Houda Darwiche

Hepatic stellate cells’ involvement in progenitor mediated liver regeneration

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-β (TGFβ)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and α-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

The role of the Wnt family of secreted proteins in rat oval "stem" cell-based liver regeneration: Wnt1 drives differentiation.

To date the molecular signals regulating activation, proliferation, and differentiation of hepatic oval cells are not fully understood. The Wnt family is essential in hepatic embryogenesis and implicated in hepatic carcinogenesis. This study elucidates novel findings implicating Wnt1 in directing oval cell differentiation during the rat 2-acetylaminofluorene (2AAF) and 2/3 partial hepatectomy (PHx) liver regeneration model. Proteins of Wnt family members were predominantly localized in pericentral hepatocytes during liver injury, oval cell activation, and hepatocyte regeneration. In addition, Wnt message increased coinciding with the rise in oval cell number, whereas protein levels peaked immediately after the height of oval cell proliferation. Immunohistochemical analysis demonstrated nuclear translocation of beta-catenin within oval cells throughout the 2AAF/PHx protocol. Furthermore, RNA interference was used in vivo to confirm the physiological requirement of Wnt1 during the oval cell induction. Ultimately, inhibition of Wnt1 resulted in failure of oval cells to differentiate into hepatocytes and alternatively induced atypical ductular hyperplasia. Taken together, these data indicate that in vivo exposure to Wnt1 shRNA inhibited rat oval cell liver regeneration. In the absence of Wnt1 signaling, oval cells failed to differentiate into hepatocytes and underwent atypical ductular hyperplasia, exhibiting epithelial metaplasia and mucin production. Furthermore, changes in Wnt1 levels are required for the efficient regeneration of the liver by oval cells during massive hepatic injury.

Inhibition of Notch signaling affects hepatic oval cell response in rat model of 2AAF-PH

Background and aims Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. Methods The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. Results Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21Waf1/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. Conclusion Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional repair of the liver by oval cells.

Biology of the Adult Hepatic Progenitor Cell: “Ghosts in the Machine”

This chapter reviews some of the basic biological principles governing adult progenitor cells of the liver and the mechanisms by which they operate. If scientists were better able to understand the conditions that govern stem cell mechanics in the liver, it may be possible to apply that understanding in a clinical setting for use in the treatment or cure of human pathologies. This chapter gives a basic introduction to hepatic progenitor cell biology and explores what is known about progenitor cell-mediated liver regeneration. We also discuss the putative stem cell niche in the liver, as well as the signaling pathways involved in stem cell regulation. Finally, the isolation and clinical application of stem cells to human diseases is reviewed, along with the current thoughts on the relationship between stem cells and cancer.

Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

Oval cell-mediated liver regeneration is a highly complex process that involves the coordination of several signaling factors, chemokines and cytokines to allow for proper maintenance of the liver architecture. When hepatocyte proliferation is inhibited, an hepatic stem cell population, often referred to as “oval cells”, is activated to aid in liver regeneration. The function of insulin-like growth factor binding protein-3 (IGFBP-3) during this process of oval cell activation is of particular interest because it is produced in liver and has been shown to induce migration and differentiation of other stem cell populations both in vitro and in vivo. Additionally, IGFBP-3 production has been linked to the transforming growth factor-β (TGF-β) superfamily, a pathway known to be induced during oval cell proliferation. In this study, we set out to determine whether IGFBP-3 plays a role in oval cell proliferation, migration and differentiation during this specific type of regeneration. Through activation of the oval cell-mediated liver regeneration in a rat model, we found that IGFBP-3 is elevated in the liver and serum of animals during peak days of oval cell activation and proliferation. Furthermore, in vitro assays found that WB-344 cells, a liver stem cell line similar to oval cells, were induced to migrate in the presence of IGFBP-3. When expression of IGFBP-3 was knocked down during oval cell activation in vivo, we found that oval cell proliferation was increased and observed the appearance of numerous atypical ductular structures, which were OV-6 and Ki67-positive. Finally, quantitative realtime PCR analysis of liver tissue from IGFBP-3 small interfering RNA (siRNA) treated animals determined that expression of TGFβ family members, including TGF-βRII and Smads 2–4, were significantly downregulated compared to animals at day 9 post-PHx alone or animals that received negative control siRNA. In conclusion, IGFBP-3 may function as a potent chemoattractant of oval cells during specific types of liver regeneration and may be involved in regulating oval cell proliferation and differentiation in vivo via the TGF-β pathway.

