These aspects were particularly concentrated in areas located at the interface between fibrotic septa and the parenchyma of cirrhotic nodules, whereas they were scarce or absent in the centre of cirrhotic nodules. expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl\2. The role of Bcl\2 was crucial as M2 ion channel blocker Bcl\2 silenced cells became susceptible to TNF\ induced apoptosis. Finally, Bcl\2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. Conclusions Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl\2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases. test or ANOVA for analysis of variance when appropriate (p<0.05 was considered significant). Results Fully activated human HSC/MFs survive most proapoptotic stimuli Fully activated human HSC/MFs (passages 4C7) survived to serum deprivation for as long as 96?hours or when exposed to TNF\ (20C100?ng/ml), NGF (10C500?ng/ml), FasL (50C200?ng/ml), doxorubicin (10C200?nM), etoposide (1C25?M), and oxidative stress mediators such as hydrogen peroxide (0.1C100?M), superoxide anion and, as already reported in detail elsewhere, 4\hydroxynonenal.25 Lack of induction of apoptosis by these agents was supported by the absence of nuclear condensation/fragmentation (fig 1A, B?B),), poly (ADP ribose) polymerase (PARP) cleavage, or activation of caspase 3\like activity (not shown). In addition, no evidence of significant cell detachment was detected within Mouse monoclonal to FOXA2 48?hours of treatment (72C96?hours for serum deprivation experiments, not shown). Only etoposide, that did not induce apoptosis at any concentration (fig 1A,B?1A,B),), caused dose dependent necrotic cell death, as shown by lactate dehydrogenase (LDH) release (fig 2?2).). Gliotoxin (0.1 and 1.0?M) represented the only exception being very effective in causing rapid detachment of cells from the culture support and nuclear condensation, in agreement with previous reports.22,26 However, these changes were neither prevented by the pan\caspase inhibitor zVAD.fmk nor associated with nuclear fragmentation or a significant increase in caspase 3\like activity (data not shown), suggesting that gliotoxin may induce a form of caspase independent, apoptosis\like programmed cell death.39 Open in a separate window Figure 1?Human activated hepatic stellate cells in myofibroblast\like phenotype (HSC/MFs) survive most proapoptotic stimuli. (A) DAPI fluorescent staining for DNA was performed in human HSC/MFs exposed for 24?hours to different proapoptotic stimuli at the indicated concentrations. Representative images, relative to the highest non\necrotic doses used, are presented in (A) (original magnification 400). Arrows indicate nuclear changes (that is, DNA fragmentation) that may be immediately indicative of classical apoptosis. (B) Data are expressed as means (SEM) (three experiments for each condition) and M2 ion channel blocker summarise evaluation of per cent of condensed/fragmented nuclei for any single tested condition. *p<0.05, **p<0.01 versus control values. Act\D, actinomycin D; CHX, cycloheximide; DAPI, 4,6\diamidine\2\phenylindole di\hydrochloride; Doxo, doxorubicin; Eto, etoposide; FasL, Fas ligand; H2O2, hydrogen peroxide; NGF, nerve growth factor; TNF\, tumour necrosis factor ; X/XO, hypoxanthine/xanthine oxidase system generating superoxide anion. Open in a separate window Figure 2?Analysis of necrotic cell death. Analysis of lactate dehydrogenase (LDH) release in culture medium was analysed in human activated hepatic stellate cells in myofibroblast\like phenotype (HSC/MFs), exposed for 24?hours to the indicated conditions. Data are expressed as means (SEM) (three experiments for each condition). *p<0.05, **p<0.01 versus control values. Act\D, actinomycin D; CHX, cycloheximide; FasL, Fas ligand; H2O2, hydrogen peroxide; NGF, nerve growth factor; TNF\, tumour necrosis factor ; X/XO, M2 ion channel blocker hypoxanthine/xanthine oxidase system generating superoxide anion. Lack of apoptosis induction could not be ascribed to the absence of the cognate receptors for some of the agents used or to an incomplete apoptotic machinery because human HSC/MFs indeed express Fas (CD\95), pro\caspase 8, and pro\caspase 9 (data not shown) as well as TNF\ receptors7 and both p75 and tyrosine kinase A receptors for NGF.40 Our results clearly indicate that caspase dependent apoptosis is inducible in human M2 ion channel blocker HSC/MFs only by actinomycin D (0.1C1.0?g/ml) or relatively high dose of cycloheximide (100?g/ml), as well as by TNF\ or FasL when used in combination with lower concentrations of cycloheximide (that is, not proapoptotic per se). This was shown by the presence of nuclear condensation/fragmentation (fig 1A,B?1A,B;; 3A), absence of significant LDH release at the time of maximal apoptosis (fig 2?2),), significant effect of pan\caspase inhibitor zVAD.fmk in preventing cell death (fig 3A?3A),), and activation of caspase 3\like activity (fig 3B?3B). Open in a separate window Figure 3?Induction of apoptosis in human activated hepatic stellate cells in myofibroblast\like phenotype (HSC/MFs).