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. Author manuscript; available in PMC: 2016 May 5.
Published in final edited form as: Dev Dyn. 2014 Nov 18;244(3):497–506. doi: 10.1002/dvdy.24222

Vascular patterning sets the stage for macro and micro hepatic architecture

Ashley E Cast 1,*, Teagan J Walter 1,*, Stacey S Huppert 1
PMCID: PMC4858172  NIHMSID: NIHMS641424  PMID: 25370311

Abstract

The liver is a complex organ with a variety of tissue components that require a precise architecture for optimal function of metabolic and detoxification processes. As a result of the delicate orchestration required between the various hepatic tissues, it is not surprising that impairment of hepatic function can be caused by a variety of factors leading to chronic liver disease. Despite the growing rate of chronic liver disease, there are currently few effective treatment options besides orthotopic liver transplantation. Better therapeutic options reside in the potential for genetic and cellular therapies that promote progenitor cell activation aiding de novo epithelial and vascular regeneration, cell replacement, or population of bioartificial hepatic devices. In order to explore this area of new therapeutic potential, it is crucial to understand the factors that promote hepatic function through regulating cell identities and tissue architecture. In this commentary, we review the signals regulating liver cell fates during development and regeneration; and highlight the importance of patterning the hepatic vascular systems to set the groundwork for the macro and micro hepatic architecture of the epithelium.

Keywords: hepatic vasculature, hepatic cell lineage plasticity, oxygen gradients

IMPORTANCE OF UNDERSTANDING THE ESTABLISHMENT AND MAINTENANCE OF LIVER CELL IDENTITIES AND ARCHITECTURE

In the United States, 1 in 2,500 infants have severe liver disease, and an estimated one quarter of Americans will suffer from a liver or biliary disease at some point during their life. Liver diseases have a variety of etiologies, including: genetic, viral, nutritional, toxicity (alcohol or medicinal), and oncogenic. For many patients with severe liver disease and liver failure, the only viable treatment option is liver transplantation. Unfortunately, the need for transplantation organs greatly exceeds the supply of donated organs each year. To further compound this dilemma, a decrease in suitable donors is projected due to the high prevalence of obesity (Ogden et al., 2014). Therefore, recent research efforts have focused on discovering new ways to treat liver disease using cell-based therapies. This work has focused on understanding the potential and molecular regulation of hepatic cellular plasticity and exploiting these cellular characteristics to aid liver regeneration under a chronic injury condition. Ideally, the present research will uncover ways to promote endogenous progenitor cell activation and drive the regeneration of specific liver tissues. For these efforts to succeed and contribute to new regenerative therapies or bioartificial organs, we must be able to reestablish the conditions that promote cell differentiation and function. Hepatic function is highly dependent on having both the correct cell identities as well as having the tissues arranged in a precise lobular architecture. Currently, there is a limited understanding of how the liver vasculature forms even though its formation is a primary and essential step in directing the hepatic architecture. In this commentary, we will describe the crucial three-dimensional hepatic architecture of the epithelial and vascular tissues, the unique mechanism of differentiation and development of each tissue, and the importance of inter-tissue interaction during development and disease; while highlighting the fundamental role the portal vein and hepatic vasculature have in establishment and maintenance of proper liver configuration.

OVERVIEW OF LIVER ARCHITECTURAL ESTABLISHMENT AND SIGNALING PATHWAYS INVOLVED

Importance of liver architecture for function

Proper liver function requires a precise spatial arrangement of liver tissues: the portal vein (PV), central vein (CV), hepatic artery (HA), intrahepatic bile duct (IHBD), and hepatocyte zonation (Figure 1). The importance of the spatial associations of liver tissues is apparent in the structure and function of the hepatic lobule. Hepatic lobules are thought to be the smallest functional unit of the liver. Two dimensionally they appear in an approximate hexagonal shape with a PV at each vertex and a CV branch in the middle. The HA and IHBD branches are arranged at the vertices in close association with PV branches in structures known as portal triads. Hepatocytes arranged in cords fill in the space between the PV and the CV. The organized arrangement of the hepatocyte cords is vital for the exocrine and endocrine functions performed by the hepatocytes. Extending along the basal side of hepatocyte cords are the sinusoids. Sinusoids are specialized capillary structures that connect the PV and HA to the CV. Hepatocytes are allowed to exchange factors with and maintain the composition of nutrients in circulation through the intricate sinusoidal network. On the apical side of hepatocytes are canaliculi, small channels that allow passive transport of bile produced by hepatocytes into the IHBDs. Apart from their relationship in the lobule (micro architecture), each of these epithelial and vascular tissues also has its own architecture and spatial organization within the liver (macro architecture).

Figure 1. Spatial architecture and hepatocyte zonal characteristics in the hepatic lobule.

