Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J-NICD-MAML complex in intron 3 of Dll4.

Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7-P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.

Intervention of glutathione in pre-mutagenic catechol-mediated DNA damage in the presence of copper(II) ions.

The catechol-mediated DNA damage in the presence of Cu(II) ions involves oxidation of guanine to 8-oxoguanine (8-oxoG) and DNA strand scission. It proceeds through the reactive oxygen species (ROS) generation. The mutagenicity of 8-oxoG lesions is due to its miscoding propensity reflected in GC→TA transversion taking place during the DNA repair process. To gain new insights into the nature of catechol-mediated DNA damage and its prevention, we have investigated the changes in DNA melting characteristics and 8-oxoG formation as the indicators of DNA damage in a model calf-thymus DNA system. A novel fluorescence method for DNA melting temperature determination, based on DAPI fluorescent-probe staining, has been proposed. The DNA melting-onset temperature has been found to be more sensitive to DNA damage than the standard melting temperature due to the increased width of the melting transition observed in oxidatively damaged DNA. We have found that the efficiency of Fenton cascade in generating DNA-damaging ROS is higher for catechol than for GSH, two strong antioxidants, mainly due to the much longer distance between ROS-generating radical group in GS to nucleobases than that of semiquinone radical group to nucleobases (2.1nm vs. 0.27nm), making the ROS transport from GSH an order of magnitude less likely to damage DNA because of short lifetime of HO radicals. The antioxidant and DNA-protecting behaviors of GSH have been elucidated. We have found that the redox potential of GSH/GSSG couple is lower than that of catechol/semiquinone couple. Hence, GSH keeps catechol in the reduced state, thereby shutting down the initial step of the catechol-mediated Fenton cascade. The catechol-induced DNA damage in the presence of Cu(II) ions has also been confirmed in studies of ON-OFF hairpin-oligonucleotide beacons.