In vitro models for liver toxicity testing

Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently the most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system.National Institutes of Health (U.S.) (P42 ES005948)National Institutes of Health (U.S.) (R01 ES01524

The Prometastatic Microenvironment of the Liver

The liver is a major metastasis-susceptible site and majority of patients with hepatic metastasis die from the disease in the absence of efficient treatments. The intrahepatic circulation and microvascular arrest of cancer cells trigger a local inflammatory reaction leading to cancer cell apoptosis and cytotoxicity via oxidative stress mediators (mainly nitric oxide and hydrogen peroxide) and hepatic natural killer cells. However, certain cancer cells that resist or even deactivate these anti-tumoral defense mechanisms still can adhere to endothelial cells of the hepatic microvasculature through proinflammatory cytokine-mediated mechanisms. During their temporary residence, some of these cancer cells ignore growth-inhibitory factors while respond to proliferation-stimulating factors released from tumor-activated hepatocytes and sinusoidal cells. This leads to avascular micrometastasis generation in periportal areas of hepatic lobules. Hepatocytes and myofibroblasts derived from portal tracts and activated hepatic stellate cells are next recruited into some of these avascular micrometastases. These create a private microenvironment that supports their development through the specific release of both proangiogenic factors and cancer cell invasion- and proliferation-stimulating factors. Moreover, both soluble factors from tumor-activated hepatocytes and myofibroblasts also contribute to the regulation of metastatic cancer cell genes. Therefore, the liver offers a prometastatic microenvironment to circulating cancer cells that supports metastasis development. The ability to resist anti-tumor hepatic defense and to take advantage of hepatic cell-derived factors are key phenotypic properties of liver-metastasizing cancer cells. Knowledge on hepatic metastasis regulation by microenvironment opens multiple opportunities for metastasis inhibition at both subclinical and advanced stages. In addition, together with metastasis-related gene profiles revealing the existence of liver metastasis potential in primary tumors, new biomarkers on the prometastatic microenvironment of the liver may be helpful for the individual assessment of hepatic metastasis risk in cancer patients

Mechanobiology of portal hypertension.

The interplay between mechanical stimuli and cellular mechanobiology orchestrates the physiology of tissues and organs in a dynamic balance characterized by constant remodelling and adaptative processes. Environmental mechanical properties can be interpreted as a complex set of information and instructions that cells read continuously, and to which they respond. In cirrhosis, chronic inflammation and injury drive liver cells dysfunction, leading to excessive extracellular matrix deposition, sinusoidal pseudocapillarization, vascular occlusion and parenchymal extinction. These pathological events result in marked remodelling of the liver microarchitecture, which is cause and result of abnormal environmental mechanical forces, triggering and sustaining the long-standing and progressive process of liver fibrosis. Multiple mechanical forces such as strain, shear stress, and hydrostatic pressure can converge at different stages of the disease until reaching a point of no return where the fibrosis is considered non-reversible. Thereafter, reciprocal communication between cells and their niches becomes the driving force for disease progression. Accumulating evidence supports the idea that, rather than being a passive consequence of fibrosis and portal hypertension (PH), mechanical force-mediated pathways could themselves represent strategic targets for novel therapeutic approaches. In this manuscript, we aim to provide a comprehensive review of the mechanobiology of PH, by furnishing an introduction on the most important mechanisms, integrating these concepts into a discussion on the pathogenesis of PH, and exploring potential therapeutic strategies

iPSC-/ESC-derived hepatic stellate cells in 2D and 3D

Liver Fibrosis is a major concern for the world as it causes chronic inflammation which again induces an unhealthy and abnormal healing response. Most common causes to liver fibrosis are alcohol consumption, non-alcoholic steatohepatitis (NASH), hepatitis B(HBV), autoimmune hepatitis, non-alcoholic fatty liver disease (NAFLD) and cholestatic liver diseases. Continuous fibrosis in the liver can lead to losing normal function of the liver and eventually end with liver failure. As of now in vitro models of hepatic stellate cells (HSCs) are needed to eventually be able to produce models that can be used to test treatments against fibrosis. By differentiating HSCs from iPSCs and hESC the derived cells were tested with both immunostaining and RT-qPCR which checked gene expression to conclude if the cells had differentiated into HSC-like cells. The iHSCs were then used to construct two types of 3D cultures where the one culture called HSC in GelTrex. GelTrex matrix were selected to mimic ECM of space of Disse, which is a mixture of reduced growth factor (RGF) and basement membrane extract (BME). The GelTrex samples were also treated with TGF-β1 to see if this would increase expression of fibrosis-associated genes and to investigate presence of fibrosis-associated proteins. The GelTrex samples showed none reproduceable results as the construction of the GelTrex droplet was unsuccessful, due to air bubbles effecting the 3D culture. The RNA isolation was performed with poor technique at the RNA extraction step, which resulted in a deviation throughout the results. The other 3D culture was called for spheroid models, which were made with co-culturing of HepG2 and iHSC to mimic the cell-cell interaction. Construction of the spheroids were done by a mixture of HepG2 and HSC with a 5:1 ratio. The cells were separated into control and cells treated with TGF-β1. The cells were sorted from the spheroid and analyzed for changed in gene expression. Sorting showed proliferation of HepG2 in spheroids, which elevated the ratio, and there for the ratio should be tweaked to lower for construction on spheroids. RT-qPCR showed error in RNA extraction in RNA isolation together with the time span on sorting cells, which might have been too low.M-K