Characterization of a novel functional protein in the pancreatic islet: islet homeostasis protein regulation of glucagon synthesis in α cells.

Objective We have identified a novel protein in bone marrow–derived insulin-producing cells. Here we characterize this protein, hereby named islet homeostasis protein (IHoP), in the pancreatic islet. Methods Detection of IHoP mRNA and protein was performed using reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in situ hybridization. Islet homeostasis protein functions were utilizing proliferation, insulin secretion by in vitro assays, and following small interfering RNA protocols for suppression of IHoP. Results We found that IHoP did not homolog with known pancreatic hormones. Islet homeostasis protein expression was seen in both bone marrow–derived insulin-producing cells and isolated pancreatic islets. Immunohistochemistry on pancreatic islet revealed that IHoP localized to the glucagon-synthesizing &agr; cells. Inhibition of IHoP by small interfering RNA resulted in the loss of glucagon expression, which induced low blood glucose levels (63–85 mg/dL). Subsequently, cellular apoptosis was observed throughout the islet, including the insulin-producing &bgr; cells. Islets of preonset diabetic patients showed normal expression of IHoP and glucagon; however, IHoP was lost upon onset of the disease. Conclusions These data suggest that IHoP could be a new functional protein in the islet and may play a role in islet homeostasis. Abbreviations IHoP - islet homeostasis protein, BM - bone marrow, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Ebola Epidemic: Using Current Events to Teach Authentic Inquiry Science

Abstract First reported in March 2014, the Ebola virus disease (EVD) outbreak in West Africa has now claimed more lives than all other known EVD outbreaks combined, making it the deadliest occurrence of the disease since it was first discovered nearly 40 years ago. In hopes of turning the outbreak into something positive from an educational standpoint, a module was developed focusing on EVD, infectious disease, and epidemiology. The module engages students in a series of inquiry-based lessons, providing accurate and up-todate information on the current outbreak of EVD in West Africa. The lessons also serve to correct popular misconceptions about the disease. The lessons include a jigsaw WebQuest using resources from the Centers for Disease Control and Prevention, a simulation based on fluid exchange to model the spread of an outbreak of infectious disease, and a “disease detective”—style mapping activity based on published data outlining the start of the current EVD outbreak in Guinea.

Using a Simulation to Illustrate Crosscutting Concepts Through a Disease Model

Abstract Using Pompe disease as a context affords the opportunity for students to consider multiple biological concepts and embraces the Next Generation Science Standards Disciplinary Core Ideas Structure and Function (LS1.A) and Inheritance of Traits (LS3.A) as well as Crosscutting Concepts Structure and Function and Cause and Effect. These crosscutting concepts are very much interrelated as we consider progression of disease from the molecular to the organismal level. The concepts are repeatedly emphasized, providing “explicit instructional support” for students to “develop a cumulative, coherent, and usable understanding of science and engineering.” DNA, proteins, enzymes, genetics, and human disease are taught together through the story of patients with Pompe disease as students engage in a simulated clinical assay and genetic analysis and present their findings in grand rounds. The activity is one of multiple lessons sequenced to scaffold student understanding of clinical and translational science, starting with a first-person perspective of a father who loses his infant son to Pompe and concluding with a role play based on actual events surrounding approval of human clinical trials of gene therapy for Pompe disease.