4B). Conversely, sorafenib—as well as the mechanistically linked compound imatinib—blocked the inducing effect of PDGF on Ang1 mRNA levels (Fig. 4C). To extend our analyses of signaling pathways responsible for PDGF-induced Ang1 production in HSCs, we treated HSCs with U0126, a MEK inhibitor or wortmannin, a phosphoinositide 3-kinase (PI3K)/Akt pathway inhibitor, in the presence or absence of PDGF, and assessed mRNA levels of Ang1 by way of RT-PCR. Whereas wortmannin markedly inhibited Ang1 synthesis,

U0126 did not (Fig. 4D). Moreover, Ang1 synthesis was not impaired in Raf-silenced HSCs (Fig. 4E). Conversely, western blot analyses revealed that fibronectin expression was inhibited in Raf-silenced HSCs (Fig. 4A, lower right panel). Hence, Ang1 expression check details in HSCs occurs through a PDGFR- and PI3K/Akt-dependent but Raf-independent mechanism,

whereas fibronectin expression in HSCs occurs through canonical PDGFR and Raf pathways. Thus, these results suggest that expression of genes that participate in remodeling of vasculature is regulated by key molecular pathways that are downstream of tyrosine Adriamycin purchase kinase receptors, such as PDGF, and that sorafenib effectively inhibits these events. We next investigated how this signaling cascade converges on nuclear transcription factors that may regulate expression of these angiogenic factors. We focused on KLF proteins because this family of proteins have emerged as key regulators of HSC function and phenotype.20-23 A systematic and family-wide screening approach of KLF proteins

revealed that KLF6, KLF7, KLF8, KLF9, and KLF15 were repressed in cells pretreated with sorafenib (data not shown). Of these, KLF6 is a molecule prominently implicated in fibrosis, thus drawing our attention to this specific KLF protein. Indeed, RT-PCR revealed a significant up-regulation of KLF6 after incubation with PDGF, an effect that was abrogated by sorafenib (Fig. 5A). To further explore participation of KLF6 in regulation of fibronectin and Ang1 in HSCs, we performed RNA interference–based knock-down in HSCs. Indeed, down-regulation of KLF6 abolished PDGF-induced induction of Ang1 mRNA and fibronectin protein levels (Fig. 5B and 5C, respectively). Corroborative cell biological studies Flucloronide also demonstrated that tubulogenesis of LECs decreased significantly after incubation with CM from KLF6 small interfering RNA (siRNA)-transfected HSCs (Fig. 5D), similar to the observation in CM derived from HSCs treated with sorafenib, suggesting that this transcription factor regulates intracellular events that participate in active endothelial tubulogenesis. Finally, KLF6 as a direct regulator of angiogenic genes was firmly established by chromatin immunoprecipitation assay, which demonstrated that this transcription factor occupies the Ang1 promoter in cultured HSCs (Fig. 5E).