Antimito- and motogenic activity of I3MO

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) play an important role in the development of cardiovascular disease. Our previous work showed that indirubin-3-monoxime, a derivative of the red isomer of indigo, exerts potent antiproliferative and antimigratory activity in platelet derived growth factor (PDGF)-stimulated VSMC. I3MO selectively interferes with STAT3 phosphorylation and signalling in VSMC and is able to reduce neointima formation in an arterial cuff model in vivo. We therefore hypothesize that (i) STAT3 phosphorylation is a crucial signalling step in VSMC proliferation and migration (ii) I3MO exerts its anti-mito- and anti-motogenic acitivity by inhibition of STAT3 phosphorylation, In the course of the proposed project we aim to (i) prove the causality between I3MO-mediated STAT3 inhibition and inhibition of proliferation and migration by I3MO in VSMC (ii) decipher the exact mode of action of indirubin-3-monoxime (I3MO) in STAT3 inhibition. We will examine whether I3MO modulates activities of kinases, phosphatases or influences levels of reactive oxygen species and cellular redox signalling. We will hereby follow candidate (Src, Abl, Shp2, PTP1B) and unbiased approaches. The latter include a kinome array and an approach comprising affinity chromatography with immobilized I3MO and VSMC lysates as well as MS analysis in order to decipher direct cellular interaction partners of I3MO in VSMC. (iii) examine how I3MO affects endothelial function and vascular inflammation and to what extent this can be traced back to STAT3 inhibition. Overall the project will provide novel insight into the detailed molecular mode of action of I3MO, a derivative of the natural red isomer of indigo, and into the role of STAT3 for VSMC proliferation and migration.

The main findings of this project are: (i) The small molecule indirubin-3- monoxime (I3MO) exerts a triple beneficial action against vascular diseases. (ii) Activation of transcription factor Nrf2 (nuclear factor 2 related 2) and inhibition of 5-lipoxygenase are novel bioactivities of I3MO. (iii) 12/15 lipoxygenase is a hitherto unknown crucial signalling hub in the signal transduction cascade to the activated transcription factor STAT3 (signal transducer and activator of transcription). The vasoprotective potential of I3MO, a more water soluble derivate of the naturally occurring compound indirubin, was investigated in cultivated cells. I3MO inhibited of proliferation and migration of primary vascular smooth muscle cells at low micromolar concentrations. I3MO further inhibits production of pro-inflammatory mediators in monocytes. These findings identified I3MO as promising hit compound for the prevention of atherosclerosis and restenosis (re-narrowing of the vessel lumen after stent placement). I3MO interferes with three major cellular responses connected with the aetiology of those vascular problems, namely with inflammation, migration and proliferation. I3MO is a compound with pronounced polypharmacology hitting on more than one target in the cell. Its antiproliferative activity is due to inhibition of STAT3 (signal transducer and activator of transcription), a protein that usually triggers expression of factors needed for cell division. Activation of the transcription factor Nrf2 causes the antimigratory effect by inducing the production of proteins that halt migration. Direct inhibition of 5-lipoxygenase, an enzyme that produces inflammatory factors accounts for the anti-inflammatory property. Digging deeper into the issue how STAT3 activation is inhibited by I3MO we identified STAT3 activation as being generally redox-sensitive and in particular dependent on the enzyme activity of 12/15 lipoxygenase. In line, I3MO reduces activation of 12/15 lipoxygenase and intracellular ROS and, thus, activation of STAT3 and proliferation in growth factor stimulated vascular smooth muscle cells. Overall, we revealed novel bioactivities for I3MO promoting the compound as starting point for further lead development and discovered so far unknown nodes and targets in cellular signal transduction.