R-4733

Pulmonary hypertension (PH) is a condition in which the smooth muscle cells (SMCs) in the pulmonary arteries multiply excessively, causing the arteries to narrow. This can ultimately result in right heart failure and premature death. Notch3 is an important factor involved in pulmonary vascular remodeling in PH. RO4929097, as a γ-secretase inhibitor that inhibits Notch3 signaling pathway, may be a potential drug for the treatment of PH, but its feasibility and related mechanism of action need to be further investigated. In vitro modeling by hypoxic incubation of human pulmonary artery SMCs (HPASMCs). RO4929097 and plasmids including overexpression-NICD3 (oe-NICD3) and NICD3 small interfering RNA (siRNA) were used to alter the expression of NICD3, and HIF-2α inhibitor PT-2385 was used to alter the expression of HIF-2α. Western blot, EdU incorporation assay was used to investigate the alteration of NICD3, HIF-2α, FoxM1 protein expression, and cell proliferation. The severity of PH in rats was assessed by measuring the weight ratio of right ventricle (RV) to left ventricle (LV) and septum (S) (RV/[LV + S]) and hematoxylin-eosin (H&E) staining of lung tissues in a hypoxia-induced PH rat model. We first determined that hypoxia induction for 48 h had the strongest induction of NICD3 and Notch3 in HPASMCs, and the strongest inhibition by 10 μM RO4929097. Treatment of HPASMCs under hypoxic conditions with RO4929097 inhibited hypoxia-induced expression of NICD3, HIF-2α, FoxM1, and proliferation of HPASMCs. The inhibitory effect of RO4929097 was reversed after overexpression of NICD3 in HPASMCs. Further, we found that PT-2385 reversed the promotional effect of overexpression of NICD3 on the proliferation of HPASMCs. In vivo experiments, hypoxia-induced PH rats treated with RO4929097 showed a reduction in right ventricular hypertrophy index (RVHI) and a return to normal pulmonary artery morphology, indicating a reduction in the severity of PH. Our data suggest that RO4929097 regulates the Notch3/HIF-2α/FoxM1 signaling pathway by inhibiting the expression of NICD3, thereby inhibiting hypoxia-induced proliferation of HPASMCs. In vivo experiments also confirmed that RO4929097 could alleviate PH as a potential therapeutic strategy.

Presenilin1 (PS1)/γ‐secretase cleaves within the transmembrane domain of numerous receptor substrates. Mutations in PS1 have implications on the catalytic subunit of γ‐secretase decreasing its activity and becoming a potential causative factor for Familial Alzheimer’s Disease (FAD). This work studies the role of PS1/γ‐secretase on the processing, angiogenic signaling, and functions of VEGFR2 and the effects of PS1 FAD mutants on the γ‐secretase‐mediated epsilon cleavage of VEGFR2.

HEK293T cells expressing WT‐PS1 or PS1‐FAD mutants (M146V, I213T, E280A, G384A, L166P, E120K, A246E) were prepared using a lentivirus‐based transduction system and used in luminescence‐based experiments. PS1‐VEGFR2 co‐immunoprecipitation (Co‐IP) experiments were analyzed on WBs. Bimolecular fluorescence complementation (BiFc) assays were carried out in HEK293T cells overexpressing PS1 or VEGFR2 tagged with half complementary portions of the mVenus and visualized by fluorescence microscopy. The ClusPro web server was used for protein‐protein docking analyses. Anti‐PS1 siRNA technology was applied to downregulate PS1 in endothelial bEnd.3 cells.

Luciferase Assay shows that PS1 FAD mutants significantly decrease the γ‐secretase‐mediated epsilon cleavage of VEGFR2 when compared to WT‐PS1, leading to the production of significantly lower amount of the VEGFR2/CTF2 peptide indicating a loss of γ‐secretase proteolytic function. This loss of function is comparable to the effect of γ‐secretase inhibitor RO4929097. Co‐IP experiments show that PS1 forms complexes with VEGFR2 and BiFc experiments show that PS1 binds to VEGFR2. Docking analysis indicates that the binding is electrostatically favored. Co‐IP between the PS1‐FAD mutants and VEGFR2 show that FAD mutants do not have differential binding to the VEGFR2 compared to WT‐PS1, showing that the effect of these mutants on VEGFR2 cleavage is not due to altered binding. PS1 downregulation of endothelial cells indicates that PS1 plays a role in the regulation of the VEGFA‐induced phosphorylation of VEGFR2 at Y1175 and Y1054‐59 as well as p‐44/42 MAPK (Erk1/2) and in VEGFA‐induced tube formation in vitro.

PS1 FAD mutants decrease production of signaling peptide VEGFR2/CTF2 probably due to reduced γ‐secretase activity of FAD mutants. Interestingly FAD mutations do not seem to affect the PS1‐VEGFR2 association. Our data support that PS1 regulates downstream signaling and angiogenic functions of VEGFR2.