Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosis.

Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-beta) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-beta bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.

Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells.

Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM alpha-actin and SM22alpha are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside-outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC-the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.

Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis.

Rat liver perisinusoidal lipocytes (PL) cultured on uncoated plastic transform spontaneously within 6-10 d to myofibroblastlike cells (MFBlC). Parallel to the transformation the TGF alpha- and TGF beta 1-mRNA expression increased and was highest in MFBlC. Competitive radioligand binding assays demonstrated that in contrast to untransformed PL the MFBlC synthesize and secrete transforming growth factor (TGF)-alpha (15 fmol/cell per 24 h) and predominantly the latent form of TGF beta 1 (0.2 fmol/cell per 24 h). Medium conditioned by MFBlC (MFBcM) significantly stimulated PL proliferation with little effect on PL proteoglycan synthesis. By transient acidification of the MFBcM, known to activate the latent form of TGF beta 1, the stimulatory effect on PL proteoglycan synthesis was enhanced and furthermore PL transformation (measured by expression of iso-alpha smooth muscle actin and loss of retinylpalmitate) was accelerated. Preincubation of this medium with neutralizing antibodies to TGF beta resulted in (a) the growth inhibitory effect was converted to a growth stimulation and (b) the stimulatory effect on proteoglycan synthesis was abolished. In summary our data indicate that progressive activation of PL on plastic (transformation to MFBlC) leads to an enhanced expression of the TGF alpha- and TGF beta 1-mRNAs and secretion of the corresponding proteins. Medium conditioned by MFBIC stimulates proliferation, transformation, and PG synthesis of untransformed PL. These mechanisms are suggested to be relevant in self perpetuation of liver fibrogenesis.

Elevated apoptosis-associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation.

OBJECTIVES: During apoptosis, intermediate filament protein cytokeratin 18 (CK18) is cleaved by caspases at Asp396 which can be specifically detected by the monoclonal antibody M30 (M30-antigen). DESIGN AND METHODS: M30-antigen serum levels were analyzed in 76 chronic liver diseases (CLD) patients and 62 healthy controls. RESULTS: M30-antigen levels were significantly elevated in CLD patients (median 296.3 U/L) compared with healthy controls (median 153.5 U/L, P<0.001) and increased with disease severity (Child-Pugh or MELD score). M30-antigen correlated with aminotransferase activities and parameters indicating cholestasis such as bile acids. Highest serum M30-antigen was associated with histologically confirmed severe intrahepatic cholestasis (median 599.1 U/L) or biliary duct inflammation (median 648.0 U/L). Furthermore, in contrast to patients with liver cirrhosis, presence of hepatocellular carcinoma was associated with elevated M30-antigen in patients without cirrhosis. CONCLUSION: Serum M30-antigen levels are elevated in CLD and correlate with hepatic inflammation as well as cholangitis and cholestasis.

Interaction of synthetic polynucleotides with small rat liver ribosomal subunits possessing low and highly phosphorylated protein S6.

The interaction of some synthetic mRNAs (polyuridylate, polyadenylate, polycytidylate) with small rat liver ribosomal subunits which have protein S6 in different states of phosphorylation was studied by BioGel column chromatography, affinity chromatography on poly(U)-Sepharose 4B, and continuous diafiltration at 4 degrees C. 40-S subunits with low phosphorylated protein S6 (isolated from normal liver) and small subunits with highly phosphorylated protein S6 (from galactosamine-, thioacetamide-, dimethylnitrosamine-, puromycin-, and cycloheximide-treated livers) bind initially equal amounts of poly(U) but the dissociation of the radioactive polyuridylate occurs much more rapidly and to a greater extent from the low than from the highly phosphorylated type of subunits. From control- and galactosamine-4-S subunits 62% and 22%, respectively, of originally bound [3H]poly(U) was removed. The release of initially bound poly(A) from 40-S subunits of galactosamine-treated liver ws retarded but reached finally the same level as with control liver ribosomal subunits (removal of 40% of bound [3H]poly(A)). No differences between low and highly phosphorylated subunits were observed with poly(C). If the dissociation reaction was performed at 22 degrees C instead of 4 degrees C the differences in the release of poly(U) described above disappeared.

Insulin-like growth factor signaling pathways in rat hepatic stellate cells: importance for deoxyribonucleic acid synthesis and hepatocyte growth factor production.

It has been shown recently that insulin-like growth factor 1 (IGF-1) increases both DNA synthesis and hepatocyte growth factor (HGF) production in cultured hepatic stellate cells. In this study, we used selective blockers to investigate crucial signaling pathways for these effects of IGF-1 in cultured rat hepatic stellate cells. Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF. In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.

