ABSTRACT
Traumatic brain injury (TBI) causes the blood-brain barrier (BBB) dysfunction and transmigration of
inflammatory immune cells into the brain, an important mechanism underlying neurovascular damage and
neuroinflammation. Adhesion of leukocytes to endothelial cells is a critical step in the migration of leukocytes
into injured tissues. Previously, it has been demonstrated that activation of leukocytes, especially neutrophils
cause the release of nuclear and granular contents to form an extensive web-like structure of DNA called
neutrophil extracellular traps (NET). However, in TBI, the mechanism of injury-induced formation of NET and
its mechanistic regulatory role in thrombosis remains elusive. Moreover, it is not clear whether blocking of
formation of NET provides better outcomes after TBI. Therefore, an approach to suppress the formation of
NET would be a valuable therapeutic strategy and to analyze the efficacy of the therapy in the functional
recovery level after TBI. Here, we hypothesize that inhibition of peptidyl arginine deiminase type 4 (PAD4), an
enzyme required for NET formation, using PAD4 antagonistic peptide (PAP) will attenuate the formation of
NET, NET-induced thrombosis, and promote neovascularization and functional recovery after TBI. In the first
aim, we test whether PAP reduces PAD4 expression, inhibits NET formation, and promotes
neovascularization. In the second specific aim, we will uncover the molecular mechanisms of the formation of
NET-induced thrombosis in TBI and we will dissect the therapeutic role of PAP in depleting NET-dependent
thrombosis. We will validate the role of PAD4 in the formation of NET and its role in thrombosis by
CRISPR/Cas9 mediated PAD4 gene deletion in human brain microvascular endothelial cells (hBMVECs) and
human neutrophil co-culture in vitro and PAD4 knockout (KO) mice (PAD4−/−) in vivo. In addition, we will study
the role of different brain cells in NET formation by creating conditional knockout mice by breeding PAD4flox
strain with specific brain cell Cre strains. In the third aim, we will use a cohort of behavioral tests that include
sensorimotor functions, memory, and psychological stress analyses to validate the role of PAP in promoting
functional recovery following TBI. Therefore, in this project, to validate the central hypothesis, these three aims
target a subset of events towards unraveling a larger picture of neurovascular remodeling and functional
recovery after TBI by attenuating PAD4 activity using a novel small peptide developed in the PI’s lab.
