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Biomedical subjects

S L Friedman

Publications and source records attributed to S L Friedman.

At least 37 records · Page 2Linked to original sources
PubMed2000

The effect of storage at 4 degrees C on the stability of ampicillin residues in raw milk.

Raw milk samples collected from tanker trucks are routinely screened for beta-lactam antibiotic drug residues using rapid screening tests. If drug residues are detected, the milk may be shipped on ice blocks to a laboratory for further analysis. A study was conducted to determine the stability of ampicillin in raw milk stored at +4 degrees C in order to predict if shipping the milk would result in the degradation of ampicillin residues. Milk samples were spiked with 20 ppb ampicillin, stored at +4 degrees C and -70 degrees C for 1-6 days, and then analysed by HPLC with fluorescence detection. No loss of ampicillin residues was found in milk stored at +4 degrees C for 1-6 days.

Ampicillin↗
PubMed2000

Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex.

Hepatic stellate cell activation is a complex process. Paradoxes and controversies include the origin(s) of hepatic stellate cells, the regulation of membrane receptor signaling and transcription, and the fate of the cells once liver injury resolves. Major themes have emerged, including the dominance of autocrine signaling and the identification of counterregulatory stimuli that oppose key features of activated cells. Advances in analytical methods including proteomics and gene array, coupled with powerful bioinformatics, promise to revolutionize how we view cellular responses. Our understanding of stellate cell activation is likely to benefit from these advances, unearthing modes of regulating cellular behavior that are not even conceivable on the basis of current paradigms.

Animals↗
PubMed2000

An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro.

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 micrometer) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARalpha, -beta, -gamma, and RXRalpha, -beta, -gamma) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10(-8)-10(-5) m) or all-trans-retinoic acid (10(-10)-10(-6) m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.

Animals↗
PubMed1999

Evaluation of fibrosis and hepatitis C.

Prognosis of hepatitis C is determined primarily by the extent and progression of fibrosis. Fibrosis, or the accumulation of extracellular matrix, is reversible, whereas cirrhosis is not. A large-scale clinical trial showed that extensive fibrosis may go undetected with little or no clinical signs or symptoms. The mean interval from time of infection to development of cirrhosis was approximately 30 years, although cirrhosis may occur in as little as 10 to 15 years or more than 50 years. Viral factors, including genotype and viral RNA levels, predict the response to therapy but do not independently correlate with rate of fibrosis. Host factors that are known to increase the likelihood of fibrosis include older age at infection, male gender, and alcohol intake. Other host factors, possibly the immune phenotype, are thought to be very important in determining rate of fibrosis, yet these factors have not yet been identified. At present, there is no substitute for liver biopsy to assess fibrosis, and there is a compelling need to develop noninvasive markers, because none currently exist. The Metavir system, a five-stage scale used for the evaluation of the extent of fibrosis, is the most carefully validated method for scoring fibrosis. Remarkable progress has been made in understanding the cellular and molecular basis of fibrosis. For example, it is now known that hepatic stellate cells are the major source of extracellular matrix after their activation, which connotes a conversion from a resting, vitamin A-rich cell to one that is proliferative, fibrogenic, and contractile. Future research should focus on better understanding the cellular basis of fibrosis and its natural history as treatments for hepatitis C continue to improve.

Disease Progression↗
PubMed1999

Activation of rat hepatic stellate cells leads to loss of glutathione S-transferases and their enzymatic activity against products of oxidative stress.

Oxidative stress, mediated partly by lipid peroxidation products, may lead to increased collagen synthesis by hepatic stellate cells (HSC). Stellate cells are protected from oxidative stress by enzymes of detoxication such as the glutathione S-transferases (GSTs), which form glutathione conjugates with lipid peroxidation products (e.g., 4-hydroxy-2-nonenal [HNE]). To better understand the role of GSTs in stellate cell biology, we examined the expression and enzymatic activity of GSTs in normal and activated (both culture- and in vivo-activated) stellate cells. Normal stellate cells contained numerous isoforms of GST including those that detoxify HNE. High levels of enzymatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) and HNE were present in normal stellate cells and were similar to levels present in whole liver. Following activation by growth in culture, the expression of several GSTs (rGSTA1/A2, A3, and M1) was lost. Also, enzymatic activities toward CDNB and HNE fell approximately 90%. However, expression of rGSTP1 was maintained. A similar loss of rGSTA1/A2, A3, and M1 with persistent expression of rGSTP1 was present after activation in vivo. Furthermore, we identified 2 subpopulations of activated stellate cells with different GST phenotypes from injured livers. In summary, activated stellate cells lose most forms of GST and associated enzymatic activities that are present in normal stellate cells. The findings raise the possibility that activated stellate cells have less ability to detoxify lipid peroxidation products and may be susceptible to oxidative stress. Additionally, we propose that the phenotypic change in GSTs is a sensitive marker of stellate cell activation.

