Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA

AbstractInduction of intrinsic liver regeneration is an unmet need that can be achieved by temporally activating key hepatocyte regenerative pathways. Here, we establish an efficient, safe, non-integrative method to transiently express hepatocyte-growth-factor (HGF) and epidermal-growth-factor (EGF) in hepatocytes via nucleoside-modified, lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) delivery in mice. We confirm specific hepatotropism of mRNA-LNP via intravenous injection of firefly luciferase encoding mRNA-LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes are transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA-LNP efficiently induce hepatocyte proliferation. In a chronic liver injury mouse model recapitulating non-alcoholic fatty liver disease, injections of both HGF and EGF mRNA-LNP sharply reverse steatosis and accelerate restoration of liver function. Likewise, HGF and EGF mRNA-LNP accelerate liver regeneration after acetaminophen-induced acute liver injury with rapid return to baseline ALT levels. This study introduces mRNA-LNP as a potentially translatable safe therapeutic intervention to harness liver regeneration via controlled expression of endogenous mitogens in vivo.

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The impact of steatosis on liver regeneration

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2018-0050 ◽

2018 ◽

Vol 0 (0) ◽

Cited By ~ 3

Author(s):

Manon Allaire ◽

Hélène Gilgenkrantz

Keyword(s):

Growth Factor ◽

Fatty Liver ◽

Liver Regeneration ◽

Liver Diseases ◽

Growth Factor Receptor ◽

Receptor Activation ◽

Liver Graft ◽

Hepatocyte Proliferation ◽

Alcoholic Fatty Liver ◽

The Impact

Abstract Alcoholic and non-alcoholic fatty liver diseases are the leading causes of cirrhosis in Western countries. These chronic liver diseases share common pathological features ranging from steatosis to steatohepatitis. Fatty liver is associated with primary liver graft dysfunction, a higher incidence of complications/mortality after surgery, in correlation with impaired liver regeneration. Liver regeneration is a multistep process including a priming phase under the control of cytokines followed by a growth factor receptor activation phase leading to hepatocyte proliferation. This process ends when the initial liver mass is restored. Deficiency in epidermal growth factor receptor (EGFR) liver expression, reduced expression of Wee1 and Myt1 kinases, oxidative stress and alteration in hepatocyte macroautophagy have been identified as mechanisms involved in the defective regeneration of fatty livers. Besides the mechanisms, we will also discuss in this review various treatments that have been investigated in the reversal of the regeneration defect, for example, omega-3 fatty acids, pioglitazone, fibroblast growth factor (FGF)19-based chimeric molecule or growth hormone (GH). Since dysbiosis impedes liver regeneration, targeting microbiota could also be an interesting therapeutic approach.

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Platelet Interactions with Liver Sinusoidal Endothelial Cells and Hepatic Stellate Cells Lead to Hepatocyte Proliferation

Cells ◽

10.3390/cells9051243 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1243 ◽

Cited By ~ 1

Author(s):

Jeremy Meyer ◽

Alexandre Balaphas ◽

Pierre Fontana ◽

Philippe Morel ◽

Simon C. Robson ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Liver Regeneration ◽

Partial Hepatectomy ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Hepatocyte Proliferation ◽

