Project Summary
Brain arteriovenous malformation (bAVM) is a high-risk factor for intracerebral hemorrhage (ICH), which leads
to a high rate of disability or death. However, the treatment options for patients are severely limited, primarily
relying on surgical methods that possibly cause intraoperative hemorrhage or even death. No pharmacologic
treatment is available, primarily because of the poor understanding of the mechanisms in bAVM
pathophysiology. Clinical studies have recently found endothelial cell (EC)-specific somatic
activating KRAS mutations in up to 76% of sporadic bAVMs, which account for over 95% of bAVM cases. By
KRASG12V overexpression in mouse brain ECs, we confirmed that KRAS mutation is sufficient for bAVM
development and established a novel animal model of bAVM that displayed salient features of human bAVM.
While the underlying mechanism by which KRAS mutation causes bAVM development remains to be defined,
endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells (ECs) acquire
mesenchymal characteristics, has been implicated in human bAVMs. We found the bAVM in our mouse model
also demonstrated increased prototypic EndMT markers (CD44, KLF4, TGFβ, Notch, and MMPs) as well as
high intensity of α-SMA and CD44 localized on ECs in the bAVMs. We also confirmed that KRASG12V induced
EndMT characteristics in cultured ECs (increased mesenchymal markers but reduced EC markers). Earlier
studies have implicated calpains (a family of Ca2+-dependent cysteine proteases) as critical modulators of
EndMT. Calpains degrade junction proteins or negative regulators of EndMT. In addition, calpain inhibition
attenuates the lung or cardiac EndMT. KRAS mutation modulates ERK activation and Ca2+ influx, the
regulators of calpain expression and activation. Supportively, we found significantly higher levels of calpain-1
and -2 in cultured ECs overexpressing KRASG12V, as well as increased calpain-2 in bAVMs from KRASG12V
mice. We also confirmed that PD150606 (an inhibitor of calpain-1 and -2) reduced (1) EndMT marker, CD44 in
cultured ECs overexpressing KRASG12V and (2) the number of bAVM and ICH in KRASG12V mice. Based on the
evidence, we hypothesized that Calpain(s)-mediated EndMT in KRAS mutant EC leads to bAVM formation
and bAVM-associated ICH. To test the hypothesis, we will address 1) the detailed mechanism and role of
calpains in mediating EndMT that advances bAVM/ICH development and 2) the mechanism by which mutant
KRAS-induced EndMT derives bAVM development. Our ultimate goal is to develop intervention strategies for
the treatment of bAVM and bAVM-associated complications. Upon successful completion of the proposed
study, we will provide a novel mechanism in bAVM pathophysiology and potential targets to treat bAVM
patients.
