Discussion

Our understanding of the spatial division of labour in the liver has remained descriptive. Here, we sought to elucidate the driving force behind hepatic angiodiversity that directs much of the spatial gene expression pattern observed in the liver. At the molecular level, the transcription factors Gata4 and c-Maf have emerged as crucial players in establishing liver sinusoidal EC identity13,14, whereas Notch and TGF-β signalling were identified as modulators of hepatic angiodiversity58,59. Hepatic EC specification is an active process involving mechanosignalling. Recently, Heg1 and Alk1, critical components of vascular mechanotransduction, have been identified as key regulators of hepatic vascular patterning and hepatic zonation via vascular Wnt factors59,60. Yet, these factors do not resolve the full extent of liver endothelial zonation and the longstanding debate over the major determinant of vascular zonation. In this regard, oxygen availability is considered a major driver of the spatial division of labour in the liver, as the central venous region is hypoxic39. However, inducing hypoxia in vitro and in vivo did not impact vascular Wnt expression and hepatic zonation, suggesting that oxygen availability is not the crucial biophysical cue that orchestrates liver zonation. In line with this, we were able to corroborate the vascular mechanotransduction concept of liver function in unique patient samples that we were able to retrieve from the pathology archive. The first case report was a patient with liver ischemia. Immunostaining analyses of liver samples from this patient did not reveal any perturbation in the hepatic GS zonation pattern. In contrast, a second case report, a patient with an anatomical heart defect and, hence, presumably altered systemic blood flow, displayed loss of pericentral GS expression in the liver. Intriguingly, a third case report, a patient with hepatic hemangiomatosis, showed loss of GS in tissue areas adjacent to the tumour where poor perfusion is expected, whereas the zonated GS expression was retained in the periphery (Supplementary Fig. 12a–c). Remarkably, the pericentral hepatic region has been shown to be a hotspot of spatial disorganisation in desmoplastic liver61. Similarly, a significant reduction in GS expressing pericentral hepatocytes was observed in cirrhotic patient samples, which was associated with sinusoidal dysregulation and a regressed central vein network62. In conclusion, albeit being observational case reports, these analytical studies of patient samples very much strengthen the conclusion that vascular Wnt expression is biomechanically activated and thereby controlling zonated liver functions.

EC are highly responsive to mechanical stretching and blood flow-mediated shear stress, resulting in morphological changes and the expression of angiocrine factors23,58,63,64. Pericentral LSEC are exposed to bidirectional blood cells of higher velocity and display a more stretched morphology, leading to the establishment of a pericentral niche that is characterised by high shear conditions27,28. At the molecular level, the zonated shear response in the hepatic vasculature may be inferred by the finding that the phosphorylation-dependent signalling activity of ITGB1 and VEGFR3, two shear responsive EC surface receptors, was biased towards the central vein, whereas their expression was not regulated spatially22. This finding was further validated by the proximity ligation assays, highlighting increased phosphorylation of ITGB1 and VEGFR3 towards the central zone (Supplementary Fig. 13a–d). Flow-dependent mechanical stimulation of hepatic EC induced vascular Wnt factors. In line with this notion, hepatic Wnt signalling was recently shown to be regulated rhythmically and culminated when hepatic blood flow is at its peak65,66. Similarly, mechano-dependent Wnt expression was also observed in ECs stimulated by oscillatory shear and for EC in the valves of the heart, pointing towards a more general mechanism24. In parallel, a recent spatiotemporal atlas of murine liver regeneration further supported the transient activation of haemodynamic response genes preceding the Wnt secretion67. Likewise, the phenotypic switch of liver EC was achieved through a flow-perturbing partial ligation of the inferior vena cava68.

The functional experiments in this study revealed that shear-induced angiocrine Wnt-expression was indispensable for maintaining a Wnt-addicted pericentral and midzonal hepatocyte progenitor niche42, which was crucial for sustaining liver mass and hepatocyte proliferation during regeneration. This finding was corroborated by previous work, showing that hepatocyte-specific deletion of Wnt receptors and modulators similarly impaired hepatic function54,69,70. Besides maintaining the pericentral hepatocyte niche, these Wnt factors are crucial for transdifferentiation of cholangiocytes into hepatocytes and, thus, are indispensable for developing, maintaining and restoring liver mass71.

Interestingly, hepatocytic Wnt- and R-spondin receptor expression is biased towards the central vein, whereas the expression of APC, a Wnt suppressor, is enriched at the portal vein54, thereby reinforcing zonated Wnt signalling. These zonated Wnt receptors and suppressors further determine the Wnt-dependency. Given that Wnt morphogens are short-lived molecules with limited diffusion distance, hepatocytes that are directly adjacent to the Wnt reservoir show more robust effects41. Prototypic Wnt downstream targets, including Glul and Aqp9 showed a marked reduction upon the loss of vascular Wnt factors, whereas CYPs were downregulated to a lesser extent. This may be due to the fact that CYPs are co-regulated by different signalling pathways, including HIFs or via pericrine Rspo342,72,73. Yet, it could also mean that thresholds to activate Wnt-regulated CYPs are much lower than for other Wnt downstream molecules. Surprisingly, the data further revealed a similar spatially distinct Wnt-machinery in LSEC with high expression of Wnt receptor complexes in the pericentral zone and the periportal bias of Wnt suppressor expression, suggesting a zonated Wnt signalling activity in the hepatic vasculature. In line with the data, a similar Wnt zonation within LSEC was observed in a recent human liver atlas. The study further highlighted that the human pericentral zonation bias is independent of lobule size74. Future work may dissect the number of liver lobes, their corresponding hepatic vascular architecture, and haemodynamics, and correlate these to liver zonation.

LSEC have dual functions in the liver, serving both as vascular and lymphatic EC, which is also reflected in their molecular makeup22,75. In lymphatic EC, oscillating shear stress was identified to trigger an autocrine canonical Wnt signalling axis that is crucial for lymphatic valve development76. Moreover, flow was shown to control the expression of the mechanosensitive gap junctional channels Cx37 and Cx43 that mediate calcium influx77,78. In the liver, endothelial Cx37 and Cx43 were expressed in the central vein and precisely modulated by both flow and the Wnt signalling pathway, highlighting the hybrid phenotype of LSEC by mimicking lymphatic valve EC. Lymphatic gene deletion of Cx37, Cx43 or both results in lymphedema and premature lymphatic valve development with disorganised lymphatic EC morphology and integrity79, which correlates with macroscopic defects in Wnt-deficient LSEC. Functionally, Cx37 regulates the vascular tone by mediating calcium-dependent intercellular communication between EC and vascular smooth muscle cells80. In the brain vasculature, gap junctional molecules are crucial for the propagation of vasodilation, thereby regulating blood flow throughout the arteriovenous axis81. In line with this, Wnt-deficient LSEC showed reduced Cx37 and Cx43 expression and consequently impaired calcium mobilisation, which reduced the vascular tone in vitro. Collectively, the data reveal that biomechanical activation, mediated by blood perfusion, serves as the zonation-enforcing parameter in the liver. EC act as a cellular decoder of biophysical parameters, hence, orchestrating overall liver function. Molecularly, biomechanical milieu activates a specialised endothelial gene programme that sustains vascular Wnt, which induces expression of gap junctional molecules, thus orchestrating instructive EC cell communication and thereby enforcing spatially distinct liver function (Supplementary Fig. 14).