Liver Sinusoidal Endothelial Cells ◽

Hepatocyte Growth

(1) Background: Platelets were postulated to constitute the trigger of liver regeneration. The aim of this study was to dissect the cellular interactions between the various liver cells involved in liver regeneration and to clarify the role of platelets. (2) Methods: Primary mouse liver sinusoidal endothelial cells (LSECs) were co-incubated with increasing numbers of resting platelets, activated platelets, or platelet releasates. Alterations in the secretion of growth factors were measured. The active fractions of platelet releasates were characterized and their effects on hepatocyte proliferation assessed. Finally, conditioned media of LSECs exposed to platelets were added to primary hepatic stellate cells (HSCs). Secretion of hepatocyte growth factor (HGF) and hepatocyte proliferation were measured. After partial hepatectomy in mice, platelet and liver sinusoidal endothelial cell (LSEC) interactions were analyzed in vivo by confocal microscopy, and interleukin-6 (IL-6) and HGF levels were determined. (3) Results: Co-incubation of increasing numbers of platelets with LSECs resulted in enhanced IL-6 secretion by LSECs. The effect was mediated by the platelet releasate, notably a thermolabile soluble factor with a molecular weight over 100 kDa. The conditioned medium of LSECs exposed to platelets did not increase proliferation of primary hepatocytes when compared to LSECs alone but stimulated hepatocyte growth factor (HGF) secretion by HSCs, which led to hepatocyte proliferation. Following partial hepatectomy, in vivo adhesion of platelets to LSECs was significantly increased when compared to sham-operated mice. Clopidogrel inhibited HGF secretion after partial hepatectomy. (4) Conclusion: Our findings indicate that platelets interact with LSECs after partial hepatectomy and activate them to release a large molecule of protein nature, which constitutes the initial trigger for liver regeneration.

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PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor

Biochemical Journal ◽

10.1042/bj20150734 ◽

2016 ◽

Vol 473 (3) ◽

pp. 257-266 ◽

Cited By ~ 16

Author(s):

Ryota Shizu ◽

Taiki Abe ◽

Satoshi Benoki ◽

Miki Takahashi ◽

Susumu Kodama ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Growth Factor ◽

Liver Injury ◽

Hepatocyte Proliferation ◽

Suppressor Genes ◽

Proliferation In Vitro ◽

Murine Hepatocyte

Activation of PXR enhanced growth factor- and liver injury-mediated murine hepatocyte proliferation in vitro and in vivo. Mechanistic analyses suggest that activated PXR down-regulates the expression of cell-cycle suppressor genes by inhibiting their FOXO3-dependent transcription.

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Impaired phosphatidylcholine biosynthesis does not attenuate liver regeneration after 70% partial hepatectomy in hepatic CTP:phosphocholine cytidylyltransferase-α deficient mice

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y2012-116 ◽

2012 ◽

Vol 90 (10) ◽

pp. 1403-1412 ◽

Cited By ~ 2

Author(s):

Ji Ling ◽

Lin Fu Zhu ◽

Dennis E. Vance ◽

René L. Jacobs

Keyword(s):

Liver Regeneration ◽

Partial Hepatectomy ◽

Hepatocyte Proliferation ◽

Phosphocholine Cytidylyltransferase ◽

Ctp:Phosphocholine Cytidylyltransferase ◽

Phosphatidylcholine Biosynthesis ◽

Murine Liver ◽

Proliferation Of Hepatocytes

Phosphatidylcholine (PC) is the major component of mammalian membranes, and the induction of PC biosynthesis has been shown to be an essential step in cell proliferation in various cell lines. Cytidine triphosphate (CTP):phosphocholine cytidylyltransferase α (CTα) regulates the primary pathway of PC biosynthesis in the liver. The targeted disruption of CTα in murine liver (LCTα−/− mice) decreases hepatic PC mass and the number of cells in the liver, suggesting CTα as an important factor for hepatocyte proliferation. To elucidate the role of CTα in hepatic cell division in vivo, we monitored liver regeneration after 70% partial hepatectomy in LCTα−/− and loxP flanked (floxed) LCTα (control) mice. To our surprise, liver re-growth, DNA synthesis, and PC mass after surgery were not impaired in LCTα−/− mice, despite reduced total PC synthesis. Furthermore, PC synthesis in the control mice was not induced after 70% partial hepatectomy. We conclude that CTα is not essential for proliferation of hepatocytes in vivo, and that basal hepatic PC biosynthesis is sufficient to sustain regeneration after 70% partial hepatectomy.

