Background Dysfunction of liver sinusoidal endothelial cells (LSECs) is permissive for the progression of liver fibrosis and cirrhosis and responsible for its clinical complications. Here, we have mapped the spatial distribution of heterogeneous liver ECs in normal vs cirrhotic mouse livers and identified zone-specific transcriptomic changes of LSECs associated with liver cirrhosis using scRNA-seq technology. Approach & Results Cirrhosis was generated in endothelial specific green fluorescent protein (GFP) reporter mice through carbon tetrachloride inhalation for 12 weeks. GFP-positive liver EC populations were isolated from control and cirrhotic mice by FACS. We identified 6 clusters of liver EC populations including 3 clusters of LSECs, 2 clusters of vascular ECs and 1 cluster of lymphatic ECs. Based on previously reported LSEC-landmarks, we mapped the 3 clusters of LSECs in zones 1, 2, and 3, and determined phenotypic changes in each zone between control and cirrhotic mice. We found genes representing capillarization of LSECs (eg, CD34) as well as extracellular matrix genes were most upregulated in LSECs of zone 3 in cirrhotic mice, which may contribute to the development of basement membranes. LSECs in cirrhotic mice also demonstrated decreased expression of endocytic receptors, most remarkably in zone 3. Transcription factors (Klf2 [Kruppel-like factor-2], Klf4 [Kruppel-like factor-4], and AP-1) that induce nitric oxide production in response to shear stress were downregulated in LSECs of all zones in cirrhotic mice, implying increased intrahepatic vascular resistance. Conclusion This study deepens our knowledge of the pathogenesis of liver cirrhosis at a spatial, cell-specific level, which is indispensable for the development of novel therapeutic strategies to target the most dysfunctional liver ECs.

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The cellular fate of nanoparticles in the liver is not fully understood. Because the effectiveness and safety of nanoparticles in liver therapy depends on targeting nanoparticles to the right cell populations, this study aimed to determine a relative distribution of PLGA-nanoparticles (sizes 271±1.4 nm) among liver cells in vivo. We found that Kupffer cells were the major cells that took up nanoparticles, followed by liver sinusoidal endothelial cells and hepatic stellate cells. Nanoparticles were found in only 7% of hepatocytes. Depletion of Kupffer cells by clodronate liposomes increased nanoparticle retention in liver sinusoidal endothelial cells and hepatic stellate cells, but not in hepatocytes. It is importantly suggested that studies of drug-loaded nanoparticle delivery to the liver have to demonstrate not only uptake of nanoparticles by the target cell type but also non-uptake by other cell types to assess their effect as well as ensure their safety.

Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B’s involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor β (TGF-β) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). Conclusion Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFβ/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.

Enhanced meningeal lymphatic drainage ameliorates neuroinflammation and hepatic encephalopathy in cirrhotic rats Short title: Meningeal lymphatics and hepatic encephalopathy