Homoeostatic regulation of cytokines to retard liver fibrosis 4

CHAPTER   94   5. RECOMMENDATIONS FOR FUTURE RESEARCH 5.1. ROLE OF TGF-β CO-REPRESSOR SnoN DURING THE HGFDEPENDENT INHIBITION OF TGF-β1 ACTIVATION As seen in Chapters and 2, TGF−β signaling plays a very important role in the activation of HSC and fibrosis progression. Strategies to target and antagonize TGF−β signaling have been researched upon extensively leading to the knowledge of activators of the signaling at the transcriptional/protein level and repressors of the signaling at the transcriptional level. Signaling by TGF−β is initiated by the binding of TGF−β to its heteromeric complex of type I & II cell surface receptors. Each receptor is a transmembrane protein that possesses a cytoplasmic serine-threonine kinase domain. Binding of TGF−β to the ectodomain of the type II receptor induces hetero-oligomerization between the type II and the type I receptors. Once the two receptor subunits are in close proximity, the constitutively active type II receptor kinase phosphorylates the type I receptor kinase (174,175). Once activated, the type I TGF−β receptor kinase phosphorylates a Smad2 or Smad3 protein, resulting in the oligomerization of Smad2 or -3 with their common partner, Smad4. The resulting heteromeric Smad protein complexes then migrate to the nucleus, where they regulate expression of a large number of target genes (176). Several nuclear proteins have been found to interact with Smad proteins. Such interactions either influence Smad function directly or, alternatively, affect the functions of its binding partners. One such nuclear protein that binds to Smad is the Ski-related protein SnoN, which when over-expressed decreases TGF−β signaling and in some cases renders the cells unresponsive to TGF−β stimulation (177). Since HGF and plasmin have been   95   demonstrated to induce SnoN expression in contexts of kidney (178,179) and liver fibrosis (126) respectively, we investigated whether SnoN expression was modulated in our HGF-treated HSC-T6 monocultures and HGF-treated fibrotic rats. Interestingly, we observed an up-regulation in HGF-treated HSC-T6 monoculture when compared to HGF-treated hepatocyte monoculture (Fig. 39) or the untreated HSC-T6 monoculture demonstrating a HGF/HGF-induced plasmin- SnoN expression relative to control (normalized to β-actin) based selective up-regulation of SnoN in ‘high TGF-β1’ contexts. 10 Untreated control cultures * HGF-treated cultures CTRL Hepatocytes HSC-T6 Figure 39: SnoN gene expression in HGF-treated liver cells in vitro as measured by RT-PCR.Freshly isolated primary rat hepatocytes or HSC-T6 cells were cultured overnight and treated with 40ng/ml HGF protein for 48 hrs and the gene expression of SnoN was evaluated by RT-PCR. * p-value < 0.05 compared to untreated control cultures of HSC-T6 cells. In DMN-induced fibrotic rats administered with Vitamin A-Liposome-HGF (VALH), we observed a large increase in SnoN expression comparable to the levels of naïve control rats (Fig. 40) demonstrating the HGF-based up-regulation of SnoN even in animal models of fibrosis. Additionally, the HSC-targeted delivery appeared to have higher levels of SnoN up-regulation compared to the   96   untargeted Liposome-HGF (Lip-HGF) therapy reiterating the importance of targeting the fibrotic foci within the diseased liver. Sno Gene expression levels in tissue homogenates (Normalized to GAPDH) CTRL DMN Lip-HGF VALH -1 -2 -3 Figure 40: SnoN expression in fibrotic rats treated with VALH particles. Gene expression of SnoN normalized to GAPDH gene in DMN-induced fibrotic rats treated with untargeted and targeted liposome-DNA complexes as measured by RT-PCR. 5.2. REGRESSION OF LIVER FIBROSIS IN TAA-INDUCED FIBROTIC RATS AFTER RETROGRADE INTRABILIARY INFUSION OF VALH PARTICLES Since our earlier treatment of DMN-induced fibrotic rats were administered 200 µg dose of therapeutic, we further investigated whether lower dose of therapeutics can also induce fibrosis regression. In order to demonstrate a more broad-based   97   application of our therapeutic in other models of liver injury we tested the VALH particles on thioacetamide (TAA)-induced fibrotic rat model. Fibrotic rats were establishment with intraperitoneal administration of 200µg/kg B.W. of thioacetamide administration consecutive days per week for 12 weeks. After 12 weeks of TAA administration and week of no injections, the rats were administered a single dose of VALH particles containing 50 µg of plasmid DNA through retrograde intrabiliary infusion as described in Chapter 3. Liver tissue samples were collected and 14 days after treatment. We observed a decline in the nodular appearance of the liver (Fig. 41) and a decline in the fibrotic index as measured form the Masson Trichrome and H&E stained images (Fig. 42). Figure 41: Appearance of rat livers after treatment. Naïve control rats (A), TAA-induced fibrotic rats (B) and VALH-treated fibrotic rats (C) after 14 days   98     LH 14 L D D LH VA VA + + A A TA TA TR C Fibrosis score 99   Figure 42: Liver histopathology and fibrosis score. Masson Trichrome and H&E stained liver tissue sections, naïve control (A), TAA only (B) and VALH-treated days (C) and 14 days after treatment (D). Scale bar: 100 µm. Decline in fibrosis score of VALH-treated fibrotic rats by day 14 (E). We also observed a decline in the fibrotic markers, α-SMA and TGF-β Receptor I (see materials & methods in Chapter 3) in the liver tissue homogenates 14 days after VALH treatment (Fig. 43). (55 kDa) (42 kDa) (47 kDa) Figure 43: Decreased α-SMA and TGF-β Receptor I expression 14 days after VALH treatment. VALH-treated TAA-induced fibrotic rats were tested for TGF-b1 and a-SMA protein levels by western blot. Benchmark protein ladder (Invitrogen) used as molecular marker. Experiments evaluating the distribution of the delivered transgene in the fibrotic liver are ongoing to test their specific localization in the HSC-rich fibrotic foci and the control of fibrosis expansion. 