Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures.

Goblet cell hyperplasia and overproduction of airway mucin are characteristic features of the lung epithelium of smokers and COPD patients. Tobacco heating products (THPs) are a potentially less risky alternative to combustible cigarettes, and through continued use solus THPs may reduce smoking-related disease risk. Using the MucilAir™ in vitro lung model, a 6-week feasibility study was conducted investigating the effect of repeated cigarette smoke (1R6F), THP aerosol and air exposure. Tissues were exposed to nicotine-matched whole aerosol doses 3 times/week. Endpoints assessed were dosimetry, tight-junction integrity, cilia beat frequency (CBF) and active area (AA), cytokine secretion and airway mucin MUC5AC expression. Comparison of incubator and air exposed controls indicated exposures did not have a significant effect on the transepithelial electrical resistance (TEER), CBF and AA of the tissues. Cytokine secretion indicated clear differences in secretion patterns in response to 1R6F and THP exposure. 1R6F exposure resulted in a significant decrease in the TEER and AA (p=0.000 and p=0.000, respectively), and an increase in MUC5AC positive cells (p=0.002). Repeated THP exposure did not result in a significant change in MUC5AC positive cells. This study demonstrates repeated cigarette smoke whole aerosol exposure can induce these morphological changes in vitro.

Application of text mining to develop AOP-based mucus hypersecretion genesets and confirmation with in vitro and clinical samples.

Mucus hypersecretion contributes to lung function impairment observed in COPD (chronic obstructive pulmonary disease), a tobacco smoking-related disease. A detailed mucus hypersecretion adverse outcome pathway (AOP) has been constructed from literature reviews, experimental and clinical data, mapping key events (KEs) across biological organisational hierarchy leading to an adverse outcome. AOPs can guide the development of biomarkers that are potentially predictive of diseases and support the assessment frameworks of nicotine products including electronic cigarettes. Here, we describe a method employing manual literature curation supported by a focused automated text mining approach to identify genes involved in 5 KEs contributing to decreased lung function observed in tobacco-related COPD. KE genesets were subsequently confirmed by unsupervised clustering against 3 different transcriptomic datasets including (1) in vitro acute cigarette smoke and e-cigarette aerosol exposure, (2) in vitro repeated incubation with IL-13, and (3) lung biopsies from COPD and healthy patients. The 5 KE genesets were demonstrated to be predictive of cigarette smoke exposure and mucus hypersecretion in vitro, and less conclusively predict the COPD status of lung biopsies. In conclusion, using a focused automated text mining and curation approach with experimental and clinical data supports the development of risk assessment strategies utilising AOPs.

Assessment of enamel discoloration in vitro following exposure to cigarette smoke and emissions from novel vapor and tobacco heating products.

PURPOSE: To evaluate in vitro enamel sample discoloration following exposure to a scientific reference cigarette (3R4F) or emissions from next generation tobacco and nicotine products (NGPs) such as electronic cigarettes (EC) and tobacco heating products (THP). METHODS: Bovine enamel blocks (6.5 × 6.5 mm) were prepared and pre-incubated with human or artificial saliva, to form a pellicle layer before exposure to either particulate matter (PM) or whole aerosols. PM was prepared by capturing 3R4F cigarette smoke (CS), a commercial THP (THP1.0) or a novel vapor product (NVP)/next generation e-cigarette aerosols on Cambridge filter pads followed by elution with dimethyl sulfoxide (DMSO). Ten enamel samples were exposed to each PM for 14 days. For aerosol exposure, 12 enamel samples were exposed (200 puffs per day, for 5 consecutive days) to 3R4F CS or THP1.0 and NVP aerosols. Control samples were incubated with DMSO (PM study) or phosphate buffered saline (PBS, aerosol study). Individual enamel sample color readings (L*, a*, b*) were measured at baseline and on each exposure day. Mean ΔL*, Δa*, Δb* and ΔE values were calculated for each product or control. A one-way ANOVA was used to assess the differences between the products and controls. The Tukey procedure for pairwise comparisons was also used. RESULTS: At all timepoints, 3R4F PM and CS induced enamel discoloration that was statistically significant (< 0.0001) when compared to THP1.0 or NVP. After 14-day PM exposure, mean ΔE values were 29.4± 3.6, 10.5 ± 2.3, 10.7 ± 2.6 and 12.6 ± 2.0 for 3R4F, THP1.0, NVP and DMSO control respectively. After 5-day CS or aerosol exposure, mean ΔE values were 26.2 ± 3.2, 3.6 ± 1.9, 3.4 ± 1.3, 5.3 ± 0.8 for 3R4F CS, THP1.0, NVP or PBS control, respectively. Both exposure methods demonstrated that THP1.0 and NVP induced minimal staining, mean ΔL* , Δa* , Δb* and ΔE values were comparable to DMSO or PBS controls. CLINICAL SIGNIFICANCE: For the first time, diverse NGPs across the risk continuum were assessed in vitro for their impact on enamel staining. CS exposure significantly increased the level of bovine enamel sample discoloration, whereas THP1.0 or NVP exposure resulted in values comparable to the controls.