Figure 1

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Hepatocyte cords run along the radius of the lobule between PVs and CVs. Bile is secreted into small canalicular channels and transported to the IHBDs. The sinusoidal capillaries carry blood from the PV and HA past the hepatocytes and ultimately into the CV. Oxygen tensions vary by vascular compartment of the liver with the highest oxygen tension found with the HA (74–104 mm Hg). In the PV, oxygen tensions are lower (34–46 mm Hg) and even lower in the CV (30–35 mm Hg). The sinusoids have a gradient of oxygen tension within periportal sinusoids at 65 mm Hg and the pericentral sinusoids at 30–35 mm Hg. The hepatocytes are arranged into three zones: zone 1 (purple) near the portal vein, zone 2 (medium blue) intermediate hepatocytes, and zone 3 (light blue) near the central vein.

Hepatic vascular development and architecture

The PV and HA architectures are essential to establish and maintain liver function. Oxygenated blood is supplied to the liver by the HA arising from the celiac artery via the descending aorta. The PV collects nutrient-rich effluent blood from the other peritoneal organs and brings it to the liver. In humans, the PV supplies approximately 75% of the afferent hepatic blood flow. The other 25% of hepatic blood flow is supplied by the HA (Tygstrup et al., 1962; Lautt, 2009). The PV and HA both empty into the hepatic sinusoids, and the CV collects the blood that has passed through the sinusoids and returns it to the inferior vena cava (Figure 1).

The PV and CV have a unique mechanism of development as extrapolated from the analysis of human fetuses (Lassau and Bastian, 1983; Gouysse et al., 2002; Collardeau-Frachon and Scoazec, 2008) and mouse embryos (Crawford et al., 2010). Both vessels derive from the fetal vitelline and umbilical veins. At the time of liver bud delamination and expansion, circa E9.5 in mouse, the hepatoblasts surround and disrupt the vitelline and umbilical veins. The veins remodel into a modified fetal circulatory network. The pre-hepatic and post-hepatic portions of the veins remain intact and give rise to the bases of the PV and CV (Figure 2) (Gouysse et al., 2002; Collardeau-Frachon and Scoazec, 2008; Crawford et al., 2010).

Figure 2. The vestigial hypothesis of PV and CV development.

Figure 2

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According to histological studies of human fetuses, the PV and CV are hypothesized to emerge from the remodeled fetal vitelline and umbilical veins. A. Prior to liver bud formation, the right umbilical vein (RUV), left umbilical vein (LUV), right vitelline vein (RVV), and left vitelline vein (LVV) empty into the sinus venosus (SV). B. As the fetal liver bud expands, it envelops these fetal vessels and causes a remodeling event, generating a new structure, the ductus venosus (DV). C. At the time of birth, the DV collapses. Hepatic circulation after birth goes only through the PV and CV, which are proposed to retain components of the fetal veins.

The PV, CV and IHBD all form hierarchical branched structures that bifurcate approximately 17 to 20 times in human forming smaller and smaller branches to achieve their macro hepatic architecture (Crawford, 2002). Whether the hierarchical branched vascular structures of the PV and CV are formed by angiogenesis and/or vasculogenesis is unknown at this time. Developmentally, the PV is the first to establish its branching architecture, followed by the IHBDs and lastly the HA. The hepatic artery is proposed to develop as an angiogenic sprout originating from the dorsal aorta (Gouysse et al., 2002; Collardeau-Frachon and Scoazec, 2008). Signals derived from the IHBD are suggested to direct the development of the HA intrahepatically. This is supported by a failure to generate mature HAs in a mouse model where IHBD morphogenesis is genetically impaired (Fabris et al., 2008). In humans, congenital hepatic vascular malformations result in redirecting blood in and around the liver. The vascular shunting can occur from the HA to the CV (arteriovenous shunt), from the HA to the PV (arterioportal shunt), or from the PV to the systemic circulation (portosystemic shunt) (DeLeve et al., 2009). Hepatic vascular malformations are rare and can be asymptomatic. Often times, the vascular malformations are diagnosed during unrelated tests or scans. Congenital portosystemic shunts or Abernathy malformations are due to abnormal development of the vitelline venous system (extrahepatic) (Morgan and Superina, 1994; Murray et al., 2003) or failure to close the ductus venosus after birth (intrahepatic) (Yoshimoto et al., 2004). In a case study of a portosystemic shunt from the main PV to the inferior vena cava (Abernathy type I), the portal triads lacked PV branches, but IHBDs and HA branches were present (Emre et al., 2007). In contrast, the portal triads were observed to have large malformed HAs and no visible patent IHBDs in an example of Abernathy type II, a portosystemic shunt from the main PV to the inferior vena cava and a hypoplastic branch connected to and draining into the liver (Schaeffer et al., 2013). Therefore, patients with Abernethy malformations do not clearly support a required sequence of PV, IHBD, and then formation of HAs. An alternative explanation for these various intrahepatic portal triad malformations is that PVs are initially formed providing a pattern for the IHBDs and HAs, but are not maintained due to a shunt resulting in a lack of sustained blood flow.

