Endothelium-driven signaling network in the development of pulmonary hypertension

ABSTRACT ______
The central focus of application is to establish a novel pulmonary hypertension (PH) model in mice by specifically
targeting the endothelium, and reveal a novel endothelium-driven signaling network of uncoupled eNOS-ER stress-
mitochondrial dysfunction axis in the pathogenesis of PH, targeting of which would result in novel therapeutics for PH.
Pulmonary hypertension is a severe human disease characterized by intensive remodeling of small arteries in the lung,
resulting in vasoconstriction, elevated vascular resistance and pulmonary arterial pressure, and eventually right heart
failure. In preliminary studies, we have generated a novel PH model by directly targeting eNOS to provoke eNOS
uncoupling (DAHP to inhibit GTPCHI) and eNOS uncoupling-dependent endothelial dysfunction. Of note, endothelial
dysfunction is one of the earliest events and may the initiating step of idiopathic pulmonary artery hypertension (IPAH).
Importantly, DAHP-treated mice developed robust PH phenotypes of increased mean pulmonary artery pressure (mPAP)
and right ventricular systolic pressure (RVSP), accompanied by extensive vascular remodel characterized by typical
human like vascular lesions of medial thickness, neointimal formation, and plexiform features. RNA-sequencing (RNA-
seq) data indicated that, comparing to human patients with PH, the DAHP model had more overlappingly and substantially
regulated genes vs. the hypoxia model (217 vs. 92). In preliminary studies we have also revealed new molecular
mechanisms mediating PH development downstream of uncoupled eNOS, involving ER stress and mitochondrial
dysfunction. Additionally, since reversal of eNOS cofactor tetrahydrobiopterin salvage enzyme dihydrofolate reductase
(DHFR) deficiency downstream of NADPH oxidase (NOX) activation is robustly effective in preserving eNOS coupling
activity, novel genetic strains specifically targeting NOX isoforms and DHFR will be examined for efficacies modulating PH
phenotypes (16 novel and unique strains, most of which made in house). In Aim 1 we aim to establish a novel human like
murine model of PH by fully characterizing phenotypes of DAHP-treated mice, and by comparing its gene regulation
profile to that of human patients with PH and of Sugen5416/Hypoxia (SuHx)-treated mice. Also to further examine roles in
PH development of novel candidate genes identified by RNA-seq analyses. In Aim 2, we will examine novel endothelium-
driven signaling network of uncoupled eNOS-ER stress-mitochondrial dysfunction axis in the pathogenesis of PH using
DAHP, hypoxia and Su/Hx models of PH. In Aim 3, we will examine whether strategies targeting endothelial DHFR, such
as endothelium-specific transgenesis of DHFR, or knockout of NOX isoforms specifically in the endothelium to preserve
DHFR function, would be of novel therapeutic potential for PH. We will also examine whether global and conditional
knockout of DHFR, or endothelium-specific overexpression of NOX isoforms, leads to PH development and exaggerated
PH. Effects on PH development of novel DHFR activators, and small molecule inhibitors for NOX, will also be examined.
These studies are highly significant and translational in potentially identifying novel therapeutic options for the treatment
and/or prevention of PH, namely via attenuation of NOX isoform activation to preserve endothelial DHFR function and
eNOS coupling activity to shut down ER stress and mitochondrial dysfunction, or via direct activation of DHFR.

Grant Number: 5R01HL154754-04
NIH Institute/Center: NIH
Principal Investigator: Hua Linda Cai