Gene expression recovery during an acute toxic damage in the liver

Specific structures and cell types in the organization of the liver are the key for its variant functions, like protein production, glucose homeostasis and detoxification. In the present work, liver damage from an acute toxic injury caused by intraperitoneal injection of a mixture of CCl4 and mineral oil in Balb/c mice and its subsequent recovery was studied using different methods to investigate specific cellular functions in the liver. The analysis by in situ hybridization and RT-qPCR showed how expression of liver specific enzymes and proteins in mouse hepatocytes is changed over a period of 6 days following injection. The genes investigated included Albumin, Arginase, Glutaminase2, Glutamine synthetase, Glucose-6-phosphatase, Glycogen synthase2, Gapdh, Cyp2e1 and Glucagon receptor genes. Interestingly, a significant change in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution in different areas of the liver were observed following CCl4 injury. Cyp2e1, an essential metabolizing enzyme in CCl4 metabolism, was strongly expressed in the pericentral zone during recovery. In comparison to hepatocytes in livers from untreated mice, liver cells from treated animals displayed distinct gene expression profiles in the damaged area around the pericentral vein during the analyzed time course and showed a complete recovery with strong albumin production at day 6 post CCl4 injection. The results obtained indicate that despite of the severe damage, liver cells in the damaged area do not simply die but instead locally adjust gene expression to deal with the damage effect and thereby ensure survival. In order to optimize the preparation of cRNA hybridization probes and enable the rapid synthetize of the large number of probes used in this study, a new rapid method for antisense cRNA preparation was established. The development of this rapid and efficient protocol for the generation of labeled cRNA probes was an important pre-requisite for the project. The new protocol is based on the preparation of DNA templates in vitro by PCR using primers that include RNA polymerase promoter sequences and size based purification of PCR fragments containing the target gene specific cDNA and promoter elements for T7 and SP6 RNA-polymerase. Purified PCR fragment based in vitro transcription enables the preparation of in situ hybridization probes, which can be used for the detection of the respective gene and visualization of the distribution of gene expression in tissue slices for any gene of interest. The optimized synthesis and purification protocols ensure high transcription efficiency and target specificity of the labeled cRNA and the obtained cRNA hybridization probes are compatible with established in situ hybridization protocols. This study proved that with a single dose of CCl4 injection in mouse, liver pericentral hepatocytes are the main cell type responsible for neutralizing the toxic agent, and the main consequence of this damage is not simply to induce cell death due to apoptosis, but instead these damaged hepatocytes seem to reduce any unnecessary activities in favor of processes needed for recovery from damage

Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro

Over recent decades, stereotactic body radiotherapy has garnered increasing popularity. Unfortunately, conventional preclinical 2D in vitro models are often insufficient for studying radiotherapy effects. Therefore, in this study, we developed a novel anthropomorphic in vitro liver phantom, which simulates the relevant hepatocellular carcinoma (HCC) tumor microenvironment and spatial organization. The liver phantom was 3D printed, filled with tissue-mimicking agarose mixture, and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. Airtight MCs induced hypoxic conditions, as verified by Hif1α staining. Irradiation was conducted with 20 Gy in one fraction using a CyberKnife, in either a 2D setup, or by irradiating MCs arranged in the 3D-printed liver model using an individually calculated treatment plan. Post-irradiation cellular damage was determined via γH2AX staining. Here, we demonstrate a new physiologically relevant approach to model HCC pathology following radiotherapy. Comparing γH2AX staining in normoxic conditions to cells grown in MCs (hypoxic conditions) revealed a reduction in cellular damage of 30.24% (p = 0.0001) in the hypoxic environment. Moreover, we compared the scattering effect of radiation on a conventional 2D in vitro model to our new 3D anthropomorphic liver phantom and observed a significant γH2AX intensity reduction of 9.6% (p = 0.0294) in HepG2 cells irradiated in the phantom. Our approach of utilizing a liver phantom takes into account the hypoxic tumor microenvironment and 3D scattering effects of tissue irradiation, thereby modeling both physical and biological parameters of HCC tumors. The use of tissue phantoms lays the groundwork for future examination of other hypoxic tumors and offers a more comprehensive approach for screening and analysis of novel cancer therapeutics

Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro

Over recent decades, stereotactic body radiotherapy has garnered increasing popularity. Unfortunately, conventional preclinical 2D in vitro models are often insufficient for studying radiotherapy effects. Therefore, in this study, we developed a novel anthropomorphic in vitro liver phantom, which simulates the relevant hepatocellular carcinoma (HCC) tumor microenvironment and spatial organization. The liver phantom was 3D printed, filled with tissue-mimicking agarose mixture, and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. Airtight MCs induced hypoxic conditions, as verified by Hif1α staining. Irradiation was conducted with 20 Gy in one fraction using a CyberKnife, in either a 2D setup, or by irradiating MCs arranged in the 3D-printed liver model using an individually calculated treatment plan. Post-irradiation cellular damage was determined via γH2AX staining. Here, we demonstrate a new physiologically relevant approach to model HCC pathology following radiotherapy. Comparing γH2AX staining in normoxic conditions to cells grown in MCs (hypoxic conditions) revealed a reduction in cellular damage of 30.24% (p = 0.0001) in the hypoxic environment. Moreover, we compared the scattering effect of radiation on a conventional 2D in vitro model to our new 3D anthropomorphic liver phantom and observed a significant γH2AX intensity reduction of 9.6% (p = 0.0294) in HepG2 cells irradiated in the phantom. Our approach of utilizing a liver phantom takes into account the hypoxic tumor microenvironment and 3D scattering effects of tissue irradiation, thereby modeling both physical and biological parameters of HCC tumors. The use of tissue phantoms lays the groundwork for future examination of other hypoxic tumors and offers a more comprehensive approach for screening and analysis of novel cancer therapeutics

