Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo.

OBJECTIVE: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. METHODS AND
RESULTS: I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells.
CONCLUSIONS: I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.