Liver Growth Factor Research Articles

Differentiated and non-differentiated HepaRG™ cells: A possible in-vitro model system for early hepatocarcinogenesis and non-genotoxic carcinogens

Many xenobiotics are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action (MoA) and health risks for humans are unclear and no in-vitro tests are available to predict NGC. Human HepaRG™ cells in the differentiated (d-HepaRG) and non-differentiated state (nd-HepaRG) were studied as new approach methodology (NAM) for NGC. Cell-biological assays were performed with d-/nd-HepaRG and human hepatoma/hepatocarcinoma cell lines to characterize the benign/malignant phenotype. Reaction of d-/nd-HepaRG to several liver growth factors and NGC (phenobarbital, PB; cyproterone acetate, CPA; WY-14643) was compared to unaltered and premalignant rat hepatocytes in ex-vivo culture. Enzyme induction by NGC was checked by RT-qPCR/oligo-arrays. Growth, anchorage-independency, migration, clonogenicity, and in-vivo tumorigenicity of nd-HepaRG ranged between benign d-HepaRG and malignant hepatoma/hepatocarcinoma cells. All growth factors elevated DNA replication of d-/nd-HepaRG cells, similarly to unaltered/premalignant rat hepatocytes. NGC induced their prototypical enzymes in the rat and human cells, but elicited a growth response only in the unaltered/premalignant rat hepatocytes and not in human d-/nd-HepaRG cells. To conclude, a benign/premalignant phenotype of d-/nd-HepaRG cells and a reactivity towards several hepatic growth factors and NGC, as known from human hepatocytes, are essential components for an in-vitro model for early stage human hepatocarcinogenesis.The potential value as new approach methodology (NAM) for NGC is discussed.

Effect of fetal liver condition media derived cytokines (IL-6 and Flt-3) on human bone marrow stem cells colony formation

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA).In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

Fibrosis and hepatic regeneration mechanism.

Liver cirrhosis is the final stage of continuous hepatic inflammatory activity derived by viral, metabolic or autoimmune origin. In the last years, cirrhosis was considered a unique and static condition; recently was accepted some patients subgroups with different liver injury degrees that coexist under the same diagnosis, with implications about the natural disease history. The liver growth factor (LGF) is a potent in vivo and in vitro mitogenic agent and an inducer of hepatic regeneration (HR) through the hepatocytes DNA synthesis. The clinical implications of the LGF levels in cirrhosis, are not clear and even with having a fundamental role in the liver regeneration processes, the studies suggest that it could be a cirrhosis severity marker, in acute liver failure and in chronic hepatitis. Its role as predictor of mortality in fulminant hepatic insufficiency patients has been suggested. HR is one of the most enigmatic and fascinating biological phenomena. The rapid volume and liver function restoration after a major hepatectomy (>70%) or severe hepatocellular damage and its strict regulation of tissue damage response after the cessation, is an exclusive property of the liver. HR is the clinical applications fundament, such as extensive hepatic resections (>70% of the liver parenchyma), segmental transplantation or living donor transplantation, sequential hepatectomies, isolated portal embolization or associated with in situ hepatic transection, temporary artificial support in acute liver failure and the possible cell therapy clinical applications.

Role of Liver Growth Factor (LGF) in Parkinson's Disease: Molecular Insights and Therapeutic Opportunities.

Parkinson's disease is the most common neurodegenerative movement disorder with unclear etiology and only symptomatic treatment to date. Toward the development of novel disease-modifying agents, neurotrophic factors represent a reasonable and promising therapeutic approach. However, despite the robust preclinical evidence, clinical trials using glial-derived neurotrophic factor (GDNF) and neurturin have been unsuccessful. In this direction, the therapeutic potential of other trophic factors in PD and the elucidation of the underlying molecular mechanisms are of paramount importance. The liver growth factor (LGF) is an albumin-bilirubin complex acting as a hepatic mitogen, which also exerts regenerative effects on several extrahepatic tissues including the brain. Accumulating evidence suggests that intracerebral and peripheral administration of LGF can enhance the outgrowth of nigrostriatal dopaminergic axonal terminals; promote the survival, migration, and differentiation of neuronal stem cells; and partially protect against dopaminergic neuronal loss in the substantia nigra of PD animal models. In most studies, these effects are accompanied by improved motor behavior of the animals. Potential underlying mechanisms involve transient microglial activation, TNF-α upregulation, and activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and of the transcription factor cyclic AMP response-element binding protein (CREB), along with anti-inflammatory and antioxidant pathways. Herein, we summarize recent preclinical evidence on the potential role of LGF in PD pathogenesis, aiming to shed more light on the underlying molecular mechanisms and reveal novel therapeutic opportunities for this debilitating disease.

