Endothelial Regulation of Vascular Calcification

血管钙化的内皮调节

• 批准号: 10363955
• 负责人: Kristina I Bostrom
• 金额: $ 54.09万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10363955
• 关键词: Angiogenesis Inhibitors; Aorta; Appearance; Area; Arteries; Blood capillaries; CD34 gene; Cardiovascular Diseases; Cartilage; Cell Lineage; Cell Maintenance; Cells; Chondrocytes; Clinical; Complication; Development; Disease; Endothelial Cells; Endothelium; Gene Deletion; Gene Expression Profile; Glycoproteins; Goals; Human; In Vitro; Intervention; Knock-out; Lead; Location; Mesenchymal; Modeling; Morbidity - disease rate; Mouse Protein; Mus; Operative Surgical Procedures; Osteogenesis; PECAM1 gene; Pathology; Peripheral; Play; Population; Prevention; Public Health; RNA analysis; Regulation; Role; Sampling; Severities; Side; Signal Transduction; Supporting Cell; Testing; Tunica Adventitia; Vascular Diseases; Vascular Endothelial Growth Factors; Vascular Endothelium; Vascular calcification; angiogenesis; bone; calcification; endothelial-specific sialomucin; experimental study; in vivo; inhibitor; matrix Gla protein; mortality; notch protein; novel; osteogenic; osteoprogenitor cell; postnatal; receptor; recruit; single-cell RNA sequencing; stem cells; therapy development; transcription factor; transcriptome sequencing; treatment strategy

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.

项目概要： 血管钙化（VC）经常使心血管疾病复杂化。它增加了发病率， 死亡率，并构成干预和手术的重大障碍。血管内皮细胞 在VC中扮演重要角色。内膜（管腔）内皮细胞（EC）通过提供 骨祖细胞通过内皮-间充质转化（EndMT）。已知外膜EC 有助于病变血管壁的新血管生成，但尚不清楚这些EC是否支持VC 是什么定义了他们。EC标志物CD 31和糖蛋白高表达的EC亚群 内粘蛋白（Emcn）已被发现在骨中支持骨形成。可能是外膜或其他 外周EC被募集到患病区域以促进钙化。初步实验，使用 基质Gla蛋白缺失（Mgp-/-）小鼠作为VC模型，显示钙化主动脉中广泛的EC参与。 我们在外膜和内膜内皮中鉴定了两种EC亚型（称为a-EC和i-EC）。 EC）。a-EC为CD 31 +Emcm+，与VC的严重程度相关，而i-EC为CD 31 +Emcm+，与VC的严重程度相关。 CD31+Emcn-。这两种内皮细胞具有不同的干细胞和成骨标记物的转录谱， EC对比a-EC中增强的Notch表达。Notch 1的内皮缺失减少了a-EC， 限制VC，同时促进Mgp-/-小鼠的软骨形成和存活。骨转录因子 Osterix在两种类型的EC中均表达。我们假设a-EC与i-EC不同， 新生VC，易受Notch破坏。我们还假设Osterix对EC谱系具有保护作用。 在目标1中，我们将表征a-EC（CD 31 +Emcn+）并与Mgp-/-中的i-EC（CD 31 +Emcn-）进行比较。 模型，并与VC的严重程度和标志物表达相关。我们将确定独特的标记， 使用来自单细胞RNA测序的转录谱，与骨相比， （scRNAseq）。我们将测试的概念，血管生成是需要VC使用血管生成抑制剂。在Aim中 2、研究内皮细胞Notch信号的缺失或获得对EC亚型和VC的影响。我们 将检查与VC相关的Notch组分的分布，并产生具有内皮细胞的Mgp-/-小鼠， Notch 1或Notch受体灭活剂Fbxw 7的特异性丧失。我们将用老鼠来确定 内皮亚型的出现、钙化和scRNAseq的转录谱。我们还将 将Notch的丢失和获得应用于体外EC，并鉴定新的Notch靶点和网络。在目标3中，我们 使用人主动脉在体外和体内确定Osterix是否有助于维持EC谱系或促进钙化 Mgp-/-小鼠中的EC和可诱导的内皮特异性Osterix基因缺失。我们将比较转录 通过scRNAseq显示具有和不具有Osterix的EC的谱，以阐明对EC谱系的影响。我... EC和涉及的信令网络。我们的研究结果可能会对VC领域产生重大影响， 特别是对内皮病理学的理解和VC的参与。