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F4/80+ Kupffer Cell-Derived Oncostatin M Sustains the Progression Phase of Liver Regeneration through Inhibition of TGF-β2 Pathway

Molecules ◽

10.3390/molecules26082231 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2231

Author(s):

Qingjun Lu ◽

Hao Shen ◽

Han Yu ◽

Jing Fu ◽

Hui Dong ◽

...

Keyword(s):

Liver Regeneration ◽

Serum Levels ◽

Inhibitory Effect ◽

Regenerative Process ◽

Oncostatin M ◽

Hepatocyte Proliferation ◽

Initiation Phase ◽

Distinct Role

The role of Kupffer cells (KCs) in liver regeneration is complicated and controversial. To investigate the distinct role of F4/80+ KCs at the different stages of the regeneration process, two-thirds partial hepatectomy (PHx) was performed in mice to induce physiological liver regeneration. In pre- or post-PHx, the clearance of KCs by intraperitoneal injection of the anti-F4/80 antibody (α-F4/80) was performed to study the distinct role of F4/80+ KCs during the regenerative process. In RNA sequencing of isolated F4/80+ KCs, the initiation phase was compared with the progression phase. Immunohistochemistry and immunofluorescence staining of Ki67, HNF-4α, CD-31, and F4/80 and Western blot of the TGF-β2 pathway were performed. Depletion of F4/80+ KCs in pre-PHx delayed the peak of hepatocyte proliferation from 48 h to 120 h, whereas depletion in post-PHx unexpectedly led to persistent inhibition of hepatocyte proliferation, indicating the distinct role of F4/80+ KCs in the initiation and progression phases of liver regeneration. F4/80+ KC depletion in post-PHx could significantly increase TGF-β2 serum levels, while TGF-βRI partially rescued the impaired proliferation of hepatocytes. Additionally, F4/80+ KC depletion in post-PHx significantly lowered the expression of oncostatin M (OSM), a key downstream mediator of interleukin-6, which is required for hepatocyte proliferation during liver regeneration. In vivo, recombinant OSM (r-OSM) treatment alleviated the inhibitory effect of α-F4/80 on the regenerative progression. Collectively, F4/80+ KCs release OSM to inhibit TGF-β2 activation, sustaining hepatocyte proliferation by releasing a proliferative brake.

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Kinetics of Expression of Connective Tissue Growth Factor Gene during Liver Regeneration after Partial Hepatectomy and -Galactosamine-Induced Liver Injury in Rats

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3693 ◽

2000 ◽

Vol 277 (2) ◽

pp. 448-454 ◽

Cited By ~ 23

Author(s):

Kozo Ujike ◽

Toshiyuki Shinji ◽

Shoji Hirasaki ◽

Hidenori Shiraha ◽

Masaki Nakamura ◽

...

Keyword(s):

Growth Factor ◽

Liver Injury ◽

Connective Tissue ◽

Liver Regeneration ◽

Partial Hepatectomy ◽

Connective Tissue Growth Factor ◽

Tissue Growth ◽

Factor Gene ◽

Growth Factor Gene ◽

Kinetics Of

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Bone Marrow-Derived Mesenchymal Stem Cells Overexpressing Akt1 Protect Against ConA-Induced Liver Injury in Mice

Blood ◽

10.1182/blood.v124.21.5805.5805 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5805-5805

Author(s):