Liver Growth Factor "LGF" as a Therapeutic Agent for Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive degenerative disorder and the most common cause of dementia in aging populations. Although the pathological hallmarks of AD are well defined, currently no effective therapy exists. Liver growth factor (LGF) is a hepatic albumin–bilirubin complex with activity as a tissue regenerating factor in several neurodegenerative disorders such as Parkinson’s disease and Friedreich’s ataxia. Our aim here was to analyze the potential therapeutic effect of LGF on the APPswe mouse model of AD. Twenty-month-old mice received intraperitoneal (i.p.) injections of 1.6 µg LGF or saline, twice a week during three weeks. Mice were sacrificed one week later, and the hippocampus and dorsal cortex were prepared for immunohistochemical and biochemical studies. LGF treatment reduced amyloid-β (Aβ) content, phospho-Tau/Tau ratio and the number of Aβ plaques with diameter larger than 25 µm. LGF administration also modulated protein ubiquitination and HSP70 protein levels, reduced glial reactivity and inflammation, and the expression of the pro-apoptotic protein Bax. Because the administration of this factor also restored cognitive damage in APPswe mice, we propose LGF as a novel therapeutic tool that may be useful for the treatment of AD.

The role of PIAS3, p-STAT3 and ALR in colorectal cancer: new translational molecular features for an old disease.

Human colorectal cancer (CRC) is characterized by a sequence of biological events that determine its induction and progression. Gut microbiota has an important role in this multistep model of carcinogenesis, as well as constitutive activation of Signal Transducer and Activator Factors 3 (p-STAT3) and Protein Inhibitor of Activated STAT3 (PIAS3), which negatively controls STAT3. It has been reported that a liver growth factor, the Augmenter of Liver Regeneration (ALR), an anti-apoptotic, anti-metastatic factor, exerts protective/cell survival and anti-metastatic activities and has been detected highly expressed in neoplastic cells. To evaluate, by immunohistochemistry, p-STAT3, PIAS3 and ALR expression in neoplastic human tissues from CRC patients, grouping the data in accordance with the histological alterations (G1, G2 and G3) and metastasis presence. Western blot (WB) analysis of ALR was also determined in neoplastic and surrounding tissues. Finally, cell proliferation (Ki-67) and apoptosis (Bcl-2) were determined. Colon cancer tissue samples showed: (1) ALR and p-STAT3 strongly over-expression in 100% of G1 tissue samples, reducing in G2 and G3 tissue samples; (2) PIAS3 immunological determination was poorly expressed in G1 tissue samples and highly expressed in the 100% of colorectal tissues from group G2 and G3. Ki-67 progressively increases with the importance of the anatomic-pathological alterations and Bcl-2 resulted higher in G3 tissue samples compared to G1 neoplastic tissues. WB data evidenced, in neoplastic tissues, compared to the tumour-surrounding tissues, ALR over-expressed in G1 neoplastic tissues and down-expressed in G3 neoplastic tissues. Our data demonstrate a different dynamism of the investigated factors in relation to the severity of CRC histological findings. We hypothesize that the positive expression of ALR and p-STAT3 in the neoplastic tissue samples from CRC G1 group, associated to the absence of PIAS3, could be useful marker to identify an early stage of the disease. Based on these data and on our previous studies on gut microbiota in precancerous intestinal lesions, we are confident that, after microbial priming, a cascade of molecular events is started. So, the detectable molecules acting in these initial steps should be considered for the study of CRC progression and therapy.

Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease.

