The use of a silicon strip detector dose magnifying glass in stereotactic radiotherapy QA and dosimetry.

PURPOSE: Stereotactic radiosurgery/therapy (SRS/SRT) is the use of radiation ablation in place of conventional surgical excision to remove or create fibrous tissue in small target volumes. The target of the SRT/SRS treatment is often located in close proximity to critical organs, hence the requirement of high geometric precision including a tight margin on the planning target volume and a sharp dose fall off. One of the major problems with quality assurance (QA) of SRT/SRS is the availability of suitable detectors with the required spatial resolution. The authors present a novel detector that they refer to as the dose magnifying glass (DMG), which has a high spatial resolution (0.2 mm) and is capable of meeting the stringent requirements of QA and dosimetry in SRS/SRT therapy. METHODS: The DMG is an array of 128 phosphor implanted n+ strips on a p-type Si wafer. The sensitive area defined by a single n+ strip is 20 x 2000 microm2. The Si wafer is 375 microm thick. It is mounted on a 0.12 mm thick Kapton substrate. The authors studied the dose per pulse (dpp) and angular response of the detector in a custom-made SRS phantom. The DMG was used to determine the centers of rotation and positioning errors for the linear accelerator's gantry, couch, and collimator rotations. They also used the DMG to measure the profiles and the total scatter factor (S(cp)) of the SRS cones. Comparisons were made with the EBT2 film and standard S(cp) values. The DMG was also used for dosimetric verification of a typical SRS treatment with various noncoplanar fields and arc treatments when applied to the phantom. RESULTS: The dose per pulse dependency of the DMG was found to be < 5% for a dpp change of 7.5 times. The angular response of the detector was investigated in the azimuthal and polar directions. The maximum polar angular response was 13.8% at the gantry angle of 320 degrees, which may be partly due to the phantom geometry. The maximum azimuthal angular response was 15.3% at gantry angles of 90 degrees and 270 degrees. The angular response at the gantry angle of 180 degrees was 6.3%. A correction function was derived to correct for the angular dependence of the detector, which takes into account the contribution of the azimuthal and polar angular response at different treatment couch positions. The maximum positioning errors due to collimator, gantry, and couch rotation were 0.2 +/- 0.1, 0.4 +/- 0.1, and 0.4 +/- 0.2 mm, respectively. The SRS cone S(cp) agrees very well with the standard data with an average difference of 1.2 +/- 1.1%. Comparison of the relative intensity profiles of the DMG and EBT2 measurements for a simulated SRS treatment shows a maximum difference of 2.5%. CONCLUSIONS: The DMG was investigated for dose per pulse and angular dependency. Its application to SRS/SRT delivery verification was demonstrated. The DMG with its high spatial resolution and real time capability allows measurement of dose profiles for cone applicators down to 5 mm in diameter, both accurately and rapidly as required in typical SRS/SRT deliveries.

Production of Ac-225 for cancer therapy by photon-induced transmutation of Ra-226.

The increasing application of Ac-225 for cancer therapy indicates the potential need for its increased production and availability. The production of Ac-225 has been achieved using bremsstrahlung photons from an 18 MV medical linear accelerator (linac) to bombard a Ra-226 target. A linac dose of 2800 Gy produced about 64 microCi of Ra-225, which decays to Ac-225. This result, while consistent with the theoretical calculations, is far too low to be of practical use. A more powerful linac is required that runs at a higher current, longer pulse length and higher frequency for practical production. This process could also lead to the reduction of the nuclear waste product Ra-226.

Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Cytokine regulation of syndecan expression in cells of liver origin.

Syndecan-1 and syndecan-2-two cell surface heparan sulfate proteoglycans-were described in normal human liver. Proteoglycans can modulate the effect of cytokines, and cytokines can influence the expression of proteoglycans. In the present work the regulatory effect of IL-1beta, IL-6, TNF-alpha, IFN-gamma and TGF-beta1 on syndecan-1 and syndecan-2 expression of hepatocytes, hepatoma cell lines, liver and skin fibroblasts has been studied. All cytokines were able to influence the steady state level of syndecan-1 and syndecan-2 mRNA. Their action was target cell specific resulting in either up- or downregulation except TGF-beta1 that was stimulatory in all cell types examined.

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate.

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA-transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor kappaB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat.

Matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) are thought to play an essential role in liver injury associated with tissue remodeling. However, their distinct expression profile in different liver repair models still remains to be established. Hepatic expression of collagenase (MMP-13), gelatinases A and B (MMP-2, -9), stromelysin-1 and -2 (MMP-3, -10), membrane-type MMP-1 (MMP-14), and TIMP-1 and -2 was studied following single and repeated CCl4-mediated injury and after partial hepatectomy. Expression was analyzed by reverse transcription-PCR (RT-PCR), northern blot analysis, zymography, and immunohistochemistry. Following a single toxic liver injury, MMPs and TIMPs were induced in a distinct time frame in that expression of most MMPs was induced during the early phase of liver injury, was maximal during the inflammatory reaction, and was diminished in the recovery phase. In contrast, TIMP and MMP-2 steady state mRNA levels remained constant in the early phase, were strongly induced during tissue inflammation, and remained increased until the recovery phase. Interestingly, hepatic TNF-alpha expression paralleled the MMP induction profile, while the increase of TGF-beta1 expression mapped to the increase of TIMPs. Chronic liver injury was accompanied by an increase in the steady state mRNA levels of MMP-2 and TIMPs, while other MMPs remained more or less unchanged or were diminished. Partial hepatectomy was followed by a dramatic increase of MMP-14 and to a lesser extent also of TIMP-1 expression; other MMPs and TIMPs were not significantly induced. Liver injury is accompanied by profound changes in hepatic MMP/TIMP expression, the latter being critically dependent on the type of injury. Single toxic injury resulting in complete restoration was characterized by a sequential induction of MMPs and TIMPs suggesting initial matrix breakdown and matrix restoration thereafter. Chronic liver injury leading to fibrosis displays overall diminished matrix degradation mainly through TIMP induction, while liver regeneration induced by partial hepatectomy caused an induction of MMP-14 and TIMP-1 only, which might be unrelated to matrix turnover but connected to pericellular fibrinolysis or fibrolysis required for hepatocellular replication.

Role of the Ets-1 transcription factor during activation of rat hepatic stellate cells in culture.

During liver tissue repair, hepatic stellate cells (HSCs), a pericyte-like nonparenchymal liver cell population, transform from a quiescent status (resting HSCs) into myofibroblast like cells (activated HSCs); the latter is the principal matrix-synthesizing cell of the liver. Although several factors have been shown to be involved in this important process, the molecular mechanisms regulating HSC activation are still under investigation. To identify key regulatory proteins involved in the HSC activation process, we used different mRNA display technologies, with cDNAs prepared from HSCs at different stages of in vitro activation. With the latter technique, the transcription factor Ets-1 was detected through its down-regulation during activation. As confirmed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNAs coding for Ets-1 were present in the highest amounts in freshly isolated HSCs and in HSCs 2 days after plating (classified as resting HSCs/early activated HSCs) and were diminished in HSCs 7 days after plating (activated cells). Ets-1 protein was present in HSC-lysates, as assessed by Western blot, and bound to an oligonucleotide containing the Ets-1 consensus cis-acting motif, as demonstrated by electrophoretic mobility shift assay. Ets-1 binding activity peaked in nuclear extracts prepared from resting/early activated cells and was diminished in extracts derived from fully activated cells. In contrast, binding activity of the transcription factors TFIID, AP-1, and SP-1 was highest in activated HSCs and only barely detectable in resting/early activated HSCs. By Northern blot and RT-PCR analysis, Ets-1-specific transcripts were present in parenchymal and other nonparenchymal liver cells too, illustrating that hepatic Ets-1 expression is not specific or restricted to HSCs. However, the unique pattern of Ets-1 binding activity present in resting versus activated HSCs and its known implications for cellular differentiation and tissue remodeling suggest that Ets-1 could be of crucial importance for HSC activation and hepatic tissue repair.

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar-parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation.

Transforming growth factor-beta1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells.

