See accompanying article on page e427

The role of vascular smooth muscle cells (SMCs) in the development and fate of atherosclerotic lesions is steadily gaining increased interest in cardiovascular research. Historically, this cell type has been attributed a distinct function in the regenerative features of the disease and a central role in intimal repair processes, highlighted by hallmark contributions from Russell Ross and Alexander Clowes.^1,2 In recent years, major advancements have provided insights that even further emphasize the central role of SMCs in cardiovascular disease.³ Genome wide association studies in coronary artery disease have facilitated the identification of several genetic variants associated with SMCs⁴ and use of lineage tracing models for identification of lesional cells of SMC origin have clarified that their ability to transdifferentiate into resemblance of other cell types leads to the formation of plaques with a much larger proportion SMC-derived cells than previously recognized.^5,6

Despite the diverse phenotypic plasticity of SMCs, their classical ability to contribute to fibrous cap formation and lesion stability is essential for the clinical course of the disease. The capacity of SMCs to provide tissue integrity in atherosclerosis is based on their ability to dedifferentiate from contractile cells in the media into proliferative and secretory cells that migrate into the intima, secrete extracellular matrix and both build and preserve the fibrous cap.⁷ Enhanced inflammation with protease activity, extracellular matrix degradation and SMC apoptosis weaken and make the cap prone to rupture, which eventually trigger atherothrombosis, myocardial infarction, and stroke.⁸ Although the importance of this fatal process has been suggested to diminish over time, likely influenced by improved medical therapy,⁹ a better understanding of the molecular pathways that operate in the formation and maintenance of the fibrous cap remains of vital significance to improve preventive pharmacotherapy in atherosclerotic cardiovascular disease.

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Martos-Rodríguez et al,¹⁰ make a significant contribution to the field by exploring the importance of Notch signaling for SMC recruitment and fibrous cap formation in atherosclerosis. The Notch signaling pathway is a well known player among those involved in the control of SMC function in development and disease. Notch signaling is a highly conserved pathway with an established and important role in vascular development and SMC biology. The pathway is named after the appearance of wings in Drosophila Notch-mutations and consists of a family of transmembrane ligands (Jagged1 and 2; Delta-like 1, 3, and 4) and receptors (Notch 1-4) with Notch extracellular (NECD) and intracellular (NICD) domains, where SMCs seem primarily dependent on signaling through Jagged1, Notch 2 and 3. Canonical Notch signaling involves protease-dependent activation and cleavage of NICD, which directly translocate to the nucleus where they act as transcriptional regulators by forming complexes with RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). This pathway differs from many others as the signal goes directly to transcriptional control and thus omits intracellular, intermediate, activation.¹¹ Nevertheless, it has been shown that the system still allows for signaling diversity by posttranslational glycosylation of the family members, which influence ligand-receptor interactions and signal intensity.¹² Noncanonical Notch signaling is less deciphered and largely unexplored in SMCs but is related to interactions with other pathways, such as platelet-derived growth factor B (PDGFB) and transforming growth factor beta (TGFβ) where the latter has been shown to cooperate with Notch in regulating SMC differentiation.¹¹

Functionally, Notch-ligands drive the differentiation of mural cells recruited by endothelial cell-derived PDGFB during vascular development and vasculogenesis with induction of alpha smooth muscle actin (ACTA2) and other contractile elements in the accumulating layers of SMCs, thus promoting differentiation, orientation, and assembly of the vessel wall media. Apart from SMC differentiation, Notch signaling may also regulate other SMC functions such as proliferation, migration, extracellular matrix synthesis, and survival.¹¹ The importance of Notch for SMC survival is emphasized in the human disorder CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), where Notch3 receptor mutations lead to apoptosis of SMCs and pericytes in the cerebral vasculature.¹¹ Because many of these functions are relevant for reparative SMC features, several studies have been conducted in vascular injury models demonstrating involvement of Notch family members and effector genes in intimal repair.^13-15