Parkinson’s disease is a neurodegenerative disorder characterized by the progressive death of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a loss of the neurotransmitter dopamine in the basal ganglia. Current treatments relieve the symptoms of the disease, but none stop or delay neuronal degeneration. Liver growth factor (LGF) is an albumin–bilirubin complex that stimulates axonal growth in the striatum and protects DA neurons in the SN of 6-hydroxydopamine-lesioned rats. Our previous results suggested that these effects observed in vivo are mediated by microglia and/or astrocytes. To determine if these cells are LGF targets, E14 (embryos from Sprague Dawley rats of 14 days) rat mesencephalic glial cultures were used. Treatment with 100 pg/mL of LGF up-regulated the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response element binding protein (CREB) phosphorylation in glial cultures, and it increased the microglia marker Iba1 and tumor necrosis factor alpha (TNF-alpha) protein levels. The treatment of E14 midbrain neurons with a glial-conditioned medium from LGF-treated glial cultures (GCM-LGF) prevented the loss of DA neurons caused by 6-hydroxy-dopamine. This neuroprotective effect was not observed when GCM-LGF was applied in the presence of a blocking antibody of TNF-alpha activity. Altogether, our findings strongly suggest the involvement of microglia and TNF-alpha in the neuroprotective action of LGF on DA neurons observed in vitro.

Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24h compared to HMEC spheroids. Supernatant fromLSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2days, which was maintained to 4days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2weeks. Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Taurocholate Induces Connective Tissue Growth Factor Expression in Hepatocytes Through ERK-YAP Signaling

Background/Aims: Cholestasis is characterized by intrahepatic accumulation of cytotoxic bile acids (BAs), ultimately leading to fibrosis and cirrhosis, but the precise role of BAs in cholestasis-induced liver fibrosis remains largely elusive. In this study, we investigated the role and the potential mechanisms of BAs during cholestasis in vivo and in vitro. Methods: The effect of BAs during cholestasis was studied in bile duct ligation (BDL) rat models in vivo. We performed immunohistochemistry, Western blotting, and quantitative RT-PCR to investigate the expression of connective tissue growth factor (CTGF/CCN2) in rat liver during cholestasis. The hepatic cell lines AML12 and BRL were stimulated with taurocholate (TC) and the level of CTGF/CCN2, and activation of ERK, Akt, p38 MAPK, JNK, YAP, and TGF-β/Smad signaling were examined using Western blotting. Next, to elucidate the mechanism underlying bile acid-induced CTGF/CCN2, we treated the cells with MEK1/2 inhibitor (U0126), YAP function inhibitor (verteporfin), p38 kinase inhibitor (SB203580), Akt inhibitor (MK2206), and small interfering RNA (siRNA) targeting mek1, erk, and yap in cooperation with TC. Besides, we confirmed the activation of these signaling pathways in BDL and sham rat livers by immunohistochemistry, Western blotting, and quantitative RT-PCR. Results: In this study, we confirmed that the expression of CTGF/CCN2 was increased in BDL-induced rodent cholestatic liver fibrosis. In addition, we showed that TC, the main component of BAs, enhanced the synthesis of CTGF/ CCN2 in AML12 and BRL hepatic cell lines. Moreover, we demonstrated that TC activated ERK, Akt, and YAP signaling in hepatocytes, but the precise roles of these signaling cascades in the expression of CTGF/CCN2 were different: TC-induced expression of CTGF/CCN2 was mediated by ERK-YAP signaling, whereas Akt signaling inhibited ERK signaling and YAP and subsequently the expression of CTGF/CCN2 in hepatocytes. Furthermore, YAP functioned as a downstream regulator of ERK signaling in TC-induced CTGF/CCN2 expression in hepatocytes. Conclusion: Our report provides evidence for the role of conjugated BAs in liver fibrosis and suggests that the production of CTGF/CCN2, induced by conjugated BAs via ERK-YAP axis activation, may be a therapeutic target in cholestasis-induced liver fibrosis.

