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.

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 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.

Response of melanoma to heat and radiation therapy--a review of the literature and experience from The Prince of Wales Hospital, Sydney.

Our review of the literature indicates that radiotherapy and/or heat therapy can provide local control of recurrent or metastatic melanoma in a large proportion of patients. This has undoubted value in the local palliation of symptoms and, in the absence of disseminated disease, can be curative. At The Prince of Wales Hospital, Sydney, we have studied the response of melanoma lesions to heat and radiation therapy and have assessed the reaction in the adjacent normal skin. Thirty-two melanoma lesions that were measurable in 12 patients received radiotherapy and heat therapy in different combinations and dose schedules (15 lesions received radiotherapy alone, six lesions received heat therapy alone, and 11 lesions received combined radiation and heat therapy). The acute normal skin reaction was compared between lesions that received single modality radiation or heat therapy and those that received the combination of heat and radiation therapy. A moderate or severe reaction developed at six of the 21 sites that were treated by a single modality, and at four of the 11 sites that received combined heat and radiation therapy (P = 0.7), and all healed within a few days. Evaluation of the melanoma response to therapy was possible only in 26 of the 32 lesions that were treated because two patients died soon after therapy and the response of their six lesions was not evaluable. A complete response occurred in 14 (54%) of 26 lesions and a partial response occurred in 10 (38%) of 26 lesions. The objective response by treatment modality was 10 of 15 lesions for radiotherapy, six of six lesions for heat therapy and eight of 11 lesions for both therapies combined. We conclude that radiotherapy and heat therapy, separately or combined, produce acceptably-low damage to normal tissue and highly-satisfactory local control of melanoma.

Gene expression and regulation of plasminogen activator inhibitor type I in hepatic stellate cells of rat liver.

BACKGROUND & AIMS: Plasminogen activator inhibitor type 1 (PAI-1) plays a crucial role in the regulation of extracellular matrix-degrading enzymes and in the production of fibrogenic mediators such as transforming growth factor beta through inhibition of plasminogen activation. Because hepatic stellate cells (HSCs), the principal matrix-producing cell of the liver, might also affect extracellular matrix degradation and growth factor activation, the aim of this study was to analyze PAI-1 expression and its regulation in HSCs compared with other liver cells. METHODS: PAI-1 synthesis of liver cells at different time points of primary culture was studied by immunoprecipitation of endogenously labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Northern blotting. RESULTS: Among the various types of liver cells, PAI-1 protein and specific transcripts were present in HSCs and endothelial cells, and no major PAI-1 synthesis was detected in Kupffer cells and hepatocytes. Transforming growth factor beta 1, tissue plasminogen activator, and dexamethasone increased PAI-1 production in HSCs. CONCLUSIONS: Apart from their role as the principal connective tissue-producing cell of the liver, HSCs might regulate extracellular matrix accumulation by modulating the activities of matrix-degrading enzymes and fibrogenic mediators through the production of PAI-1.

Accumulation and cellular localization of fibrinogen/fibrin during short-term and long-term rat liver injury.

BACKGROUND/AIMS: During liver fibrosis, there is a putative pacemaker role of fibronectin. Fibrinogen is closely linked to fibronectin during clotting processes. The aim of this study was to show fibrinogen gene expression during liver damage. METHODS: Fibrinogen/fibrin deposition in damaged livers was studied by immunohistology. Fibrinogen gene expression was analyzed in vivo in a model of CCl4-induced rat liver damage and in vitro in isolated liver cells by means of Northern blot analysis and in situ hybridization. RESULTS: Immunohistology showed striking amounts of fibrinogen and fibrin deposits in pericentral necrotic areas (short-term damage) and within fibrotic septa (long-term damage). Total RNA extracted from short-term-damaged livers contained an increased fibrinogen messenger RNA level. By in situ hybridization, fibrinogen transcripts were localized in cells of the nonnecrotic areas (short-term damage) and outside fibrotic septa (long-term damage). In vitro studies showed fibrinogen de novo synthesis restricted to hepatocytes. CONCLUSIONS: The results show fibrinogen/fibrin deposition during short-term liver injury and liver fibrogenesis, which may suggest the involvement of a "clotting-like process" in short-term liver damage and liver fibrosis. The data might indicate that fibrin/fibronectin constitute a "provisional matrix," which affects the attraction and proliferation of inflammatory and matrix-producing cells.

A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development.

