Endothelial Regulation of Vascular Calcification

项目总结： 血管钙化(VC)常合并心血管疾病。它增加了发病率和 死亡率高，是干预和外科手术的一个重大障碍。血管内皮细胞发挥作用 在风投中扮演着重要的角色。内膜(腔)内皮细胞(ECs)通过提供 成骨祖细胞通过内皮-间充质转化(EndMTs)。外膜内皮细胞是已知的 促进病变血管壁的新生血管生成，但这些内皮细胞是否支持血管内皮细胞尚不清楚 以及它们的定义。EC标志物CD31和糖蛋白高表达的内皮细胞亚群 内粘蛋白(Emdomucin，EMCN)被发现存在于骨骼中，可支持骨形成。有可能是外星人或其他 外周内皮细胞被招募到病变区域以促进钙化。初步实验，使用 基质GLA蛋白缺失(MGP-/-)小鼠作为VC模型，表现为内皮细胞广泛参与钙化的主动脉。 我们鉴定了外膜和内膜内皮细胞的两种亚型(称为a-ECs和i-ECs)。 ECS)。A-ECs为CD31+Emcm+，与VC的严重程度相关，而i-ECs与VC的严重程度相关 CD31+Emcn-。这两种内皮细胞具有不同的转录图谱，在I-ECs中具有干细胞和成骨标记。 ECS与增强的Notch在a-ECs中的表达。内皮细胞Notch1缺失减少了a-ECs和 在促进MGP-/-小鼠软骨形成和存活的同时限制VC。骨转录因子 Osterix在两种类型的内皮细胞中均有表达。我们假设a-ECs不同于i-ecs，招募来 新生的风投，很容易受到Notch的颠覆。我们还假设Osterix是EC血统的保护者。 在目标1中，我们将表征a-ECs(CD31+Emcn+)，并与MGP-/-中的i-ECs(CD31+Emcn-)进行比较 模型，并与VC的严重程度和标志物的表达相关。我们将确定唯一的标记，用于 使用来自单细胞RNA测序的转录图谱，将各自的内皮细胞与骨骼进行比较 (ScRNAseq)。我们将使用血管生成抑制剂来测试VC需要血管生成这一概念。在AIM 2、我们将确定内皮细胞Notch信号的丢失或增加对EC亚型和VC的影响。我们 将检测与VC相关的Notch成分的分布，并产生MGP-/-小鼠的内皮- Notch1或Notch受体失活子Fbxw7的特异性丢失。我们将使用小鼠来确定对 ScRNAseq检测血管内皮细胞亚型、钙化及转录谱。我们还将 将Notch的损失和增益应用于体外培养的内皮细胞，并鉴定新的Notch靶点和网络。在《目标3》中，我们将 确定Osterix在体外和体内是否有助于维持EC谱系或促进钙化 MGP-/-小鼠ECS和可诱导的内皮特异性Osterix基因缺失。我们会将转录后的 用scRNAseq技术分析有无Osterix对EC谱系a-ECs vc的影响。我- ECS，并涉及信令网络。我们的结果可能会对风险投资领域产生重大影响，在 特别是对血管内皮细胞的病理及在VC中的参与的了解。

PROJECT SUMMARY: Vascular calcification (VC) frequently complicates cardiovascular disease. It increases the morbidity and mortality and constitutes a significant obstacle in interventions and surgeries. The vascular endothelium plays an important role in VC. The intimal (luminal) endothelial cells (ECs) contribute to VC by providing osteoprogenitor cells through endothelial-mesenchymal transitions (EndMTs). The adventitial ECs are known to contribute to neo-angiogenesis in diseased vascular wall, but it is unknown whether such ECs support VC and what defines them. A subset of ECs with high expression of the EC marker CD31 and the glycoprotein Endomucin (Emcn) has been found in bone to support bone formation. It is possible that adventitial or other peripheral ECs are recruited to diseased areas to promote calcification. Preliminary experiments, using the Matrix Gla Protein null (Mgp-/-) mouse as a VC model, showed extensive EC involvement in the calcified aorta. We identified two subtypes of ECs in the adventitial vs. the intimal endothelium (referred to as a-ECs and i- ECs). The a-ECs were CD31+Emcm+ and correlated with the severity of the VC, whereas the i-ECs were CD31+Emcn-. The two ECs had distinct transcriptional profiles with stem cell and osteogenic markers in the i- ECs vs. enhanced Notch expression in the a-ECs. Endothelial deletion of Notch1 reduced the a-ECs and limited VC while promoting cartilage formation and survival in the Mgp-/- mice. The bone transcription factor Osterix was expressed in both types of ECs. We hypothesize that a-ECs are distinct from i-ECs, recruited to nascent VC, and susceptible to Notch disruption. We also hypothesize that Osterix is protective of EC lineage. In Aim 1, we will characterize the a-ECs (CD31+Emcn+) and compare to the i-ECs (CD31+Emcn-) in the Mgp-/- model, and correlate with severity of VC and marker expression. We will identify unique markers for the respective ECs, with comparison to bone, using transcriptional profiles from single cell RNA sequencing (scRNAseq). We will test the concept that angiogenesis is required for VC using angiogenic inhibitors. In Aim 2, we will determine the effect of loss or gain of endothelial Notch signaling on the EC subtypes and VC. We will examine the distribution of Notch components in relation to VC, and generate Mgp-/- mice with endothelial- specific loss of Notch1 or the Notch receptor inactivator Fbxw7. We will use the mice to determine the effect on the appearance of the endothelial subtypes, calcification and transcriptional profiles by scRNAseq. We will also apply loss and gain of Notch to ECs in vitro and identify novel Notch targets and networks. In Aim 3, we will determine if Osterix helps maintain EC lineage or promotes calcification in vitro and in vivo using human aortic ECs and inducible endothelial-specific Osterix gene deletion in Mgp-/- mice. We will compare the transcriptional profiles of ECs with and without Osterix by scRNAseq in order to clarify the effect on EC lineage, a-ECs vc. I- ECs, and involved signaling networks. Our results may have a significant impact on the field of VC, in particular on the understanding of the endothelial pathology and involvement in VC.