Oxygen Gradient Induced in Microfluidic Chips Can Be Used as a Model for Liver Zonation

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production

HIV protease inhibitor-induced cardiac dysfunction and fibrosis is mediated by platelet-derived TGF-β1 and can be suppressed by exogenous carbon monoxide.

Human immunodeficiency virus (HIV) infection is an independent risk factor for cardiovascular disease. This risk is magnified by certain antiretrovirals, particularly the protease inhibitor ritonavir, but the pathophysiology of this connection is unknown. We postulated that a major mechanism for antiretroviral-associated cardiac disease is pathologic fibrosis linked to platelet activation with release and activation of transforming growth factor (TGF)-β1, and that these changes could be modeled in a murine system. We also sought to intervene utilizing inhaled carbon monoxide (CO) as proof-of-concept for therapeutics capable of regulating TGF-β1 signaling and collagen autophagy. We demonstrate decreased cardiac function indices, including cardiac output, ejection fraction and stroke volume, and prominent cardiac fibrosis, in mice exposed to pharmacological doses of ritonavir. Cardiac output and fibrosis correlated with plasma TGF-β1 levels. Mice with targeted deletion of TGF-β1 in megakaryocytes/platelets (PF4CreTgfb1flox/flox) were partially protected from ritonavir-induced cardiac dysfunction and fibrosis. Inhalation of low dose CO (250ppm), used as a surrogate for upregulation of inducible heme oxygenase/endogenous CO pathways, suppressed ritonavir-induced cardiac fibrosis. This occurred in association with modulation of canonical (Smad2) and non-canonical (p38) TGF-β1 signaling pathways. In addition, CO treatment suppressed the M1 pro-inflammatory subset of macrophages and increased M2c regulatory cells in the hearts of RTV-exposed animals. The effects of CO were dependent upon autophagy as CO did not mitigate ritonavir-induced fibrosis in autophagy-deficient LC3-/- mice. These results suggest that platelet-derived TGF-β1 contributes to ritonavir-associated cardiac dysfunction and fibrosis, extending the relevance of our findings to other antiretrovirals that also activate platelets. The anti-fibrotic effects of CO are linked to alterations in TGF-β1 signaling and autophagy, suggesting a proof-of-concept for novel interventions in HIV/antiretroviral therapy-mediated cardiovascular disease

Scheme of enzymatic reactions involved in basic liver functions and metabolism.

<p>(<b>A</b>) Function of key enzymes of nitrogen metabolism and their zonation in healthy liver hepatocytes. (<b>B</b>) Enzymatic reactions involved in glucose storage and release in hepatocytes. (<b>C</b>) Metabolic activation of CCl₄ in pericentral hepatocytes.</p

Graphical summary of the area specific gene expression patterns.

<p>Results from the ISH analysis are summarized into 4 conditions. Untreated liver (normal), and after 3h and 6h, 2 days (2d) and 3 days (3d) upon CCl₄ treatment. Intensity of the color reflects the relative intensity of gene expression for each gene (intense/dark color: strong/high signal). The intensities only visualize the relative distribution of the respective mRNA along the axis from the periportal area to the pericentral vein (blue: higher periportal: green; higher pericentral). Open cells (circular light to dark coloring) are used to indicate non homogenous speckled patterns observed in the ISH images. </p

<i>In situ</i> hybridization for genes from carbohydrate metabolism.

<p><i>In </i><i>situ</i> hybridization of mouse liver sections with probes for selected genes of carbohydrate metabolism at different time points post CCl₄ injection. Genes were visualized by dual staining with yellow and violet dye precipitation. Gene names are indicated on the left in the respective color. Co-staining for both genes in the same area resulted in dark “brown” staining. Pictures were captured with 4x objective.</p

<i>In situ</i> hybridization for genes from nitrogen metabolism.

<p><i>In </i><i>situ</i> hybridization of mouse liver sections with probes for selected genes involved in nitrogen metabolism at different time points post CCl₄ injection. In each panel, genes were visualized by dual staining with yellow and violet dye precipitation. Gene names are indicated on the left in the respective color. Co-staining for both genes in the same area resulted in dark “brown” staining. Pictures were captured with 4x objective. </p

Higher resolution <i>in situ</i> hybridization images.

<p><i>In </i><i>situ</i> hybridization of mouse liver sections from untreated animals and form days 1 to 3 after CCl₄ injection, with higher magnification (20x objective). Genes analyzed are indicated at the left in the respective color for each row. Co-staining for both genes in the same area resulted in dark “brown” staining. Specific areas of the liver tissue (acini) are marked: central vein (cv), portal vein/area (pv).</p