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Potential conflict of interest: Nothing to report.
Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal-binding protein-Jκ (RBP-J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP-J resulted in LSEC proliferation and a veno-occlusive disease–like phenotype in the liver, as manifested by liver congestion, deposition of fibrin-like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP-J–deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma-membrane of LSs to hamper the microcirculation and intensify veno-occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules—such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin-6, and hepatocyte growth factor—was disturbed. Conclusion: Notch/RBP-J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation. (HEPATOLOGY 2009;49:268-277.)
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Liver sinusoid (LS) endothelial cells (LSECs) are specialized endothelial cells lining the surface of LSs, which function to filler serum, regulate microcirculation, and participate in metabolism with its endocytic ability.1 LSECs have complicated communications with hepatocytes, and play critical roles in supporting liver development and regeneration.2 During liver regeneration in adults, for example in the rat or mouse partial hepatectomy (PHx) model, hepatocytes proliferate to form avascular parenchymal islands, and stimulate and attract LSECs,3-5 through vascular endothelial growth factor (VEGF) and other angiogenic signals.6-8 On the other hand, LSECs can play an angiogenesis-independent protective role on hepatocytes through vascular endothelial growth factor receptor 1 (VEGFR1) signaling. LeCouter et al.9 showed that PHx-induced VEGF up-regulation can induce LSEC proliferation by binding to VEGFR2, and can induce LSECs to secrete interleukin-6 (IL-6) and hepatocyte growth factor (HGF) to promote hepatocyte proliferation through VEGFR1. These results have established critical roles of LSECs in liver development and regeneration, but how the functions of LSECs are regulated is unclear.
LSECs also participate in the pathogenesis of many liver diseases.10 Bone marrow transplantation, alcoholic liver insults, and other chemical offenses may result in sinusoidal obstructive syndrome (SOS) or veno-occlusive disease, which is characterized by congested LSs, fibrin deposition, subendothelial edema, and hepatocellular abnormalities.11, 12 Evidence has shown that damage of LSECs is an initial step leading to SOS.13-16 Although surgery17 and matrix metalloproteinase inhibitors18 have been shown to be effective in the treatment of SOS, understanding the molecular mechanisms underlying SOS is essential for effective therapy of this disease, which is frequently lethal.
Notch signaling regulates cell proliferation, apoptosis, and cell fate decisions in a broad range of tissues during embryonic and postnatal development.19 The DNA-binding protein recombination signal binding protein-Jκ (RBP-J) mediates signaling from all four mammalian Notch receptors by associating with the Notch intracellular domains. This protein interaction transactivates genes downstream to RBP-J, such as the Hes family members.19 Notch signaling plays a pivotal role in the vascular system20 by restricting endothelial tip cell specification21 and specifying arterio-venous identity.22 The disease genes responsible for CADASIL and Allagilles syndrome, both of which show inherited vascular anomalies, have been mapped to Notch3 and Jagged1, respectively,20 suggesting a role in pathological angiogenesis. Indeed, blockade of Dll4 increases EC proliferation and tumor vascular density but inhibits tumor growth due to poor function of new vessels, a phenomenon defined as the Delta paradox.23 Recently, using an RBP-J conditional knockout mouse model, we showed that Notch signaling is critical in the maintenance of vascular homeostasis.24
Although Notch signaling has been implicated in the development of liver biliary system and vasculature,25-27 the actual roles and mechanisms of the Notch pathway in the regulation of hepatic development and regeneration are obscure. Whereas some reports have indicated that Notch signaling promoted hepatocellular proliferation,28 Croquelois et al.29 showed that there was an increase in spontaneous proliferation of hepatocytes in Notch1-deficient mice. We report that conditional deletion of RBP-J, the common downstream transcription factor for all Notch receptors, mainly leads to defects in LSECs, which results in an SOS-like disease and interfered liver regeneration.
The RBP-J–floxed mouse strain was constructed as described30 and was maintained in our laboratory. The Mx-Cre transgenic mouse, in which the Cre gene is under the control of the interferon-α–inducible Mx promoter, was a gift from K. Rajewsky. RBP-J–floxed mice were crossed with Mx-Cre mice to obtain heterozygous and homozygous mice bearing the Mx-Cre transgene (RBP+/f-MxCre and RBPf/f-MxCre, respectively) as genotyped via polymerase chain reaction (PCR).30 To induce Cre-mediated deletion of RBP-J, 8- to 10-week-old female mice were injected intraperitoneally with 300 μg/100 μL of poly(I)-poly(C) (Sigma, St. Louis, MO) four times at 2-day intervals. Mice were then injected with the same amount of poly(I)-poly(C) four more times at 1-week intervals (eight injections in total). Mice were subjected to further analysis 1 week after the last injection. PHx was performed as described.29 All animal experiments were approved by the Animal Experiment Administration Committee of our institution.
