In Vitro Human Tissue-Engineered Blood Vessel Disease Model of Progeria

摘要 Hutchinson-Gilford早衰综合征（HGPS）是一种罕见的加速衰老的常染色体显性遗传病 导致7到20岁之间的死亡这种疾病是由点突变引起的， 核蛋白核纤层蛋白A的交替剪接形式，称为早老蛋白，在细胞核中积累。 HGPS的小鼠模型表现出与HGPS核纤层蛋白基因突变的许多表型相似性，但 动脉粥样硬化不会发展，这表明动物模型的适用性有限。由于心血管 疾病是HGPS患者死亡的主要原因，我们建议使用一种新的组织 工程血管微生理学系统，以开发疾病的生物标志物，并评估 治疗对相关生理测量的有效性。我们已经开发出了小动脉规模的 内皮化组织工程血管（TEBV）使用平滑肌细胞（SMC），来源于 使用健康和HGPS细胞的诱导多能干细胞（iPSC）。可以生产TEBV， 在制备的几小时内在生理流动条件下灌注，并表现出内皮介导的 血管活性和对炎症介质的反应。我们可以进行标准的功能测试， 炎症信号的影响，从而跟踪同一血管中疾病的进展。的 HGPS-TEBV提供了比在塑料上培养的细胞更真实的体外环境， 发现新疗法和鉴定生物标志物的过程。在这个项目中，我们将测试 假设组织工程血管是用来自HGPS患者的细胞制成的， 在体外概括体内发现的结构和活性，并有助于评估有效性， 作用方式适合临床研究的候选药物。在目标1中，我们将检验TEBV 来自HGPS患者的细胞具有与HGPS小鼠模型相同的表型。我们将评估 (1)细胞数量减少和氧化应激对HGPS功能改变的相对贡献- TEBV，（2）流量对EC NRF 2活性及其调节的氧化基因的影响，以及（3）比较TEBV 结构和功能与HGPS的鼠标模型。对照条件将包括用以下物质制备的TEBV： 来源于HGPS患者之一的父母的细胞。在目标2中，我们将修改系统以运行多个 TEBV同时进行，并检验联合治疗无效的假设，因为它们 没有恢复SMC的数量、分化和血管活性。在目标3中，我们将评估 用于改变TEBV中HGPS表型的早衰症的新治疗。我们将研究代理人的影响 其单独或与洛那法尼组合改善线粒体功能和/或蛋白质降解， 抑制早老蛋白产生的反义寡核苷酸。将在小鼠中进行相应的研究， 评估HGPS-TEBV模型是否再现了HGPS小鼠模型中发现的血管变化。

Abstract Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal dominant disease of accelerated aging which leads to death between 7 and 20 years of age. The disease arises from point mutations that produce an alternately spliced form of the nuclear protein lamin A, known as progerin, that accumulates in the cell nucleus. Mouse models of HGPS exhibit many phenotypical similarities with the HGPS lamin gene mutation, but atherosclerosis does not develop, suggesting a limit to the suitability of animal models. Since cardiovascular disease represents the primary cause of death among those with HGPS, we propose to use a novel tissue engineered blood vessel microphysiological system to develop biomarkers for the disease and assess the effectiveness of treatment against relevant physiological measurements. We have developed arteriolar-scale endothelialized tissue-engineered blood vessels (TEBVs) using smooth muscle cells (SMCs) derived from induced pluripotent stem cells (iPSCs) using healthy and HGPS cells. The TEBVs can be produced and perfused at physiological flow conditions within a few hours of preparation and exhibit endothelial-mediated vasoactivity and respond to inflammatory mediators. We can perform standard functional tests and examine the effects of inflammatory signals, thus tracking the progression of the disease in the same vessel. The HGPS-TEBVs provide a more realistic in vitro environment than cells cultured on plastic and can help advance the process of discovering novel therapeutics and identification of biomarkers. In this project, we will test the hypotheses that tissue-engineered blood vessels made with cells derived from individuals with HGPS recapitulate in vitro the structure and activity found in vivo and can aid in assessing the effectiveness and mode of action suitable drug candidates for clinical studies. In Aim 1, we will test the hypothesis that TEBVs with cells derived from HGPS patients have the same phenotype as a mouse model of HGPS. We will assess (1) the relative contribution of reduced cell number and oxidative stress on the altered function of HGPS- TEBVs, (2) the effect of flow on EC NRF2 activity and oxidative genes it regulates, and (3) compare TEBV structure and function with the mouse model for HGPS. Control conditions will consist of TEBVs prepared with cells derived from a parent of one of the HGPS patients. In Aim 2, we will modify our system to run multiple TEBVs simultaneously and test the hypothesis that combination therapies have been ineffective because they have not restored SMC number, differentiation, and vasoactivity. In Aim 3, we will assess the suitability of novel treatments for progeria to alter the HGPS phenotype in the TEBVs. We will examine the effect of agents which improve mitochondrial function and or protein degradation, alone or in combination with lonafarnib and anti-sense oligonucleotides that inhibit progerin production. Corresponding studies in mice will be performed to assess whether the HGPS-TEBV model reproduces changes to vessels found in mouse model of HGPS.