Lukun Zhou ◽

Shuang Liu ◽

Chuanyi M. Lu ◽

Jianfeng Yao ◽

Yuyan Shen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Growth Factor ◽

Liver Injury ◽

Lethal Dose ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Ifn Γ

Abstract Liver injury associated with veno-occlusive disease and graft-versus-host disease (GVHD) is a frequent and severe complication of hematopoietic stem cell transplantation, and remains an important cause of transplant-related mortality. Bone marrow derived mesenchymal stem cells (MSCs) have been evaluated for the prevention and treatment of refractory GVHD. However, poor cell viability has limited the therapeutic capacity of mesenchymal stromal cell therapy in vivo. In this study, we genetically engineered C57BL/6 mouse bone marrow MSCs using ex vivo retroviral transduction to overexpress Akt1, a serine threonine kinase and pro-survival signal protein, and tested the hypothesis that Akt1-expressing MSCs (Akt1-MSCs) are more resistant to apoptosis and can ameliorate acute liver injury induced by concanavalin A (ConA) in BALB/c mice. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ (100 ng/mL) stimulation conditions, Akt1-GFP-MSCs had proliferation and survival (anti-apoptotic) advantages with down-regulated apoptosis pathways, compared to control GFP-MSCs. Twenty-four hours after receiving lethal dose of ConA (40 mg/kg, intravenous) (N=10 each group), no mouse survived, with or without 1x106 Akt1-MSCs or GFP-MSCs administration (intravenous); however, 3 and 1 survived in the 5×106 Akt1-MSCs group and 5×106 GFP-MSCs groups, respectively. In subsequent sub-lethal dose ConA (20 mg/kg) experiments, compared to GFP-MSCs, mice received Akt1-MSCs administration had significantly lower serum AST, ALT, TNF-α and IFN-γ levels and less histopathological abnormalities. In addition, Akt1-MSCs treated mice had significantly higher serum concentrations of IL-10, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). In vivo imaging showed that, hepatic fluorescence signal in sub-lethal ConA+Akt1-MSCs group was significantly stronger than ConA+GFP-MSCs group on day 0, and persisted up to 14 days, whereas the signal in ConA+GFP-MSCs, Akt1-MSCs and GFP-MSCs groups was negligible on both day 7 and day 14. Thus, bone marrow derived MSCs genetically enhanced with Akt1 had survival advantage in vitro and in vivo, and have the potential to be a potent therapy for prevention and amelioration of GVHD-associated liver impairment. Further translational pre-clinical studies are ongoing to further determine the efficacy, dosage and timing of administration of Akt1-MSCs in animal models. Disclosures No relevant conflicts of interest to declare.

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Platelet-induced cell signaling during liver regeneration

British Journal of Surgery ◽

10.1093/bjs/znab202.081 ◽

2021 ◽

Vol 108 (Supplement_4) ◽

Author(s):

A Balaphas ◽

J Meyer ◽

R Perozzo ◽

M Zeisser Labouebe ◽

S Berndt ◽

...

Keyword(s):

Growth Factor ◽

Liver Regeneration ◽

Partial Hepatectomy ◽

Transforming Growth Factor ◽

Fold Increase ◽

Transforming Growth Factor Β1 ◽

Remnant Liver ◽

Liver Sinusoidal Endothelial Cells

Abstract Objective To investigate the mechanisms driving the interaction of platelets with liver sinusoidal endothelial cells (LSEC) during liver regeneration. Methods Platelets were tracked in vivo in mice by intravital confocal microscopy after partial hepatectomy. In vitro, we isolated highly pure mouse LSEC and analyzed their interactions with platelets, hepatic stellate cells (HSC), Kupffer cells and hepatocytes. Results Recruited platelets adhered to LSEC in vivo within the remnant liver segments following partial hepatectomy and were necessary for the interleukin 6 (IL-6) burst that occurred afterwards. In vitro, platelets were activated after incubation with LSEC and released transforming growth factor β1 (TGF-β1), which stimulated LSEC to secrete IL-6 (fold increase of 9.8±0.73 relative to baseline). Antibody-mediated neutralization of TGF-B1 or its downstream SMAD signalling pathway prevented the effects of activated platelets on LSEC. We also demonstrated that IL-6 released by LSEC stimulates HSC to produce hepatocyte growth factor (HGF) a main mitogen for hepatocytes. Conclusion Our results suggest that after hepatectomy, platelets initiate liver regeneration by interacting with LSEC and stimulate IL-6 release, which in turn stimulates HSC to produce HGF.

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