LSECs were magnetically sorted from collagenase D (2 mg/mL, Roche Diagnostics)–treated liver tissues using anti-LSEC MicroBeads (ME-9F1, Miltenyi Biotec GmbH, Germany) following the manufacturer's instructions. Cells were cultured in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 40 μg/mL ECGS (Sigma). In some experiments, γ-secretase inhibitor IX (GSI, Calbiochem, La Jolla, CA) was added at a concentration of 75 μM. For the isolation of hepatocytes, liver was perfused with preheated collagenase IV (0.05%, Sigma). The resulting cell suspension was subjected to density gradient centrifugation. Purified hepatocytes were cultured in Dulbecco's modified Eagle's medium in the presence of 10% fetal bovine serum, 10 ng/mL HGF (eBiosicence), 0.5 U/mL insulin, and 10 μg/mL hydrocortisone. The cells were more than 90% pure, as evaluated by immunostaining and morphology.
Liver was removed and fixed in Bouin's fixative overnight. Tissues were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin-eosin (H&E) by standard methods for routine morphological analysis. Perls staining was also performed according to standard procedures.
Immunohistochemistry was performed using standard procedures with rat anti-mouse CD31 (Chemicon International), rabbit polyclonal anti-Hif-1α, rabbit anti-mouse Ki67, or rabbit anti-mouse VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) as primary antibodies. Secondary antibodies included horseradish peroxidase–conjugated rabbit anti-rat immunoglobulin G (Zhongshanxinqiao, China) and goat anti-rabbit immunoglobulin G (Boster Bio Tec, China). Images were taken under a microscope with a CCD camera.
Liver embedded in OCT was sectioned at 10 μm. For staining, sections were fixed with 4% paraformaldehyde and stained with fluorescein isothiocyanate–conjugated anti-VEGFR2 (Chemicon International), fluorescein isothiocyanate–conjugated anti–proliferation cell nuclear antigen (PCNA) (Sigma, St. Louis, MI), or Rhodamine-agglutinin l (Vector Laboratories, Burlingame, CA). Images were taken using a fluorescence microscope (Olympus BX51, Japan) with a CCD camera (Olympus DP70) or confocal microscope (FV1000, Olympus). Flow cytometry was performed essentially as described.30
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed using a TUNEL kit (Promega) according to the manufacturer's protocol.
Sample preparation and observation with transmission electron microscope (TEM) or scanning electron microscopy (SEM) were performed as described.24
Enzyme-Linked Immunosorbent Assay.
Sera were collected from RBP-Jf/f-MxCre and control mice at different time points after PHx and were used to detect albumin using a kit (Bioresun, China). For the detection of cytokines, sera or LSEC culture supernatants were collected and assayed using HGF, IL-6, and VEGF enzyme-linked immunosorbent assay (ELISA) kits (Jingmei Biotech, Shenzhen, China) following the recommended protocols.
Real-Time Reverse-Transcription PCR.
Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Complementary DNA was prepared using a reverse-transcription kit from TOYOBO (Osaka, Japan). Real-time reverse-transcription PCR (RT-PCR) was performed using a kit (SYBR Premix EX Taq, Takara) and the ABI PRISM 7300 real-time PCR system, with β-actin as a reference control. Primers used in real-time PCR are listed in Supplementary Fig. 1.
Images were imported into Image Pro Plus 5.1 software, and pixels for each color were analyzed to quantitatively represent positively stained cells. Statistical analysis was performed with the SPSS 12.0 program. Results are expressed as the mean ± standard deviation. Comparisons between groups were undertaken using an unpaired Student t test. P < 0.05 was considered statistically significant.
RBP-J Deletion Leads to Liver Congestion.
In RBPf/f-MxCre and the control RBP+/f-MxCre mice, administration of the interferon-α inducer poly(I)-poly(C) induced more than 90% deletion of the floxed RBP-J gene in endothelial cells and liver.30 Compared with the Notch1 conditional inactivation,29 livers of the induced RBPf/f-MxCre mice appeared normal except for a reddish color (Fig. 1A). The reddish color of the liver of RBPf/f-MxCre mice did not fade significantly even after flushing with phosphate-buffered saline (Supplementary Fig. 2). Perls staining revealed that in the liver of control mice, no hemin deposition was visible. In the liver of RBP-J knockout mice, however, hemin deposition increased greatly (Fig. 1B). These results suggest that deletion of RBP-J led to liver congestion.