Whereas abundant literature has identified a central role for Notch signaling in the control of SMC function in development and intimal repair, much less is known about its role in the development, progression, and fate of SMCs in atherosclerotic lesions. In the work by Martos-Rodríguez et al, in ATVB, we get novel insights into the resemblance between atherosclerotic fibrous cap formation and embryonic media development with respect to the overarching control of these processes exerted by the Notch pathway. The authors report observations of Notch family member expression of in fibrous caps of human plaques, using mouse models including strains permitting lineage tracing of SMCs, atherosclerosis models, and mice with loss, as well as gain, of Notch signaling, and make confirmatory observations of SMCs in vitro. The mouse strains were either developed using conditional, SMC-specific, ablation of RBPJ, or incorporated SMC-specific constitutive Notch signaling by over-expression of NICD. The authors report how loss of Notch signaling did not affect the accumulation of SMC-derived cells in the fibrous cap but could show that these cells were prevented from reacquisition of ACTA2 expression, whereas constitutive Notch signaling instead prevented medial SMCs from contributing to plaque development (Figure).¹⁰

Figure. Schematic summary of the role of Notch signaling in fibrous cap formation in atherosclerotic plaques. Notch activity (blue arrows), promotes reacquisition of alpha smooth muscle actin (ACTA2) expression in modulated cap smooth muscle cells (SMCs), which is prevented in atherosclerotic mice with SMC-specific deletion of the Notch co-activating transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). In addition, Notch activity also suppresses medial SMCs recruitment to the fibrous cap.

Although the work by Martos-Rodríguez et al suggests that Notch signaling both plays a central role in investment of medial SMCs in fibrous cap formation and in reacquisition of ACTA2 expression, in a manner very similar to vessel wall development, the relevance of these processes for maintenance of fibrous cap integrity and plaque stability remains unknown. Genetic links with cardiovascular disease would strengthen the significance of Notch signaling for atherosclerotic plaque vulnerability. Previously, other target genes and pathways have been identified in GWAS of coronary artery disease cohorts and linked to control of SMC function with implications for fibrous cap stability, such as CDKN2B, TCF21, SMAD3, MIA3, CHI3L1, and LMOD1.^4,16,17 Possibly, similar associations also apply to the Notch pathway since a coronary artery disease risk locus affecting a binding site for RBPJ in the TWIST1 gene has recently been reported.¹⁸

A significant proportion of ACTA2 positive cells in the fibrous cap of both mice and humans were recently shown to be of non-SMC origin and were also associated with a reduced capacity to maintain cap stability.¹⁹ Whereas Martos-Rodríguez et al rather convincingly demonstrate how Notch signaling contributes to the maintenance of a differentiated SMC phenotype in mouse atherosclerosis, either by preserving medial SMCs or by promoting ACTA2 expression in cap cells of SMC origin, it is unknown if this pathway differently influences transdifferentiated plaque cells of SMC- and non-SMC origin, such as those derived through endothelial- or macrophage- mesenchymal transition, and how this ultimately influences lesion biology.

By exploring the role of the Notch pathway in fibrous cap formation, Martos-Rodríguez et al have provided yet another SMC-related molecular pathway for consideration in future atherosclerosis research. To make an impact of clinical relevance, such efforts should mechanistically address how this pathway influences fibrous cap stability and ultimately explore its potential as pharmacological target for intervention using agents capable of modulating Notch signaling and promoting SMC differentiation.²⁰

The author is supported by funding from the Swedish Heart-Lung Foundation (20180036, 20170584, 20180244, 201602877, 20180247), the Swedish Research Council (2017-01070, 2019-02027), and Karolinska Institutet.

Disclosures None.

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.

For Sources of Funding and Disclosures, see page 2386.

