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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Vascularization of the Intrahepatic Biliary Tree and Its Role in the Regulation of Cholangiocyte Growth

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The structure of the peribiliary plexus (PBP) in the normal liver has been the subject of a number of studies using both light microscopy, and scanning electron microscopy observations. The PBP, that stems from the hepatic artery branches and flows into the hepatic sinusoids, nourishes the biliary tree and plays a fundamental role in supporting the secretory and absorptive functions of the biliary epithelium. Little information is available on the rearrangement of the PBP in human liver diseases and in experimental models characterized by proliferation of bile ducts. Bile duct ligation in rats induces marked proliferation of bile ducts. Our group investigated the relationships between proliferation of bile duct cells and peribiliary plexus after common bile duct ligation (BDL) in the rat.

After one week of BDL, a normal organization of the sinusoidal network persists despite significant proliferation of the bile ducts, whereas after three weeks of BDL a typical, well-developed peribiliary plexus is present. The peribiliary plexus runs at the periphery of the liver lobule and consists as bundles of vessels, composed of capillaries of homogeneous diameter with a typical loose round mesh structure. There is no evidence of vascular proliferation or other morphological modifications at the level of sinusoids. Considering the enormous expansion of the PBP during BDL, we evaluated if vascular endothelial growth factor (VEGF) acts as an angiogenic factor in BDL rats. In normal rat livers, VEGF is expressed only in scattered hepatocytes within the periportal zone. As a novel observation, our group observed that in BDL rats, VEGF was expressed by a large number of hepatocytes near the areas of cholangiocyte proliferation, and a strong VEGF immunolocalization was observed in proliferating cholangiocytes. These data were also confirmed in vitro by Western-Blot and RT-PCR analysis. Our findings indicate that the intrahepatic biliary epithelium expresses VEGF particularly during cholangiocyte proliferation. These novel data can be helpful in clarifying the mechanisms triggering the intense vascular proliferative response occurring during BDL.

Background and Experimental Approach

The concept that liver microcirculation influences hepatic function extensively and it is fundamental for the zonal organization of the hepatic structure was first developed in 1666 by the Italian anatomist, Marcello Malpighi.1 He described a hexagonal shaped structure, that he termed “acinus”, as the glandular unit of the liver; this description correlates with the current concept of the liver morpho-functional unit also termed “acinus” by Rappaport in 1952.2 In order to study the hepatic microvascular organization and the changes occurring in liver pathology, our group has used the method of Scanning Electron Microscope (SEM) of vascular corrosion casts. The technique consists of injecting a liquid polymerizing resin into the vascular hepatic tree to form a cast, followed by the corrosion of the surrounding tissue, thus revealing the microcirculation cast of the liver vascular spaces that shows the vascular lumina in the tiniest detail.3,4

Rearrangement of PBP during Cholangiocyte Proliferation

Under very low magnification, scanning electron microscopic observation of the normal extrahepatic biliary tree shows a rich and complex network of vessels of varying diameter. At high magnification, extrahepatic biliary tree vascular casts show the presence of two main vascular layers: an outer arterial and venous layer, and an inner richer capillary layer. On the lumenal side there are many round vascular empty pits within the casts that are associated with small glands distributed in the lamina propria of the common bile duct.5

The rich and bi-laminar vascular network of the rat extrahepatic biliary tree continues with two similar vascular layers of the intrahepatic biliary plexus, at least near the hilus of the organ (fig. 1). In fact, the intrahepatic peribiliary plexus shows a complex vascular network only in the larger portal spaces, whereas in the smaller portal spaces it is composed of fewer connected vessels.

Figure 1. SEM of vascular corrosion cast.

Figure 1

SEM of vascular corrosion cast. Original magnification 130x. Vascular plexus of an intrahepatic biliary duct close to the hilus, showing an outer (arrows) arterial and venous layer and an inner (arrowhead) capillary layer of vessels. Branches of the hepatic (more...)