Abnormal LS Structure Following Deletion of RBP-J.
Liver congestion might be attributed to LSEC abnormalities.13-16 We stained LSECs with anti-CD31, agglutinin, and anti-VEGFR2 via immunohistochemistry or immunofluorescence. The results confirmed that in the liver of RBPf/f-MxCre mice, there was a significant increase of LSECs (Fig. 1C,D, and data not shown). Time course analyses showed that although VEGFR2+ signals in the liver increased 4 weeks after the starting of induction, statistically significant change was detected 6 weeks after the first injection of poly(I)-poly(C) (Fig. 1E).
We further examined the ultra-structure of LSs. SEM examination demonstrated the deposition of fibrin-like materials in LSs following RBP-J deletion (Fig. 2A, upper). In addition, a significant increase of endothelial fenestrations was noticed in RBP-J knockout mice (Fig. 2A, lower). TEM observation revealed that the space of Disse in the liver of RBPf/f-MxCre mice was significantly widened compared with the control (Fig. 2B,C), suggesting edema of the space of Disse. Therefore, although RBP-J deletion induced LSEC proliferation, the proliferated LSECs appeared abnormal and led to an SOS-like disease in the liver of mice, as evidenced by liver congestion, deposition of fibrin-like materials in LSs, and edema in the space of Disse.
RBP-J Deletion Induces Hepatocyte Proliferation and Apoptosis.
H&E staining showed an approximately normal lobular structure in the liver parenchyma of RBPf/f-MxCre mice (Fig. 3A, upper). However, immunohistochemical staining for Ki67 antigen showed a mildly increased proliferation of hepatocytes in RBP-J knockout mice (Fig. 3A, middle; Fig. 3B), consistent with the Notch1 deletion.29 We also examined hepatocyte apoptosis in the livers of RBP-J knockout mice via TUNEL combined with agglutinin staining. The results indicated that apoptotic hepatocytes increased in RBPf/f-MxCre mice compared with the control (Fig. 3A, lower; Fig. 3C), suggesting that RBP-J deletion induced hepatocyte proliferation and apoptosis.
We then cultured LSECs and hepatocytes in vitro to observe their proliferation and their interactions. Cell proliferation was monitored by counting cell numbers or via 5-carboxyfluorescein diacetate succinimidyl ester dilution assay.24 The results showed that blockade of Notch signaling, by treatment with a γ-secretase inhibitor and/or by RBP-J disruption, promoted both LSEC and hepatocyte proliferation in vitro (Fig. 4A-C; Supplementary Fig. 3A-C), suggesting that Notch signaling could repress proliferation of these cells. The culture supernatant of RBP-J–deficient and control LSECs were collected and were used to stimulate the proliferation of wild-type hepatocytes. The results showed that compared with the control, supernatant from RBP-J–deficient LSECs produced significantly less activities of promoting hepatocyte proliferation (Fig. 4D; Supplementary Fig. 3D). Consistently, RBP-J–deficient LSECs secreted lower levels of VEGF, HGF, and IL-6 (Fig. 4E). These results indicate that RBP-J–mediated Notch signaling normally promoted LSECs to protect hepatocytes via paracrine pathways.
RBP-J Ablation Leads to Abnormal LS Regeneration After PHx.
PHx induces a regenerative response in both hepatocytes and LSECs. In RBPf/f-MxCre mice, liver regeneration after PHx appeared abnormal, as evidenced by the increased liver/body weight ratio (Fig. 5A) and decreased number of nuclei in H&E-stained tissues (Fig. 5B,C), on 2 and 5 days after the surgery. H&E staining also revealed that on day 2 and day 5 after PHx, regenerating liver of RBPf/f-MxCre mice could form a roughly normal lobular structure (Fig. 5B). However, the arrangement of LSs appeared abnormal. On day 5 after PHx, radially arranged LSs did not appear in the livers of RBPf/f-MxCre mice, in contrast with controls (Fig. 5B, middle). This was shown more clearly by a sketch of the H&E-stained tissues, which was generated by processing the images of H&E staining using Adobe Photoshop (Fig. 5B, lower). We further stained the samples with Rhodamine-agglutinin. The result (Fig. 5B, bottom) confirmed that the arrangement of LS was abnormal, and the width of LS was significantly narrowed in RBP-J–deleted mice. These findings suggested that in RBPf/f-MxCre mice, regenerating liver could not form the regular structure of LSs 5 days after PHx.