The sinusoids, lined by liver sinusoidal endothelial cells (LSECs), traverse the lobule, bridging the PV and the CV. The origin of LSECs in the liver is not known. LSECs are presumed to arise from endothelial cells (ECs) resident in the septum transversum mesenchyme (STM), including the ECs that surround that hepatic bud at the time of evagination and delamination (Matsumoto et al., 2001). LSECs provide a lenient selective barrier between the sinusoidal blood and the hepatocytes, allowing for particles to pass through and access the hepatocytes. Sensitivity of LSECs to radiation, ischemia, toxins, chemotherapy and various medication leads to sinusoidal obstructive syndrome. The change in LSECs morphology causes obstructions or gaps in the endothelial barrier hindering sinusoidal blood flow (DeLeve, 211).

Signaling pathways controlling vascular morphogenesis

The main pathway implicated in regulating vascular morphogenesis is vascular endothelial growth factor (VEGF) signaling. VEGF is a crucial signaling pathway governing vascular development and behavior throughout the body. In mammals, there is one principal Vegf ligand and two principal receptors, VEGF receptor 1 (VEGFR1, also known as Flt1) and VEGFR2 (also known as KDR and Flk1) (Ferrara and Davis-Smyth, 1997). The VEGF signaling pathway is essential during development. Mice homozygous for a VEGFR1 or VEGFR2 deletion are embryonic lethal between embryonic day 8.5–9.5 due to impaired or altered vascular development and failed blood-island formation (Fong et al., 1995; Shalaby et al., 1995; Carmeliet et al., 1996; Ferrara et al., 1996). Important to the liver, the timing of lethality associated with a deficiency in either VEGF receptor corresponds to the formation of the hepatic diverticulum, after hepatic specification and prior to hepatic vascularization (Gualdi et al., 1996; Crawford et al., 2010). Therefore, new mouse models need to be generated for investigating the role of VEGF signaling in hepatic vascular development and macro architectural patterning.

In general, VEGF signaling has several known functions in vascular development and homeostasis, including: angiogenic directional growth and branching behavior, vascular permeability, endothelial fenestration, endothelial proliferation, and endothelial cell survival (Connolly et al., 1989; Gerber et al., 2002; Gerhardt et al., 2003; Carpenter et al., 2005; Lee et al., 2007; Krueger et al., 2011; Leung et al., 2013). Angiogenesis is the process of vascular development by which new vessels are generated and grow by sprouting from an existing vessel and extending directionally. The directionality of the angiogenic growth is directed by the graded concentration of Vegf protein (Figure 3). During angiogenesis, ECs in the growing vessel can take on one of two identities: tip or stalk (Phng and Gerhardt, 2009). Tip cells are located at the growing end of blood vessels. They display filopodial extensions and are responsible for pathfinding and directionality (Gerhardt et al., 2003). Stalk cells are all cells of the new vessel that trail behind the tip cell; these form a luminal structure and perform the proliferation required for vessel extension and growth.

Figure 3. Notch and VEGF signaling collaborate to specify tip and stalk cells during angiogenesis.

Figure 3

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Angiogenic growth is directed by a gradient of Vegf protein (blue). The tip cell (light red) expresses VEGF receptor (VEGFR), allowing it to respond to Vegf protein. Activation of VEGFR causes the upregulation of Dll4 and VEGFR in the tip cell. Dll4 signals to activate Notch in stalk cells (dark red). The activation of Notch causes the down-regulation of VEGFR, making the stalk cells less responsive to environmental Vegf protein. Together, Notch and VEGF signaling generate positive feedback loops that reinforce the separate tip and stalk cell identities.

Tip and stalk cells engage in lateral inhibition through VEGF and Notch signaling. Both tip and stalk cells express VEGFR2. Upon exposure to Vegf protein, VEGFR2 is activated, leading to the upregulation of the Notch ligand Delta-like4 (Dll4) and VEGFR2, in a positive feedback loop (Phng and Gerhardt, 2009). Dll4 interacts activates Notch receptors expressed on surrounding cells. The activation of Notch inhibits the expression of VEGFR2 and upregulates VEGFR1, making the Notch-activated cell less sensitive to Vegf protein (Phng and Gerhardt, 2009). Through these feedback loops, one cell adopts the tip cell identity while the surrounding cells are designated as stalk cells. The functions of Notch and VEGF signaling in the tip/stalk designation have been defined in detail in several papers, mainly using the cultured mouse retina as a model system (Gerhardt et al., 2003; Hellstrom et al., 2007; Lobov et al., 2007; Suchting et al., 2007; Trindade et al., 2008; Benedito et al., 2009).