Apoptotic Effects of Reduced Brain Derived Neurotrophic Factor (BDNF) on Mouse Liver and Kidney

Objective: Brain-derived neurotrophic factor (BDNF) promotes the development and differentiation of neurons and synapses, as well as neuronal survival, by acting on specific neuronal groups in the central and peripheral nervous systems. However, the direct effect of BDNF on apoptosis in peripheral tissues is not known. The aim of this study was to investigate the relationship between BDNF and apoptosis, and the density and distribution of BDNF receptors in liver and kidney tissues by histological and immunehistochemical methods. Methods: Seven wild-type and 7 BDNF heterozygous (reduced BDNF levels) male mice were used in the study. Caspase-3 and TUNEL immunehistochemical stainings were performed in order to investigate the presence of apoptosis in the liver and kidney tissues of the studied groups. Apoptosis-entering cells were counted and the groups were compared. Concentration and distribution of BDNF receptors, tropomyosin-related kinase B (TrkB) and nerve growth factor receptor p75 (NGFR p75), in liver and kidney tissues were also examined by immunehistochemical analyzes. Results: As a result of Caspase-3 and TUNEL immunehistochemical staining, more cells were counted to enter the apoptotic process in sections of BDNF heterozygous group compared to control group (p<0.0001). In both groups TrkB and NGFR p75 receptors in liver and kidney tissues were determined in trace amounts, but there was no difference in intensity and distribution between the studied groups. Conclusion: According to our histological and immunehistochemical stainings and statistical analysis of cell count between groups, it was found that BDNF is protective against apoptosis in liver and kidney. The lack of difference between the studied groups in terms of intensity and distribution of BDNF receptors, suggests that BDNF receptor distribution in the liver and kidney tissues may be different from the nervous system or that BDNF may differ in affinity for these receptors.

A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of the therapeutic effect of liver growth factor in emphysema

Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The current study has examined differences in metabolite profiles (fingerprints) of plasma from mice (strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months. One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was established, whereas the other group did not receive the treatment. Age and sex-matched mice not exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints (untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The signals were processed and a large number of possible metabolites were found (23944). Multivariate data analysis provided models that highlighted the differences between control and smoke exposed mice in both conditions. Accurate masses of features (possible compounds) representing significant differences were searched using online public databases. Lipid mediators, related to intracellular signaling in inflammation, were found among the metabolites putatively identified as markers of the different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids point at the relevance of such metabolites in the regulation of the processes related to tissue regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could potentially guide the characterization of relevant metabolites leading the regeneration of lungs in emphysema disease.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich’s ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 μg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Puerarin protects against CCl4-induced liver fibrosis in mice: possible role of PARP-1 inhibition

Liver fibrosis, which is the pathophysiologic process of the liver due to sustained wound healing in response to chronic liver injury, will eventually progress to cirrhosis. Puerarin, a bioactive isoflavone glucoside derived from the traditional Chinese medicine pueraria, has been reported to have many anti-inflammatory and anti-fibrosis properties. However, the detailed mechanisms are not well studied yet. This study aimed to investigate the effects of puerarin on liver function and fibrosis process in mice induced by CCl4. C57BL/6J mice were intraperitoneally injected with 10% CCl4 in olive oil(2mL/kg) with or without puerarin co-administration (100 and 200mg/kg intraperitoneally once daily) for four consecutive weeks. As indicated by the ameliorative serum hepatic enzymes and the reduced histopathologic abnormalities, the data collected showed that puerarin can protect against CCl4-induced chronic liver injury. Moreover, CCl4-induced development of fibrosis, as evidenced by increasing expression of alpha smooth muscle actin(α-SMA), collagen-1, transforming growth factor (TGF)-β and connective tissue growth factor(CTGF) in liver, were suppressed by puerarin. Possible mechanisms related to these suppressive effects were realized by inhibition on NF-κB signaling pathway, reactive oxygen species(ROS) production and mitochondrial dysfunction in vivo. In addition, these protective inhibition mentioned above were driven by down-regulation of PARP-1 due to puerarin because puerarin can attenuate the PARP-1 expression in CCl4-damaged liver and PJ34, a kind of PARP-1 inhibitor, mimicked puerarin's protection. In conclusion, puerarin played a protective role in CCl4-induced liver fibrosis probably through inhibition of PARP-1 and subsequent attenuation of NF-κB, ROS production and mitochondrial dysfunction.

