Balakrishnan Chakrapani Narmada

Cell-delivery therapeutics for liver regeneration☆

For acute, chronic, or hereditary diseases of the liver, cell transplantation therapies can stimulate liver regeneration or serve as a bridge until liver transplantation can be performed. Recently, fetal hepatocytes, stem cells, liver progenitor cells, or other primitive and proliferative cell types have been employed for cell transplantation therapies, in an effort to improve the survival, proliferation, and engraftment of the transplanted cells. Reviewing earlier studies, which achieved success by transplanting mature hepatocytes, we propose that there is a switch-like regulation of liver regeneration that changes state according to a stimulus threshold of extracellular influences such as cytokines, matrices and neighboring cells. Important determinants of a successful clinical outcome include sufficient quantities and functional levels of the transplanted cells (even for short periods to alter the environment), rather than just engraftment levels or survival durations of the exogenously transplanted cells. The relative importance of these determining factors will impact future choices of cell sources, delivery vehicles, and sites of cell transplantation to stimulate liver regeneration for patients with severe liver diseases.

HGF regulates the activation of TGF-β1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor‐beta 1 (TGF‐β1). Specifically, we tested whether HGF decreases the levels of active TGF‐β1, and whether such decrease depends on the predominantly hepatocyte‐secreted protease plasmin, and whether it depends on the TGF‐β1 activator thrombospondin‐1 (TSP‐1). With hepatocyte monocultures, we found HGF‐induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI‐1, TGF‐β1, and TIMP‐2). With in vitro models of fibrotic liver (HSC‐T6 hepatic stellate cells, or co‐cultures of HSC‐T6 and hepatocytes), we found high levels of fibrosis‐associated proteins such as TSP‐1, active TGF‐β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP‐1 protein cleavage; and decreased the levels of active TGF‐β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF‐β1 activation and Collagen I. Meanwhile, the TSP‐1‐specific peptide inhibitor, LSKL, reduced TGF‐β1 to the same level as in the HGF‐treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP‐1 dependent pathway of TGF‐β1 activation. Therefore, HGF can decrease TGF‐β1 activation and TGF‐β1‐dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP‐1‐dependent activation of TGF‐β1. J. Cell. Physiol. 228: 393–401, 2013.

Hepatic Stellate Cell–Targeted Delivery of Hepatocyte Growth Factor Transgene via Bile Duct Infusion Enhances Its Expression at Fibrotic Foci to Regress Dimethylnitrosamine-Induced Liver Fibrosis

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites From Nutraceuticals

Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small‐molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell‐derived endothelial cells (hPSC‐ECs) and human pluripotent stem cell‐derived hepatocytes (hPSC‐HEPs) for predicting vascular‐protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti‐inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin‐1β owing to limited metabolic activity of hPSC‐ECs. Conversely, hPSC‐HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC‐HEPs were cocultured with hPSC‐ECs to permit paracrine interactions, the continuous turnover of metabolites mitigated interleukin‐1β stimulation on hPSC‐ECs. We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor κB, and interleukin‐8 production. Thus, integration of hPSC‐HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial‐hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular‐targeting candidate compounds. Stem Cells Translational Medicine 2017;6:851–863

Computational analysis reveals the coupling between bistability and the sign of a feedback loop in a TGF-β1 activation model

BackgroundBistable behaviors are prevalent in cell signaling and can be modeled by ordinary differential equations (ODEs) with kinetic parameters. A bistable switch has recently been found to regulate the activation of transforming growth factor-β1 (TGF-β1) in the context of liver fibrosis, and an ordinary differential equation (ODE) model was published showing that the net activation of TGF-β1 depends on the balance between two antagonistic sub-pathways.ResultsThrough modeling the effects of perturbations that affect both sub-pathways, we revealed that bistability is coupled with the signs of feedback loops in the model. We extended the model to include calcium and Krüppel-like factor 2 (KLF2), both regulators of Thrombospondin-1 (TSP1) and Plasmin (PLS). Increased levels of extracellular calcium, which alters the TSP1-PLS balance, would cause high levels of TGF-β1, resembling a fibrotic state. KLF2, which suppresses production of TSP1 and plasminogen activator inhibitor-1 (PAI1), would eradicate bistability and preclude the fibrotic steady-state. Finally, the loop PLS − TGF-β1 − PAI1 had previously been reported as negative feedback, but the model suggested a stronger indirect effect of PLS down-regulating PAI1 to produce positive (double-negative) feedback in a fibrotic state. Further simulations showed that activation of KLF2 was able to restore negative feedback in the PLS − TGF-β1 − PAI1 loop.ConclusionsUsing the TGF-β1 activation model as a case study, we showed that external factors such as calcium or KLF2 can induce or eradicate bistability, accompanied by a switch in the sign of a feedback loop (PLS − TGF-β1 − PAI1) in the model. The coupling between bistability and positive/negative feedback suggests an alternative way of characterizing a dynamical system and its biological implications.