Improved predictive models for pre-clinical drug toxicity studies

Increasingly, drug-induced liver injury is one of the main reason for drugs to be withdrawn from the market even after passing toxicity studies in pre-clinical and clinical trials because of risks of toxicity and ineffective treatments. Human immortalised hepatocyte cell lines used in drug testing are widely available, inexpensive and easy to culture. However, these cell lines are commonly known to have poor predictive capabilities and improved in vitro hepatic models are required for predicting hepatotoxicity of large numbers of compounds in drug discovery. In this study, the primary goal was to develop an improved in vitro human hepatic model using a combination of the C3A human hepatic cell line and human umbilical vein endothelial cells (HUVECs), for prediction of acetaminophen (APAP) hepatotoxicity. Initial experiments showed that co-culture of HUVEC:C3A in EGM-2, an endothelial medium, was essential to support both cell types, and that co-cultures maintained the initial cell seeding ratio of 1:1 (HUVEC:C3A) after 3 days. Phenotyping of co-cultured cells using platelet endothelial cell adhesion molecule (PECAM-1/CD31) for HUVECs, and hepatic epithelial (EpCAM) markers for C3As demonstrated that at ratio 1:1 (HUVEC:C3A), there is cross-talk between HUVECs and C3As and cells in co-culture showed properties of self-organisation. This interaction resulted in improved hepatic metabolic activity in vitro in respect of albumin synthesis and cytochrome P450 activity. Treatment with low (5 mM), intermediate (10 mM) and high doses (20 mM) of APAP, showed that prediction of hepatotoxicity using specific kits for cell viability and mitochondria function, was significantly improved in C3As in the presence of HUVECs, thus demonstrating an in vitro human hepatic co-culture could be an invaluable model for drug toxicity studies. We observed that the intermediate APAP dose had no effect on cell viability and mitochondrial function in co-cultures, whilst by comparison both lactate levels and oxidative stress were perturbed in mono-cultures. Co-cultures also up-regulated expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in HUVECs following APAP exposure, which may be important in modulating the toxic effect of APAP on C3As. To further improve the in vitro liver-like model, Matrigel™ was incorporated to promote vascular formation by HUVECs and support hepatic organization, migration and function of C3As. In HUVEC mono-cultures, Matrigel™-promoted vascularization, haptotaxis and self-organization and in HUVEC:C3A co-cultures formation of structures reminiscent of liver sinusoids and maintenance of hepatic albumin synthesis and CYP3A4 activity. Time-lapse imaging showed haptotactic migration of hepatocytes towards endothelial cells, with Matrigel™ likely having a chemotactic effect on HUVECs and C3As, resulting in interconnected vascular network. APAP inhibited angiogenesis in HUVEC mono-cultures whereas APAP had no effect in HUVEC:C3A co-cultures. In conclusion, the development of an in vitro human organotypic co-culture model of HUVECs and C3As significantly enhanced hepatic function, demonstrated by significant improvement in hepatic metabolism, evidence of greater resistance to APAP toxicity, and improved cell-cell communication. Co-cultures markedly modulated APAP hepatotoxicity compared with C3A mono-cultures. Furthermore, co-culture of HUVECs and C3As using a complex basement membrane biomatrix (Matrigel™) produced a self-assembling interconnected vascular network, improved hepatocyte function as well as reproducibility of responses to APAP toxicity. The application of the described co-culture models may improve the accuracy, efficacy and predictive power of drug toxicity testing strategies in drug development

