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

背景：肺动脉高压是一种依赖于多种血管细胞的致命性疾病。 类型。但是，分子串扰的关键系统仍然是个谜。在之前的奖项中，我们定义了一个关键的监管 转录共激活因子YAP/TAZ与谷氨酰胺酶(GLS1)之间的轴，建立了一个新的 PAH中谷氨酰胺代谢如何与血管僵硬有关的范例。然而，关键问题依然存在。 激活YAP/TAZ以启动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着血管僵硬和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通过 衰老相关分泌表型(SASP)因子-在PAH中被报道，但其后果 多环芳烃中的衰老还没有被研究。我们推测EC衰老诱导炎性SASP信号转导 PA成纤维细胞，重新编程丝氨酸和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衰老在控制成纤维细胞谷氨酰胺和丝氨酸中的作用 新陈代谢、血管僵硬和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及其对成纤维细胞YAP和下游代谢重编程的影响。通过EC- 有PAH的特定分泌组跟踪小鼠，我们将定义源自以下来源的SASP蛋白因子的完整图谱 PAH相关的衰老内皮细胞。谷氨酰胺/丝氨酸标记的人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)检测GLS1和丝氨酸分解代谢酶SHMT1的变化 对于血管僵硬和PAH是必不可少的。在这里，我们将确定成纤维细胞特异性的GLS1基因敲除 或SHMT1逆转PAH小鼠的血管硬化，如果AAV特异性地传递SHMT1和GLS1驱动 血管硬化和PAH。利用小分子抑制PLGA包裹的YAP/GLS1/SHMT1基因 纳米颗粒用于吸入治疗，我们将确定这种治疗方法逆转血管僵硬和 啊哈。目的3)应用18F-氟谷氨酰胺PET显像检测SSc-PAH与正常对照的谷氨酰胺摄取。 控制。我们将在系统性硬化症依赖的PAH(SSC-PAH)和SSC中测试18F-FGln PET成像 患者早期表现为PAH，运动PH。这项研究将定义谷氨酰胺的相关性 人多环芳烃发育(不只是终末期)的代谢和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，能够定义 EC从衰老到成纤维细胞的代谢途径，对诱导血管僵硬和PAH至关重要。我们会 测试一种新型的吸入联合代谢疗法，我们将开始一项首个人类诊断研究 18F-FGln PET/CT。因此，我们的目标是建立汇聚成纤维细胞氨基的广泛的细胞间轴。 酸代谢作为PAH的关键调节因子，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.