Differential expression of 39 genes was confirmed by qRT-PCR analysis in all groups with high statistical significance, whereas seven genes were only partially confirmed (Supporting Information Table 4). In addition, 449 genes (LSECup) were also found to be up-regulated in

culture with FC >2 when comparing LSEC0h with LSEC42h (not shown), indicating that LSEC dedifferentiation in culture is not just a process of cellular deterioration. Using qRT-PCR, several of these genes were shown to reach expression levels also seen in LMEC0h (Cxcr4, Apelin), whereas others were specifically induced in cultured LSEC (Esm1, Aatf) (Fig. 2C). As Wnt-2 and Flt-4/Vegfr3 were confirmed to be overexpressed in LSEC compared to LMEC and to be considerably down-regulated in LSEC in vitro by qRT-PCR (Fig. 3A), the Gefitinib solubility dmso Wnt and VEGF signaling pathways were scrutinized for broader impairment in LSEC transdifferentiation in vitro. qRT-PCR analysis showed that VegfA was

highly overexpressed in LMEC versus LSEC and did not decline in culture. Vegf B, C, D did not show any significant differences. Vegfr2 shown by us to be induced in LSEC by Wnt-2, but not Vegfr1, was overexpressed in LSEC versus LMEC; both receptors were down-regulated in LSEC in vitro. VEGF coreceptor Nrp2 (venous/lymphatic EC), but not Nrp1 (arterial EC), paralleled expression of Vegfr2/3. Tmem24 involved in Flt-4/Vegfr3 Cabozantinib signaling was also found to be down-regulated upon culture (Fig. 3B). Previously, we have shown that Wnt2 is strongly overexpressed in LSEC and that it acts as an autocrine growth factor by way of Wnt receptors Fzd4, Fzd5, and Fzd9. Differential expression of Wnt signaling components in LSEC and LMEC was also previously demonstrated by us including Wntless homolog (Wls), an intracellular

wnt transporter, and wnt-inhibitory factor Bacterial neuraminidase (Wif-1), whereas expression of most Wnt ligands except for Wnt-7b and Wnt-10b was relatively weak in LSEC as well as in LMEC.8 Upon transdifferentiation in culture (42 hours), wnt2 receptors Fzd4, Fzd5, and Fzd9 and Wnt-10b and Wls were considerably down-regulated, whereas Wnt-7b, Wif1, and Devl did not change significantly (Fig. 3C). Organ-specific blood vascular EC differentiation is thought to be mediated by specific combinations of otherwise non-EC-specific transcription factors. In this respect, identification and confirmation by qRT-PCR of the three transcription factors Tfec, Gata-4, and Maf in the LSECspecific+down gene signature was quite intriguing (Fig. 4A). In contrast to Gata-4, expression of Gata family members Gata-2, -3, and -5 was much lower in LSEC versus LMEC and did not change significantly upon culture (Fig. 4B,C). Similarly, basic helix-loop-helix (bHLH) transcription factors Mitf, Tfe3, and Tfeb showed a much lower expression level in LSEC than Tfec and grossly remained unaltered throughout cultivation (not shown).