Vegf expression and consequent angiogenesis is often promoted by hypoxia. Under hypoxic conditions, cells stabilize the hypoxia inducible factor1α (HIF1α) protein, and the HIF1α/β heterodimer binds enhancer sequences of the Vegfa gene, stimulating the production of Vegf protein (Jones et al., 2001). Secretion of Vegf from a region of hypoxic tissue directs angiogenesis towards that area, ultimately increasing blood flow to alleviate the hypoxic condition.

VEGF signaling has also been shown to be important for the maintenance of LSEC characteristics (DeLeve et al., 2004; Walter et al., 2014a). In experiments with cultured primary LSECs, the loss of fenestrae and upregulation of the platelet-derived endothelial cell adhesion molecule (PECAM) expression occur within days after isolation and culture. Treatment of cultured LSECs with exogenous Vegf or co-cultures of LSECs with a Vegf-producing cell type such as hepatocytes or stellate cells is sufficient to maintain LSEC characteristics (DeLeve et al., 2004). Similar results are observed in vivo. Liver-specific Vegf deficient mice exhibit an expansion of cells expressing PECAM and laminin deposition, demonstrating a loss of LSEC identity (Walter et al., 2014a). Due to the close spatial relationship between LSECs and hepatocytes, it is likely that the hepatocytes are a source of Vegf that maintains LSEC identity in vivo.

Another important signaling pathway in hepatic vascularization is Notch. Notch signaling components are necessary for arteriovenous specification during embryogenesis (Gridley, 2010). Overexpression of Notch4 in the adult hepatic endothelium produces enlarged vessels and arterialization (Carlson et al., 2005). Interestingly, when constitutive activation of Notch4 was ceased, the vessels returned to their normal state. This suggests a role for Notch4 in adult arteriovenous specification and maintenance.

IHBD development and architecture depends on PV establishment

The IHBD has a hierarchical branching architecture that follows the branching pattern of the PV, stemming from a developmental connection (Figure 4). In the first step of IHBD morphogenesis, ductal plate formation, hepatoblasts adjacent to the portal vein mesenchyme activate expression of biliary epithelial cell (BEC) marker genes such as Sry-related HMG box 9 (Sox9). The specified BECs then undergo a remodeling event during which the cells become polarized and generate a lumen adjacent to the PV. The ductal plate cells that are not incorporated into IHBDs regress, turn off BEC markers, and differentiate as periportal hepatocytes (Antoniou et al., 2009; Si-Tayeb et al., 2010; Carpentier et al., 2011). Ductal plate structures are first observed in the proximal hilar regions of the liver and appear in the more distal regions progressively following the PV during embryonic and early postnatal development.

Figure 4. IHBD has a hierarchical branching architecture with a close association with the PV.

Figure 4

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Ink injection highlights the hierarchical branching architecture and close proximity of the IHBD (black) and PV (white) systems in the mouse left lobe. This close association stems from a developmental process of ductal plate formation around PVs, culminating in the emergence of lumenal IHBDs spatially associated with PVs.

The smallest components of the biliary system are the canaliculi. The apical surfaces of adjacent hepatocytes are joined to form a narrow canalicular channel. Hepatocytes secrete bile into the canaliculi that form a connected network extending throughout the parenchymal epithelium (Treyer and Musch, 2013). The canaliculi are eventually connected to the small peripheral IHBD branches that merge into fewer, larger branches, until finally one single branch carries the bile out of the liver and transports it into the gallbladder for storage and ultimately into the intestine to aid in digestion. The IHBD system relies on its highly regulated three-dimensional structure to access all of the hepatocytes and effectively clear bile out of the liver. IHBD architectural formation is a highly complex and regulated process wherein ductal plate regression and tube establishment must occur in a coordinated fashion along the three-dimensional PV network to form a connected IHBD network.

Signaling pathways regulating IHBD morphogenesis

The primary pathway that has been implicated in IHBD morphogenesis is Notch signaling. Notch is a conserved signaling pathway that plays a role in reiterative cell fate decisions in several organs during development. Notch signaling has known roles in stem cell maintenance, cell fate decisions, proliferation, and tissue patterning (Andersson et al., 2011). In mammals, there are five canonical Notch ligands across two families (Delta1, 3, and 4, and Jagged1 and 2) and four Notch receptors (Notch1, 2, 3, and 4). Notch ligands and receptors are both membrane bound and participate in juxtacrine signaling between adjacent cells. Therefore, the Notch intercellular signaling pathway disposes the proximal spatial localization of the PV and IHBD.