The role of relaxin in the regulation of human liver and kidney fibrosis

Liver fibrosis has a range of aetiologies and is a global cause of mortality. Acritical effect of liver fibrosis which also increases mortality is portalhypertension. The hepatic stellate cell is accepted as a major progenitor of livermyofibroblasts, which have been shown to be a major source of collagen andextracellular matrix proteins that disrupt liver architecture and function. Relaxinis a hormone involved in remodelling of extracellular matrix in the uterus andcervix and is known to increase renal blood flow in pregnancy. It has beenimplicated in the regulation of fibrosis in animal models and to modify the cellbiology of hepatic stellate cells in vitro. I have demonstrated the profile ofexpression of relaxin receptors in primary human stellate cells (HSC), showingthem to express RXFP-1, 3 and 4. Using a cAMP assay I confirm these receptorsto be functional, with RXFP-1 positively and RXFP-3 and 4 negatively couplingto cAMP. The expression of RXFP-1 is coupled with the level of activation,demonstrating a possible role for H2-relaxin in the regulation of HSC. I haveestablished a dynamic regulation of fibrotic mediators and HSC activationmarkers, including a reduction in ?-SMA, TIMP-1 and TGF-? with increases inMMP-1 and MMP-2, consistent with H2-relaxin having potentially therapeuticantifibrotic effects by increasing the fibrolytic phenotype. In addition throughthe use of gel contraction assays I demonstrate that H2-relaxin reduces serum orendothelin-1 induced HSC contraction. Through the use of siRNA I haveconfirmed that H2-relaxin mediates its regulation of fibrotic mediators and HSCactivation markers as well as the inhibition of gel contraction through the relaxinreceptor RXFP-1. I have evidence to suggest that the inhibition of contractionmay in part be via nitric oxide release in HSC. In conclusion I propose thatRXFP-1 is a potential therapeutic target in end stage human liver disease,targeting fibrosis and portal blood hypertension via both resolution of thephenotypic collagen deposition and vascular constriction associated with thehuman hepatic stellate cell

Role of nonmuscle myosin II isoforms in hepatic stellate cell activation

The hepatic stellate cell (HSC) plays a pivotal role in the development of hepatic fibrosis. Regulation of liver microcirculation is a complex system where blood flow is under systemic and sinusoidal control. Upon a fibrogenic stimulus, quiescent HSCs transdifferentiates into activated myofibroblast-like cell and exert a sustained contractile force, resulting in hyperconstricted vessels. Early phenotypic changes observed during the transdifferentiation process include cell stretching and elongation of cytoplasmic processes. These changes facilitate proliferation and migration of HSCs to the areas of injury where excess amounts of collagen are deposited altering normal liver architecture and leading to liver dysfunction. Under chronic liver injury, this wound healing response perpetuates and impairs blood flow through the liver resulting in portal hypertension, which is one of the main clinical manifestations of liver fibrosis. Nonmuscle myosin II (NMM II) has been shown to be involved in cellular contraction, proliferation and migration. Using a whole-cell contraction assay and a selective myosin II inhibitor, we have demonstrated that myosin II is essential for HSC contraction. Additionally, the expression of NMM II isoforms in activated HSCs was upregulated. To further explore the mechanism of regulation, we altered in vitro conditions to more closely depict the in vivo environment using a matrix stiffness assay. Under these conditions, a minimal increase in NMM II-A and II-B mRNA expression was detected, which suggests that mechanical properties of the liver may regulate NMM II isoforms in HSCs. siRNA mediated isoform knockdown had no effect on culture-activated HSC contraction and proliferation but cell migration was inhibited by 25%. Overall, our results demonstrate that myosin II isoforms play a critical role in the development of portal hypertension and HSC migration; however, the specific isoform responsible for this hypercontractile response has yet to be identified. Supported by NIH Grant AA14891

Redox mechanisms in hepatic chronic wound healing and fibrogenesis

Reactive oxygen species (ROS) generated within cells or, more generally, in a tissue environment, may easily turn into a source of cell and tissue injury. Aerobic organisms have developed evolutionarily conserved mechanisms and strategies to carefully control the generation of ROS and other oxidative stress-related radical or non-radical reactive intermediates (that is, to maintain redox homeostasis), as well as to 'make use' of these molecules under physiological conditions as tools to modulate signal transduction, gene expression and cellular functional responses (that is, redox signalling). However, a derangement in redox homeostasis, resulting in sustained levels of oxidative stress and related mediators, can play a significant role in the pathogenesis of major human diseases characterized by chronic inflammation, chronic activation of wound healing and tissue fibrogenesis. This review has been designed to first offer a critical introduction to current knowledge in the field of redox research in order to introduce readers to the complexity of redox signalling and redox homeostasis. This will include ready-to-use key information and concepts on ROS, free radicals and oxidative stress-related reactive intermediates and reactions, sources of ROS in mammalian cells and tissues, antioxidant defences, redox sensors and, more generally, the major principles of redox signalling and redox-dependent transcriptional regulation of mammalian cells. This information will serve as a basis of knowledge to introduce the role of ROS and other oxidative stress-related intermediates in contributing to essential events, such as the induction of cell death, the perpetuation of chronic inflammatory responses, fibrogenesis and much more, with a major focus on hepatic chronic wound healing and liver fibrogenesis