Cassia angustifolia extract is not hepatotoxic in an in vitro and in vivo study.

BACKGROUND: Cassia angustifolia L. (senna) is traditionally used as a laxative. Its major components are sennosides that are responsible for the laxative effect. Senna is recommended for the short-term treatment of acute constipation. Nevertheless people use its preparations as self-medication, often for long periods, to treat chronic constipation thus exposing themselves to adverse reactions. Most reactions were associated with hepatotoxicity. AIMS: The present study was aimed to evaluate the toxicity of a C. angustifolia leafextract (standardized at 60% of sennosides) on rat liver cells and the long-term effects on liver functions, in Wistar rats. METHODS: Cytotoxicity was assessed in a buffalo normal rat liver cell line (BRL-3A) by the trypan blue assay and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction test. In vivo effects were observed after oral administration of the extract for 4 or 8 weeks at doses of 12 and 58 mg/kg/day. At the end of treatment, animals were sacrificed, the postmortem examination was performed and serum was used for biochemical analysis. Liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters. RESULTS AND CONCLUSION: In BRL-3A cells, the extract was cytotoxic at concentrations that appear largely higher than those attainable in humans. In Wistar rats, the extract did not induce any significant change in all of the parameters tested. In summary, the present study indicates a lack of hepatotoxicity of senna at doses higher than those generally used in humans.

Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle.

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.

Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation.

BACKGROUND: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease. AIMS: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation. METHODS: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis. RESULTS: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups. CONCLUSIONS: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.

Alfa and beta estrogen receptors and the biliary tree.

This manuscript summarizes recent data showing that estrogens and their receptors play an important role in modulating cholangiocyte proliferation. We have recently demonstrated that rat cholangiocytes express both estrogen receptors (ER)-alpha and -beta subtypes, while hepatocytes only express ER-alpha. ER and especially the ER-beta subtype, are overexpressed in cholangiocytes proliferating after bile duct ligation (BDL) in the rat, in association with enlarged bile duct mass and with enhanced estradiol serum levels. Cholangiocyte proliferation, during BDL, is impaired by estrogen antagonists (tamoxifen, ICI 182,780) which furthermore, induce the overexpression of Fas antigen and activate apoptosis of proliferating cholangiocytes. 17beta-estradiol stimulates, in vitro cholangiocyte proliferation, and this effect is individually blocked by tamoxifen or ICI 182,780. Cholangiocyte proliferation during BDL was associated with an enhanced protein expression of phosphorylated extracellular regulated kinases (ERK)1/2 which is, in contrast, negatively modulated by tamoxifen in association with its antiproliferative effect. This indicates a major involvement of the ERK system in the estrogen modulation of cholangiocyte proliferation.

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle.

PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.

Investigation of G2-phase chromosomal radiosensitivity in hereditary non-polyposis colorectal cancer cells.

PURPOSE: To investigate whether cells from hereditary nonpolyposis colorectal cancer (HNPCC) patients, a genetic condition characterized by constitutional mutations in DNA mismatch repair genes and associated with predisposition to colorectal carcinoma (CRC), could present a higher G2 chromosomal radiosensitivity. It is generally hypothesized that cancer predisposition in HNPCC is associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair but, to date, no data on G2 radiosensitivity have been reported for HNPCC. MATERIALS AND METHODS: Lymphoblastoid cell lines derived from six HNPCC patients heterozygous for MLH1, one HNPCC patient carrying a mutant MSH2 allele and three healthy controls were treated with 50 cGy of X-rays and sampled at various harvesting times, monitoring cell-cycle progression by 5-bromo-2-deoxyuridine (BrdUrd) incorporation in order to analyse chromosomal damage in the homogeneous G2 population. RESULTS: There were no differences between lymphoblasts derived from patients in the frequency of G2 chromosomal aberrations induced by X-rays when compared with control cell lines. However, despite the absence of G2 radiosensitivity in HNPCC cells, lymphoblasts from patients heterozygous for MLH1 mutations showed a higher induction of chromatid exchanges. CONCLUSIONS: The observed possible incorrect rejoining of double-strand breaks in MLH1 heterozygotes would be an additional and important factor contributing to loss of heterozygosity in HNPCC patients.