Ductal plate morphogenesis begins at E13.5 in mouse, at which time the mesenchymal cells surrounding the PV express the Notch ligand Jagged1. Jagged1 interacts with the fundamental hepatic Notch receptor, Notch2, expressed on bipotential hepatoblasts to activate the Notch pathway (Geisler et al., 2008; Hofmann et al., 2010). The expression of a constitutively activated form of Notch2 or Notch1 is sufficient to promote BEC differentiation (Tchorz et al., 2009; Sparks et al., 2010). Additionally, Notch signaling is required for remodeling of the specified BECs into mature, lumenal IHBDs (McCright et al., 2002; Tanimizu and Miyajima, 2004; Geisler et al., 2008; Lozier et al., 2008; Hofmann et al., 2010; Sparks et al., 2010; Jeliazkova et al., 2013). Reduced Notch activity, accomplished through hepatic specific loss of Notch2, results in BEC specification, but inability to remodel into communicating IHBDs (Sparks et al., 2010). Further reduction of Notch activity via hepatic specific loss of Rbpj, the DNA-binding co-factor for all four Notch receptors, results in reduced BEC specification (Zong et al., 2009; Sparks et al., 2010).

In addition to Notch signaling, the transcription factors Sox9, Hnf1β, and Hnf6 are important for biliary morphogenesis; without each of these genes, delays or disruptions in biliary development are observed in mice (Clotman et al., 2002; Coffinier et al., 2002; Antoniou et al., 2009). It is likely that Notch acts upstream of Sox9 during biliary morphogenesis, as Sox9 is a known target of Notch signaling in several organs (Muto et al., 2009; Haller et al., 2012; Chen et al., 2013). The control of Hnf1β and Hnf6 by Notch signaling is less clear, occurring either in parallel or as an epistatic relationship. Hnf1β and Hnf6 expression are both increased with hepatic loss of Rbpj at embryonic day 16.5 (Vanderpool et al., 2012). Therefore, the increase in Hnf6 is likely compensatory and may contribute to the observed increase in its target Hnf1β expression (Clotman et al., 2002). Initial regulation of both Hnf1β and Sox9 by Hnf6 appears to occur during early embryonic time points, with expression of both factors approaching or equaling control mice by E17.5 in an Hnf6 global loss model (Clotman et al., 2002; Antoniou et al., 2009). An alternate possibility would be an epistatic model in which Notch signaling occurs upstream of Hnf6, acting as an attenuator of Hnf6. However, previous experimental models have shown that constitutive Notch activation does not down-regulate expression of Hnf6 (Tanimizu and Miyajima, 2004; Zong et al., 2009). The possibility of Hnf6 occurring upstream of Notch signaling is unlikely, given that Sox9 is a Notch target (Zong et al., 2009) and isolated hepatoblast-specific loss of Hnf6 did not result in any changes in Sox9 at embryonic and early postnatal stages (Vanderpool et al., 2012). Although mechanistic studies remain to be performed, published genetic data suggest that control of factors essential for early IHBD development occurs along parallel mechanisms through Hnf6 and Notch signaling.

Hepatocyte zonal organization is tightly correlated to the architecture of hepatic vascular systems

Within the lobule, there is spatial organization of hepatocytes that is important for function. Approximately 15–25 hepatocytes span the distance between the portal and central veins in the rat liver, forming a series of hepatocyte plates that radiate between the central vein and the portal vein vertices (Colnot and Perret, 2011). The hepatocytes collectively perform a wide variety of tasks, including gluconeogenesis, urea genesis, β-oxidation, and liponeogenesis (Bhatia et al., 1996). These functions are segregated between different subpopulations of hepatocytes. The lobule is organized into three spatial zones: zone 1 is the periportal zone, zone 2 is the intermediate zone, and zone 3 is the pericentral zone (Figure 1). Zone 1 hepatocytes specialize in gluconeogenesis and urea formation, while zone 3 hepatocytes specialize in liponeogenesis, glutamine synthesis, and glycolysis (Colnot and Perret, 2011). Accordingly, the expression of metabolic genes varies between the zones. For example, in rats and mice, one enzyme involved in urea synthesis, glutamine synthetase, is only expressed in the 1–2 cell layers of hepatocytes that immediately juxtapose the central veins. Cell morphologies also change zonally in the rat liver; periportal hepatocytes are smaller, approximately 7–15 μm in diameter, than pericentral hepatocytes, measuring approximately 30–40 μm (Bhatia et al., 1996).

