Project Summary
Pulmonary arterial hypertension (PAH) is a rare, devastating disease with the initial pathognomonic instigator
being a combination of genetic and environmental (injurious) factors including hypoxemia and reactive oxygen
species (ROS) signaling. PAH is characterized by aggressive lung endothelial cell (EC)/smooth muscle cell
(SMC) proliferation/migration (remodeling) in balance with apoptosis, senescence and several other factors all
leading to ensuing right ventricle (RV) failure. Hypoxia and EC injury in rodents spawn physiological and cellular
signaling changes resembling hallmark changes observed in human PAH. We postulated that NADPH oxidase
1 (NOX1) and attendant ROS mediate EC proliferation and hemodynamic changes occurring in PAH. Findings
from our lab have shown induction of NOX1 and ROS in tissue from PAH patients and in human pulmonary
artery endothelial cells (HPAECs). Furthermore, the laboratory’s preliminary findings implicate AMPK isoform
alpha-1 (AMPKa1) as a potential lynchpin in the NOX1 pro-proliferative pathway. Mechanistically speaking, we
deduced that redox-sensitive cysteines (Cys 299 & 304) in AMPKa1 would be susceptible to oxidation, which
would, in turn, augment AMPKa1 activity. Using the bioinformatic tools of STRING, TRANSFAC and Orange, I
discovered that phosphorylated/activated AMPKa1 is associated with cyclin-dependent kinase inhibitor 1
(p21cip1), a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors. Importantly, the subcellular
localization of p21cip1 dictates its cellular function. In the nucleus, the Cip/Kip family promotes apoptosis and
senescence through retinoblastoma (Rb)-mediated suppression of the cell cycle. In contrast, when
phosphorylated and largely localized in the cytosol, p21cip1 purportedly indirectly actuates (disinhibits) cyclin D
and CDK4/6 leading to Rb inhibition (by phosphorylation) and a permissive cell cycle progression. Therefore, we
postulate that NOX1 mediates oxidative activation of AMPKa1 subunit via oxidizing cysteines 229 and/or 304
(Cys299/304). This is expected to phosphorylate p21cip1 leading to disinhibited binding from cyclin/CDK and cell
cycle progression and EC hyperproliferation in PAH. To test this overarching hypothesis, the following aims will
be tested: (1) To determine whether Nox1-induced oxidation of AMPKa1 elicits p21cip1 family disinhibition of
CyclinD/CDK4/6 and EC proliferation/migration; (2) To Interrogate the effect of EC NOX1 in the promotion of
vascular remodeling and hemodynamic changes in PAH animal models (Mouse and Rat Su/Hx). This proposal
is expected to reveal previously unidentified pathways controlled by NOX1 that participate in the pathology of
PAH. Additionally, an F31 award at this time will enhance my ability to grow as a young scientist and develop
the skills needed to obtain my PhD and pursue a career in the biomedical sciences.
