Petra E. de Ruiter

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.

Hepatocyte‐derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation

Recent animal and human studies have highlighted the potential of hepatocyte‐derived microRNAs (HDmiRs) in serum as early, stable, sensitive, and specific biomarkers of liver injury. Their usefulness in human liver transplantation, however, has not been addressed. The aim of this study was to investigate serum HDmiRs as markers of hepatic injury and rejection in liver transplantation. Serum samples from healthy controls and liver transplant recipients (n = 107) and peritransplant liver allograft biopsy samples (n = 45) were analyzed via the real‐time polymerase chain reaction quantification of HDmiRs (miR‐122, miR‐148a, and miR‐194). The expression of miR‐122 and miR‐148a in liver tissue was significantly reduced with prolonged graft warm ischemia times. Conversely, the serum levels of these HDmiRs were elevated in patients with liver injury and positively correlated with aminotransferase levels. HDmiRs appear to be very sensitive because patients with normal aminotransferase values (<50 IU/L) had 6‐ to 17‐fold higher HDmiR levels in comparison with healthy controls (P < 0.005). During an episode of acute rejection, serum HDmiRs were elevated up to 20‐fold, and their levels appeared to rise earlier than aminotransferase levels. HDmiRs in serum were stable during repeated freezing and thawing. In conclusion, this study shows that liver injury is associated with the release of HDmiRs into the circulation. HDmiRs are promising candidates as early, stable, and sensitive biomarkers of rejection and hepatic injury after liver transplantation. Liver Transpl 18:290–297, 2012.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Background/aims RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. Methods Transmission of RNAi was investigated in culture by assessing silencing of HCV replication and expression of viral entry receptor CD81 using a human hepatic cell line and primary B lymphocytes transduced with siRNA-expressing vectors. In vivo transmission between hepatic cells was investigated in NOD/SCID mice. Involvement of exosomes was demonstrated by purification, uptake and mass spectrometric analysis. Results Human and mouse liver cells, as well as primary human B cells, were found to have the ability to exchange small RNAs, including cellular endogenous microRNA and delivered siRNA targeting HCV or CD81. The transmission of RNAi was largely independent of cell contact and partially mediated by exosomes. Evidence of RNAi transmission in vivo was observed in NOD/SCID mice engrafted with human hepatoma cells producing CD81 siRNA, causing suppression of CD81 expression in mouse hepatocytes. Conclusion Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases.

Secreted Factors of Human Liver-Derived Mesenchymal Stem Cells Promote Liver Regeneration Early After Partial Hepatectomy

Rapid liver regeneration is required after living-donor liver transplantation and oncologic liver resections to warrant sufficient liver function and prevent small-for-size syndrome. Recent evidence highlights the therapeutic potential of mesenchymal stem cells (MSC) for treatment of toxic liver injury, but whether MSC and their secreted factors stimulate liver regeneration after surgical injury remains unknown. Therefore, the aim of this study is to investigate the effect of human liver-derived MSC-secreted factors in an experimental liver resection model. C57BL/6 mice were subjected to a 70% partial hepatectomy and treated with either concentrated MSC-conditioned culture medium (MSC-CM) or vehicle control. Animals were analyzed for liver and body weight, hepatocyte proliferation, and hepatic gene expression. Effects of MSC-CM on gene expression in a human hepatocyte-like cell line (Huh7 cells) were analyzed using genome-wide gene expression arrays. Liver regeneration was significantly stimulated by MSC-CM as shown by an increase in liver to body weight ratio and hepatocyte proliferation. MSC-CM upregulated hepatic gene expression of cytokines and growth factors relevant for cell proliferation, angiogenesis, and anti-inflammatory responses. In vitro, treatment of Huh7 cells with MSC-CM significantly altered expression levels of ~3,000 genes. Functional analysis revealed strong effects on networks associated with protein synthesis, cell survival, and cell proliferation. This study shows that treatment with MSC-derived factors can promote hepatocyte proliferation and regenerative responses in the early phase after surgical resection. MSC-CM may represent a feasible new strategy to promote liver regeneration in patients undergoing extensive liver resection or after transplantation of small liver grafts.

Mycophenolic acid augments interferon‐stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo

Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon‐α (IFN‐α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN‐α. The role of IMPDH and interferon‐stimulated genes (ISGs) was investigated in two HCV models using gain‐ or loss‐of‐function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1‐derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA‐resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN‐α resulted in additive effects on ISG expression and enhanced IFN‐induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. Conclusion: MPA exerts a potent anti‐HCV effect in vitro and in mice and acts in synergy with IFN‐α. MPAs antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN‐α. (HEPATOLOGY 2012;55:1673–1683)

Phosphorylation of androgen receptor isoforms

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.