The murine homeobox-containing gene Hoxa-7 is expressed in restricted patterns during embryogenesis and plays an important role in the control of region-specific differentiation. Previous studies have shown that separate elements specify lineage restriction and expression boundaries of Hoxa-7. In particular 3.6 kb of 5' flanking sequences were sufficient to establish an anterior boundary of Hoxa-7 gene expression. To identify the minimal regulatory element specifying the anterior boundary of expression, transgenic mice were generated carrying chimeric constructs with deletions of 5' flanking sequences fused to a thymidine kinase minimal promoter/E. coli lacZ reporter construct. By deletion analysis, a 470 bp long control element (AX 470) located 1.6 kb upstream of the transcription start site was identified that directed expression of the beta-galactosidase protein in a pattern reflecting the anterior boundary of expression of the endogenous Hoxa-7 gene. This element was active in either orientation and conferred region-specific expression to unrelated promoters, thereby behaving like an enhancer element. In contrast, transgenic mice carrying further 5' and 3' deletions of the 470 bp long element did not exhibit an anterior boundary of Hoxa-7 expression. Based on these results the minimal control element (AX 470) specifying the anterior boundary of Hox expression was designated as Hoxa-7 enhancer. Furthermore, 3 kb of the human HOXA7 upstream region were sequenced and compared to its mouse homologue in order to identify conserved regions. Sequence comparison revealed motifs that were strongly conserved between both species. The human homologue of the mouse Hoxa-7 enhancer was 70% identical at the nucleotide level and was also capable of directing an anterior boundary in transgenic mice. Using transgenic lines a detailed analysis of the Hoxa-7 enhancer-directed expression during embryogenesis was performed. lacZ expression was first detected in the allantois at day 7.5 p.c. and in mesoderm and ectoderm at day 8.5 of gestation. Between gestational ages E8.5 to E12.5 beta-gal expression was observed in the somites, spinal cord, spinal ganglia and paraxial mesoderm as well as in mesenchymal layers of the kidney. A distinct anterior limit of expression was noted in transgenic lines at level C4 (neural tube) and C5 (spinal ganglia). Our deletion experiments defined a minimal enhancer element specifying the anterior boundary of Hox gene expression in early and late phases of development. Further studies aim at characterizing the trans-acting factors that mediate the spatial and temporal expression of Hox genes in the developing embryo.

Expression of von Willebrand factor in normal and diseased rat livers and in cultivated liver cells.

Von Willebrand factor (vWf) is an adhesive glycoprotein known to play an important role in hemostasis and in tissue injury. Because the latter process resembles hepatic fibrogenesis, we studied the tissue distribution of vWf in diseased livers. In normal rat liver vWf was strongly expressed in the vascular endothelium and as small spots or fiber-like structures in the hepatic parenchyma. During acute liver injury, pronounced staining was observed within the area of necrosis. In fibrotic livers vWf deposits were distributed predominantly at the scar-parenchyma interface but also within the septum and in sinusoidal lining cells. Testing different liver cell populations in vitro demonstrated that vWf gene expression was limited to endothelial cells (ECs) and, therefore, the latter cell population might represent the vWf-positive cells detected in vivo. The distribution of vWf within fibrotic septa suggests that vWf becomes a component of the extracellular matrix (ECM) in fibrotic livers.

Bone morphogenetic protein-6 is expressed in nonparenchymal liver cells and upregulated by transforming growth factor-beta 1.

Bone morphogenetic protein-6 (BMP-6) is a member of the TGF-beta superfamily, which controls growth and differentiation during embryogenesis and acts as an osteoinductive factor in the adult organism. In order to gain further insights into the role of BMP-6, the present study analyzed the expression pattern of BMP-6 in adult rat tissues with special emphasis to the liver, since TGF-beta 1, another member of the TGF-beta superfamily, has been shown to play a fundamental role in liver physiology. Rat BMP-6-coding cDNAs were generated by homology cloning using RT-PCR and displayed 89.6 and 83.4% homology to mouse and human BMP-6, respectively. By Northern blotting BMP-6-specific transcripts 3.7 kb in size were detected in major amounts in lung and in minor quantities in spleen, kidney, heart, brain, and liver. Among the different hepatic cell populations tested BMP-6 expression was confined to nonparenchymal liver cells, namely rat hepatic stellate cells (HSC) and Kupffer cells (KC). During primary culture BMP-6 expression was increased in HSC but declined in KC. Interestingly, TGF-beta 1 stimulated BMP-6 expression of HSC especially at an early time point of culture, while interferon-gamma downregulated BMP-6 expression. The detection of BMP-6 transcripts in the liver, the cell-type-restricted expression pattern, and its regulation propose that, in addition to its osteoinductive properties, BMP-6 might play a role in liver growth and differentiation, in particular after tissue damage.

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells.

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.

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.

Reactivities of tyrosine, histidine, tryptophan, and methionine in radical pair formation in flavin triplet induced protein nuclear magnetic polarization.

