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

动脉粥样硬化血栓形成，由不稳定的动脉粥样硬化斑块破裂或侵蚀引起，是 世界范围内的死亡。然而，调节晚期动脉粥样硬化病变稳定性的机制仍然存在。 人们对此知之甚少。我们实验室的最新研究(2021年自然代谢)表明，平滑肌细胞(SMC) 在载脂蛋白E-/-小鼠中，血小板衍生生长因子受体-(PDGFR)的条件性基因敲除(KO)与 SMC几乎完全不能包埋病变或纤维帽。然而，尽管几乎完成了 在SMC PDGFRKO小鼠的皮损中没有发现平滑肌细胞，令人惊讶的是，我们没有观察到病变大小或指数的变化 在西方饮食(WD)18周后，斑块稳定性的变化，包括ACTA2+纤维帽的厚度。 进一步的研究表明：1)与Acta2+纤维帽细胞几乎完全来源于 SMC，我们发现它们只占晚期ApoE-/-头臂(BCA)或人类ACTA2+细胞的60%-75% 冠状动脉(CA)病变，其余来自内皮细胞(EC)到间充质细胞到肌成纤维细胞 转化(EndoMT-MFT，15-20%)和巨噬细胞向肌成纤维细胞的转化(MMFT，10-15%)；2)SMC丢失 投资于SMC PDGFRKO或骨髓移植的病变与2-3倍相关 EndoMT-MFT和MMFT增加；3)EndoMT-MFT增加，MMFT不能维持稳定性指数 WD饲养延长至26周，表明纤维帽细胞维持能力存在质的差异 病变的稳定性取决于其来源；4)EC对ACTA2+纤维帽的贡献随着病变的增加而增加 通过给小鼠喂食18周的WD和12周的饲料来回归；5)雌性而不是雄性 表现出IL1R1依赖的EndoMT。这里的研究将检验EC对ACTA2+的贡献 纤维帽随致死性照射而增加-骨髓移植、SMC PDGFRKO、年龄、女性和/或斑块消退 结果：1)内皮细胞增殖和复制衰老增加；2)内皮细胞完整性丧失和/或抗 血栓性质；以及3)增加斑块侵蚀或破裂的易感性。目标一号将确定被标记的 致死性照射后EndoMT-MFT对ACTA2+纤维帽的贡献增加-BMT，SMC PDGFRKO或伴有斑块消退与增殖增加、克隆性扩张、复制性相关 衰老和/或EC功能障碍。目标2将决定是否治疗我们的SMC-或EC-谱系追踪载脂蛋白E-/- 或者我们的新型MYH11-CRERT2/ROSA-EYFP/SR-BICT/CT/LDLRCT-(CT)小鼠患有晚期BCA和CA病变 抗衰老药物与斑块稳定性指数的有益改变、改善存活率、减少MI或 卒中，减少对维持ACTA2+纤维帽的EndoMT-MFT的依赖，和/或改善EC 完整性和/或功能。所有目标包括稳定与高风险颈动脉内膜切除术的人体验证研究 样本，以及对晚期病变发病的性别相关决定因素的评估。建议进行的研究 将为调节细胞和细胞外基质(ECM)组成的机制提供新的见解 并可能导致开发新的治疗方法来增强斑块的稳定性。

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.