Angiocrine Wnt signaling controls liver growth and metabolic maturation in mice

Abstract

Postnatal liver development is characterized by hepatocyte growth, proliferation, and functional maturation. Notably, canonical Wnt signaling in hepatocytes has been identified as an important regulator of final adult liver size and metabolic liver zonation. The cellular origin of Wnt ligands responsible for homeostatic liver/body weight ratio (LW/BW) remained unclear, which was also attributable to a lack of suitable endothelial Cre driver mice. To comprehensively analyze the effects of hepatic angiocrine Wnt signaling on liver development and metabolic functions, we used endothelial subtype-specific Stab2-Cre driver mice to delete Wls from hepatic endothelial cells (HECs). The resultant Stab2-Cre^tg/wt ;Wls^fl/fl (Wls-HECKO) mice were viable, but showed a significantly reduced LW/BW. Specifically, ablation of angiocrine Wnt signaling impaired metabolic zonation in the liver, as shown by loss of pericentral, β-catenin-dependent target genes such as glutamine synthase (Glul), RhBg, Axin2, and cytochrome P450 2E1, as well as by extended expression of periportal genes such as arginase 1. Furthermore, endothelial subtype-specific expression of a c-terminally YFP-tagged Wls fusion protein in Wls-HECKO mice (Stab2-Cre^tg/wt ;Wls^fl/fl ;Rosa26:Wls-YFP^fl/wt [Wls-rescue]) restored metabolic liver zonation. Interestingly, lipid metabolism was altered in Wls-HECKO mice exhibiting significantly reduced plasma cholesterol levels, while maintaining normal plasma triglyceride and blood glucose concentrations. On the contrary, zonal expression of Endomucin, LYVE1, and other markers of HEC heterogeneity were not altered in Wls-HECKO livers.

Conclusion: Angiocrine Wnt signaling controls liver growth as well as development of metabolic liver zonation in mice, whereas intrahepatic HEC zonation is not affected. (Hepatology 2017).

Figure 1

Wls‐HECKO mice are viable and show a reduction of endothelial Wls (^* P < 0.05; ^** P < 0.01; n.s. = not significant). (A) Genotype distribution of male (left) and female (right) mice at P28 (bars, 95% confidence intervals). (B) Reduction of Wls mRNA in HECs shown by qRT‐PCR. Fold change relative to β‐Actin is shown; Ctrl was set to 1. Bars represent SEM. Mean fold reduction in Wls‐HECKO is 12.5 (P = 0.0495; n = 3). Mice used were 12‐week‐old males. (C) Body weight differs significantly between male Ctrl and male Wls‐HECKO mice (26.8 vs. 24.6 g; P = 0.0132) at 13 weeks. In female mice, no significance in body weight is found between Ctrl and Wls‐HECKO, although there is a trend (P = 0.0759) toward a slightly lower body weight. (D) LW/BW at 13 weeks differs significantly between both male Ctrl and male Wls‐HECKO (5.1% vs. 4.1%; P < 0.01), as well as female Ctrl and female Wls‐HECKO (4.2% vs. 3.1%; P < 0.01), respectively. (E) Representative pictures of sectioned livers from male (left) and female (right) Ctrl and Wls‐HECKO mice (12 weeks old). Abbreviation: P, postnatal day.

Figure 2

Disturbed metabolic zonation in Wls‐HECKO livers. Immunohistochemistry (10× objective) shows Glul, RhBg, Arg1, and CYP2E1 staining pattern in Ctrl pericentral HCs (left) in comparison to Wls‐HECKO (right). Scale bar = 100 μm; n = 7.

