👤 Roxana Ola

TF (transcription factor) Prdm16 (positive regulatory domain-containing protein 16) regulates hematopoietic and neuronal stem cell homeostasis, adipose differentiation, and cardiac development. Its role in the circulatory system extends beyond the heart, as Prdm16 loss in arterial endothelial cells (ECs) impairs arterial reperfusion of ischemic mouse limbs due to endothelial dysfunction, and Zebrafish were used to analyze vascular development, arteriovenous endothelial specification, and the emergence of arteriovenous malformations in the absence or presence of Prdm16 or Notch signaling. Lentiviral-mediated Prdm16 overexpression in human endothelial (progenitor) cells was coupled to qRT-PCR (real-time quantitative polymerase chain reaction), Western blot, and transcriptional profiling to document Prdm16's importance for arterial lineage specification. Coimmunoprecipitation in HEK293 (human embryonic kidney 293) cells was performed to assess physical interaction between Prdm16 and the Notch pathway. Existing mouse and human data sets were reanalyzed to evaluate Prdm16 expression in mammalian arteriovenous malformations. Prdm16 actively promotes arterial EC identity while suppressing venous fate. Like in mice, Prdm16 is expressed by arterial ECs early during vascular development in zebrafish, where it synergistically coordinates arterial development together with canonical notch signaling, as their combined loss in zebrafish leads to arteriovenous malformations. PRDM16's arterializing effect on human ECs is dependent on canonical Notch activity, as it is blunted in the presence of canonical Notch inhibitors and potentiated in the presence of delta-like ligand 4. Mechanistically, Prdm16 does not increase the protein levels of the cleaved intracellular domain of Notch receptors (notch intracellular domain) but rather potentiates the effect of the latter via physical and functional interaction. Prdm16 further finetunes Notch signaling and arterial development by complexing with Hey2 (Hes-related family bHLH TF with YRPW motif 2), the basic helix-loop-helix TF acting downstream of canonical Notch during arterial lineage specification and development. Together, our data demonstrate an intricate interplay between Prdm16 and Notch in ECs and indicate that Prdm16 signaling may constitute a novel therapeutic target for arteriovenous malformations. Show less

To determine whether taurine transporter (TauT) activity and expression are regulated by hyperosmolarity in RPE, ganglion, and Müller cells. Uptake of taurine was measured in ARPE-19 cells cultured in DMEM-F12 medium without or with the addition of 50 mM NaCl or 100 mM mannitol. The kinetics of the transport were analyzed. RT-PCR and Northern and Western blot analyses were used to assess TauT mRNA and protein levels. The influence of hyperosmolarity on the uptake of taurine, myo-inositol, and gamma-aminobutyric acid GABA was studied in RPE, RGC-5, and rMC1 cells. TauT activity was abundant in RPE and was stimulated (3.5-fold) when the cells were exposed to hyperosmolar conditions (DMEM-F12 culture medium plus 50 mM NaCl or 100 mM mannitol). Peak stimulation of taurine uptake occurred after 17 hours of exposure to hyperosmolar medium. Kinetic analysis revealed that the hyperosmolarity-induced stimulation was associated with an increase in V(max) of TauT with no change in K(m). TauT mRNA and protein levels increased in RPE cells exposed to hyperosmolar conditions. Hyperosmolarity also stimulated the uptake of myo-inositol ( approximately 15-fold); GABA uptake was influenced less markedly. Immunofluorescence and functional studies showed that TauT is present in cultured RGC-5 and rMC1 cells. TauT activity was robust in these cells in normal osmolar conditions and increased by approximately twofold in hyperosmolar conditions. These studies provide the first evidence that hyperosmolarity regulates TauT activity and expression in RPE and that TauT is present in ganglion and Müller cells and is regulated by hypertonicity. The data are relevant to diseases such as diabetes, macular degeneration, and neurodegeneration, in which retinal cell volumes may fluctuate dramatically. Show less