Signaling pathways controlling lobular hepatocyte zonation

The main factor implicated in the formation and maintenance of hepatocyte zonation is Wnt/β-catenin signaling. β-catenin stabilization is both necessary and sufficient for expression of the pericentral enzyme glutamine synthetase (GS) in vivo in the mouse liver (Colnot and Perret, 2011). In a normal liver, stabilized β-catenin is observed only in the zone 3 pericentral hepatocytes. In a liver-specific β-catenin knockout mouse model, the expression of GS is completely absent from the liver. In contrast, the liver-specific knockout of Apc, a negative regulator of β-catenin stabilization, activates the expression of GS in all hepatocytes in the liver (Benhamouche et al., 2006; Colnot and Perret, 2011). The expression of Wnt ligands has been found in several liver cell types, including: hepatocytes, BECs, LSECs, stellate cells (pericytes in the liver sinusoids), and Kupffer cells (resident macrophages in the liver) (Zeng et al., 2007). It has been hypothesized, although without any concrete evidence, that Wnt/β-catenin signaling in the pericentral zone is directed by LSECs (Colnot and Perret, 2011). To investigate which hepatic cell type is responsible for providing Wnt ligands for proper hepatic zonation, a Wntless mouse line was used (Yang et al., 2014). Wntless is required to facilitate the secretion of all Wnt ligands from the cell membrane to signal to other cells. Targeted deletion of Wntless from hepatic epithelial cells, endothelial cells and macrophages were examined by using various Cre lines to eliminate the ability of each cell lineage to secrete Wnt ligands. The results indicate that hepatic epithelial cells and macrophages are not the source of Wnt proteins for β-catenin activation necessary for hepatic zonation. The complete loss of Wntless from hepatic endothelial cells results in embryonic lethality, preventing assessment of proper hepatic zonation. Therefore, further studies will be required to determine whether LSECs provide the source of Wnt ligands in the pericentral zone.

An additional key molecule mediating hepatic zonation is Hnf4α. Hnf4α opposes Wnt signaling to promote the expression of zone 1 periportal hepatocyte genes and inhibit the expression of zone 3 pericentral hepatocyte genes (Colletti et al., 2009). In an Hnf4α deficient liver, the expression of pericentral hepatocyte genes, including GS, is expanded into the periportal zone (Stanulovic et al., 2007). Evidence indicates that Hnf4α and β-catenin, along with its DNA-binding co-factors Tcf/Lef, directly compete for binding on the same consensus enhancer motifs thereby regulating zonal hepatocyte gene expression (Colletti et al., 2009; Colnot and Perret, 2011).

However, what sets up the localized expression pattern of molecular signals may be a more basic physiological principle and intimately regulated by vascular patterning. Oxygen pressure regulates hepatic zonation. The oxygen pressure both in the sinusoids and in the hepatocytes is graded along the axis of the hepatic lobule between the PV and the CV. The blood oxygen tension in the rat sinusoid ranges from approximately 65 mm Hg in the periportal zone to approximately 30–35 mm Hg in the pericentral zone (Figure 1) (Jungermann and Kietzmann, 1996; Lautt, 2009). The oxygen gradient within the sinusoid also generates a gradient in hepatocyte intracellular oxygen tension. The hepatocyte oxygen tensions are considered to range from 45–50 mm Hg in the periportal hepatocytes to 15–20 mm Hg in the pericentral hepatocytes (Jungermann and Kietzmann, 2000). There is evidence that hepatocytes are able to sense oxygen tension through a non-respiratory chain ferro-heme protein, revealing a potential pathway for oxygen to regulate hepatocyte zonal gene expression (Kietzmann et al., 1992; Kietzmann et al., 1993). In cultured primary rat hepatocytes, oxygen tensions influence the gene expression of some zonal genes, including the periportal gene phosphoenolpyruvate carboxykinase (PEPCK, involved in gluconeogenesis) and the pericentral gene glucokinase (GK, involved in glycolysis) (Wolfle and Jungermann, 1985; Jungermann and Kietzmann, 1996). However, it appears that only certain classes of zonal hepatocyte genes are sensitive to oxygen tension. Genes involved in glucose and drug metabolism are more readily influenced by blood flow and oxygen tension, while genes involved in ammonia detoxification and glutamine synthesis have a more stable and defined expression pattern in the face of oxygenation manipulations (Wagenaar et al., 1993; Wagenaar et al., 1994; Bhatia et al., 1996; Jungermann and Kietzmann, 1997; Allen and Bhatia, 2003; Colnot and Perret, 2011). While it is clear that oxygen pressure can influence hepatocyte zonation, an in vivo mechanism through which the regulation occurs and the importance of oxygen pressure during developmental zone establishment and homeostasis remains unknown.

