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

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.

Grant Number: 5R01HL124021-10
NIH Institute/Center: NIH
Principal Investigator: Stephen Chan