Vladimir I. Khaoustov

Induction of three-dimensional assembly of human liver cells by simulated microgravity

SummaryThe establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

MICROARRAY ANALYSIS OF GENES DIFFERENTIALLY EXPRESSED IN HEPG2 CELLS CULTURED IN SIMULATED MICROGRAVITY: PRELIMINARY REPORT

SummaryDeveloped at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conductive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Cultures of human liver cells in simulated microgravity environment

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

MDM2/p53 protein expression in the development of colorectal adenocarcinoma☆☆☆

The murine double minutes 2 (MDM2) oncoprotein inhibits p53-mediated tumor suppression. MDM2 has been shown to be overexpressed in sarcomas and more recently was implicated in the pathogenesis of carcinomas. The purpose of this study was to determine the expression pattern of MDM2 in adenomas and colorectal adenocarcinomas and decide whether there is a correlation between MDM2 and p53 protein status. Paraffin-embedded tissues from 52 colorectal cancer (CRC) specimens and their adjacent normal tissue (N-CRC) were studied. In addition, 56 sporadic adenomas were investigated for the immunohistochemical expression of MDM2 and p53 proteins. Immunoreactivity of p53 indicating p53 gene mutation (p53 +) was significantly higher in CRC (44%) compared to adenomas (23.2%) (P <0.01). None of the N-CRC specimens expressed the immunoreactive p53 protein. MDM2 overexpression (MDM2+) was similar in adenomas (30.3%) and CRC (25%), but only 2 (3.8%) of 52 N-CRC specimens showed overexpression of MDM2. In most cases MDM2 expression was associated with negative p53 expression (wild-type p53) in both adenomas (r = 0.59, P <0.001) and CRC (r = 0.69, P <0.0001). No correlation was found between MDM2, p53 expression, and either the histologic grade, nodal stage or morphology of the tumors. There is greater p53 mutation in CRC compared to adenomas and N-CRC. The data indicate that MDM2 is overexpressed in CRC and is significantly associated with wild-type p53 compared to N-CRC specimens from the same patient. The MDM2 expression pattern is similar in adenomas and CRC, which may suggest that MDM2 overexpression is an early event in the progression of CRC.

The effect of ursodeoxycholic acid on the survivin in thapsigargin-induced apoptosis

Endoplasmic reticulum (ER) was recently suggested as a third subcellular compartment in apoptotic execution. Survivin is a member of inhibitors of apoptosis and ursodeoxycholic acid (UDCA) prevents apoptosis from various apoptotic stimuli. To assess the activity of survivin and the effect of UDCA on the survivin in ER stress-mediated apoptosis, we treated hepatoma cell lines with thapsigargin (TG). TG-induced apoptosis was assessed by morphological changes, DNA fragmentation, cleavages of poly(ADP-ribose)polymerase (PARP), and activation of calpain and caspase-12. The level of survivin was decreased after TG treatment in hepatoma cell lines indicating that survivin play an important role in ER stress-mediated apoptosis. UDCA prevented decrease in survivin levels and inhibited TG-induced apoptosis and caspase-12 activation suggesting an anti-apoptotic effect of UDCA.

Hyperlipasemia Associated with Hepatitis C Virus

Extrahepatic manifestations of chronic hepatitis C virus (HCV) infection have been well described. However, hyperlipasemia and/or pancreatitis have not been reported. Following the observation that several HCV patients had elevated lipase levels, this retrospective study was conducted to assess the association between hyperlipasemia and/or pancreatitis with hepatitis C infection. Of 204 subjects who underwent evaluation for hepatitis C, 103 had lipase levels determined at baseline. The control group consisted of 41 nonHCV subjects with a variety of gastrointestinal diseases including 18 with nonalcoholic liver disease. Twenty-five percent of HCV patients had elevated lipase at baseline as compared to 10% of controls (P = 0.04; OR = 3.1; 95% CI: 1.02–9.60). Mean lipase levels were 253 ± 72 units/liter (normal range 114–286 units/liter and 210 ± 42 units/liter for the HCV and control groups, respectively (P = 0.002). No significant difference in amylase was found between the groups. There was a significant association between ALT (>1.5 times the upper limit of normal) and lipase (P = 0.02; OR = 3.0; 95% CI: 1.1–7.5). Among 30 patients who received interferon-based therapy ± ribavirin, 11 had elevated lipase at baseline. Six of these patients responded to therapy and demonstrated normalization of lipase levels. In contrast, allnonresponders with baseline hyperlipasemia continued to have high lipase levels (P = 0.17; OR = 4.0; 95% CI: 0.6–28.4). Furthermore, only 3 of 8 (37.5%) patients with normal lipase responded to treatment as compared to 6 of 10 (60%) of hyperlipasemic patients (P = 0.36; OR = 2.5; 95% CI: 0.4–16.9). In conclusion, hyperlipasemia and/or subclinical pancreatitis may represent extrahepatic manifestations of HCV infection and should not preclude treatment.