The intrahepatic peribiliary plexus originates from hepatic artery branches and flows into the hepatic sinusoids by means of connecting small venules with portal vein branches.3,5 Little information exists regarding the modifications of the hepatic microvasculature in experimental animal models of cholangiopathies and in human liver disorders. Furthermore, the rearrangement of the peribiliary plexus around proliferating bile ducts following common bile duct ligation has not been defined. In the last twenty years, our group has investigated the modification of the intrahepatic biliary tree and peribiliary plexus proliferation after BDL. Observations in the rat have been performed by light microscopy, SEM and immunohistochemical techniques.4,6,7

Scanning electron microscopic vascular corrosion casts of one-week BDL livers show the presence of a normal organization of the sinusoidal network. Near the liver capsule, hepatic vein branches originate from the confluence of sinusoids, and a small number of arterial and portal branches. Deep within the liver parenchyma, the typical structure of a classic lobule, in which sinusoids run towards the central vein is apparent.8-10 A well-developed peribilary network that runs together with large branches of portal vein and hepatic artery4 (fig. 2).

Figure 2. SEM of vascular corrosion cast.

Figure 2

SEM of vascular corrosion cast. Original magnification 120x. Peribiliary plexus (arrows) running adjacent to a large branch of the portal vein (V) in normal rat.

At the periphery of the lobule there is a very thin empty zone without any characteristic vascular element. Three weeks after common BDL, a typical well-developed peribiliary microvascular plexus, originating from arterioles derived from hepatic arterial branches is observed. The plexus runs at the periphery of the lobule and appears hypertrophic but otherwise normal in its arrangement (fig. 3). The plexus is composed of many layers and shows an intimate meshed network, characterized by round loops, resembling the organization of the inner vascular layer of the extrahepatic peribiliary plexus (fig. 4). Between the peribiliary plexus and the sinusoidal network there is an empty space, which corresponds to proliferating connective tissue, digested during the casting procedure (fig. 5). Infrequent vascular communications between the peribiliary plexus and sinusoids are also visible. The efferent vessels that arise from the confluence of the capillaries form a small vein that tends to drain into the interlobular vein. In some areas of the liver the peribiliary plexus does not seem to develop completely in 3 week BDL rats. In these areas there is a typical neovascular organization with interrupted vascular loops and dead-end vessels (fig. 6). These structures may represent an attempt to increase metabolic exchange within the ductular lumen and peribiliary plexus.6

Figure 3. SEM of vascular corrosion cast.

Figure 3

SEM of vascular corrosion cast. Original magnification 25x. The peribiliary plexus (P) runs at the periphery of the hepatic lobules, characterized by sinusoids (s), in BDL rat liver.

Figure 4. SEM of vascular corrosion cast.

Figure 4

SEM of vascular corrosion cast. Original magnification 110x. The proliferating peribiliary plexus in BDL rat liver, made up of a capillary network (arrows), connected with the hepatic sinusoids by small vessels (arrowhead), and by an outer layer of larger (more...)

Figure 5. SEM of vascular corrosion cast.

Figure 5

SEM of vascular corrosion cast. Original magnification 50x. An empty space (*) is present in the cast between the peribiliary plexus (P) and the hepatic sinusoids (s). The two microvascular networks are connected by some small vessels (arrows) that cross (more...)

Figure 6. SEM of vascular corrosion cast.

Figure 6

SEM of vascular corrosion cast. Original magnification 150x. Proliferated peribiliary plexus (P) and its supplying artery (A) are evident in rat liver two weeks after BDL.

In contrast to the profound modification of the peribiliary plexus, the sinusoid organization remains quite normal in BDL rats. In previous studies following BDL,6 the increase of blood bilirubin levels starts early, and remains at high levels until four weeks after BDL (fig. 7). This finding suggests that hepatocytes retain their capacity to conjugate bilirubin.

Figure 7. Total (TOT) and direct (DIR) blood bilirubin levels after 3 days and 1-4 weeks from BDL.

Figure 7

Total (TOT) and direct (DIR) blood bilirubin levels after 3 days and 1-4 weeks from BDL.

Role of VEGF in the Regulation of Vascular Proliferation during Cholangiocyte Growth

Since the peribiliary plexus nourishes the biliary tree, it has been proposed a countercurrent flow of substances reabsorbed from the bile is returned to the hepatocytes (cholehepatic shunt) (fig. 8).

Figure 8. Schematic representation of ductal secretion by functional interaction between bile salts and cholangiocytes.

Figure 8

Schematic representation of ductal secretion by functional interaction between bile salts and cholangiocytes.

Considering the enormous extension of the peribiliary plexus during BDL, it is interesting to evaluate the possible role played by one of the most potent, well known angiogenic factors, VEGF (fig. 9). In fact, its role in vascular proliferation associated with tumour growth or wound healing has been widely documented in different organs.11 The production of VEGF has also been demonstrated in a large number of normal epithelial cells, such as keratinocytes,12 goblet cells in nasal polyps,13 pulmonary cells,14 prostate cells,15 ductal cells derived from normal pancreas16 and also in normal hepatocytes.17 In particular VEGF has been detected in normal perivenular hepatocytes (Rappaport's zone 3) as well as in large numbers of hepatocytes from BDL rats, but in previous studies have not demonstrated VEGF in cholangiocytes.

