Effect of sorafenib on murine liver regeneration

Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib prolongs survival of patients with advanced disease and is approved for the systemic treatment of unresectable HCC. It possesses antiangiogenic and antiproliferative properties by way of inhibition of the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR-2) and platelet-derived growth factor receptor-beta 1/2 (PDGFR-β) and the kinase RAF. Sorafenib represents a candidate compound for adjuvant therapy in HCC patients. The aim of our study was to investigate whether sorafenib affects liver regeneration. C57BL6 mice received sorafenib orally at 30 mg/kg/day or its vehicle either for 14 days until the day before hepatectomy or starting the day after surgery or both. Animals were sacrificed 24, 72, and 120 hours after hepatectomy. Liver regeneration was calculated as a percent of initial liver weight. Bromodeoxyuridine (BrdU) incorporation and phospho-extracellular signal-regulated kinase (pERK1/2) were determined by immunohistochemistry on liver sections. VEGF-A, PDGF-BB, and hepatocyte growth factor (HGF) levels were measured in liver tissue homogenates. Histological analysis of scar tissue was performed. Treatment stopped 1 day before surgery had no impact on liver regeneration. Continuous sorafenib treatment and treatment started 1 day after surgery had statistically significant effects on liver regeneration at 120 hours compared to vehicle-treated control animals (72% ± 12 versus control 88% ± 15 and 70% ± 13 versus control 86% ± 5 at 120 hours, both P ≤ 0.02). BrdU incorporation showed decreased numbers of positive nuclei in both groups receiving sorafenib after surgery. Phospho-ERK levels were reduced in sorafenib-treated animals. An increase of VEGF-A levels was observed in mice receiving sorafenib. Wound-healing complications were observed in animals receiving sorafenib after surgery and confirmed on histological sections. CONCLUSION: This preclinical study shows that sorafenib did not impact on liver regeneration when ceased before surgery; however, administration after hepatectomy affected late liver regeneration.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice

Conditioning with granulocyte colony-stimulating factor (G-CSF) promotes liver regeneration in an experimental small-for-size liver remnant mouse model. The mechanisms involved in this extraordinary G-CSF effect are unknown. The aim of this study was to investigate the influence of G-CSF on the hepatic microvasculature in the regenerating liver. The hepatic sinusoidal microvasculature and microarchitecture of the regenerating liver were evaluated by intravital microscopy in mice. Three experimental groups were compared: (1) unoperated unconditioned animals (control; n = 5), (2) animals conditioned with G-CSF 48 h after 60% partial hepatectomy (G-CSF-PH; n = 6), and (3) animals sham conditioned 48 h after 60% PH (sham-PH; n = 6). PH led to hepatocyte hypertrophy and increased hepatic sinusoidal velocity in the sham-PH and G-CSF-PH groups. Increased sinusoidal diameter and increased hepatic blood flow were observed in the G-CSF-PH group compared to the sham-PH and control groups. Furthermore, there was a strong positive correlation between spleen weight and hepatic sinusoidal diameter in the G-CSF-PH group. The increased hepatic blood flow could explain the observed benefit of G-CSF conditioning during liver regeneration. These results elucidate an unexplored aspect of pharmacological modulation of liver regeneration and motivate further experiments.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

Elevated liver regeneration in response to pharmacological reduction of elevated portal venous pressure by terlipressin after partial hepatectomy.

BACKGROUND Liver regeneration is of crucial importance for patients undergoing living liver transplantations or extended liver resections and can be associated with elevated portal venous pressure, impaired hepatic regeneration, and postoperative morbidity. The aim of this study was to assess whether reduction of portal venous pressure by terlipressin improves postoperative liver regeneration in normal and steatotic livers after partial hepatectomy in a rodent model. METHODS Portal venous pressure was assessed after minor (30%), standard (60%), or extended (80%) partial hepatectomy (PH) in mice with and without liver steatosis. Liver regeneration was assessed by BrdU incorporation and Ki-67 immunostaining. RESULTS Portal venous pressure was significantly elevated post-PH in mice with normal and steatotic livers compared to sham-operated mice. Reduction of elevated portal pressure after 80% PH by terlipressin was associated with an increase of hepatocellular proliferation. In steatotic livers, animals treated with terlipressin had an increase in liver regeneration after 30% PH and increased survival after 60% PH. Mechanistically, terlipressin alleviated IL-6 mRNA expression following PH and down-regulated p21 and GADD45 mRNA suggesting a reduction of cell cycle inhibition and cellular stress. CONCLUSIONS Reduction of elevated portal pressure post-PH by the use of terlipressin improves liver regeneration after PH in lean and steatotic mouse livers.