An example demonstrating the importance of proper PV and HA architecture to pattern hepatocyte zonation is observed in a mouse model where Vegf was specifically deleted from the liver epithelial lineages at midgestation (Walter et al., 2014a). Epithelial derived-Vegf is required for the progressive postnatal elaboration of the PV and HA architecture. The changes in hepatic vasculature correlate with observed changes in hypoxic regions across the hepatic epithelium. These changes culminate in perturbation of the hepatocyte zones. Localization of glutamine synthetase, a pericentral hepatocyte marker, is expanded along with carbamoyl phosphate synthestase 1 (CPS1), expressed around the portal vein and intermediate hepatocytes. These results demonstrate the requirement for VEGF signaling in the hepatic epithelium to establish the proper liver vasculature, hepatic oxygenation, and hepatocyte zonal architecture.

KEY ISSUES OF HEPATIC CELL PLASTICITY THAT REMAIN TO BE ADDRESSED FOR REGENERATIVE AND CELL REPLACEMENT THERAPIES

Relationship of hepatocyte zonal identities to cell plasticity and progenitor activity

To further demonstrate the importance of PV establishment, ductular reactions are only observed in periportal regions of chemical-injury and genetic-driven experimental models (Desmet, 2011; Walter et al., 2014b). Additionally, in a variety of experimental injury models and human chronic liver diseases, there is evidence of coincident hepatocyte-to-BEC lineage conversion with the presence of ductular reactions (Michalopoulos et al., 2005; Limaye et al., 2008; Yanger et al., 2013; Sekiya and Suzuki, 2014; Walter et al., 2014b). Together, these data indicate the biological phenomenon of hepatocyte-to-BEC conversion is restricted to zone 1 and zone 2 hepatocytes during liver injury. Even in experimental mouse models where activated Notch is overexpressed in all hepatoblasts or hepatocytes, conversion of hepatoblasts/hepatocytes into BECs only occurs in zone 1 and zone 2 hepatocytes; zone 3 hepatocytes do not fully activate a biliary program expressing cytokeratin 19 despite expressing activated Notch (Sparks et al., 2010; Jeliazkova et al., 2013; Yanger et al., 2013). Potential mechanisms for the spatial restriction in BEC differentiation include 1) a difference in competency of hepatocytes in different zones to receive and/or respond to a pro-BEC differentiation signal or 2) the spatial restriction of a pro-BEC signal to periportal areas. Additionally, differences in the oxygen tension gradient may directly, within the hepatocyte, or indirectly, through vascular macro architecture, influence hepatocyte competency. If an endogenous hepatocyte zonal identity is crucial for cell plasticity, focusing either exclusively on periportal hepatocytes or attempting to identify a zonal signal or oxygen level required to activate hepatocyte-to-BEC conversion may aid current research efforts.

Often times, the signaling pathways and mechanisms that control embryonic cell fates and tissue architecture retain function during adult homeostasis and are re-activated during organ regeneration. In the liver, several signaling pathways with known roles during hepatogenesis, including Notch and Wnt, have roles during injury and regeneration as well. Sox9, a direct target of Notch signaling in the hepatic ductal plate, has been implicated as a marker of hepatocyte-to-BEC conversion during mouse liver injury (Yanger et al., 2013). Thus, the ductal plate differentiation program may be utilized for the generation of new BECs in injured livers of adult mice. During liver injury in adult mice, disrupting Notch signaling is demonstrated to reduce the expression of Sox9 in hepatocytes and to consequently restrict the process of hepatocyte-to-BEC conversion, confirming the link between Notch signaling and Sox9 in cell lineage decisions and plasticity in both the embryonic and adult liver (Yanger et al., 2013; Sekiya and Suzuki, 2014). Studies have demonstrated that Notch signaling plays a role in the generation of the ductular reaction in rats and in mice, as inhibiting Notch signaling can reduce or delay the formation of the ductular reaction (Darwiche et al., 2011; Fiorotto et al., 2013). However, loss of Hnf6 within the background of deficient Notch signaling, via Rbpj, results in initial ductal insufficiency and progressive regeneration of communicating IHBDs (Vanderpool et al., 2012; Walter et al., 2014b). This demonstrates the plasticity of the liver in a ductopenic model and its ability to regenerate bile ducts without the presence Hnf6 or Rbpj.

In addition to Notch, Wnt signaling has also been examined for its role in directing cell fate decisions in the regenerating adult rodent liver. Current thinking suggests that Notch and Wnt may have opposing roles in directing cell fate decisions during liver regeneration, with Notch promoting BEC specification and Wnt promoting hepatocyte fates (Boulter et al., 2012; Strazzabosco and Fabris, 2013). When acute and chronic human liver diseases were analyzed for activation of Notch or Wnt signaling, Wnt signaling was found to be upregulated in hepatic progenitor cells (HPCs) in a parenchymal disease, while Notch was activated in the HPCs of a biliary disease (Spee et al., 2010). These findings suggest different regenerative responses occur in the liver depending on the affected tissue and extent of the injury.