Identification of components of grape powder with anti-apoptotic effects

This study is to investigate the mechanism underlying the anti-apoptotic effects of freeze-dried grape powder (FDGP) and identify the polyphenolic compounds involved. We examined apoptotic signaling pathways affected by FDGP and by its active components, including epicatechin, cyanidin, quercetin, and resveratrol, in human Huh7 hepatoma cells by assaying cell viability assays, the activities of caspase 3 and caspase 7, and the expression of endoplasmic reticulum stress-associated proteins. FDGP dramatically decreased taurodeoxycholic acid (TDCA)-induced production of reactive oxygen species (ROS). Assessment of individual active components revealed that at concentrations corresponding to 300 μg/mL FDGP, only quercetin demonstrated cytoprotective effects against mitochondrial-mediated apoptosis. In contrast, increased concentrations of other individual polyphenolic compounds were required to produce measurable cytoprotective effect. Only combinations of all four polyphenolic compounds (epicatechin, cyanidin, quercetin, and resveratrol) restored a degree of the anti-apoptotic effects seen with FDGP. The pretreatment of FDGP at 30 μg/mL concentration could reverse the thapsigargin-induced effects on the expression of endoplasmic reticulum stress-associated proteins. In conclusion, FDGP reduced oxidative stress, endoplasmic reticulum stress, and apoptosis. The mechanisms involved in the anti-apoptotic effects of FDGP included reduced generation of ROS, and reduced processing of certain caspases. We demonstrated that quercetin, epicatechin, and cyanidin are active compounds within FDGP that attenuate apoptosis. These findings contribute to our understanding of the molecular mechanisms of anti-apoptotic and anti-oxidant effects of grape and are expected to assist in developing clinical protocols to treat a variety of stress-mediated conditions.

Freeze-dried grape powder attenuates mitochondria- and oxidative stress-mediated apoptosis in liver cells.

The beneficial effects of grape consumption have been attributed to the antioxidant activity of its polyphenols. This study was conducted to investigate the cytoprotective effects of a freeze-dried grape powder (FDGP) on liver cells. FDGP treatment of primary hepatocytes and hepatoma cells revealed increased metabolic activity of cells and phosphorylation of Akt and IkappaBalpha, as well as up-regulation of proliferating cell nuclear antigen (PCNA) level. To study the molecular mechanisms of FDGP effects, cells were treated with TNF-related apoptosis-inducing ligand (TRAIL); taurodeoxycholic acid (TDCA); thapsigargin (TG), to induce cell apoptosis through death receptor-, mitochondria-, or ER-mediated pathway; and H(2)O(2), to induce oxidative stress, respectively. TDCA-induced activation of caspase-3, caspase-7, caspase-9, and Bax was dramatically decreased with cotreatment of FDGP. Furthermore, FDGP reduced levels of annexin V positive cells by 4-fold. Also, FDGP pretreatment restored cellular glutathione content by 71% in cells treated with H(2)O(2). However, FDGP did not inhibit ER-mediated apoptosis. In conclusion, FDGP increased the viability and metabolic activity of liver cells and attenuated oxidative stress- and mitochondria-mediated apoptosis. These data may contribute to the understanding of the mechanisms involved in protective effects of grape in a variety of liver conditions associated with cellular stress.

Role of the long cytoplasmic domain of the SIV Env glycoprotein in early and late stages of infection

BackgroundThe Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity.ResultsWe compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly.ConclusionThe results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.

Induction of hepatitis B virus gene expression at low temperature

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.