Detection of spontaneous tumorigenic transformation during culture expansion of human mesenchymal stromal cells

Human mesenchymal stem/stromal cells (MSCs) have been explored in a number of clinical trials as a possible method of treating various diseases. However, the effect of long-term cell expansion in vitro on physiological function and genetic stability is still poorly understood. In this study, MSC cultures derived from bone marrow and liver were evaluated for the presence of aberrant cells following long-term expansion. In 46 independent cultures, four batches of transformed MSCs (TMCs) were found, which were all beyond the culture period of five weeks. These aberrant cells were first identified based on the appearance of abnormal cytology and the acquirement of growth advantage. Despite common MSC markers being diminished or absent, TMCs remain highly susceptible to lysis by allogenic natural killer (NK) cells. When transplanted into immunodeficient mice, TMCs formed sarcoma-like tumors, whereas parental MSCs did not form tumors in mice. Using a combination of high-resolution genome-wide DNA array and short-tandem repeat profiling, we confirmed the origin of TMCs and excluded the possibility of human cell line contamination. Additional genomic duplication and deletions were observed in TMCs, which may be associated with the transformation event. Using gene and microRNA expression arrays, a number of genes were identified that were differentially expressed between TMCs and their normal parental counterparts, which may potentially serve as biomarkers to screen cultures for evidence of early transformation events. In conclusion, the spontaneous transformation of MSCs resulting in tumorigenesis is rare and occurs after relatively long-term (beyond five weeks) culture. However, as an added safety measure, cultures of MSCs can potentially be screened based on a novel gene expression signature.

Synthesis and post-translational modification of the androgen receptor in LNCaP cells

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting with a specific polyclonal antibody showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Most of the receptor protein is present in the higher molecular mass form. Pulse labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Alkaline phosphatase treatment of cytosols from [35S]methionine pulse labelled cells caused a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform. This indicates that the observed 110 to 112 kDa upshift of the newly synthesized androgen receptor reflects receptor phosphorylation. Both isoforms can bind hormone and can undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.

Progesterone-Induced Inhibition of Growth and Differential Regulation of Gene Expression in PRA- and/or PRB-Expressing Endometrial Cancer Cell Lines:

Objective: Progesterone plays an important role in controlling proliferation and diferentiation of the human endometrium. Because there are two progesterone receptor isoforms (PRA and PRB), it was important to generate tools to be able to study the role of these two progesterone receptors separately. Methods: Using stable transfection techniques, both human progesterone receptor isoforms (hPRA and hPRB) were reintroduced into a hPR-negative subclone of the well-differentiated endometrial cancer cell line Ishikawa. Several Ishikawa subcell lines were constructed, each expressing different levels of hPRA, hPRB, or hPRA and hPRB, respectively. Results: These Ishikawa subcell lines showed a marked progesterone-induced growth inhibition with induction of apoptosis after long-term culture in the presence of hormone. Upon measuring gene regulation, a clear diference in regulation of expression of the selected genes by progesterone treatment was observed between the PRA-, PRB-, or PRA/B-expressing cell lines. Integrin β34 (ITGB4) was only regulated in PRA-expressing cells; amphiregulin was highly regulated in PRB-expressing cells; inuslin-like gwoth factor binding protein 3 (IGFBP3) was only regulated in PRBand PRA /B-expressing cells; and metallothionein 1L (MT1L) was highly regulated in PRA/B-expressing cells. Interestingly, based on literature data, these genes can be implicated in induction of apoptosis, but are modulated here in such a way that suggests induction of resistance against apoptosis. Conclusion: Reintroduction of PRs into Ishikawa cells rescued progesterone responsiveness in these cells. Furthermore, using these human endometrial cancer subcell lines, clear and distinct functional diferences between the PR isoforms were observed.

MicroRNA profiles in graft preservation solution are predictive of ischemic-type biliary lesions after liver transplantation

BACKGROUND & AIMS Ischemic-type biliary lesions (ITBL) are the second most common cause of graft loss after liver transplantation. Though the exact pathophysiology of ITBL is unknown, bile duct injury during graft preservation is considered to be a major cause. Here we investigated whether the release of cholangiocyte-derived microRNAs (CDmiRs) during graft preservation is predictive of the development of ITBL after liver transplantation. METHODS Graft preservation solutions (perfusates) and paired liver biopsies collected at the end of cold ischemia were analysed by RT-qPCR for CDmiR-30e, CDmiR-222, and CDmiR-296 and hepatocyte-derived miRNAs (HDmiRs) HDmiR-122 and HDmiR-148a. MicroRNAs in perfusates were evaluated on their stability by incubation and fractionation experiments. MicroRNA profiles in perfusates from grafts that developed ITBL (n=20) and grafts without biliary strictures (n=37) were compared. RESULTS MicroRNAs in perfusates were proven to be stable and protected against degradation by interacting proteins. Ratios between HDmiRs/CDmiRs were significantly higher in perfusates obtained from grafts that developed ITBL (p<0.01) and were identified as an independent risk factor by multivariate analysis (p<0.01, HR: 6.89). The discriminative power of HDmiRs/CDmiRs in perfusates was validated by analysis of separate brain death- (DBD) and cardiac death donors (DBD; p ≤ 0.016) and was superior to expression in liver biopsies (C=0.77 in perfusates vs. C<0.50 in biopsies). CONCLUSIONS This study demonstrates that differential release of CDmiRs during graft preservation is predictive of the development of ITBL after liver transplantation. This provides new evidence for the link between graft-related bile duct injury and the risk for later development of ITBL.