Figure 9. Schematic representation of the VEGF regulation, mediated by different factors, and effect on endothelial cells.

Figure 9

Schematic representation of the VEGF regulation, mediated by different factors, and effect on endothelial cells.

We have recently investigated the expression of VEGF in BDL rat livers by immunohistochemistry and by using a mouse monoclonal antibody. Our preliminary data indicate that proliferating cholangiocytes of BDL rat livers show a marked expression of VEGF (fig. 10), which was confirmed by RT-PCR (fig. 11) and western-blot analysis (fig. 12) in cholangiocytes isolated from BDL rats there was a significantly higher expression of VEGF compared to cholangiocytes isolated from normal rats. VEGF expression was also investigated by western-blot analysis in proliferating cholangiocytes isolated from partial hepatectomized rats, where VEGF expression was 3-fold higher in large cholangiocytes than small cholangiocytes. These findings indicate VEGF may play a role in peribiliary plexus demonstrated by scanning electron microscopic vascular corrosion cast following BDL.7 During BDL, the development of the peribiliary plexus is closely correlated with an increase in bile duct mass due to cholangiocyte proliferation. Cholangiocyte proliferation is regulated and modulated by different factors such as the cholinergic system, gastrointestinal hormones,18 inflammatory mediators (TNF alpha and interleukin-1) and many growth factors. In particular, upregulation of secretin receptors and the secretin-stimulated cholangiocyte cyclic adenosine-mono-phosphate levels as well as biliary salts (taurocholic and taurolithocholic acid) may modulate the secretory and proliferative events.19

Figure 10. Immunohistochemistry for VEGF.

Figure 10

Immunohistochemistry for VEGF. Original magnification 100x. Immunolocalization of VEGF in proliferating cholangiocytes and in hepatocytes of BDL rat liver.

Figure 11. Diagram of the first demonstration by western blot analysis of VEGF expression in isolated cholangiocytes from normal rat and after partial hepatectomy.

Figure 11

Diagram of the first demonstration by western blot analysis of VEGF expression in isolated cholangiocytes from normal rat and after partial hepatectomy.

Figure 12. RT-PCR demonstration of VEGF expression in normal and BDL rat cholangiocytes.

Figure 12

RT-PCR demonstration of VEGF expression in normal and BDL rat cholangiocytes.

Recently our group has also demonstrated that cholangiocyte proliferation in BDL rats is regulated both in vivo and in vitro by estrogens and their receptors, the latter being represented mainly by the ER-beta subtype in cholangiocytes of both normal and BDL rats.7

Summary/Future Perspectives

In conclusion, during BDL the marked proliferation of peribiliary plexus supports the increased nutritional and functional needs of proliferating cholangiocytes and the demonstrated overexpression of VEGF may be the stimulus of vascular endothelial proliferation, which may consequently maintain growth of cholangiocytes (fig. 13). Cholangiocytes produce VEGF and as a paracrine signaling mechanism, VEGF can stimulate vascular proliferation supporting the progressive extension of the peribiliary plexus. The specific role of increasing biliary pressure and increasing biliary acids recircle sustained by the peribilary plexus which occurs after ligation of the intrahepatic common bile duct may be important in the regulation of cholangiocyte proliferation (fig. 14). The potential cross-talk between proliferating cholangiocytes and the adjacent vascular peribiliary plexus needs to be studied in the future.

Figure 13. Immunohistochemistry for VEGF.

Figure 13

Immunohistochemistry for VEGF. Original magnification 400x.Positive immunostaining for VEGF in proliferating cholangiocytes of BDL rat liver.

Figure 14. Hypothesis of the role of increasing biliary pressure during BDL, biliary acids recircle and peribiliary plexus on regulation of cholangiocytes proliferation.

Figure 14

Hypothesis of the role of increasing biliary pressure during BDL, biliary acids recircle and peribiliary plexus on regulation of cholangiocytes proliferation.

Acknowledgments

Portions of the studies outlined in this chapter were supported by a grant award from Scott & White Hospital and Texas A&M University, by an NIH grant DK58411 and by VA Merit Award to Dr. Alpini.

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