5.3. ALTERNATIVE SOURCE OF HEPATOCYTES FOR TRANSPLANTATION PURPOSES IN END-STAGE LIVER DISEASES 5.3.1. iPSC-Derived Hepatocytes   100   Liver cell transplantation (LCT), an experimental procedure designed to reconstitute the liver mass with functional hepatocytes, is based on transplantation of isolated hepatocytes from a cadaver or from a liver portion from a living donor (180). This experimental procedure has been successfully used in patients to correct certain metabolic disorders (180). Transplantation of fetal hepatocytes has also been considered as an alternative treatment (181). Liver cell transplantation (LCT) has been attempted in patients with acute liver failure, chronic liver disease with endstage cirrhosis, and children with metabolic disease (180). Since metabolic deficiencies are often associated with the severe damage to hepatocytes, transplanted hepatocytes have a growth advantage over recipient hepatocytes. Under these conditions, donor hepatocytes have a selective pressure and LCT has been reported in patients to correct ornithine trans-carbamylase deficiency, α-1-anti-trypsin deficiency, glycogen storage disease type Ia, infantile Refsum’s disease, factor VII deficiency, bile salt export protein deficiency, and Crigler-Najjar syndrome type (180,182). Immunological rejection of hepatocytes requires prolonged immunosuppressive therapy in patients suitable for LCT (183). Identification and characterization of hepatocyte progenitor/stem cells and their differentiation into functionally mature liver cells is an evolving goal for the stem cell-based therapy for liver diseases. In recent times, there has been a lot of interest in the development of induced pluripotent stem cells from adult human somatic cells by the forced expression of genes otherwise known as Yamanaka factors i.e., Oct3/4, Sox2, c-Myc and Klf-4 (184). Further research showed that iPS can be generated even without the oncogene c-Myc thus reducing the risk of tumor development (185). In our study, we differentiated iPSF4 (WiCell; Fig. 44) into hepatocytes employing a modified directed differentiation protocol adapted from Roelandt et al. (186).   101   Figure 44: Undifferentiated feeder-free iPSF4 colonies. iPSF4 cell colonies maintained in mTESR1 medium showing undifferentiated morphology under phase contrast microscope (10x magnification). We cultured the iPS cells in matrigel-coated dishes in APEL medium and treated them with different cytokines for different time periods as described in Fig. 45. Oncostatin-M Figure 45: Schematic representation of the protocol for directed differentiation of pluripotent stem cells into mature hepatocytes. Protocol adapted from (182). Initial phase of differentiation until day 10 is to induce the pluripotent stem cells into the definitive endoderm lineage by inducing the Activin/Wnt signaling pathway. The   102   canonical Wnt signaling involves Wnt proteins and their interactions with cell-surface receptors of the Frizzled family on target cells and further signaling that regulates the amount of B-catenin that enters the nucleus in turn regulates physiological responses such as cell growth and morphogensis (187). As seen in Fig. 46, 10 days from start of differentiation there was a strong increase in the marker for endoderm, Foxa2 as compared to the ectoderm or mesoderm markers Pax6 and Brachury/T respectively in feeder-free cultures. A   103   Fold change over day control (Normalized to GAPDH) B Day 100 Day 10 80 60 40 20 10 -5 -10 Oct3/4 Foxa2 T Pax6 Figure 46: Definitive endoderm induction of hiPSCs. Morphology of iPSF4 cell colonies in feeder and feeder-free configurations (A; 4x magnification). At day and day 10, the feeder-free cultures show a gradual progression in definite endoderm marker Foxa2 The next phase of hepatic induction leads to highly specific epithelial morphology similar to mature hepatocytes (Fig. 47A) and significant increases in the hepatocytespecific genes such as albumin, AAT, HNF4A and CYP3A4 (Fig. 47B). A   Feeder culture Feeder-free culture 104   B Figure 47: Expression of hepatocyte-like markers in differentiated hiPSCs by day 20. Photomicrographs showing hepatocyte-like morphology of differentiated hiPSCs (A; upper panel: 4x magnification, lower panel: 40x magnification). Increased expression of hepatocyte specific genes as assessed from RT-PCR measurements (B). We also observed a significant increase in the expression of hepatic markers albumin and MRP-2 (Fig. 48) and increase in albumin secretion (140.57 ± 7.33ng/million cells) in the hepatocyte-like cells derived from hiPSCs. Figure 48: Expression of mature hepatic markers by the hepatocyte-like cells derived from hiPSCs. Albumin (green) and MRP-2 (red). 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