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-FG...