Compromised LSEC Regeneration After PHx in RBP-J Knockout Mice.
Immunofluorescent staining showed that compared with controls, LSEC regeneration was severely reduced after PHx, as shown by significantly reduced VEGFR2-positive cells in the livers of RBPf/f-MxCre mice 2 or 5 days following the operation (Fig. 6A,B). Agglutinin staining further confirmed the reduction of LSECs in the livers of RBPf/f-MxCre mice after PHx (Fig. 6A,B). Costaining with agglutinin and anti-PCNA revealed that PCNA-positive LSECs decreased remarkably in the livers of RBPf/f-MxCre mice (Fig. 6C,D). Thus, deletion of RBP-J attenuated LSEC proliferation in regenerating liver after PHx.
Disruption of RBP-J Leads to Obstruction of LS Microcirculation During Regeneration After PHx.
Under SEM, the most remarkable change was that a large number of red blood cells were clustered within LSs in RBP-J knockout mice (Fig. 7A,B), suggesting that the microcirculation of LSs was obstructed. At a higher magnification, compared with controls, LSECs in the regenerating liver of RBPf/f-MxCre mice appeared degenerative (Fig. 7C). Under TEM, some LSECs lost their cytoplasm and organelles, and the remnant plasma membrane formed bulge-like loops protruding into the lumen of LSs to embolize the blood flow (Fig. 7D, middle). In the lower panel of Fig. 7D, we could see that mitochondria and ribosomes were liberated into the sinusoidal lumen in RBPf/f-MxCre mice during liver regeneration. These data suggest that in RBP-J knockout mice, regeneration of LSECs was damaged, leading to further obstruction of microcirculation in liver.
RBP-J Disruption Causes Decreased Proliferation and Increased Apoptosis of Hepatocytes After PHx.
We then investigated hepatocyte proliferation in RBP-J knockout mice via immunohistochemical staining for Ki67. The results showed that on both day 2 and day 5 after PHx, Ki67+ hepatocytes decreased significantly in RBPf/f-MxCre mice (Fig. 8A, upper; Fig. 8B). Apoptotic hepatocytes, as estimated via TUNEL combined with agglutinin staining, increased in the livers of RBPf/f-MxCre mice after PHx (Fig. 8A, middle; Fig. 8C). Apoptotic LSECs also increased, as shown by TUNEL-positive and agglutinin-stained cells (Fig. 8A, middle). Under TEM, we found that endoplasmic reticulum was abnormal with excessive dilation, suggesting hepatocyte degeneration in the livers of RBPf/f-MxCre mice during regeneration (Fig. 8A, lower). These results suggest that liver regeneration after PHx was interfered in RBPf/f-MxCre mice, which was supported by the decreased serum albumin level in RBPf/f-MxCre mice compared with the control (Fig. 8D).
Interfered Dynamic Expression of Molecules Involved in Liver Regeneration in RBP-J Knockout Mice.
Using real-time RT-PCR, we found that the expression tendency of Notch1, Notch2, Notch4, and Jagged1 were comparable between RBP-J knockout and control mice, although the expression level of some molecules was different at certain time points (Fig. 9A, upper line). Hes1 decreased in both resting and regenerating livers of RBPf/f-MxCre mice and showed a subverted tendency of dynamic changes during liver regeneration compared with controls.
Hif-1α and VEGF messenger RNA (mRNA) showed mild and parallel changes in resting and regenerating livers. However, the protein level of Hif-1α and VEGF was quite dramatic and different from the mRNA level, as assessed by immunohistochemistry. In resting RBPf/f-MxCre mice, both Hif-1α and VEGF proteins were significantly down-regulated, regardless the abnormal proliferation of LSECs and the obstruction of LSs (Fig. 1). During the regeneration, both Hif-1α and VEGF proteins showed a sharp increase on day 2 and reduced to a very low level on day 5 after the operation, in contrast to controls (Fig. 9B; Supplementary Fig. 4).