Insulin-like growth factors stimulate expression of hepatocyte growth factor but not transforming growth factor beta1 in cultured hepatic stellate cells.

Hepatic stellate cells (HSC) are located adjacent to hepatocytes and produce hepatocyte growth factor (HGF) in the normal liver, whereas transformed HSC in fibrotic livers produce transforming growth factor beta1 (TGFbeta1), an inhibitor ofhepatocyte proliferation. In addition to the endocrine actions of hepatic insulin-like growth factor-I (IGF-I), it also stimulates the proliferation of HSC. In this study we found that addition of IGF-1 (20-500 ng/ml) for 48 h to 2- to 7-day-old primary cultures of rat HSC resulted in a time- and dose-dependent increase by 50-190% of the concentrations of immunoreactive HGF in the medium. The levels of HGF as well as DNA synthesis measured as thymidine incorporation were also enhanced by IGF-II and des(1-3)IGF-I, which has reduced binding to IGF binding proteins. There was no consistent effect of the IGFs on the levels of immunoreactive TGFbeta1 or on the total DNA content of the cultures. There was no effect of human GH on medium levels of HGF or TGFbeta1, thymidine incorporation, or total DNA content. IGF-I increased the abundance of HGF messenger RNA, as measured by the RNase protection/solution hybridization technique, whereas there was no effect on TGFbeta1 or glyceraldehyde phosphate dehydrogenase messenger RNA. The results suggest that IGFs stimulate the production of HGF but not TGFbeta1 by HSC in vitro.

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts.

BACKGROUND: The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. RESULTS: With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. CONCLUSIONS: Our results indicate that FuGENE6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.

Differential expression of keratan sulphate proteoglycans fibromodulin, lumican and aggrecan in normal and fibrotic rat liver.

In this study we investigated in rat liver the expression of genes coding for the core proteins of fibromodulin, lumican and aggrecan. By means of Northern analysis and in situ hybridization we present evidence for their differential transcription during liver fibrogenesis. Whereas no fibromodulin expression could been detected, both lumican and aggrecan transcripts were found displaying different time-courses of expression during the fibrogenic process. Based on studies performed in non-hepatic tissues, these proteoglycans are considered to have keratan sulphate glycosaminoglycan side chains. The expression of the respective core protein genes in liver is unexpected since published data have shown neither keratan sulphate nor its synthesis de novo in this tissue. The results also point to a putative role of aggrecan in the modulation of the inflammatory process in the liver.

Biosynthesis of small proteoglycans by hepatic lipocytes in primary culture.

Chondroitin sulfate/dermatan sulfate proteoglycans were obtained from the secretions of cultured rat hepatic lipocytes. The collagen-binding small proteoglycan II represented only a minor species (less than 10%), whereas similar amounts of small proteoglycan I and of a novel collagen-binding proteoglycan with a core protein of 101 kDa were found. These results support the concept of a special role of lipocytes in the pathogenesis of liver fibrosis.

Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro.

The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form.

Transforming growth factors (TGF alpha and TGF beta 1) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells.

The synthesis of total sulfated glycosaminoglycans (GAG) was stimulated by transforming growth factors (TGF alpha 1.4-fold at 5 ng/ml, and TGF beta 1 2.05-fold at 2.5 ng/ml) in primary cultures of rat liver fat storing cells (FSC). The combination of both TGFs resulted in an additively stimulated synthesis of total sulfated GAG (more than 3-fold), chondroitin sulfate (more than 15-fold) and hyaluronate (3.8-fold), respectively, whereas the formation of dermatan sulfate was unchanged and that of heparan sulfate was slightly reduced. In summary, TGFs were identified as important mediators of stimulated GAG synthesis in those cells of the liver (FSC), which are the primary site of matrix glycoconjugate production.

Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells.

To current knowledge, transforming growth factor beta (TGFbeta) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFbeta system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFbeta, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFbeta response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFbeta downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFbeta, leading to, e.g., induction of alpha2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFbeta-dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFbeta sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFbeta response. In transdifferentiated MFB, expression of a constitutively active TGFbeta receptor I, but not treatment with TGFbeta1, resulted in transcriptional activation of a TGFbeta responsive promoter, thereby demonstrating completely restored TGFbeta signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFbeta signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

Developmental changes of proteoglycan synthesis in rat liver and isolated hepatocytes.