Plumbagin Ameliorates CCl 4 -Induced Hepatic Fibrosis in Rats via the Epidermal Growth Factor Receptor Signaling Pathway.

Epidermal growth factor (EGF) and its signaling molecules, EGFreceptor (EGFR) and signal transducer and activator of transcription factor 3 (STAT3), have been considered to play a role in liver fibrosis and cirrhosis. Plumbagin (PL) is an extracted component from the plant and has been used to treat different kinds of cancer. However, its role in regulation of EGFR and STAT3 during liver fibrosis has not been investigated. In this study, the effects of PL on the regulation of EGFR and STAT3 were investigated in carbon tetrachloride (CCl4) induced liver fibrosis and hepatic stellate cells (HSC-T6). PL significantly attenuated liver injury and fibrosis in CCl4 treated rats. At concentrations of 2 to 6 μM, PL did not induce significant cytotoxicity of HSC-T6 cells. Moreover, PL reduced phosphorylation of EGFR and STAT3 in both fibrotic liver and heparin-binding EGF-like growth factor (HB-EGF) treated HSC-T6 cells. Furthermore, PL reduced the expression of α-SMA, EGFR, and STAT3 in both fibrotic liver and HB-EGF treated HSC-T6 cells. In conclusion, plumbagin could ameliorate the development of hepatic fibrosis through its downregulation of EGFR and STAT3 in the liver, especially in hepatic stellate cells.

Liver growth factor as a tissue regenerating factor in neurodegenerative diseases.

Liver growth factor (LGF) is a hepatic mitogen purified by our group in 1986. In the following years we demonstrated its activity both in “in vivo” and “in vitro” systems, stimulating hepatocytes mitogenesis as well as liver regeneration in several models of liver injury. Furthermore, we established its chemical composition (albumin-bilirubin complex) and its mitogenic actions in liver. From 2000 onwards we used LGF as a tissue regenerating factor in several models of extrahepatic diseases.The use of Liver growth factor as a neural tissue regenerator has been recently protected (Patent No US 2014/8,642,551 B2).LGF administration stimulates neurogenesis and neuron survival, promotes migration of newly generated neurons, and induces the outgrowth of striatal dopaminergic terminals in 6-hidroxydopamine-lesioned rats. Furthermore, LGF treatment raises striatal dopamine levels and protects dopaminergic neurons in hemiparkinsonian animals. LGF also stimulates survival of grafted foetal neural stem cells in the damaged striatum, reduces rotational behaviour and improves motor coordination. Interestingly, LGF also exerts a neuroprotective role both in an experimental model of cerebellar ataxia and in a model of Friedrich´s ataxia. Microglia seem to be the cellular target of LGF in the CNS. Moreover, the activity of the factor could be mediated by the stimulation of MAPK´s signalling pathway and by regulating critical proteins for cell survival, such as Bcl-2 and phospho-CREB. Since the factor shows neuroprotective and neurorestorative effects we propose LGF as a patented novel therapeutic tool that may be useful for the treatment of Parkinson´s disease and cerebellar ataxias. Currently, our studies have been extended to other neurological disorders such as Alzheimer’s disease (Patent No: US 2014/0113859 A1).

Proliferative activity of liver growth factor is associated with an improvement of cigarette smoke-induced emphysema in mice.

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression.

Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3β-hydroxysteroid dehydrogenase (3β-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3β-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery.

Corticosterone mediates the inhibitory effect of restraint stress on the migration of mesenchymal stem cell to carbon tetrachloride-induced fibrotic liver by downregulating CXCR4/7 expression.

