Considering the enormous extension of the PBP during BDL, it is interesting to evaluate the possible role played by one of the most potent and well known angiogenic factors, the VEGF. VEGF is a member of a family of related growth factors that includes VEGF-A, -B, -C, -D, and -E, and placenta growth factor[59-62]. VEGF’s role in vascular proliferation associated with tumour growth or wound healing has been widely documented in different organs. The expression of VEGF and its receptors is not restricted to vascular endothelial cells, since their expression has been detected in vascular smooth muscle cells, osteoblasts, regenerating myotubes and hematopoietic stem cells[60-62,64]. Moreover, VEGF has also been secreted in a large number of normal epithelial cells, such as keratinocytes, goblet cells in nasal polyps, pulmonary cells, prostate cells, ductal cells derived from normal pancreas and also in normal hepatocytes[67-69]. Because cholangiocyte proliferation, as described above, is regulated by growth hormones, we investigated the expression of VEGF and its receptors in normal and proliferating biliary epithelia.
After immunohistochemistry of liver sections from normal and BDL rats, we found that intrahepatic cholangiocytes from both normal and BDL rats express VEGFR-2 and VEGFR-3 but not VEGFR-1
. This data was confirmed by immunoblot analysis in a purified cholangiocyte cell culture. Moreover, we found that intrahepatic cholangiocytes from normal and BDL rats express the protein for VEGF-A and VEGF-C
. Immunohistochemistry shows that the expression of VEGF-A and VEGF-C was higher in bile ducts from BDL rats (Figure 4
) compared to bile ducts from normal rats
. In addition, we found VEGF in the supernatant of primary cultures of normal cholangiocytes, indicating that the intrahepatic cholangiocytes secrete VEGF. Following BDL, the amount of VEGF secreted by cholangiocytes into the supernatant significantly increased compared to the amount of VEGF secreted by normal cholangiocytes. In normal rat liver, pericentral hepatocytes show positivity for VEGF-A (Figure 5
In order to confirm that VEGF plays an important role in the regulation of cholangiocyte proliferation, we also tested the effect of the administration of an anti-VEGF-A or an anti-VEGFC antibody to BDL rats. The administration of anti-VEGF to BDL rats induced an increased number of apoptotic cholangiocytes compared with BDL and a decrease in the number of PCNA and CK-19 positive cholangiocytes. In order to demonstrate that the proliferation of the PBP following BDL only occurs after cholangiocyte proliferation, we measured the number of PCNA-positive vascular endothelial cells and cholangiocytes in liver sections from rats with BDL for 1 or 2 wk. After BDL for 1 wk, PCNA was expressed only by proliferating cholangiocytes (and in rare hepatocytes), whereas endothelial cells were negative. In contrast, after BDL for 2 wk, both cholangiocytes and endothelial cells became positive for PCNA. This was confirmed by the immunohistochemical expression of Factor VIII-related antigen (a marker of endothelial cells) in liver sections from normal, 1- and 2-wk BDL rats. The number of Factor VIII- related antigen positive cells increase, in comparison with normal rats, only after 2-wk BDL but it was unchanged after 1-wk BDL, thus confirming that the proliferation of PBP occurs after that of the biliary epithelium.
In addition, to better define the role of VEGF-A in the regulation of cholangiocyte and PBP proliferation during BDL we performed in: (1) normal rats and normal rats + hepatic artery ligation (HAL); (2) 1 wk BDL[4,5] rats; and (3) rats that (immediately after BDL or BDI + HAL) were treated by IP implanted with BSA or r-VEGF-A for 1 wk[71-74]. With the aim of evaluating PBP organization and the cholangiocyte VEGF protein expression, SEMvcc were performed. The peribiliary plexus, observed in BDL rats, was not demonstrated in BDL + HAL rats by the SEMvcc technique. We did not observe PBP in BDL + HAL rats because the PBP is nourished by the HA. In normal rats, PBP was observed more easily in large portal tracts. In small portal tracts, the PBP was characterized by single layer of capillaries or even by a single capillary; in BDL + HAL we do not observed PBP. Administration of r-VEGF-A to BDL + HAL rats prevented the HAL-induced microvascular modification (absence of PBP). The microvascular pattern observed after administration of r-VEGF-A to BDL + HAL rats demonstrated the presence of a PBP with similar characteristics previously described in BDL rats[9,71].
Immunohistochemistry in BDL liver sections shows that bile ducts express VEGF-A, VEGFR-2 and VEGFR-3. HAL induced a decrease in the number of cholangiocytes positive for VEGF-A and VEGFR-2 and VEGFR-3 receptors compared to liver sections from 1 wk BDL rats. Following the administration of r-VEGF-A to BDL + HAL rats, the expression of VEGF-A and VEGFR-2 and VEGFR-3 was similar or higher than that of BDL rats. VEGFR-1 was not expressed by cholangiocytes. These indicate that VEGF, predominantly by an autocrine mechanism, plays an important role in modulating cholangiocyte proliferation
. Furthermore, if we drastically reduced the HA blood flow in BDL rats by the HAL, we induced the absence of PBP, reduction of both VEGF and VEGF receptors at the cholangiocyte level and a reduction of bile duct mass with no induced effect in the liver of normal rats (Figure 6
). Evidently, when the intrahepatic bile duct mass is expanded, as occurs after BDL
, blood supply through the hepatic artery becomes fundamental in sustaining the enhanced nutritional and functional demands of proliferating intrahepatic biliary epithelium
. VEGF has been demonstrated to participate in a complex that sustains cholangiocyte proliferation after BDL
; therefore, concerning the effect of HAL on bile duct mass, the fall of synthesis and release of VEGF by cholangiocytes after HAL certainly has a role in compromising cell proliferation.
In conclusion, recent data highlights the role of arterial blood supply of biliary tree in conditions of cholangiocyte proliferation, such occurs during chronic cholestasis. On the other hand, the role played by VEGF as a tool of cross-talk between cholangiocytes and PBP endothelial cells suggests that manipulation of VEGF release and function could represent a therapeutic strategy for human pathological conditions characterized by damage of hepatic artery or the biliary tree.