Animals↗
PubMed1999

CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells.

Hepatic stellate cells (HSCs) are a major source of extracellular matrix, which, during fibrogenesis, undergo a process of "activation" characterized by increased proliferation and collagen synthesis. Oxidative stress can stimulate HSC proliferation and collagen synthesis in vitro. Cytochrome P4502E1 (CYP2E1) is an effective producer of reactive oxygen species. To study how intracellular oxidative stress modulates alpha 2 collagen type I (COL1A2) gene induction, a rat HSC line (HSC-T6) was transfected with human CYP2E1 complementary DNA in the sense and antisense orientation and with empty vector, and stable cell lines were generated. The cells expressing CYP2E1 displayed elevated production of reactive oxygen species and showed a 4-fold increase in COL1A2 messenger RNA (mRNA) levels; expression of this mRNA among different clones appeared to correlate with the level of CYP2E1. COL1A2 expression was decreased by vitamin E treatment or transfection with manganese superoxide dismutase, and was further increased after treatment with L-buthionine sulfoximine (BSO) to lower GSH levels. Thus, CYP2E1-dependent oxidative stress plays a major role in the elevation of COL1A2 mRNA levels in this system. Nuclear run-on assay showed a 3-and-a-half-fold increase in COL1A2 transcription in the cells expressing CYP2E1; stabilization of COL1A2 mRNA was also observed. These results indicate that under oxidative stress conditions, COL1A2 mRNA expression is regulated both transcriptionally and through mRNA stabilization. The CYP2E1-expressing HSC appear to be a valuable model for the sustained generation of reactive oxygen species and may allow the elucidation of signaling pathways responsible for oxidant stress-mediated collagen gene induction.

Animals↗
PubMed1999

Increased 9,13-di-cis-retinoic acid in rat hepatic fibrosis: implication for a potential link between retinoid loss and TGF-beta mediated fibrogenesis in vivo.

BACKGROUND/AIMS: During hepatic fibrosis, hepatic stellate cells (HSCs) transform into myofibroblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Recently, we have demonstrated that 9,13-di-cis-retinoic acid (RA), a geometric isomer identified as a stable and major metabolite of vitamin A in circulation, stimulates the synthesis of plasminogen activator (PA) and induces PA/plasmin-dependent latent transforming growth factor (TGF)-beta activation in HSC cultures, probably via induction and activation of RA receptor (RAR) alpha. The aim of the present study was to address a potential link between the loss of retinyl esters to increased formation of RA(s), which might play a role in facilitating TGF-beta-mediated liver fibrogenesis in vivo. METHODS: We examined the effect of 9,13-di-cis-RA on transactivating activity of RARalpha in HeLa cells as well as its effect on PA- and TGF-beta-dependent collagen synthesis in rat and human HSC cultures. We measured the changes in 9,13-di-cis-RA levels both during activation of rat HSCs in vitro and during porcine serum-induced rat hepatic fibrosis in vivo and correlated this with RAR alpha/beta, PA, TGF-beta and type I procollagen mRNA expression in the fibrotic liver. RESULTS: 9,13-di-cis-RA transactivated RARalpha, and provoked PA/plasmin and TGF-beta-dependent procollagen synthesis in HSCs. 9,13-di-cis-RA levels were increased both in activated HSCs in vitro and in fibrotic liver accompanying the enhanced expression of RAR alpha/beta, PA, TGF-beta and procollagen in vivo. CONCLUSIONS: These findings suggest a potential link between 9,13-di-cis RA formation and hepatic fibrosis via formation of TGF-beta in vivo, and thus provide further insight into the biologic role of retinoids during hepatic fibrogenesis.

Animals↗
PubMed1999

Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy.

