Liver sinusoidal endothelial cells (LSECs) critically regulate liver homeostasis and diseases through angiocrine factors. Notch is critical in endothelial cells (ECs). In the current study, Notch signaling was activated by inducible EC‐specific expression of the Notch intracellular domain (NIC). We found that endothelial Notch activation damaged liver homeostasis. Notch activation resulted in decreased fenestration and increased basement membrane, and a gene expression profile with decreased LSEC‐associated genes and increased continuous EC‐associated genes, suggesting LSEC dedifferentiation. Consistently, endothelial Notch activation enhanced hepatic fibrosis (HF) induced by CCl4. Notch activation attenuated endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) signaling, and activation of sGC by 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole (YC‐1) reversed the dedifferentiation phenotype. In addition, Notch activation subverted the hepatocyte‐supporting angiocrine profile of LSECs by down‐regulating critical hepatocyte mitogens, including Wnt2a, Wnt9b, and hepatocyte growth factor (HGF). This led to compromised hepatocyte proliferation under both quiescent and regenerating conditions. Whereas expression of Wnt2a and Wnt9b was dependent on eNOS‐sGC signaling, HGF expression was not rescued by the sGC activator, suggesting heterogeneous mechanisms of LSECs to maintain hepatocyte homeostasis. Conclusion: Endothelial Notch activation results in LSEC dedifferentiation and accelerated liver fibrogenesis through eNOS‐sGC signaling, and alters the angiocrine profile of LSECs to compromise hepatocyte proliferation and liver regeneration (LR). (Hepatology 2018).

BackgroundEndothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor β, and other signals, but the detailed molecular mechanisms remain unclear.Methods and ResultsSmall RNA sequencing initially identified miR‐342‐5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR‐342‐5p targeted endoglin and modulated transforming growth factor β signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR‐342‐5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads–based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR‐342‐5p agomir in P3 pups. Moreover, miR‐342‐5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial–mesenchymal transition. Transfection of endoglin at least partially reversed endothelial–mesenchymal transition induced by miR‐342‐5p. The expression of miR‐342‐5p was upregulated by transforming growth factor β, and inhibition of miR‐342‐5p attenuated the inhibitory effects of transforming growth factor β on lumen formation and sprouting by ECs. In addition, VEGF repressed miR‐342‐5p expression, and transfection of miR‐342‐5p repressed VEGFR2 and VEGFR3 expression and VEGF‐triggered Akt phosphorylation in ECs. miR‐342‐5p repressed angiogenesis in a laser‐induced choroidal neovascularization model in mice, highlighting its clinical potential.ConclusionsmiR‐342‐5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways.

SummaryNotch signaling is critically involved in neural development, but the downstream effectors remain incompletely understood. In this study, we cultured neurospheres from Nestin-Cre-mediated conditional Rbp-j knockout (Rbp-j cKO) and control embryos and compared their miRNA expression profiles using microarray. Among differentially expressed miRNAs, miR-342-5p showed upregulated expression as Notch signaling was genetically or pharmaceutically interrupted. Consistently, the promoter of the miR-342-5p host gene, the Ena-vasodilator stimulated phosphoprotein-like (Evl), was negatively regulated by Notch signaling, probably through HES5. Transfection of miR-342-5p promoted the differentiation of neural stem cells (NSCs) into intermediate neural progenitors (INPs) in vitro and reduced the stemness of NSCs in vivo. Furthermore, miR-342-5p inhibited the differentiation of neural stem/intermediate progenitor cells into astrocytes, likely mediated by targeting GFAP directly. Our results indicated that miR-342-5p could function as a downstream effector of Notch signaling to regulate the differentiation of NSCs into INPs and astrocytes commitment.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.