Microarchitecture of the liver: A Jigsaw puzzle
Authors: Marcos Rojkind, George Philips, Anna Mae Diehl
Journal: Journal of Hepatology (2010)

COMMENTARY ON: Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration. Hoehme S, Brulport M, Bauer A, Bedawy E, Schormann W, Hermes M, Puppe V, Gebhardt R, Zellmer S, Schwarz M, Bockamp E, Timmel T, Hengstler JG, Drasdo D. Proc Natl Acad Sci USA 2010 Jun 8;107(23):10371–10376. Copyright (2010) by the National Academy of Sciences; USA. Abstract reprinted with permission from the National Academy of Sciences. http://www.ncbi.nlm.nih.gov/pubmed/20484673 Abstract: Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl(4), a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16day regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named “hepatocyte-sinusoid alignment” (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues. Multiple studies have been performed to understand the molecular mechanisms involved in the regulation of liver regeneration [[1]Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1283) Google Scholar]. However, little is known regarding the changes in the three-dimensional organization of the liver’s microarchitecture occurring during this process. These changes may be critical in determining whether a certain type of injury will result in liver regeneration or scar formation. In the excellent manuscript by Hoehme et al. [[2]Hoehme S. Brulport M. Bauer A. Bedawy E. Schormann W. Hermes M. et al.Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.Proc Natl Acad Sci USA. 2010; 107: 10371-10376Crossref PubMed Scopus (260) Google Scholar] the authors used immunostaining, fluorescent reporter mice, confocal microscopy, and computer/mathematical modeling to develop a model that predicts how hepatic microarchitecture is re-established after acute CCl4-induced liver damage. The results support a process that includes transient loss of polarity and increased migration by recently replicated hepatocytes which allow the daughter hepatocytes to quickly align along the closest hepatic sinusoids. The authors term this process hepatocyte sinusoidal alignment (HSA) and conclude that recovery of normal hepatic microarchitecture (i.e., regeneration) is not achieved unless an HSA occurs. These results stress the fact that cell–cell and cell–matrix interactions are critical for the completion of liver regeneration. The nature and quantity of the extracellular matrix (EM) in the space of Disse results from the contribution of several cell types. Accordingly, if the correct cell–cell interactions are formed and the proper geometry is established (cell polarization), the correct EM will be produced, and therefore, normal regeneration should occur [[3]Rudolph K.L. Trautwein C. Kubicka S. Rakemann T. Bahr M.J. Sedlaczek N. et al.Differential regulation of extracellular matrix synthesis during liver regeneration after partial hepatectomy in rats.Hepatology. 1999; 30: 1159-1166Crossref PubMed Scopus (66) Google Scholar]. If an HSA does not take place, the establishment of the wrong cell–cell or cell–EM interactions could lead to an abnormal deposition of EM (i.e. liver fibrosis). Furthermore changes in the microarchitecture could determine whether fibrosis is reversible or not. Although this study was not designed to characterize the mechanisms that initiate or terminate HSA, the authors speculate that the process is guided by factors released by dead hepatocytes and sinusoidal cells that survive in the necrotic area. A few potential mediators are suggested, namely hepatocyte growth factor (HGF), tumor necrosis factor alpha (TNFα), and interleukin 6 (IL6), because these factors have been shown to promote liver regeneration and are produced by sinusoidal cells [4Yamada Y. Kirillova I. Peschon J.J. Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1446Crossref PubMed Scopus (842) Google Scholar, 5Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. et al.Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1325) Google Scholar]. However, there are other factors that could play a role in organizing the microarchitecture of the liver. These include among others, the induction of endothelial cell proliferation by VEGF or NO [6Shergill U. Das A. Langer D. Adluri R. Maulik N. Shah V.H. Inhibition of VEGF-and NO-dependent angiogenesis does not impair liver regeneration.Am J Physiol Regul Integr Comp Physiol. 2010; 298: R1279-R1287Crossref PubMed Scopus (15) Google Scholar, 7Donahower B.C. McCullough S.S. Hennings L. Simpson P.M. Stowe C.D. Saad A.G. et al.Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.J Pharmacol Exp Ther. 2010; 334: 33-43Crossref PubMed Scopus (34) Google Scholar], the production of Wingless (Wnt) and hedgehog (Hh) ligands by hepatocytes and liver sinusoidal cells [[8]Yang L. Wang Y. Mao H. Fleig S. Omenetti A. Brown K.D. et al.Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells.J Hepatol. 2008; 48: 98-106Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar], and the secretion of TGF-β, a negative regulator of hepatocyte proliferation [[1]Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1283) Google Scholar]. In addition, EM produced could play a key role in harboring growth factors (i.e. HGF) needed for liver regeneration [[9]Schuppan D. Schmid M. Somasundaram R. Ackermann R. Ruehl M. Nakamura T. et al.Collagens in the liver extracellular matrix bind hepatocyte growth factor.Gastroenterology. 1998; 114: 139-152Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Despite the significant progress made, additional questions need to be addressed:1.Is the HSA occurring after acute injury with CCl4 also occurring after partial hepatectomy? Lobular architecture in the remnant liver lobes is not grossly altered acutely after PH, but must ultimately adapt to accommodate the mass of new hepatocytes that are regenerated. Little is known about the mechanisms that orchestrate this process.2.What is the role of VEGF in sustaining sinusoidal endothelial cells? Shergill et al. [[6]Shergill U. Das A. Langer D. Adluri R. Maulik N. Shah V.H. Inhibition of VEGF-and NO-dependent angiogenesis does not impair liver regeneration.Am J Physiol Regul Integr Comp Physiol. 2010; 298: R1279-R1287Crossref PubMed Scopus (15) Google Scholar] showed that in a mouse model of liver regeneration post-hepatectomy, inhibition of VEGF and NO-dependent angiogenesis did not impair liver regeneration. However, in a model of regeneration post-acetaminophen toxicity, Donahower et al. [[7]Donahower B.C. McCullough S.S. Hennings L. Simpson P.M. Stowe C.D. Saad A.G. et al.Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.J Pharmacol Exp Ther. 2010; 334: 33-43Crossref PubMed Scopus (34) Google Scholar] showed that the administration of recombinant VEGF reduced necrosis and enhanced hepatocyte regeneration. Thus, although there may be redundant pathways to stimulate vascular proliferation, this appears to be a key event in liver regeneration.3.What is the role of epithelial/mesenchymal (EMT) transitions in liver regeneration? When the cells detach and proliferate, are they undergoing EMT [[10]Sicklick J.K. Choi S.S. Bustamante M. McCall S.J. Pérez E.H. Huang J. et al.Evidence for epithelial-mesenchymal transitions in adult liver cells.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G575-G583Crossref PubMed Scopus (108) Google Scholar] or do they simply round up, detach from the EM, divide, and reattach to the EM by modifying the expression of cell surface receptors (i.e. fibronectin, integrins)?4.Hoehme et al. [[2]Hoehme S. Brulport M. Bauer A. Bedawy E. Schormann W. Hermes M. et al.Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.Proc Natl Acad Sci USA. 2010; 107: 10371-10376Crossref PubMed Scopus (260) Google Scholar] did not establish which sinusoidal cells are in direct contact with the hepatocytes. In healthy livers, HSC residing in the space of Disse are interposed between hepatocytes and SEC. Additional research is needed to determine how HSC respond to regenerative stimuli. Do they proliferate and/or migrate along with the hepatocytes during the process of HSA?5.Is there a functional unit of liver that contains a specific number (ratios) of hepatocytes and liver sinusoidal cells? If there is one, what are these ratios? When we induce liver injury do we destroy functional units or individual hepatocytes? Understanding microarchitecture of the liver could help define pathways involved in determining whether regeneration or scar formation will be induced.6.Finally, it will be important to determine whether the production of HGF, IL-6, TNF-α, Hh ligands, and other factors are responsible for HAS or whether the formation of HAS stimulates the production of the correct mediators that will induce liver regeneration [[11]Choi S.S. Omenetti A. Witek R.P. Moylan C.A. Syn W.K. Jung Y. et al.Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis.Am J Physiol Gastrointest Liver Physiol. 2009; 297: G1093-G1106Crossref PubMed Scopus (194) Google Scholar]. Overall, the excellent paper by Hoehme et al. [[2]Hoehme S. Brulport M. Bauer A. Bedawy E. Schormann W. Hermes M. et al.Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.Proc Natl Acad Sci USA. 2010; 107: 10371-10376Crossref PubMed Scopus (260) Google Scholar] opens new avenues that could help us in answering some of the questions raised. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.