Characterisation of the borgwaldt LM4E system for in vitro exposures to undiluted aerosols from next generation tobacco and nicotine products (NGPs).

Traditional in vitro exposure to combustible tobacco products utilise exposure systems that include the use of smoking machines to generate, dilute and deliver smoke to in vitro cell cultures. With reported lower emissions from next generation tobacco and nicotine products (NGPs), including e-cigarettes and tobacco heating products (THPs), diluting the aerosol is potentially not required. Herein we present a simplified exposure scenario to undiluted NGP aerosols, using a new puffing system called the LM4E. Nicotine delivery from an e-cigarette was used as a dosimetry marker, and was measured at source across 4 LM4E ports and in the exposure chamber. Cell viability studies, using Neutral Red Uptake (NRU) assay, were performed using H292 human lung epithelial cells, testing undiluted aerosols from an e-cigarette and a THP. E-cigarette mean nicotine generated at source was measured at 0.084 ±â€¯0.005 mg/puff with no significant differences in delivery across the 4 different ports, p = 0.268 (n = 10/port). Mean nicotine delivery from the e-cigarette to the in vitro exposure chamber (measured up to 100 puffs) was 0.046 ±â€¯0.006 mg/puff, p = 0.061. Aerosol penetration within the LM4E was 55% from source to chamber. H292 cells were exposed to undiluted e-cigarette aerosol for 2 h (240 puffs) or undiluted THP aerosol for 1 h (120 puffs). There were positive correlations between puff number and nicotine in the exposed culture media, R2 = 0.764 for the e-cigarette and R2 = 0.970 for the THP. NRU determined cell viability for e-cigarettes after 2 h' exposure resulted in 21.5 ±â€¯17.0% cell survival, however for the THP, full cytotoxicity was reached after 1-h exposure.

In vitro models for assessing the potential cardiovascular disease risk associated with cigarette smoking.

Atherosclerotic cardiovascular disease is a prevalent human disorder and a significant cause of human morbidity and mortality. A number of risk factors may predispose an individual to developing atherosclerosis, and of these factors, cigarette smoking is strongly associated with the development of cardiovascular disease. Current thinking suggests that exposure to toxicants found in cigarette smoke may be responsible for this elevated disease likelihood, and this gives rise to the idea that reductions in the levels of some smoke toxicants may reduce the harm associated with cigarette smoking. To assess the disease risk of individuals who smoke cigarettes with altered toxicant levels, a weight-of-evidence approach is required examining both exposure and disease-related endpoints. A key element of such an assessment framework are data derived from the use of in vitro models of cardiovascular disease, which when considered alongside other forms of data (e.g. from clinical studies) may support evidence of potential reduced risk. Importantly, such models may also be used to provide mechanistic insight into the effects of smoking and of smoke toxicant exposure in cardiovascular disease development. In this review the use of in vitro models of cardiovascular disease and one of the contributory factors, oxidative stress, is discussed in the context of assessing the risk potential of both conventional and modified cigarettes. Practical issues concerning the use of these models for cardiovascular disease understanding and risk assessment are highlighted and areas of development necessary to enhance the power and predictive capacity of in vitro disease models in risk assessment are discussed.