In order to check the validity of several basic assumptions of protein photochemically induced nuclear polarization (protein photo-CIDNP), we have investigated the quenching processes of the dye triplets by the side chains of tyrosine, histidine, and tryptophan in a variety of molecular systems and environments. The quenching (H atom or electron transfer) is the generating process of the triplet electron-spin-correlated radical pair, the evolution of which gives rise to nuclear polarization. At pH 7 the quenching of 10-(carboxyethyl)flavin triplets by tyrosine and tryptophan is almost diffusion controlled. Quenching by histidine is slower. We have also investigated the slow quenching (by electron transfer) by the side chains of methionine and could show that quenching by cysteine S derivatives is negligible. Quenching by tyrosine and histidine peptides and by the tyrosines of the pancreatic trypsin inhibitor protein is slightly slower than by free side chains. Quenching is strongly viscosity controlled, to be expected of a process requiring bimolecular contact. Reactivity trends at high viscosities resemble those observed in fluid aqueous solutions. Activation energies of quenching by tyrosine, tryptophan, and histidine are similar. No difference could be detected in the mechanism of quenching by these side chains. No fast static quenching was observed that could compete with the diffusional 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.

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 extracellular matrix proteoglycans perlecan and decorin in carbon-tetrachloride-injured rat liver and in isolated liver cells.

Proteoglycans are important components of the extracellular matrix. They are involved in liver regeneration as well as in liver fibrosis. The distribution and cellular source of proteoglycans under normal as well as pathological conditions is still under debate. Localization of decorin and perlecan was studied in normal, acutely damaged, and cirrhotic liver by histochemistry. Furthermore, their synthesis was analyzed in different liver cell populations isolated from normal rat liver. In normal liver, decorin positivity was observed in the perisinusoidal space and in the portal area. Perlecan was clearly detectable in the portal area (blood vessels and bile ducts); a moderate reaction was also seen along the sinusoids. Strong positivity for both proteoglycans was detectable in the necrotic areas of acutely damaged liver. Chronic liver damage was characterized by the deposition of decorin and perlecan in the fibrotic septa. Immunocytochemical reactions were positive for perlecan and decorin in cultured Ito and endothelial cells but not in hepatocytes and Kupffer cells. Northern hybridization confirmed the capacity of Ito cells and endothelial cells to express the two genes. Interestingly, although rat skin fibroblasts expressed strong messages for both proteoglycans, rat aortic smooth muscle cells did not synthesize decorin.

Entactin gene expression in normal and fibrotic rat liver and in rat liver cells.

BACKGROUND: Entactin, a constituent of basement membranes, is a sulfated glycoprotein with binding sites for laminin, collagen type IV, fibronectin, and cell surfaces. As it is known that excess matrix deposition and sinusoidal basement membrane formation is a central characteristic of liver fibrogenesis, we investigated whether the entactin gene is expressed in normal and in damaged rat liver and which cell types are able to express this gene. In addition, we were interested in the cellular origin and time course of laminin synthesis, a matrix protein closely associated with entactin. EXPERIMENTAL DESIGN: Entactin gene expression was analyzed in normal, acutely and chronically damaged rat livers (CCl4-model) by immunohistochemistry and in situ detection of specific transcripts. Rat fat-storing cells (FSC) (Ito), hepatocytes, Kupffer cells, liver endothelial cells, arterial smooth muscle cells (SMC), and skin fibroblasts (SF) were isolated according to standard techniques. Entactin gene expression in cultured cells was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates, Northern blot analysis, and immunocytochemistry. RESULTS: In normal liver, entactin was detected in the vessel walls as continuous deposits and in a spot-like fashion along the sinusoids. Entactin was detectable among the cells of the inflammatory infiltrates of acutely damaged liver and in connective tissue septa, in the walls of newly formed vessels and bile ducts of fibrotic liver. Laminin distribution in the vessels was similar, but it was additionally present in the space of Dissé of damaged liver. By in situ hybridization, few entactin-positive cells were found in normal liver sections. Strongly positive cells were scattered over the injured parenchyma of acutely and chronically damaged livers. Northern blot analysis of total RNA extracted from normal and damaged liver tissue showed a distinct increase of entactin specific transcripts during development of fibrosis. Hybridization of total RNA from cultured FSC, hepatocytes, Kupffer cells, endothelial cells, SMC, and SF revealed entactin specific mRNA in FSC, SMC, SF, and endothelial cells; laminin mRNA was found in FSC and SF. Synthesis and secretion of both proteins were found in FSC, SMC and SF. Entactin and laminin gene expression increased in parallel to FSC during time in culture. CONCLUSIONS: Among the main liver cells, FSC show the highest entactin gene expression and might therefore play the dominant role in the synthesis of this protein in normal and fibrotic liver. However, endothelial cells and liver myofibroblasts could also contribute to entactin production.