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent leads to cirrhosis and liver failure. Exciting progress has been made in understanding the mechanisms of hepatic fibrosis. Major advances include: (i) characterization of the components of extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of hepatic stellate cells as the primary source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation, and signalling pathways involved in liver fibrogenesis; (iv) characterization of key matrix proteases and their inhibitors; (v) identification of apoptotic mediators in stellate cells and exploration of their roles during the resolution of liver injury. These advances have helped delineate a more comprehensive picture of liver fibrosis in which the central event is the activation of stellate cells, a transformation from quiescent vitamin A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of antifibrotic therapy. Points of therapeutic intervention may include: (i) removing the injurious stimuli; (ii) suppressing hepatic inflammation; (iii) down-regulating stellate cell activation; and (iv) promoting matrix degradation. The future prospects for effective antifibrotic treatment are more promising than ever for the millions of patients with chronic liver disease worldwide.

Animals↗
PubMed1999

Cytokines and fibrogenesis.

Cytokines play a major role in the development of hepatic fibrosis, the wound-healing response of the liver to chronic injury. Major concepts in defining the role of cytokines in fibrogenesis include (1) Cytokines may be pro- or antifibrogenic; (2) autocrine, paracrine, and matrix-bound sources of cytokines are the most important; and (3) multiple mechanisms of cytokine regulation are essential to fine-tune their effects. The hepatic stellate cell is the key effector of the fibrotic response and both a principal source and target of cytokines. Activation of stellate cells connotes the conversion of a resting vitamin A-rich cell to one which is proliferative, contractile, fibrogenic, and devoid of vitamin A. The features of stellate cell activation provide a framework in which to understand how cytokines drive fibrosis. These features include (1) proliferation; (2) contractility; (3) fibrogenesis; (4) extracellular matrix degradation; (5) chemotaxis; (6) cytokine release; and (7) retinoid loss. The insights gained from illuminating the role of stellate cells has engendered realistic hopes for treating hepatic fibrosis through modulation of cytokine actions.

Adipocytes↗
PubMed1999

Stellate cell activation in alcoholic fibrosis--an overview.

There has been remarkable progress in our understanding of how chronic alcohol ingestion may lead to hepatic injury and scarring, or fibrosis. Hepatic fibrosis represents the liver's wound healing response and is characterized by accumulation of interstitial matrix, or scar. Fibrosis in the liver results from the activation of stellate cells, or resident mesenchymal cells. Stellate cell activation is a dramatic phenotype transition whose net effect is the replacement of normal liver matrix by scar. Features of stellate cell activation include increased cell accumulation from proliferation and directed migration, increased matrix production, enhanced contractility, accelerated degradation of the normal liver matrix, release of profibrogenic cytokines, and loss of cellular vitamin A. Alcohol may enhance fibrogenesis through stimulation of stellate cells by hypoxia, generation of lipid peroxides from damaged hepatocytes, production of acetaldehyde that may have direct fibrogenic activity, and through activation of Kupffer cells or resident macrophages. Unanswered questions remain to be studied, but the clarification of underlying mechanisms of fibrosis portends continued progress in our ability to treat alcoholic liver fibrosis.

Alcohol Drinking↗
PubMed1998

Transcriptional activation of transforming growth factor beta1 and its receptors by the Kruppel-like factor Zf9/core promoter-binding protein and Sp1. Potential mechanisms for autocrine fibrogenesis in response to injury.

We have explored the regulation of transforming growth factor beta (TGF-beta) activity in tissue repair by examining the interactions of Zf9/core promoter-binding protein, a Kruppel-like zinc finger transcription factor induced early in hepatic stellate cell (HSC) activation, with promoters for TGF-beta1 and TGF-beta receptors, types I and II. Nuclear extracts from culture-activated HSCs bound avidly by electrophoretic mobility shift assay to two tandem GC boxes within the TGF-beta1 promoter but minimally to a single GC box; these results correlated with transactivation by Zf9 of TGF-beta1 promoter-reporters. Zf9 transactivated the full-length TGF-beta1 promoter in either primary HSCs, HSC-T6 cells (an SV40-immortalized rat HSC line), Hep G2 cells, or Drosophila Schneider (S2) cells. Recombinant Zf9-GST also bound to GC box sequences within the promoters for the types I and II TGF-beta receptors. Both type I and type II TGF-beta receptor promoters were also transactivated by Zf9 in mammalian cells but not in S2 cells. In contrast, Sp1 significantly transactivated both receptor promoters in S2 cells. These results suggest that (a) Zf9/core promoter-binding protein may enhance TGF-beta activity through transactivation of both the TGF-beta1 gene and its key signaling receptors, and (b) transactivating potential of Zf9 and Sp1 toward promoters for TGF-beta1 and its receptors are not identical and depend on the cellular context.