Real-time assessment of cigarette smoke particle deposition in vitro.

BACKGROUND: Recently there has been a rapid increase in approaches to assess the effects of cigarette smoke in vitro. Despite a range of gravimetric and chemical methods, there is a requirement to identify simpler and more reliable methods to quantify in vitro whole smoke dose, to support extrapolation and comparisons to human/in vivo dose. We have previously characterised an in vitro exposure system using a Borgwaldt RM20S smoking machine and a chamber exposing cellular cultures to whole smoke at the air-liquid interface. In this study we demonstrate the utility of a quartz crystal microbalance (QCM), using this exposure system, to assess real-time cigarette smoke particulate deposition during a 30 minute smoke exposure. Smoke was generated at various dilutions (1:5-1:400, smoke:air) using two cigarette products, 3R4F Kentucky reference and 1 mg commercially available cigarettes. The QCM, integrated into the chamber, assessed particulate deposition and data generated were compared to traditional chemical spectrofluorometric analysis. RESULTS: The QCM chamber was able to detect mass differences between the different products within the nanogram range. 3R4F reference cigarette smoke deposition ranged from 25.75 ±2.30 µg/cm2 (1:5) to 0.22 ±0.03 µg/cm2 (1:400). 1 mg cigarette smoke deposition was less and ranged from 1.42 ±0.26 µg/cm2 (1:5), to 0.13 ±0.02 µg/cm2 (1:100). Spectrofluorometric analysis demonstrated statistically significant correlation of particulate deposition with the QCM (p < 0.05), and regression R2 value were 97.4 %. The fitted equation for the linear model which describes the relationship is: QCM = -0.6796 + 0.9744 chemical spectrofluorescence. CONCLUSIONS: We suggest the QCM is a reliable, effective and simple tool that can be used to quantify smoke particulate deposition in real-time, in vitro and can be used to quantify other aerosols delivered to our chamber for assessment.

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine.

BACKGROUND: There have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber. RESULTS: The syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 µg/cm2)) demonstrated uniform smoke delivery within the chamber. CONCLUSIONS: These results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.

Hepatic stellate cells require a stiff environment for myofibroblastic differentiation.

The myofibroblastic differentiation of hepatic stellate cells (HSC) is a critical event in liver fibrosis and is part of the final common pathway to cirrhosis in chronic liver disease from all causes. The molecular mechanisms driving HSC differentiation are not fully understood. Because macroscopic tissue stiffening is a feature of fibrotic disease, we hypothesized that mechanical properties of the underlying matrix are a principal determinant of HSC activation. Primary rat HSC were cultured on inert polyacrylamide supports of variable but precisely defined shear modulus (stiffness) coated with different extracellular matrix proteins or poly-L-lysine. HSC differentiation was determined by cell morphology, immunofluorescence staining, and gene expression. HSC became progressively myofibroblastic as substrate stiffness increased on all coating matrices, including Matrigel. The degree rather than speed of HSC activation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. Quiescent cells on soft supports were able to undergo myofibroblastic differentiation with exposure to stiff supports. Stiffness-dependent differentiation required adhesion to matrix proteins and the generation of mechanical tension. Transforming growth factor-ß treatment enhanced differentiation on stiff supports, but was not required. HSC differentiate to myofibroblasts in vitro primarily as a function of the physical rather than the chemical properties of the substrate. HSC require a mechanically stiff substrate, with adhesion to matrix proteins and the generation of mechanical tension, to differentiate. These findings suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis.