Figure 3

β‐catenin distribution and Axin2 expression in the hepatic lobule. (A) Representative pictures of immunofluorescence (63× objective) showing membranous β‐catenin staining in HCs and cholangiocytes and EMCN (red) staining in CVECs and pericentral LSECs in both Ctrl (left) and Wls‐HECKO (right). RhBg (blue) is expressed in HCs adjacent to CVECs expressing EMCN in Ctrl (upper left); RhBg is missing in Wls‐HECKO (upper right). Arg1 is not expressed in pericentral HCs in Ctrl (blue, lower left), but positive in Wls‐HECKO (blue, lower right). Scale bar = 50 μm; n = 5. (B) Axin2 is highly expressed in pericentral HCs in Ctrl; low‐level activity in some cells can be seen in Wls‐HECKO. 20× objective; Scale bar = 50 μm; n = 3.

Figure 4

No major differences in routine stainings of Wls‐HECKO livers. H&E, Sirius Red, PAS, Prussian blue, and Oil Red O staining of Ctrl (left) and Wls‐HECKO (right) liver sections (10× objective). PAS, Prussian Blue, and Oil Red O staining show no enhanced carbohydrate, iron, or fat depositions in sections of Wls‐HECKO livers in comparison to Ctrl. Scale bar = 100 μm; n = 7; 13‐week‐old males were used.

Figure 5

HEC markers remain unchanged in Wls‐HECKO. Representative pictures of immunofluorescence (20× objective) showing strong EMCN (green) staining in CVECs and pericentral LSECs; LYVE1 (red) is low in this area and highly positive in midlobular LSECs in both Ctrl (left) and Wls‐HECKO (right). Glul (blue) is expressed in HCs adjacent to CVECs expressing EMCN in Ctrl (inset, left), whereas Glul is missing in Wls‐HECKO (inset, right). Representative pictures of IHC (10× objective) for LSEC markers Stab1 and Stab2, which are expressed in all LSECs, whereas CD32b is stronger in midlobular LSECs. Wls‐HECKO livers show a similar staining pattern to Ctrl for all markers. Scale bar = 100 μm; n = 5. Abbreviation: IHC, immunohistochemistry.

Figure 6

Slightly altered fat metabolism in Wls‐HECKO animals (11 weeks old; ^* P < 0.05; ^** P < 0.01; n.s. = not significant [P > 0.05]). (A) Plasma cholesterol is lower in both male (101 mg/dL in Ctrl vs. 73 mg/dL in Wls‐HECKO) and female (59 mg/dL in Ctrl vs. 47 mg/dL in Wls‐HECKO) Wls‐HECKO mice. (B) Liver cholesterol did not differ significantly in both sexes. (C) Dry blood total acylcarnitines are significantly elevated in male Wls‐HECKO mice (3.27 vs. 3.94 nmol/mg protein). (D) Plasma triglycerides remain unchanged in all subgroups. (E) Blood glucose in male mice does not differ significantly after 4 hours of fasting. (F) Left: Bilirubin levels exceed 0.145 mg/dL in male Wls‐HECKO mice more often than in Ctrl; female mice showed no significance. Black dots represent single animal Ctrl, gray dots single animal Wls‐HECKO bilirubin levels. Right: induction of hepatic HMOX1 shown by qRT‐PCR. Fold change relative to β‐Actin is shown; Ctrl was set to 1. Bars represent SEM. Mean fold induction in Wls‐HECKO is 1.66 (P = 0.0409; n = 7).

Figure 7

RosaEvi‐YFP expression in Wls‐rescue livers. Representative pictures of immunofluorescence (63× objective) showing focal YFP (green) staining in HECs of Wls‐rescue livers (A, n = 2), strong YFP staining in HECs of Wls‐HECKO;eYFP (C, n = 5), and no YFP signal in Ctrl liver (E, n = 5). Ctrl (n = 5) and Wls‐rescue (n = 2) livers show a pericentral, membranous RhBg staining pattern in HCs (A,E), which is absent in pericentral HCs in Wls‐HECKO;eYFP (C). EMCN (green) staining is positive in CVECs and pericentral LSECs, whereas LYVE1 (red) is highly positive in midlobular LSECs of all groups (B,D,F). Glul (blue) is expressed in HCs adjacent to CVECs expressing EMCN in Wls‐rescue (B) and Ctrl (F). Wls‐HECKO;eYFP shows no signal for Glul. (F). Scale bar = 50 μm.