请参阅第 e427 页上的随附文章

血管平滑肌细胞 (SMC) 在动脉粥样硬化病变的发展和命运中的作用正逐渐引起心血管研究的兴趣。从历史上看，这种细胞类型在疾病的再生特征中具有独特的功能，并在内膜修复过程中发挥核心作用，Russell Ross 和 Alexander Clowes 的标志性贡献突出了这一点。^1,2近年来，重大进展提供了进一步强调 SMC 在心血管疾病中的核心作用的见解。³冠状动脉疾病的全基因组关联研究促进了与 SMC 相关的几种遗传变异的鉴定⁴和使用谱系追踪模型来识别 SMC 起源的病变细胞已经阐明，它们转分化为与其他细胞类型相似的能力导致形成斑块，其中 SMC 衍生细胞的比例比以前认识到的要大得多。^5,6

尽管 SMC 具有多种表型可塑性，但它们有助于纤维帽形成和病变稳定性的经典能力对于疾病的临床过程至关重要。SMC 在动脉粥样硬化中提供组织完整性的能力是基于它们从培养基中的收缩细胞去分化为增殖和分泌细胞的能力，这些细胞迁移到内膜、分泌细胞外基质以及构建和保存纤维帽。⁷蛋白酶活性增强的炎症、细胞外基质降解和 SMC 凋亡减弱，使帽容易破裂，最终引发动脉粥样硬化血栓形成、心肌梗塞和中风。⁸尽管有人认为这种致命过程的重要性会随着时间的推移而减弱，这可能会受到改进药物治疗的影响^9，但更好地了解在纤维帽形成和维持中起作用的分子途径对于改进预防性药物治疗仍然具有重要意义。动脉粥样硬化心血管疾病。

在本期动脉硬化、血栓形成和血管生物学中，Martos-Rodríguez 等人，¹⁰通过探索 Notch 信号对动脉粥样硬化中 SMC 募集和纤维帽形成的重要性，对该领域做出了重大贡献。Notch 信号通路是参与控制发育和疾病中 SMC 功能的那些中的一个众所周知的参与者。Notch 信号传导是一种高度保守的通路，在血管发育和 SMC 生物学中具有既定的重要作用。该途径以果蝇 Notch 突变中翅膀的出现命名，由一系列跨膜配体（Jagged1 和 2；Delta 样 1、3 和 4）和受体（Notch 1-4）和 Notch 细胞外（NECD）组成。 ) 和细胞内 (NICD) 结构域，其中 SMC 似乎主要依赖于通过 Jagged1、Notch 2 和 3 的信号传导。典型的 Notch 信号传导涉及 NICD 的蛋白酶依赖性激活和裂解，它们通过与 RBPJ（免疫球蛋白 kappa J 区的重组信号结合蛋白）形成复合物直接转移到细胞核中作为转录调节因子。该途径与许多其他途径不同，因为信号直接进入转录控制，因此省略了细胞内的中间激活。¹¹尽管如此，该系统仍然允许通过家族成员的翻译后糖基化产生信号多样性，这会影响配体 - 受体相互作用和信号强度。^{12 非}经典 Notch 信号在 SMC 中较少被破译，并且在很大程度上未被探索，但与其他途径的相互作用有关，例如血小板衍生生长因子 B (PDGFB) 和转化生长因子β (TGFβ)，后者已被证明与 Notch 合作在调节 SMC 分化中。¹¹

在功能上，Notch-配体在血管发育和血管生成过程中通过诱导 α 平滑肌肌动蛋白 (ACTA2) 和 SMC 堆积层中的其他收缩元件驱动由内皮细胞衍生的 PDGFB 募集的壁细胞分化，从而促进分化、定向、和血管壁介质的组装。除了 SMC 分化外，Notch 信号还可以调节其他 SMC 功能，例如增殖、迁移、细胞外基质合成和存活。¹¹ Notch 对 SMC 存活的重要性在人类疾病 CADASIL（伴有皮质下梗塞和白质脑病的常染色体显性脑病）中得到强调，其中 Notch3 受体突变导致脑血管系统中的 SMC 和周细胞凋亡。¹¹由于其中许多功能与修复性 SMC 特征相关，因此在血管损伤模型中进行了几项研究，证明 Notch 家族成员和效应基因参与内膜修复。^13-15

