Project Details
Description
The aim of this grant is to elucidate the role of mitochondrial dynamics protein Drp1 as a novel redox sensor
that transmits VEGF-derived H2O2 signaling to enhance angiogenesis via regulation of endothelial cell
(EC) glycolysis. The induction of new blood vessels is critical for tissue repair in response to injury such as
peripheral arterial disease (PAD), which is impaired in diabetes. Reactive oxygen species (ROS) such as H2O2
derived from NADPH oxidase (NOX) and mitochondria at normal level act as signaling molecules to promote
VEGF-induced angiogenesis in endothelial cells (ECs) and reparative neovascularization. However, it remains
unclear “how diffusible H2O2 signal can be specifically transmitted to promote therapeutic angiogenesis”.
Signaling function of ROS is mainly through oxidation of reactive Cys residues to generate “Cysteine sulfenic
acid (Cys-OH)” (sulfenylation) which is involved in disulfide bond formation with target protein and redox
signaling. In addition, ECs utilize glycolysis as a major source of ATP to promote angiogenesis. However, the
mechanistic link between NOX-mitochondrial ROS (mitoROS)/redox signaling and EC metabolism (glycolysis)
in VEGF-induced angiogenesis is entirely unknown. Drp1 GTPase is key regulator of mitochondrial (mito) fission
via its post translational modification, but its role in ROS dependent VEGFR2 signaling and angiogenesis in ECs
and in vivo has never been reported. Our preliminary data are consistent with the hypothesis that VEGF
induces sulfenylation of Drp1 via NOX-derived H2O2, which drives mito fission-mitoROS axis that
promotes oxidative activation of key metabolic enzyme AMPK via disulfide bond formation (early phase)
as well as PFKFB3 expression (late phase) in ECs. This in turn enhances endothelial glycolysis and
angiogenesis required for restoring neovascularization in ischemic vascular disease. Aim1 will
characterize the VEGF-induced Drp1 sulfenylation and establish its role in ROS-dependent angiogenic
responses in ECs. Aim2 will determine the molecular mechanism by which VEGF-induced Drp1 sulfenylation
promotes glycolysis via mitochondrial ROS-dependent manner in ECs. Aim 3 will determine the functional role
of endothelial Drp1 in ROS-dependent reparative neovascularization and address underlying mechanisms in
vivo using animal model of PAD (hindlimb ischemia model). We will also address how diabetes -induced excess
ROS impair angiogenesis in ECs and in vivo by focusing on Drp1 phosphorylation at S616, but not Drp1-CysOH.
We will use various innovative reagents, methods and mice including biotin-labelled Cys-OH trapping probe;
BiFC-based protein-protein interaction in situ; real-time imaging of cytosol- and mitoROS using redox-sensitive
biosensors; newly developed EC-specific Drp1-/- mice and CRISPR/Cas9-generated “redox dead” Cys
oxidation-defective Drp1 or AMPK knock-in mutant mice. Our proposal will provide novel mechanistic
insights into Cys oxidized mitochondrial fission protein Drp1 that orchestrates NOX/mito ROS signaling and
glycolysis as a potential therapeutic target for treatment of ischemic cardiovascular diseases.
Status | Active |
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Effective start/end date | 9/1/21 → 7/31/24 |
Funding
- National Heart, Lung, and Blood Institute: $495,249.00
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