Graphical AbstractHighlights d STAT activation hampers Ascl1's ability to reprogram M€ uller glia into retinal neurons.d Progenitor-like cells from Ascl1-expressing M€ uller glia have high STAT signaling.d Ascl1 ChIP-seq shows that STAT potentially directs Ascl1 to inappropriate targets.d STAT inhibitors, along with Ascl1 and TSA, cause an increase in neuron regeneration. SUMMARYM€ uller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of G i -coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.Sphingosine 1-phosphate (S1P) 2 is a plasma-borne lysosphingolipid that has been shown to regulate endothelial barrier integrity (1). For example, treatment of cultured endothelial cells with S1P associated with the carrier albumin acts to increase endothelial barrier activity as indicated by increased transendothelial electrical resistance (2, 3). Moreover, S1P administration greatly reduces lung capillary leakage induced in mice by lipopolysaccharide treatment (4). Mechanistically, S1P acts to enhance tight junction formation in neighboring endothelial cells by influencing subcellular distributions of tight junction components including ZO-1 and claudin-5 (5). In addition, S1P induces endothelial cortical actin assembly (1) and relocation of endothelial cell junctional adhesion molecules including platelet endothelial cell adhesion molecule and vascular endothelial-cadherin to cell-cell junctional areas (5).In plasma, S1P is found associated with multiple lipoproteins including low density lipoproteins, very low density lipoproteins, and high density lipoproteins (HDL). However, the bulk of the lipoprotein particle-associated S1P (54%) is bound to HDL (6). A number of recent studies point to the S1P cargo of HDL as being a mediator of many of the cardiovascular effects of HDL including the ability to promote vasodilation, vasoconstriction, and angiogenesis, protect against ischemia/reperfusion injury, and inhibit/reverse atherosclerosis (7). One important cardiovascular-related effect of S1P that has not yet been attributed to HDL is regulation of endothelial barrier activity, a major physiological function of the endothelium. Here we investigate the role of HDL as a regulator of endothelial barrier integrity processes known to be dependent on S1P signaling. EXPERIMEN...

Background: S1P promotes endothelial barrier and is associated with HDL and albumin in blood. Results: HDL-S1P sustained barrier longer than albumin-S1P, reduced S1P receptor (S1P1) degradation, increased S1P1 cell surface and lipid raft levels, and persistently activated PI3K-Akt and eNOS. Conclusion: HDL-S1P persistently activates S1P-S1P1-PI3K-Akt-eNOS signaling. Significance: The findings highlight the basis for HDL-S1P as a mediator of sustained endothelial barrier.

BackgroundThe lysosphingolipid sphingosine 1-phosphate (S1P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence suggests that many of the effects of HDL on cardiovascular function may be attributable to its S1P cargo.MethodsHere we have evaluated how levels of S1P and related sphingolipids in an HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S1P levels in the HDL-containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:≥73.5 mg/dL; males:≥61.9 mg/dL) and verified IHD; subjects with high HDL-C and no IHD; individuals with low HDL-C (females:≤38.7 mg/dL; males:≤34.1 mg/dL) and IHD, and subjects with low HDL-C and no IHD.ResultsThe results show a highly significant inverse relationship between the level of S1P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, an inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S1P and C24:1-ceramide, in the HDL-containing fraction of serum. Additionally, we demonstrated that the amount of S1P on HDL correlates with the magnitude of HDL-induced endothelial cell barrier signaling.ConclusionsThese findings indicate that compositional differences of sphingolipids in the HDL-containing fraction of human serum are related to the occurrence of IHD, and may contribute to the putative protective role of HDL in IHD.

Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P 2 is expressed in conjunction with S1P 1 and/or S1P 3 . Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1 ؉ /PE-CAM ؉ ), angioblasts (Flk1 ؉ /PECAM ؊ ), or undifferentiated mesodermal cells (Flk1 ؊ /PECAM ؊ ). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P 1 , S1P 2 , and S1P 3 .Sphingosine-1-phosphate (S1P) 1 is a bioactive sphingolipid generated by the action of sphingosine kinase on sphingosine.