Chromosome radiosensitivity in human G2 lymphocytes and cell-cycle progression.

PURPOSE: To investigate the possibility that the differential G2-phase radiosensitivity of human peripheral blood lymphocytes, found in normal individuals using the 'G2-phase chromosome radiosensitivity assay', could be attributed to heterogeneity in cellular progression to mitosis rather than differences in radiosensitivity. MATERIALS AND METHODS: Human peripheral blood lymphocytes, from four different donors, were exposed to 50 cGy X-rays and sampled at different times. The progression of cells into mitosis was monitored by 5-bromo 2'-deoxyuridine (BrdUrd) incorporation. RESULTS: The heterogeneous G2-phase chromosome radiosensitivity among different donors was abolished when homogeneous G2-phase cell populations were scored; they contained similar frequencies of cells in early or late G2-phase. CONCLUSIONS: The heterogeneous G2-phase chromosome radiosensitivity, usually found in different normal donors, is caused by the analysis of different cell populations rather than reflecting intrinsic differences in radiosensitivity.

Lack of effect of caffeine post-treatment on X-ray-induced chromosomal aberrations in Werner's syndrome lymphoblastoid cell lines: a preliminary report.

PURPOSE: To investigate whether in Werner's syndrome cells the G2 phase of the cell cycle has some abnormal response to post-treatment with agents such as caffeine and hydroxyurea known to interfere with cellular response to DNA damage. MATERIALS AND METHODS: Two Werner's syndrome lymphoblastoid cell lines (KO375 and DJG) and the normal cell line SNW646 were exposed to 50 cGy of X-rays or mitomycin-C and posttreated with caffeine or hydroxyurea in the G2 phase of the cell cycle. RESULTS: Hydroxyurea post-treatment potentiated the X-ray-induced aberration levels both in the normal and Werner's syndrome (KO375 and DJG) cell lines; in contrast caffeine was only effective in the normal cell line. Similar results were observed when Werner's syndrome cells were treated in the G1 phase with the S-dependent agent mitomycin-C and post-treated with caffeine in G2, extending the observation that Werner's syndrome cells are unaffected by caffeine G2 post-treatment. CONCLUSIONS: These results show a lack of caffeine effect in Werner's syndrome cells, suggesting an involvement of the Werner's syndrome protein in the signal transduction pathway by which caffeine could override the DNA damage induced G2 checkpoint.

Evidence that camptothecin-induced aberrations in the G(2) phase of cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription.

It is widely accepted that camptothecin (CPT) is an S-dependent genotoxin. In this study, we aimed to elucidate the 'puzzling' induction of chromosomal damage by CPT in the G(2) phase of CHO cells, where no DNA synthesis is expected, focusing the attention on the possible role of the ongoing RNA synthesis, supposed to cause the conversion of CPT-single stranded cleavage complexes spaced closely on opposite DNA strands into DNA double strand breaks (DSB's) by the action of traversing RNA polymerase.CHO AA8 and its parental mutant EM9 cell lines were pre-treated with alpha-amanitin, which prevents transcription to pre-m-RNA and challenged cells with CPT for the last hour in culture to evaluate whether G(2)-CPT-induced aberrations would have been reduced or abolished in the absence of RNA synthesis compared with G(2)-CPT treatment alone. The results obtained indicated a marked and significant reduction of aberration yields, to almost the control values (alpha-amanitin alone) when inhibition of RNA synthesis was substantial (3h total alpha-amanitin). Partial inhibition of RNA synthesis (2h total alpha-amanitin) slightly reduced the CPT-induced aberrations yield only at the high dose-level employed of CPT (20mM). This finding strongly supports the hypothesis that CPT-single stranded cleavages complexes spaced closely on opposite DNA strands are converted into DNA double strand breaks by the action of traversing RNA polymerase.

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage.

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle.

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.

Effects of low power 904 nm radiation on rat fibroblasts explanted and in vitro cultured.

In this study, 904 nm radiation was used on rat fibroblasts, explanted and in vitro cultured, to verify the action of various factors on cell growth. Parameters which can modify the behaviour of cultured cells, including the pulse repetition rate and the intensity of the radiation, the distance between the source and the irradiated dishes, the daily duration of exposure and the length of treatment, were studied. The most effective values of the intensity and pulse repetition rate which stimulate cell growth were 3 x 10(-4) W m-2 and 1.6 kHz respectively; the best stimulation was obtained at a sample-source distance of 0.1 m. The duration of daily exposure had no significant effect, whereas the best stimulus of cell growth was obtained by extending the treatment to 12 days.