Animals↗
PubMed1998

p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs.

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53(-/-)) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53(-/-) Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

Antineoplastic Agents↗
PubMed1998

Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis.

Wound repair in the liver induces altered gene expression in stellate cells (resident mesenchymal cells) in a process known as "activation." A zinc finger transcription factor cDNA, zf9, was cloned from rat stellate cells activated in vivo. Zf9 expression and biosynthesis are increased markedly in activated cells in vivo compared with cells from normal rats ("quiescent" cells). The factor is localized to the nucleus and the perinuclear zone in activated but not quiescent cells. Zf9 mRNA also is expressed widely in nonhepatic adult rat tissues and the fetal liver. The zf9 nucleotide sequence predicts a member of the Kruppel-like family with a unique N-terminal domain rich in serine-proline clusters and leucines. The human zf9 gene maps to chromosome 10P near the telomere. Zf9 binds specifically to a DNA oligonucleotide containing a GC box motif. The N-terminal domain of Zf9 (amino acids 1-201) is transactivating in the chimeric GAL4 hybrid system. In Drosophila schneider cells, full length Zf9 transactivates a reporter construct driven by the SV40 promoter/enhancer, which contains several GC boxes. A physiologic role for Zf9 is suggested by its transactivation of a collagen alpha1(I) promoter reporter. Transactivation of collagen alpha1(I) by Zf9 is context-dependent, occurring strongly in stellate cells, modestly in Hep G2 cells, and not at all in D. schneider cells. Our results suggest that Zf9 may be an important signal in hepatic stellate cell activation after liver injury.

Amino Acid Sequence↗
PubMed1998

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen 1, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Animals↗
PubMed1997

p53 does not repress hypoxia-induced transcription of the vascular endothelial growth factor gene.

Hypoxia-induced neovascularization mediated by vascular endothelial growth factor (VEGF) contributes to tumor progression. Based on its effects when overexpressed in transient transfection assays, p53 has been proposed to repress VEGF transcription. To investigate this hypothesis, we have analyzed endogenous VEGF mRNA levels in Hep3B cells stably expressing an inducible p53-estrogen receptor fusion protein and in irradiated RKO cells expressing endogenous wild-type p53. In both cell lines, VEGF mRNA levels increased in response to hypoxia, either in the presence or absence of functional p53. Our data provide no evidence for a causal relationship between the loss of p53 activity and increased VEGF expression that is observed during tumor progression. Studies that attribute repressor functions to p53 based on analysis of cells transiently overexpressing this protein should be interpreted cautiously.

Carcinoma, Hepatocellular↗
PubMed1997

Early genes induced in hepatic stellate cells during wound healing.

Activation of mesenchymal cells is a central event in the wound healing response of most tissues. In liver, the mesenchymal element responsible for organ fibrosis is the hepatic stellate cell (HSC) (formerly known as lipocyte or Ito cell). The phenotypic cascade of stellate cell activation in liver fibrosis has been well documented and involves both marked morphologic changes and upregulation of several functional components including extracellular matrix, cytokine receptors, contractile filaments and metalloproteinases. However, the genetic regulation of stellate cell activation is poorly understood. In an attempt to clone genes that are involved in the regulation of HSC activation we have combined cDNA library amplification by PCR with subtraction hybridization/differential screening, and have successfully identified genes induced in vivo during early stellate cell activation in a rat model of liver fibrosis. The subtracted cDNA library comprised less than 100 unique sequences. Of these, 13 clones with sizes ranging from 322 to 745 were sequenced and characterized. Gene induction in HSCs was monitored by RNAse protection assay during early liver injury induced by the hepatotoxin CCl4. The sequenced cDNAs corresponding to the known genes included type II transforming growth factor beta receptor, glutathione peroxidase I, transferrin and several clones encoding cellular retrotransposons, whose expression was not previously identified in non-parenchymal liver cells. In addition, one partial cDNA predicted a zinc-finger motif, suggesting a possible role of a novel transcriptional regulator. Our approach represents a valuable strategy for clarifying in vivo regulatory mechanisms of mesenchymal cell activation in wound healing.

Animals↗