The expression of VEGFR1 and VEGFR2 changed discrepantly. Deletion of RBP-J down-regulated VEGFR1 and up-regulated VEGFR2 in resting livers. During liver regeneration in control mice, the mRNA levels of VEGFR1 and VEGFR2 increased. In RBPf/f-MxCre mice, we noticed stronger up-regulation of VEGFR1 and a failure of VEGFR2 up-regulation after PHx (Fig. 9A, middle line). The mRNA levels of IL-6 and HGF were decreased in resting and early regenerating livers (day 2). On day 5, however, their levels were higher than those of controls (Fig. 9A, lower line). We also examined the levels of IL-6, HGF, and VEGF in circulation via ELISA. In resting animals, RBP-J deletion led to increased VEGF and IL-6 but decreased HGF in circulation. During liver regeneration, these cytokines showed a similar tendency of change as their mRNA level (Supplementary Fig. 5).
Conditional deletion of Notch1 in the liver has been reported.29 In both RBP-J deletion and Notch1 deletion mice, increased hepatocyte proliferation was detected in resting livers. However, only in the Notch1 knockout mice did hepatocyte proliferation lead to a nodular hyperplasia phenotype. In contrast, remarkable changes in LSECs and LSs were more prominent in the RBP-J knockout mice. Deletion of RBP-J induced significant increase of LSECs and SOS-like pathological changes, including significant liver congestion, LSEC proliferation and deposition of fibrin-like materials in LS, and edema in the space of Disse. Although hepatocyte proliferation was increased, apoptosis of hepatocytes increased as well. Because LSECs and LSs are important for nourishing hepatocytes, it is reasonable to speculate that increased hepatocyte apoptosis results from malnutrition due to abnormal LS microcirculation. This might not be true, however, because in the resting livers of RBP-J knockout mice, Hif-1α expression was significantly reduced at both mRNA and protein levels, suggesting that hypoxia did not exist in liver parenchyma. It is possible that abnormal proliferating LSECs failed to provide growth factors for hepatocytes. Reduced expression of VEGFR1, which transduces a signal for LSECs to produce IL-6 and HGF,9 in the livers of RBP-J deletion mice supports this speculation. Consistently, HGF and IL-6 were down-regulated in resting livers and cultured LSECs of RBP-J-deleted mice. Therefore, in addition to repressing hepatocyte proliferation in resting livers, Notch/RBP-J signaling supports hepatocyte survival, at least partially through VEGFR1-HGF signaling in LSECs.
During liver regeneration after PHx, the proliferation response of LSECs was significantly reduced, and degeneration of LSECs further resulted in LS embolization and intensified SOS-like changes, which might contribute to increased liver/body weight ratio during liver regeneration. This is reminiscent of angiogenesis in tumors in the absence of Notch/RBP-J signaling.23 Our results suggest that the phenomenon called Delta paradox (increased angiogenesis but decreased vessel function) may also exist in normal organs when Notch signaling is blocked. The important role of Notch signaling in angiogenesis is not only the regulation of endothelial cell proliferation, but also endothelial cell differentiation to form morphologically and functionally intact vessels.20 LSECs are specialized endothelial cells. During liver regeneration, Notch/RBP-J signaling might also be important for LSEC differentiation to form functional microcirculation in LSs. This is supported by the finding that EphrinB2, which has been considered as a marker of arterial endothelial cell differentiation,20 was not up-regulated during liver regeneration in the absence of RBP-J (data not shown) in contrast to controls.
In the absence of Notch/RBP-J signaling, PHx-induced liver regeneration was interfered but not blocked completely. Indeed, 10 days after PHx, liver structure was almost completely regenerated regardless of RBP-J deficiency, although liver function has not been fully recovered as shown by the serum albumin (Supplementary Fig. 6). Hepatocytes and LSECs have sophisticated communications. In RBP-J knockout mice, both hepatocytes and LSECs are influenced. RBP-J deletion down-regulated Hif-1α and VEGF mRNA in resting livers and in the early stage after PHx, but up-regulated them at the late stage during regeneration. At the protein level, the dynamics of Hif-1α and VEGF protein expression were profoundly interfered (Fig. 9B). VEGF receptors were involved in the regulation of both LSECs and hepatocytes. VEGFR2 mediates the proliferation signal for LSECs, while VEGFR1 inhibits VEGFR2 and mediates the signal for producing IL-6 and HGF for hepatocytes.9RBP-J deletion also led to dynamic changes in the expression of VEGFR1 and VEGFR2 in resting and regenerating livers. These results suggest that RBP-J deletion changes the rhythm of liver regeneration-related molecules, and this rhythm may be important for coordinated proliferation and/or differentiation of LSECs and hepatocytes during liver regeneration.
We thank Hui Wang for her critical correction of the English in the manuscript. We also thank K. Rajewsky for Mx-Cre mouse.