BACKGROUND/AIMS: It is suggested that during fibrogenesis as well as during carcinogenesis of the liver, the hepatic microvascular phenotype is transformed from sinusoids - which lack a basement membrane--into continuous capillaries which rest on a basement membrane. As transforming growth factor (TGF)-beta1 seems to be the most effective mediator in the stimulation of matrix protein synthesis, we were interested in the modulation of basement membrane proteins collagen type IV, laminin, and entactin expression by TGF-beta1 in liver sinusoidal endothelial cells (SECs), especially since a stimulation of the synthesis of collagen type IV but not of entactin and laminin by TGF-beta1 has been demonstrated in a fibrosarcoma cell line. METHODS: The synthesis of the basement membrane (BM) proteins entactin, laminin, and collagen type IV and of the extracellular matrix (ECM) proteins tenascin and fibronectin with or without TGF-beta1--stimulation was analyzed by immunostaining, immunoprecipitation of endogenously labeled proteins and Northern blot analysis of total RNA extracted from freshly isolated or cultured SECs from rat or guinea pig livers. Furthermore, SECs were isolated from acutely and chronically CCl4-damaged rat livers and were analyzed for matrix protein expression. RESULTS: SECs were adherent 24 h after isolation and formed confluent monolayers on day 4 of primary culture. Specific immunoprecipitates and specific transcripts for the BM proteins entactin, laminin, and collagen type IV and for ECM proteins tenascin and fibronectin were detectable in freshly isolated or cultured SECs. The synthesis of all tested BM proteins and ECM proteins was stimulated at least 3-fold by TGF-beta1. In SECs isolated after CCl4-induced acute and chronic liver damage, increased levels of matrix protein transcripts were detectable. CONCLUSIONS: The stimulation of the synthesis of all BM-proteins by TGF-beta1 in vitro and the accumulation of ECM transcripts in SECs isolated from CCl4-treated livers, suggests that SECs are involved in the formation of a basement membrane during the "capillarization" of the sinusoids during liver disease.

Matrix metalloproteinases in early human liver development.

Matrix metalloproteinases (MMPs) regulate matrix deposition in tissues. Collagens I, III, and IV are involved in early human liver development. To establish whether MMPs specific for these collagens participate in early human liver development, we localized immunohistochemically MMP-1 and MMP-13 (for collagens I and III) and MMP-2 and MMP-7 (for collagen IV) in the early human liver anlage [6th-10th gestational week (GW)]. MMP-1 was found from the 6th GW onward in hepatocytes and later also in outer limiting plate hepatocytes, early bile ducts, and periportal mesenchymal cells. In the 6th GW, MMP-2 was found only in microvascular endothelium. In the 7th GW, MMP-2 was also detected in hepatocytes. From the 9th GW onward, MMP-2 was detectable in all hepatocytes and erythropoietic, endothelial, and periportal mesenchymal cells. MMP-7 was present in the 6th GW in some hepatocytes and endothelial cells, but from the 7th GW onward, only in hematopoietic cells. MMP-13 was found exclusively in hematopoietic cells. This study has shown that production of MMP -1, MMP-2, MMP-7, and MMP-13 during human liver development already occurs from the 6th GW. At this time-point their substrates are only traces or are not yet present in the tissue. A possible role of MMPs in early liver development is discussed.

Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS: We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS: HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS: The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.

Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells.

Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.

Cellular localization of hepatic cytochrome 1B1 expression and its regulation by aromatic hydrocarbons and inflammatory cytokines.

Cytochrome P450 1B1 (CYP1B1) is an activator of several xenobiotics and is induced in the liver upon experimental exposure to aromatic hydrocarbons. Since its cellular localization and regulation are incompletely clarified, Cyp1B1 expression and inducibility by 9,10-dimethyl-1,2-benzanthracene (DMBA) and inflammatory cytokines were investigated in different rat liver cell populations in vitro and in the liver during hepatocellular injury. Expression of Cyp1B1 was studied by Northern blot analysis in hepatic stellate cells (HSCs), myofibroblasts (MFs), Kupffer cells (KCs), and hepatocytes at various time points of primary cultures and in acutely damaged rat liver (carbon tetrachloride model). Enzyme inducibility was assessed by incubation of cells with DMBA as well as, in the case of HSCs, with tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor beta1 (TGFbeta1). Cyp1B1 messengers were expressed at high levels by HSCs and MFs, whereas constitutive expression was not detectable in KCs or in hepatocytes. Cyp1B1-specific mRNA were expressed at highest levels in HSCs at an early stage of activation (2 days after plating) and were diminished upon further activation. DMBA strongly enhanced Cyp1B1 gene expression in HSCs, MFs, and in hepatocytes at day 3 of primary cultures, but not in hepatocytes at day 1, or in KCs. The inflammatory cytokine TNF-alpha enhanced the Cyp1B1 gene expression in HSCs, either when administered alone or in addition to DMBA, while TGFbeta1 did not affect Cyp1B1 expression, even after DMBA induction. We conclude that HSCs and MFs seem to be the major cellular sources of hepatic Cyp1B1 expression and that the constitutive expression of the Cyp1B1 gene and the responsiveness to DMBA stimulation differ between mesenchymal and parenchymal liver cells, indicating a cell-specific regulation of Cyp1B1 gene expression. Interestingly, TNF-alpha is a potent stimulator of the Cyp1B1 gene in HSCs and acts in concert with DMBA.

Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1.

BACKGROUND/AIMS: Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. METHODS: Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. RESULTS: MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-alpha stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-beta1 induced TIMP expression only. CONCLUSIONS: Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-alpha, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-beta1 through TIMP induction.

Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair.

Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.

Characterization of the IGF axis components in isolated rat hepatic stellate cells.

The insulin-like growth factors I and II (IGF-I, -II) are circulating peptides known to participate in the regulation of metabolism, growth, and cellular differentiation. In the present study, "early cultured" (days 2-3 of culture) and "culture-activated" (days 6-7 of culture) rat hepatic stellate cells (HSCs) were analyzed for expression of individual components of the IGF axis. Northern blot analysis of IGF-I messenger RNA (mRNA) revealed transcripts of 7.5, 4, 2, and 1.0 to 1.5 kb in culture-activated HSCs, while early cultured HSCs did not express IGF-I mRNA. In culture-activated HSCs, an IGF-I secretion of 8.3+/-2.5 ng/10(6) cells per 24 hours was determined radioimmunologically. In media from early cultured HSCs, IGF-I was not detectable. The IGF-I receptor (IGF-I-R) mRNA expression was three-fold higher in early cultured HSCs than in culture-activated HSCs. By immunohistochemistry, a decrease of IGF-I-R expression of HSCs in vivo following CCl4-induced liver damage was noted as well. IGF binding proteins (IGFBPs) were detected in conditioned media from HSCs by 125I-IGF-I ligand blotting at apparent molecular masses of 24 and 41 to 45 kd that were immunologically identified as IGFBP-4 and -3, respectively. Synthesis of these IGFBPs increased with time of culture. At neutral pH, no IGFBP proteolysis was observed in conditioned media of early cultured and culture-activated HSCs, whereas at acidic pH, protease activities against IGFBP-3 and -4 were detectable. IGFBP protease activities were completely abolished by inhibitors of aspartyl and cysteine proteases. Addition of 100 nmol/L IGF-I stimulated cell proliferation of early cultured HSCs 5.6+/-1.1- and 4.6+/-0.2-fold as measured by [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation, respectively. In culture-activated HSCs, proliferation was increased 1.2+/-0.1-fold in the presence of 100 nmol/L IGF-I in both proliferation assays. It can be concluded that due to a higher expression of the IGF-I-R and lower levels of IGFBPs, early cultured HSCs are more susceptible to the mitogenic actions of IGFs than the culture-activated HSCs. The present data suggest a role for the IGF axis components in the initiation rather than the perpetuation of HSC proliferation during hepatic fibrogenesis.

CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable in parallel with HSC activation, which suggests that apoptosis might represent an important mechanism terminating proliferation of activated HSC.

The complement-activating protease P100 is expressed by hepatocytes and is induced by IL-6 in vitro and during the acute phase reaction in vivo.

Ra reactive factor, a lectin present in the sera of a wide variety of vertebrates, is composed of mannan-binding proteins and a serine protease termed P100, which is known to activate complement. Using differential mRNA display technology to study the "activation"-dependent gene expression of hepatic stellate cells (HSC), we partially cloned a cDNA encoding the rat homolog of P100, which displayed 94% and 88% homology to mouse and human P100 cDNA, respectively. In the rat P100, specific transcripts 5.4, 4.0, and 3.3 kb in size were detected in major amounts in normal liver, but were absent or near the detection limit in other organs. Among the different liver cell populations studied during primary culture, P100-specific transcripts of 4.0 kb were prominent in HSC and present in hepatocytes and hepatoma cells, whereas Kupffer cells and sinusoidal endothelial cells were P100-negative. In addition to 4.0-kb mRNA, freshly isolated hepatocytes also contained transcripts of 5.4 and 3.3 kb, which were down-regulated during primary culture. In situ hybridization of normal liver tissue confirmed the in vitro data in that P100 was expressed by hepatocytes and nonparenchymal liver cells, which probably represent HSC. In vitro P100 steady-state mRNA levels of hepatocytes were stimulated by IL-6 and/or dexamethasone. During the acute phase reaction induced by turpentine injection, P100 steady-state mRNA levels were up-regulated in rat liver. The data demonstrate that: (a) the liver is the primary site for P100 expression in the rat; (b) HSC and hepatocytes appear to represent the cellular sources; and (c) P100 steady-state mRNA levels are up-regulated by the acute phase mediators IL-6 and dexamethasone in vitro and during the acute phase reaction in vivo, suggesting that P100 represents a novel, positive acute-phase gene in the rat.