The synthesis of sulfated proteoglycans in late fetal (19th to 22nd day of intrauterine life), early postnatal, and adult liver tissue as well as in hepatocytes and their distribution in plasma membranes were studied. Overall proteoglycan production is enhanced two-fold in fetal as compared with adult liver tissue. In contrast to slices from adult liver, in which the synthesis of heparan [35S]-sulfate comprises more than 80% and chondroitin sulfate less than 5% of total glycosaminoglycans, chondroitin [35S]sulfate is the major type of glycosaminoglycans synthesized in fetal liver representing about 50% of total sulfated glycosaminoglycans. Thus, the synthesis of chondroitin sulfate is elevated nearly 30-fold in fetal liver as compared with the adult counterpart. Immediately after birth chondroitin sulfate formation decreases rapidly reaching adult levels between the 10th and 15th day of postnatal life. The production of heparan sulfate is almost unchanged during perinatal liver development due to a relatively low fractional synthesis of heparan [35S]sulfate in fetal liver. Hepatocytes were identified as the cell type responsible for elevated chondroitin sulfate production in fetal liver. Erythroblasts, which synthesize chondroitin sulfate, contribute less than 10% to total glycosaminoglycan synthesis in embryonic liver. Plasma membranes of adult liver contain exclusively heparan sulfate whereas in neonatal liver cell membranes 25% of labeled glycosaminoglycans is represented by chondroitin sulfate, a fraction which decreases rapidly after birth. In parallel to the postnatal shut down of chondroitin sulfate synthesis the activity of the UDPxylose:coreprotein xylosyltransferase (EC. 2.4.2.26) decreases from 4.8 +/- 0.5 dpm/h per microgram protein to 0.3 +/- 0.1 dpm/h per microgram protein suggesting a regulatory function of the enzyme for proteochondroitin sulfate synthesis in developing liver. The formation of both heparan sulfate and chondroitin sulfate is dependent on functioning protein synthesis, which indicates, together with double labeling experiments using [3H]serine and [14C]glucosamine as isotopic precursors, their synthesis as proteoglycans. The positive correlation (r = 0.949) between the incorporation of [3H]thymidine into DNA and chondroitin [35S]sulfate production supports the assumption of a cell growth promoting activity of chondroitin sulfate and points to a significant role of the glycosaminoglycan in the process of cellular proliferation and tissue differentiation.

The synthesis of glycosaminoglycans in aging rat liver. A brief note.

The synthesis of glycosaminoglycans (GAG) was studied in liver slices from postnatal (9 days), young (140 days), adult (490 days) and senescent (940 days) rats. It was found that the rate of synthesis was highest in postnatal rat liver and decreased to about half in young rats with no further reduction in adult and senescent age groups. The specific radioactivity of the precursors of GAG synthesis did not change with age. The synthesis pattern of specific types of GAG in postnatal liver was characterized by a significant higher percentage of chondroitin sulfate and hyaluronic acid. In the following age classes the profile of specific GAG synthesis did not change significantly (heparin sulfate: chondroitin sulfate" hyaluronic acid: "keratin sulfate" = 84%:8.3%:1.5%:1.6%).

Age-related decrease in the activity of UDP-xylose:core protein xylosyltransferase in rat costal cartilage.

The activity of UDP-xylose:core protein xylosyltransferase (EC 2.4.2.26) in costal cartilage of young rats (3 months) and old rats (36 months) was measured. The enzyme activity in cartilage of young rats (mean +/- S.D.) is 3370 +/- 1440 Bq h-1 mg-1 DNA, which is about three times higher than that determined in cartilage of old rats (1090 +/- 520 Bq h-1 mg-1 protein). The amount of galactosamine-containing proteoglycosaminoglycans that are extractable with 4 M guanidinium chloride from cartilage is significantly higher in young rats (29.1 +/- 4.8 nmol GalN per mg cartilage wet weight) than in old animals (5.8 +/- 3.0 nmol GalN per mg cartilage wet weight). Thus, if xylosyltransferase activity is referred to the amount of galactosamine-containing proteoglycans in cartilage, nearly identical values are obtained (young rats, 80 +/- 30 Bq h-1 mumol-1 GalN; old rats, 85 +/- 35 Bq h-1 mumol-1 GalN). The results support the assumption that the synthesis of proteochondroitin sulfate is diminished in costal cartilage of old rats by a mechanism involving a reduced activity of xylosyltransferase.

The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria.

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

The effect of trifluoperazine on the phosphorylation of ribosomal small subunit protein S6 in D-galactosamine-injured and normal rat liver.

