Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death

Abstract

Objectives: Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury. Methods & Results: The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern in vitro . Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation. Conclusions: In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.