Besides the known capacity of mature hepatocytes to proliferate and produce large numbers of hepatocytes, the process of liver regeneration has long implicated facultative stem cells to rescue hepatic function in conditions where hepatocytes are unable to meet the required demands (Miyajima et al., 2014). Recently there has been much debate on the topic of whether liver stem cells exist in mouse models of hepatic injury associated with ductular reactions (Grompe, 2014). Clonally derived cells isolated from the reactive ductular populations demonstrate bipotentiality in culture and the ability to engraft and generate hepatocytes upon transplantation (Dorrell et al., 2011; Huch et al., 2013). However, in vivo lineage tracing using genetic tools in mouse models of hepatic injury does not support the presence of a facultative liver stem cell (Schaub et al., 2014; Yanger et al., 2014). A caveat to the current mouse injury models of ductular reactions is the fact that some hepatocytes retain the ability to proliferate and therefore may be able to meet the demands of hepatic function without instigating the facultative stem cell compartment. Thus, unveiling a facultative liver stem cell in mice conceivably awaits development of a novel hepatic injury model.

Vascular implications for de novo liver regenerative therapies and generation of bioartificial organs

The PV structure appears to be the first occurrence of an organized three-dimensional architecture during development of the liver. Based on the observed sequence of hepatogenesis in humans (Gouysse et al., 2002; Collardeau-Frachon and Scoazec, 2008; Fabris et al., 2008) and experimental mouse models (Fabris et al., 2008; Hofmann et al., 2010), it can be extrapolated that the establishment of the PV is crucial for directing the development of the IHBD and the HA, and together, the spatial relationship of the macro architecture. The PV, HA, and CV are then involved in generating and maintaining the micro structure of the lobule and the spatial identities of hepatocytes by establishing the oxygen gradient across the lobule. As such, cell replacement therapies aimed at regenerating large portions of the liver after, for example, resection, should carefully consider the vascular architecture. It is likely that the implantation of hepatocytes without an appropriate and regulated vascular architecture may not result in proper hepatocyte survival and function, as well as establishment and maintenance of an IHBD system.

Additionally, studies aimed at generating bioartificial organs from decellularized liver extracellular matrices obtain the most success from protocols utilizing reagents that decellularize the liver while maintaining the native vascular architecture (Uygun et al., 2010). The liver vasculature is a primary step for directing hepatocyte zonal identity as well as providing nutrients and oxygen to nourish the hepatocytes seeded onto the decellularized liver matrix. The extracellular matrix (ECM) of the liver provides structural support, however, it also aids the liver tissue by releasing biochemical cues such as variety of growth factors. Although there have been many advances in bioartificial livers, there are still challenges that remain, one of which is low efficiency of hepatocyte engraftment. To try and overcome this limitation, one research group designed liver scaffolds that encompassed liver-derived ECM and growth factors that were then implanted into the rat liver (Hammond et al., 2011). They found that with this technique they had increased infiltration of the hepatocytes into the scaffold as well as an increase in proliferation around the implanted scaffold (Hammond et al., 2011). Future work in this field will be focused on understanding the composition of native liver ECM for effective synthesis in the lab, as well as, uncovering the complexities of the growth factors, how they promote migration and induce proliferation into the matrix, and what concentrations are optimal for these processes (Bhatia et al., 2014; Handa et al., 2014).

CONCLUSIONS

In order to make advances in future therapeutic options for chronic liver disease, a greater understanding of hepatic vasculature development is crucial. As discussed, the proper establishment of the PV and other hepatic vasculature networks are central for directing subsequent liver tissue architecture, hepatocyte zonation, and ultimately liver function. In addition to hepatic vascular architecture, a more thorough understanding of signaling pathways directing hepatic vasculature formation and endothelial diversity is needed. Endothelial cells from different organs harbor combinations of transcription factors and other factors important for microvascular specification such as angiocrine growth factors and chemokines that are distinguishable by organ type (Nolan et al., 2013). The microvasculature depends on these cues from their environment for proper specification and functionality (Nolan et al., 2013). A greater understanding of these unique factors for each organ will be beneficial in optimizing bioartifical organs of all kinds. All of these areas that direct macro and micro hepatic architecture need to be understood to ultimately drive better therapeutic options and optimal innovation of bioartifical livers to address chronic liver disease.

Acknowledgments

Financial Support: This work was supported by grants from the National Institutes of Health (NIH) to S.S.H (R01DK078640).

Footnotes

Disclosures: The authors have nothing to disclose.

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