The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation.

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

The RAD9-RAD1-HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling.

The WRN protein belongs to the RecQ family of DNA helicases and is implicated in replication fork restart, but how its function is regulated remains unknown. We show that WRN interacts with the 9.1.1 complex, one of the central factors of the replication checkpoint. This interaction is mediated by the binding of the RAD1 subunit to the N-terminal region of WRN and is instrumental for WRN relocalization in nuclear foci and its phosphorylation in response to replication arrest. We also find that ATR-dependent WRN phosphorylation depends on TopBP1, which is recruited by the 9.1.1 complex in response to replication arrest. Finally, we provide evidence for a cooperation between WRN and 9.1.1 complex in preventing accumulation of DNA breakage and maintaining genome integrity at naturally occurring replication fork stalling sites. Taken together, our data unveil a novel functional interplay between WRN helicase and the replication checkpoint, contributing to shed light into the molecular mechanism underlying the response to replication fork arrest.

Polycystic liver diseases.

Polycystic liver diseases (PCLDs) are genetic disorders with heterogeneous etiologies and a range of phenotypic presentations. PCLD exhibits both autosomal or recessive dominant pattern of inheritance and is characterized by the progressive development of multiple cysts, isolated or associated with polycystic kidney disease, that appear more extensive in women. Cholangiocytes have primary cilia, functionally important organelles (act as mechanosensors) that are involved in both normal developmental and pathological processes. The absence of polycystin-1, 2, and fibrocystin/polyductin, normally localized to primary cilia, represent a potential mechanism leading to cyst formation, associated with increased cell proliferation and apoptosis, enhanced fluid secretion, abnormal cell-matrix interactions, and alterations in cell polarity. Proliferative and secretive activities of cystic epithelium can be regulated by estrogens either directly or by synergizing growth factors including nerve growth factor, IGF1, FSH and VEGF. The abnormalities of primary cilia and the sensitivity to proliferative effects of estrogens and different growth factors in PCLD cystic epithelium provide the morpho-functional basis for future treatment targets, based on the possible modulation of the formation and progression of hepatic cysts.

New insights into liver stem cells.

Hepatic progenitor cells are bi-potential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic and the cholangiocytic lineages. In adult livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, representing a reserve compartment that is activated only when the mature epithelial cells of the liver are continuously damaged or inhibited in their replication, or in cases of severe cell loss. Hepatic progenitor cell activation has been described in various acute and chronic liver diseases. Their niche is composed by numerous cells such as Hepatic Stellate Cells, endothelial cells, hepatocytes, cholangiocytes, Kupffer cells, pit cells and inflammatory cells. All these cells, numerous hormones and growth factors could interact and cross-talk with progenitor cells influencing their proliferative and differentiative processes. Hepatic progenitor cells and their niche could represent, in the near future, a target for therapeutic approaches to liver disease based on cell-specific drug delivery systems. Isolation and transplantation of hepatic progenitor cells could represent a new approach for therapy of end-stage chronic liver diseases, as they offer many advantages to transplantation of mature hepatocytes. The possibility of applying stem cell therapy to liver diseases will represent a major goal in this field.

Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor.

BACKGROUND: Estrogens may induce the proliferation of neoplastic cells by activating neo-angiogenesis. AIM: To evaluate the effect of estrogens on the expression of vascular endothelial growth factor (VEGF) and related receptors (VEGF-R) in human cholangiocarcinoma and the role played by VEGF in mediating the proliferative effects of estrogens. METHODS: Seven biopsies of intra-hepatic cholangiocarcinoma and the HuH-28 cell lines were investigated. Cell proliferation was measured by both PCNA Western blot and MTS proliferation assay. RESULTS: By immunohistochemistry, biopsies of human cholangiocarcinoma stained positively for VEGF-A and VEGF-C and related receptors. HuH-28 cells expressed VEGF-A, -C, and VEGFR-1, -2, -3 and, their protein level was enhanced by 17beta-estradiol in association with the stimulation of cell proliferation. 17beta-Estradiol-stimulated proliferation of HuH-28 cells was blocked by 70% by VEGF-TRAP, a receptor-based VEGF inhibitor. 17beta-Estradiol induced the secretion of VEGF in the supernatant of HuH-28 cells. The stimulatory effect of 17beta-estradiol on the protein expression of VEGF-A, VEGF-C and VEGFR-1, -2, -3 was blocked by antagonists of ER (Ici182,780) or insulin-like growth factor 1-receptor (alphaIR3). CONCLUSIONS: With the limitations of experiments performed in a cell line, our study indicates that VEGF plays a major role in mediating the proliferative effects of estrogens on human cholangiocarcinoma.

