EndMT as a target to treat human brain arteriovenous malformations - 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. A recent study reported that endothelial-to-mesenchymal transition (EndMT) is a common feature in human bAVM, irrespective of the underlying genetic background. Our proteomic analyses also confirmed increased EndMT markers in human bAVMs. EndMT is involved in physiologic and pathologic angiogenesis, and endothelial cells (ECs) undergoing EndMT showed decreased junctional proteins. KRAS mutations are detected in human bAVMs at a high frequency (~76%), and KRAS-mutant bAVMs are more prone to rupture (1.7 fold). These results suggest that KRAS mutations may induce more severe EndMT, leading to exacerbated bAVM development and rupture. In this exploratory grant, we propose to use our existing and prospective biorepository of human bAVMs and cultured endothelium we earlier established using fresh human bAVMs. The study will be guided by our animal translational studies that used our recently established mouse model of bAVMs by brain EC-specific overexpression of KRASG12V (KRASG12V mice). The bAVMs in KRASG12V mice recapitulated salient features of human bAVMs including EndMT (e.g., increased α- SMA, CD44, CDH2). Our in vitro study demonstrates enhanced EndMT markers in cultured ECs overexpressing KRASG12V (ECG12V), which were accompanied by dysregulated EC functions. Previous studies have implicated calpains (a family of Ca2+-dependent cysteine proteases) as critical modulators of EndMT by showing that calpain inhibition attenuates EndMT in cardiac and lung tissues. In our preliminary studies, we found higher levels of calpain-1 and -2 in ECG12V and human bAVM tissues and confirmed that PD150606 (an inhibitor of calpain-1 and 2) reverses EndMT marker in ECG12V and ICHs and bAVMs in KRASG12V mice. The evidence led us to hypothesize that KRAS mutation exacerbates bAVM pathology through exaggerated EndMT, and calpain-mediated EndMT is a ubiquitous and essential mechanism for human bAVM, regardless of the etiology. Using our human materials from local surgeries (by Dr. Chen, multi-PI), Aim 1 will determine if KRAS mutation exacerbates EndMT and pathology in human bAVM by comparing EndMT features between human KRAS-mutant bAVMs (bAVMKmt) vs. KRAS-wild type bAVMs (bAVMKwt), and Aim 2 will probe the role of calpains in EndMT and EC functions in cultured human bAVM ECs by determining EndMT markers and EC functions in primary cultured bAVM ECs with calpain inhibition. Our study will provide preliminary data to justify further therapeutic experiments in preclinical mouse models of bAVM and may yield a new therapeutic target for treating human bAVMs.