尽管大量文献已经确定了 Notch 信号在控制发育和内膜修复中 SMC 功能中的核心作用，但对其在动脉粥样硬化病变中 SMC 的发育、进展和命运中的作用知之甚少。在 Martos-Rodríguez 等人的作品中，在ATVB，我们对动脉粥样硬化纤维帽形成和胚胎培养基发育之间的相似之处获得了新的见解，这些相似之处在于 Notch 途径对这些过程的总体控制。作者使用小鼠模型报告了人类斑块纤维帽中 Notch 家族成员表达的观察结果，包括允许谱系追踪 SMC 的品系、动脉粥样硬化模型以及具有 Notch 信号丢失和获得的小鼠，并对SMC 体外。小鼠品系要么是使用有条件的、SMC 特异性的、RBPJ 消融来开发的，要么是通过 NICD 的过度表达来加入 SMC 特异性的组成型 Notch 信号。¹⁰

数字。 Notch 信号在动脉粥样硬化斑块中纤维帽形成中的作用示意图。Notch 活性（蓝色箭头）促进调节帽平滑肌细胞 (SMC) 中 α 平滑肌肌动蛋白 (ACTA2) 表达的重新获得，这在具有 SMC 特异性缺失 Notch 共激活转录因子 RBPJ（重组）的动脉粥样硬化小鼠中被阻止免疫球蛋白κJ区的信号结合蛋白）。此外，Notch 活动还抑制了内侧 SMC 向纤维帽的募集。

尽管 Martos-Rodríguez 等人的工作表明 Notch 信号在内侧 SMC 的纤维帽形成和重新获得 ACTA2 表达方面发挥着核心作用，其方式与血管壁发育非常相似，但这些过程与纤维帽完整性和斑块稳定性的维持仍然未知。与心血管疾病的遗传联系将加强 Notch 信号对动脉粥样硬化斑块易损性的重要性。以前，在冠状动脉疾病队列的 GWAS 中已经确定了其他靶基因和通路，这些基因和通路与 SMC 功能的控制有关，对纤维帽稳定性有影响，例如CDKN2B、TCF21、SMAD3、MIA3、CHI3L1和LMOD1。^4,16,17可能类似的关联也适用于 Notch 通路，因为最近报道了冠状动脉疾病风险位点影响 TWIST1 基因中 RBPJ 的结合位点。¹⁸

最近显示小鼠和人类纤维帽中很大比例的 ACTA2 阳性细胞是非 SMC 来源的，并且还与维持帽稳定性的能力降低有关。¹⁹虽然 Martos-Rodríguez 等人相当令人信服地证明了 Notch 信号如何通过保留内侧 SMC 或通过促进 SMC 起源的帽细胞中的 ACTA2 表达来维持小鼠动脉粥样硬化中分化的 SMC 表型，但不知道这种途径是否不同影响 SMC 和非 SMC 来源的转分化斑块细胞，例如通过内皮细胞或巨噬细胞间充质转化衍生的那些细胞，以及这如何最终影响病变生物学。

通过探索 Notch 通路在纤维帽形成中的作用，Martos-Rodríguez 等人提供了另一种与 SMC 相关的分子通路，供未来动脉粥样硬化研究考虑。为了产生临床相关性的影响，这些努力应该从机制上解决该途径如何影响纤维帽稳定性，并最终探索其作为使用能够调节 Notch 信号传导和促进 SMC 分化的药物干预的药理学靶点的潜力。²⁰

作者得到了瑞典心肺基金会 (20180036、20170584、20180244、201602877、20180247)、瑞典研究委员会 (2017-01070、2019-02027) 和 Karolinska 研究所的资助。

披露无。

本文中表达的观点不一定是编辑或美国心脏协会的观点。

有关资金来源和披露信息，请参见第 2386 页。