Recent studies have revealed that mesenchymal stem cells (MSCs) have a great potential in therapeutic applications. The low efficiency of MSC recruitment and homing to sites of diseased organ tissue, however, remains a major hurdle in their application for treatment of diseases. Stress is commonly associated with various diseases. At the present time, little information is available about the effect of stress on MSC function. Here, we employed a carbon tetrachloride (CCl4)-induced mouse liver fibrosis model to investigate whether constraint stress affects the migration of MSCs to fibrotic liver. MSC homing to the fibrotic liver was significantly inhibited in mice with restraint stress. Restraint stress induced an elevation of corticosterone level in the serum. Blocking glucocorticoid signaling with either corticosterone-synthesis inhibitor metyrapone (MET) or glucocorticoid receptor antagonist RU486 attenuated restraint stress-induced inhibition of MSCs migration. The serum concentration of stromal cell-derived factor-1 (SDF-1) increased in mice treated with CCl4. Restraint stress had no influence on expression of SDF-1 and hepatocyte growth factor (HGF) in the fibrotic liver. Culture with the serum of CCl4-treated mice or SDF-1 promoted MSC migration, which was suppressed by corticosterone. Exposure of MSCs to corticosterone decreased their expression of C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine receptor type 7 (CXCR7). These results demonstrate that the inhibitory effect of corticosterone on MSC migration might be mediated via decreasing the expression of CXCR4 and CXCR7 in MSCs. Interventions targeting the interaction between corticosterone and its receptor improve migration and homing of MSCs in hosts receiving transplantation of these cells.

Neuroprotective role of liver growth factor "LGF" in an experimental model of cerebellar ataxia.

Cerebellar ataxias (CA) comprise a heterogeneous group of neurodegenerative diseases characterized by a lack of motor coordination. They are caused by disturbances in the cerebellum and its associated circuitries, so the major therapeutic goal is to correct cerebellar dysfunction. Neurotrophic factors enhance the survival and differentiation of selected types of neurons. Liver growth factor (LGF) is a hepatic mitogen that shows biological activity in neuroregenerative therapies. We investigate the potential therapeutic activity of LGF in the 3-acetylpiridine (3-AP) rat model of CA. This model of CA consists in the lesion of the inferior olive-induced by 3-AP (40 mg/kg). Ataxic rats were treated with 5 µg/rat LGF or vehicle during 3 weeks, analyzing: (a) motor coordination by using the rota-rod test; and (b) the immunohistochemical and biochemical evolution of several parameters related with the olivo-cerebellar function. Motor coordination improved in 3-AP-lesioned rats that received LGF treatment. LGF up-regulated NeuN and Bcl-2 protein levels in the brainstem, and increased calbindin expression and the number of neurons receiving calbindin-positive projections in the cerebellum. LGF also reduced extracellular glutamate and GABA concentrations and microglia activation in the cerebellum. In view of these results, we propose LGF as a potential therapeutic agent in cerebellar ataxias.

Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease largely associated with cigarette smoke exposure (CSE) and characterized by pulmonary and extrapulmonary manifestations, including systemic inflammation. Liver growth factor (LGF) is an albumin-bilirubin complex with demonstrated antifibrotic, antioxidant, and antihypertensive actions even at extrahepatic sites. We aimed to determine whether short LGF treatment (1.7 μg/mouse ip; 2 times, 2 wk), once the lung damage was established through the chronic CSE, contributes to improvement of the regeneration of damaged lung tissue, reducing systemic inflammation. We studied AKR/J mice, divided into three groups: control (air-exposed), CSE (chronic CSE), and CSE + LGF (LGF-treated CSE mice). We assessed pulmonary function, morphometric data, and levels of various systemic inflammatory markers to test the LGF regenerative capacity in this system. Our results revealed that the lungs of the CSE animals showed pulmonary emphysema and inflammation, characterized by increased lung compliance, enlargement of alveolar airspaces, systemic inflammation (circulating leukocytes and serum TNF-α level), and in vivo lung matrix metalloproteinase activity. LGF treatment was able to reverse all these parameters, decreasing total cell count in bronchoalveolar lavage fluid and T-lymphocyte infiltration in peripheral blood observed in emphysematous mice and reversing the decrease in monocytes observed in chronic CSE mice, and tends to reduce the neutrophil population and serum TNF-α level. In conclusion, LGF treatment normalizes the physiological and morphological parameters and levels of various systemic inflammatory biomarkers in a chronic CSE AKR/J model, which may have important pathophysiological and therapeutic implications for subjects with stable COPD.