The effect of the Ca2+-calmodulin antagonist trifluoperazine on the elevation of phosphorylation of rat liver small ribosomal subunit protein S6 induced by the hepatotoxic agent D-galactosamine has been studied. Trifluoperazine applied in various doses (1-100 mg/kg body wt) before injection of D-galactosamine into the rat does not reverse the strong increase in phosphorylation promoted by D-galactosamine. Instead, trifluoperazine has been identified as a potent stimulator of S6 phosphorylation in normal rat liver in vivo without causing significant changes in the cyclic AMP content of liver and the overall rate of liver protein synthesis. Both drugs, however, were not effective in stimulating the incorporation of [32P]phosphate into microsomes or crude ribosomes in liver slices in vitro. The results suggest that a calmodulin-activated protein kinase probably is not primarily engaged in S6 phosphorylation produced by D-galactosamine. However, further in vitro studies are needed to reach a definite conclusion.

Questioning the reliability of p-nitrophenyl-beta-D-xyloside as probe to study the metabolic effects of abrogated proteoglycan synthesis in cultured cells.

p-Nitrophenyl-beta-D-xylopyranoside (PNP-Xyl) and similar aglycone derivatives of xylosides are proposed selective inhibitors of proteoglycan synthesis which are used frequently to analyse the metabolic and cellular effects of abrogated proteoglycan formation and, hence, tentatively, the functions of these complex molecules. Using rat liver fat storing cell (FSC) cultures as a model, the possibility was tested that p-nitrophenol (PNP), which might be generated by the enzymatic hydrolysis of PNP-Xyl, could mediate some of those effects ascribed previously to PNP-Xyl induced inhibition of proteoglycan synthesis. PNP-Xyl and PNP inhibited dose-dependently the proliferation of FSC reaching 50% inhibition at about 1.9 and 0.6 mM, respectively. The inhibition of proliferation was not accompanied by signs of toxic cell damage and was fully reversible after withdrawal of the drugs. After an initial 4-fold stimulation of the formation of [35S]sulfate-labeled medium glycosaminoglycans (GAG) by PNP-Xyl at 0.1 mM, higher concentrations of this compound (about 0.5 mM) but also PNP decreased progressively the synthesis of sulfated medium GAG. A proliferation inhibiting concentration of PNP (0.75 mM) induced disorganization and reduced the expression of desmin- and smooth muscle iso-alpha-actin containing cytoskeletal filaments. These effects were similar to related effects reported previously for PNP-Xyl. Incubation of FSC with 5 mM PNP-Xyl resulted in a time-dependent increase of PNP in medium and cells; intracellular concentrations of PNP were reached sufficient to inhibit the mitotic activity of FSC. In lysates of FSC 0.65 nmol PNP/hr/micrograms DNA or 1 x 10(5) cells were generated from PNP-Xyl (5 mM) added as substrate. Exemplified with PNP-Xyl-treated FSC cultures, the results suggest for other cell and organ systems also that PNP, which is enzymatically cleaved from PNP-Xyl, might mediate at least some of the major effects attributed previously to the inhibition of proteoglycan synthesis. The aglycone may interfere with the effects of PNP-Xyl on proteoglycan metabolism and, therefore, could complicate in an unpredictable manner the interpretation of metabolic inhibitory studies using these compounds.

Effect of pentoxifylline on the fibrogenic functions of cultured rat liver fat-storing cells and myofibroblasts.

The effects of pentoxifylline (PTX) an analogue of the methylxanthine theobromine, on basic fibrogenic reactions of cultured fat-storing cells and myofibroblasts (MFB), the cell types most important for the excessive production of extracellular matrix components in fibrosing liver, were studied. The proliferation of MFB (i.e., activated, transdifferentiated fat-storing cells) was more dose-dependently inhibited by pentoxifylline than that of unactivated fat-storing cells (ED50 50 microgram/mL). In addition, PTX retarded the transdifferentiation of fat-storing cells into smooth muscle alpha-actin positive MFB, a 50% reduction in actin-positive cells being reached with concentrations of 0.5 mg PTX/mL medium. The transdifferentiation-associated decrease in retinyl palmitate of cultured fat-storing cells was delayed by PTX. The synthesis of [35S] sulfate-labeled glycosaminoglycans (GAG) and total and cellular fibronectin was not significantly reduced by treatment of MFB with PTX up to 1.0 mg/mL. It is concluded that PTX reduces the transdifferentiation of fat-storing cells to MFB and the proliferation of MFB, but leaves the synthesis of extracellular matrix components GAG and fibronectin unaffected. The effect of PTX on the former reactions might account for the reported antifibrogenic properties of this drug in experimental hepatic fibrogenesis.