Cigarette smoke total particulate matter increases mucous secreting cell numbers in vitro: a potential model of goblet cell hyperplasia.

Cigarette smoking is associated with chronic obstructive pulmonary disease (COPD)--a term encompassing chronic lung inflammation, chronic bronchitis and emphysema. Goblet cell hyperplasia is a characteristic feature of the lung epithelium in COPD patients contributing to the overproduction of airway mucus, including mucin MUC5AC. Using an in vitro model of differentiated lung epithelium we have investigated morphological and cellular changes in response to interleukin (IL)-13 (2.5-20 ng/ml), cigarette smoke total particulate matter (TPM; 0.31-20 microg/ml) and three mainstream cigarette smoke constituents: acrolein, formaldehyde and acetaldehyde (all at 0.001-1 microM) over 28 days during differentiation (agents replaced daily Monday-Friday). Control cultures of human bronchial epithelial cells (HBECs) underwent mucociliary differentiation producing a columnar epithelium containing goblet, ciliated and basal cells. Non-cytotoxic doses of IL-13 induced a significant increase in the percentage of MUC5AC positive cells. Using both flow cytometry and immunocytochemical techniques for identification of MUC5AC positive cells, TPM treatment induced an increase in MUC5AC positive cells, becoming maximal at 5 microg/ml. Acrolein treatment also increased the percentage of MUC5AC positive cells. However, formaldehyde or acetaldehyde had little effect. This study demonstrates that lung toxicants can induce a profound effect on cellular differentiation in an in vitro model of the human lung epithelium.

An in vitro perfusion system to examine the responses of endothelial cells to simulated flow and inflammatory stimulation.

Atherosclerosis is a disease process which develops at the arterial branches and curvatures of medium to large arteries. Local haemodynamic flow patterns in these vessels play an essential role in the formation of atherosclerotic lesions. To simulate pro-atherogenic blood flow patterns, we have developed a perfusion system with the ability to simulate in vivo patterns of blood flow in vitro. In this system, human umbilical vein endothelial cells were seeded in y-shaped microslides, in which they were exposed to a variety of flow patterns. Besides being able to reproduce the disturbed flow involved in the development of pro-atherogenic events within the arterial wall, the system also permitted the assessment of the pre-conditioning/priming effect of oscillatory flow on endothelial cells. The system was further capable of integrating multi-endpoint assays relevant to cardiovascular disease. We show that oscillatory flow primed endothelial cells, making them more sensitive to subsequent treatments. The treatment of oscillatory flow primed cells with TNFalpha resulted in the detection of enhanced levels of pro-inflammatory and chemoattractant factors such as IL-8 and MCP-1. These measurements were facilitated by the small volumes of medium circulating within the perfusion system. Oscillatory flow also altered the characteristics of monocyte adhesion to the endothelial layer. In summary, this system allows the monitoring of multiple endpoints and biomarkers, and provides an alternative to the use of in vivo and ex vivo models of cardiovascular disease.

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization.

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.

Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking.

BACKGROUND & AIMS: Liver fibrosis and cirrhosis result from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs). Previously considered irreversible, we have studied a model of cirrhosis to determine the mechanisms mediating and limiting spontaneous recovery. METHODS: A micronodular cirrhosis was induced in rats after 12 weeks of CCl(4) intoxication. Livers were analyzed for evidence of matrix degradation, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression, stellate cell apoptosis, tissue transglutaminase (tTg) expression, and matrix cross-linking during spontaneous recovery of up to 366 days. RESULTS: Over 366 days of recovery, micronodular cirrhosis underwent significant remodeling to a macronodular cirrhosis. Expression of collagen-1 and TIMP messenger RNA (mRNA) decreased significantly and active MMPs were shown in livers during remodeling of fibrosis. Resolution also was characterized by apoptosis of HSCs, predominantly at the margins of fibrotic septa. Residual septa, not remodeled at 366 days, were characterized by tTg-mediated cross-linking and relative hypocellularity. CONCLUSION: Recovery from comparatively advanced cirrhosis is possible and results in remodeling from a micronodular cirrhosis to a macronodular cirrhosis. We suggest resolution is limited by tTg-mediated matrix cross-linking and a failure of HSC apoptosis.

Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells.

Extracellular nucleotides regulate a variety of cellular activities, including proliferation of fibrogenic cells outside of the liver. However, the expression of receptors for extracellular nucleotides in hepatic stellate cells (HSC) is unknown. Thus our aims were to investigate the expression of mediators of nucleotide signaling in HSC and to determine whether extracellular nucleotides regulate HSC function. Confocal video microscopy was used to observe nucleotide-induced changes in cytosolic Ca(2+) (Ca(i)(2+)) in live HSC. P2Y receptor subtype expression and ectonucleotidase expression in quiescent and activated HSC were determined using RT-PCR, Northern blot, immunoblot, and confocal immunofluorescence. Functional ectonucleotidase activity was assessed using a colorimetric method. Nucleotide-sensitive procollagen-1 mRNA expression in activated HSC was assessed using real-time RT-PCR. Extracellular ATP increased Ca(i)(2+) in HSC; this was inhibited by the P2 receptor inhibitor suramin. Quiescent HSC expressed the P2Y subtypes P2Y(2) and P2Y(4) and were activated by ATP and UTP, whereas activated HSC expressed the P2Y subtype P2Y(6) and were activated by UDP and ATP. Activated but not quiescent HSC expressed the ectonucleotidase nucleoside triphosphate diphosphohydrolase 2, extracellular UDP tripled procollagen-1 mRNA expression in activated HSC, and this was inhibited by the P2Y receptor inhibitor suramin. HSC express functional P2Y receptors and switch the expression of P2Y receptor subtypes on activation. Moreover, HSC differentially regulate nucleoside triphosphate diphosphohydrolase expression after activation. Because activation of P2Y receptors in activated HSC regulates procollagen-1 transcription, P2Y receptors may be an attractive target to prevent or treat liver fibrosis.

Inhibitors of tryptase as mast cell-stabilizing agents in the human airways: effects of tryptase and other agonists of proteinase-activated receptor 2 on histamine release.

Tryptase, the major secretory product of human mast cells, is emerging as a new target for therapeutic intervention in allergic airways disease. We have investigated the ability of tryptase and inhibitors of tryptase to modulate histamine release from human lung mast cells and have examined the potential contribution of proteinase-activated receptor 2 (PAR2). The tryptase inhibitor APC366 [N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride] was highly effective at inhibiting histamine release stimulated by anti-IgE antibody or calcium ionophore from enzymatically dispersed human lung cells. A concentration of APC366 as low as 10 microM was able to inhibit anti-IgE-dependent histamine release by some 50%. Addition of leupeptin or the tryptic substrate N-benzoyl-D,L-arginine-p-nitroanilide also inhibited IgE-dependent histamine release. Purified tryptase in the presence of heparin stimulated a small but significant release of histamine from lung cells, suggesting that tryptase may provide an amplification signal from activated cells that may be susceptible to proteinase inhibitors. Trypsin was also able to induce histamine release apparently by a catalytic mechanism. Moreover, pretreatment of cells with metabolic inhibitors or with pertussis toxin reduced responses, indicating a noncytoxic pertussis toxin-sensitive G protein-mediated signaling process. Addition to cells of the PAR2 agonists SLIGKV-NH(2) or tc-LIGRLO-NH(2) or appropriate control peptides were without effect on histamine release, and PAR2 was not detected by immunohistochemistry in tissue mast cells. The potent actions of tryptase inhibitors as mast cell-stabilizing agents could be of value in the treatment of allergic inflammation of the respiratory tract, possibly by targeting the non-PAR2-mediated actions of tryptase.

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent.

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.

Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors.

BACKGROUND/AIMS: Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation. METHODS: Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. RESULTS: Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells. CONCLUSIONS: This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.