (EndoMT-MFT, 15-20%) and macrophage-to-myofibroblast transitions (MMFT, 10-15%); 2) loss of SMC investment into lesions with SMC PDGFR KO or bone marrow transplantation (BMT) was associated with a 2-3 fold increase in EndoMT-MFT and MMFT; 3) increased EndoMT-MFT, and MMFT did not sustain indices of stability when WD feeding was extended to 26 weeks suggesting qualitative differences in the ability of fibrous cap cells to sustain lesion stability depending on their origin; 4) the contribution of EC to the ACTA2+ fibrous cap increased with lesion regression by feeding mice 18 weeks of WD followed by 12 weeks of chow diet; and 5) female but not male mice exhibit IL1R1-dependent EndoMT. Studies herein will test the hypothesis that the contribution of EC to the ACTA2+ fibrous cap increases with lethal irradiation-BMT, SMC PDGFR KO, aging, female sex, and/or plaque regression resulting in: 1) increased EC proliferation and replicative senescence; 2) loss of EC integrity and/or reduced anti- thrombotic properties; and 3) increased susceptibility to plaque erosion or rupture. Aim 1 will determine if the marked increase in the contribution of EndoMT-MFT to the ACTA2+ fibrous cap following lethal irradiation-BMT, SMC PDGFR KO, or with plaque regression is associated with increased proliferation, clonal expansion, replicative senescence, and/or dysfunction of EC. Aim 2 will determine if treatment of our SMC- or EC-lineage tracing ApoE-/- or our novel Myh11-CreERT2/Rosa-eYFP/SR-BICT/CT/LDLR- (CT) mice with advanced BCA and CA lesions with senolytic drugs is associated with beneficial changes in indices of plaque stability, improved survival, reduced MI or stroke, reduced dependence on EndoMT-MFT for maintenance of the ACTA2+ fibrous cap, and/or improved EC integrity and/or function. All aims include human validation studies on stable versus high risk carotid endarterectomy samples, and assessment of sex-dependent determinants of late-stage lesion pathogenesis. The proposed studies will provide novel insights regarding mechanisms that regulate the cellular and extracellular matrix (ECM) composition of the fibrous cap and may lead to development of novel therapeutic approaches for enhancing plaque stability.