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.


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.
Schwaiberger
Andrea V
AV
Department of Pharmacognosy, University of Vienna, A-1090 Vienna, Austria.
Heiss
Elke H
EH
Cabaravdic
Muris
M
Oberan
Tina
T
Zaujec
Jan
J
Schachner
Daniel
D
Uhrin
Pavel
P
Atanasov
Atanas G
AG
Breuss
Johannes M
JM
Binder
Bernd R
BR
Dirsch
Verena M
VM
D000818:Animals; D001157:Arterial Occlusive Diseases / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D000077214:Becaplermin; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation; D002478:Cells, Cultured; D003251:Constriction, Pathologic; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005263:Femoral Artery / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D006965:Hyperplasia; D007211:Indoles / Q000494:pharmacology*; D007371:Interferon-gamma / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010091:Oximes / Q000494:pharmacology*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D007211:Indoles; D019394:Ki-67 Antigen; D010091:Oximes; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; C494088:Stat3 protein, rat; C474021:indirubin-3'-monoxime; D000077214:Becaplermin; D007371:Interferon-gamma; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin