An endothelial-fibroblast axis connecting senescence to amino acid metabolism for control of vascular stiffness in PAH

背景：肺动脉高压（Pulmonary arterial hypertension，PAH）是一种依赖于多种血管细胞的致死性疾病 类型但是，关键的分子串扰系统仍然是个谜。在之前的裁决中，我们定义了一个关键的监管 轴之间的转录辅激活因子雅普/TAZ与酶谷氨酰胺酶（GLS 1），建立一个新的 谷氨酰胺代谢如何与PAH中的血管僵硬度相关的范例。然而，关键问题依然存在。 激活雅普/TAZ以引发PAH的触发因素是什么？它们是否来自不同的细胞类型？ 在这些触发因素的下游，其他氨基酸的代谢是否控制血管硬化和PAH？ 最近，内皮细胞（EC）衰老稳定的细胞周期停滞导致炎症信号转导， 衰老相关分泌表型（SASP）因子在PAH中有报道，但 PAH中的衰老尚未探索。我们推测EC衰老诱导炎性SASP信号传导， PA成纤维细胞，重新编程丝氨酸沿着谷氨酰胺代谢以控制胶原沉积，血管 硬度和PAH。目的1）明确EC衰老对成纤维细胞谷氨酰胺和丝氨酸的调控作用 代谢、血管硬化和PAH。我们计划研究携带EC特异性缺陷的PAH小鼠， 衰老驱动因子p16以及对成纤维细胞雅普和下游代谢重编程的影响。通过EC- 特异性分泌组跟踪PAH小鼠，我们将定义SASP蛋白因子的整个谱， PAH相关衰老EC。通过标记谷氨酰胺/丝氨酸后的人PAH肺的单细胞RNA测序 摄取和光谱（MIMS）成像，我们将确定EC衰老是否与成纤维细胞相关 谷氨酰胺/丝氨酸摄取。目的2）确定GLS 1和丝氨酸催化酶SHMT 1是否改变 对血管硬化和PAH至关重要。在这里，我们将确定是否成纤维细胞特异性敲除GLS 1 或SHMT 1逆转PAH小鼠中的血管硬化，并且如果SHMT 1和GLS 1的AAV特异性递送驱动 血管硬化和PAH。PLGA包裹小分子抑制雅普/GLS 1/SHMT 1的研究 纳米颗粒用于吸入治疗，我们将定义这种治疗逆转血管硬化的疗效， 呸。目的3）利用18F-荧光谷氨酰胺PET成像来测量SSc-PAH与SSc-PAH的谷氨酰胺摄取。 对照我们将在系统性硬化症依赖性PAH（SSc-PAH）和SSc 患有PAH早期形式的患者，运动PH。本研究将确定谷氨酰胺的相关性 在人类PAH的发展（不仅仅是终末期）中的代谢以及18F-FGln作为 一种新的SSc-PAH诊断示踪剂。意义：我们的多学科团队具有独特的定位， EC衰老至成纤维细胞代谢途径，对诱导血管硬化和PAH至关重要。我们将 测试一种新的吸入组合代谢疗法，我们将着手进行一项首次人体诊断研究， 18F-FGln PET/CT。因此，我们的目标是建立广泛的细胞间轴，汇聚成纤维细胞氨基， 酸代谢作为PAH的关键调节剂，从而提供新的靶向治疗和诊断。

Background: Pulmonary arterial hypertension (PAH) is a deadly disease dependent on several vascular cell types. But, key systems of molecular cross-talk remain enigmatic. In the prior award, we defined a key regulatory axis between the transcriptional coactivators YAP/TAZ with the enzyme glutaminase (GLS1), establishing a new paradigm of how glutamine metabolism is related to vascular stiffness in PAH. Yet, crucial questions remain. What are the triggers that activate YAP/TAZ to initiate PAH and do they originate from separate cell types? Downstream of those triggers, does metabolism of other amino acids control vascular stiffening and PAH? Recently, endothelial cell (EC) senescence–stable cell cycle arrest resulting in inflammatory signaling via senescence associated secretory phenotype (SASP) factors–was reported in PAH, but the consequences of senescence in PAH are unexplored. We postulate that EC senescence induces inflammatory SASP signaling to PA fibroblasts, reprogramming serine along with glutamine metabolism to control collagen deposition, vascular stiffness, and PAH. Aim 1) Define the role of EC senescence in controlling fibroblast glutamine and serine metabolism, vascular stiffening, and PAH. We plan to study PAH mice carrying EC-specific deficiency of the senescence driver p16 and the effects on fibroblast YAP and downstream metabolic reprogramming. Via EC- specific secretome-tracking mice with PAH, we will define the entire profile of SASP protein factors derived from PAH-relevant senescent ECs. By single cell RNA sequencing of human PAH lung after labeled glutamine/serine ingestion and spectral (MIMS) imaging, we will determine if EC senescence correlates with fibroblast glutamine/serine uptake. Aim 2) Determine if alterations of GLS1 and the serine catabolism enzyme SHMT1 are essential for vascular stiffening and PAH. Here, we will determine if fibroblast-specific knockout of GLS1 or SHMT1 reverses vascular stiffening in PAH mice and if AAV-specific delivery of SHMT1 and GLS1 drives vascular stiffening and PAH. Using small molecules to inhibit YAP/GLS1/SHMT1 encapsulated in PLGA nanoparticles for inhaled therapy, we will define the efficacy of such therapy to reverse vascular stiffening and PAH. Aim 3) Utilize 18F-fluoroglutamine PET imaging to measure glutamine uptake in SSc-PAH vs. controls. We will test 18F-FGln PET imaging in systemic sclerosis-dependent PAH (SSc-PAH) and in SSc patients with an early-stage form of the PAH, exercise PH. This study will define the relevance of glutamine metabolism in the development (not merely end-stage) of human PAH and the potential of 18F-FGln to serve as a novel diagnostic tracer for SSc-PAH. Significance: Our multi-disciplinary team is uniquely positioned to define an EC senescence-to-fibroblast metabolism pathway critical for inducing vascular stiffening and PAH. We will test a novel inhaled combinatorial metabolic therapy, and we will embark on a first-in-human diagnostic study of 18F-FGln PET/CT. Thus, we aim to establish the broad intercellular axes that converge upon fibroblast amino acid metabolism as a crucial regulator of PAH, thereby offering novel targeted therapeutics and diagnostics.