The logic and regulation of cell cycle exit and reentry.

Tissue repair and regeneration are very complex biological events, whose successful attainment requires far more than mere cell division. However, almost unavoidably they entail cell proliferation as a fundamental premise. Full regeneration or repair cannot be achieved without replacing cells lost to disease or injury, replacement that can only take place via proliferation of surviving cells. This review endeavors to outline the molecular bases of exit from and reentry into the cell cycle. In recent years, the decision to proliferate or not has been seen as mostly the concern of cyclins and cyclin-dependent kinases. This account tries to show that cell cycle inhibitors are as important as the positive regulators in the making of this decision. Finally, the authors wish to suggest that the molecular knowledge of the cell cycle can be harnessed to the benefit of many aspects of regenerative medicine.

Effects of Cimicifuga racemosa extract on liver morphology and hepatic function indices.

UNLABELLED: Cimicifuga racemosa (black cohosh) is a herbaceous perennial plant, that has been traditionally used for a variety of ailments (dyspepsia, climacteric complaints, muscular rheumatisms, menstrual cramps). From laboratory and clinical studies, black cohosh seems to have a relatively good safety profile, even if a number of case reports of hepatotoxicity were a matter of recent concern. AIM: A number of case reports indicated that C. racemosa could induce hepatotoxicity. We evaluated the effects of black cohosh extract on liver morphology, and on levels of various hepatic function indices in rats. METHODS: Wistar rats received 300mg/kg/day of C. racemosa extract by gavage, for 30 days. Biochemical analysis of serum was conducted by an automated, random-access clinical chemistry analyzer. Liver samples were used for hystomorphological and immunohistochemical examination, for the detection of apoptosis (TUNEL assay), and for the determination of GSH level (spectrophotometrical analysis). RESULTS: C. racemosa extract does not affect liver morphology and hepatic function indices, in rats. CONCLUSIONS: On the basis of experimental data, the use of 300mg/kg/day of black cohosh appears quite safe in rats. Nevertheless, in humans the safety of C. racemosa should be further monitored, in terms of patient-related factors.

Hepatic microcirculation and cholangiocyte physiopathology.

BACKGROUND: The peribiliary plexus (PBP) plays a fundamental role in supporting the functions of the biliary epithelium. After common bile duct ligation (BDL) progressive PBP proliferation is demonstrated. We have, recently, demonstrated that the biliary epithelium express Vascular Endothelial Growth Factor (VEGF), both subtype -A and -B and VEGF receptors. Taking in consideration the wide extension of PBP during BDL, aim of our study is to investigate the role of VEGF in stimulating angiogenesis and also in the modulation of epithelial cells proliferation. MATERIAL AND METHODS: Experimental studies were performed by evaluating the effects of: a) endogenous VEGF neutralization by chronic administration of anti VEGF-C antibody on cholangiocyte proliferation in BDL rats and; b) the hepatic artery ligation (HAL) immediately after BDL followed by treatment (7 days) with a recombinant of VEGF-A (administered through IP implanted minipumps) on cholangiocyte proliferative activities. RESULTS: Both administration of antiVEGF-C antibody and HAL decreases cholangiocyte proliferation. The decrease of cholangiocyte proliferation was associated with depressed VEGF-A protein expression. The administration of rVEGF-A to BDL, hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats. CONCLUSION: These data suggest that VEGF-C modulates the proliferative activities of cholangiocytes in experimental cholestasis and that circulating factors (i.e., VEGF) in the blood supply of the intra-hepatic biliary epithelium, play an important role in the balance between cholangiocyte proliferation/loss.