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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response. Results: (1) the effects of ethanol and AA on KC and HSC were as follows: Cell proliferation, lipid peroxidation, H2O2, O2·, nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-α) were higher in KCethanol than in KCcontrol, and were enhanced by AA; HSCethanol proliferated faster, increased collagen, and showed higher GSH than HSCcontrol, with modest effects by AA. (2) AA effects on the control co-culture: We previously reported the ability of KC to induce a pro-fibrogenic response in HSC via reactive oxygen species (ROS)-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). (3) Ethanol effects on the co-cultures: Co-culture with KCcontrol or KCethanol induced HSCcontrol and HSCethanol proliferation; however, the pro-fibrogenic response in HSCethanol was suppressed because of up-regulation of TNF-α and GSH, which was prevented by a TNF-α neutralizing antibody (Ab) and by L-buthionine-sulfoximine, a GSH-depleting agent. (4) Ethanol plus AA effects on the co-cultures: AA lowered TNF-α in the HSCcontrol co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSCethanol co-cultures by counteracting the up-regulation of TNF-α and GSH with a significant increase in GSSG and in pro-fibrogenic transforming growth factor beta (TGF-β). Conclusion: These results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM remodeling and suggest that even if chronic ethanol consumption sensitizes HSC to up-regulate anti-fibrogenic signals, their effects are blunted by a second “hit” such as AA. (HEPATOLOGY 2008;48:2027-2039.)

Under normal conditions, the space of Disse contains a non–electron-dense basement membrane–like matrix, which is essential for maintaining the differentiated function of all resident liver cells.1 As the liver becomes fibrotic, the total content of collagens increases and it is accompanied by a shift in the subendothelial space, from normal low-density basement membrane to one enriched in fibrillar collagens type I and III.1 Decades of elegant studies have defined that up-regulation of collagen I synthesis during hepatic stellate cell (HSC) activation is among the most remarkable molecular responses to injury mediated by multiple signals, many of which remain unknown.2–11

Paracrine stimuli initiating HSC activation derive from injured hepatocytes9, 10, 12 and neighboring Kupffer cells (KC)2, 13 in addition to swift and subtle changes in extracellular matrix (ECM) composition. Evidence for a role of KC early on in ethanol-induced liver injury springs from their elevated production of reactive oxygen species (ROS), cytokines, and growth factors that provide pivotal effects on all other liver cells.14–24 Recruitment of pro-inflammatory cells also occurs, and on arriving at the site of injury they release multiple mediators that further activate HSC and enhance collagen I deposition.25 In the final step, maturation and organization of ECM occurs, connective tissue fibers retract, and most of the cellular infiltrates disappear.26 At this stage, when the liver becomes cirrhotic, the end-stage of fibrosis, there is insufficient remodeling, and liver injury becomes nearly irreversible.27 Thus, efforts to understand fibrosis focus primarily on the early events or “hits” that lead to accumulation of scar in hope of identifying therapeutic targets to slow its progression or help its resolution.1

Chronic injury leading to liver fibrosis takes place in response to alcohol abuse. Indeed, generation of ROS by KC increases under ethanol treatment, and a role for n-3 and n-6 series polyunsaturated fatty acids (PUFAs)11, 28–30 needs to be considered as they accelerate the development of alcoholic liver disease. In fact, pathological changes occur mostly in rats fed ethanol with PUFAs.28 Therefore, both ethanol and PUFAs add an additional layer of complexity to the elaborate crosstalk between KC and HSC.

Understanding the dynamics of how alcohol and PUFAs regulate KC effects on HSC behavior is of great value for pharmacological design to help prevent liver injury. We have previously shown that KC modulate the fibrogenic response in HSC through ROS-dependent mechanisms.2 To gain additional insight, a co-culture model was established using KC and HSC from control and from ethanol-fed rats and arachidonic acid (AA), as a representative n-6 series PUFA, was added. The results described herein advance the field by unveiling potential synergism between ethanol and AA to the mechanism whereby KC modulate ECM deposition and remodeling. The data suggest that chronic ethanol consumption sensitizes HSC in the presence of KC to up-regulate two powerful anti-fibrogenic mediators such as tumor necrosis factor alpha (TNF-α) and reduced glutathione (GSH). These signals may be switched on in HSC in the early stages of alcohol-induced liver injury, even though uncoordinated HSC proliferation may coexist, likely reflecting that KC could drive the HSC phenotype to a more pro-inflammatory or proliferative and to a less fibrogenic behavior. However, a second “hit” such as PUFAs (like AA) may tip the balance favoring scarring by lowering TNF-α, depleting GSH stores, increasing oxidized glutathione (GSSG), and significantly up-regulating transforming growth factor beta (TGF-β), a powerful pro-fibrogenic mediator.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chronic Alcohol Feeding Model.

Rats (300-g female Sprague-Dawley, n = 10/group) were fed the control or ethanol Lieber-DeCarli diets for 8 months.31 Animals received humane care according to the criteria outlined in the Guide for Care and Use of Laboratory Animals. Alanine aminotransferase, aspartate aminotransferase, ethanol, and nonesterified fatty acids were assayed using kits from Thermo Electron Corporation (Waltham, MA), Sigma (St. Louis, MO), and Wako Chemicals (Richmond, VA), respectively. Hematoxylin and eosin and transmission electron microscopy sections were evaluated by a liver pathologist.

Co-cultures.

Primary rat KC and HSC were isolated by differential centrifugation and elutriation as previously described.2 Buoyancy was similar in HSCcontrol and in HSCethanol (Fig. 1C, bottom), allowing for isolation of similar HSC populations in the density gradient. KC were additionally purified by cell sorting using CD163-fluorescein isothiocyanate, a KC-specific marker, and by selective attachment. Details on the co-culture model can be found in an earlier publication.2

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Figure 1. Pathology and ultrastructural studies. Hematoxylin and eosin staining in livers from control rats showed minimal steatosis (top), whereas rats fed ethanol showed periportal and pericentral microvesicular and macrovesicular steatosis (original magnification 200×) (A). Aspartate aminotransferase and alanine aminotransferase activities, plasma ethanol levels, and plasma nonesterified fatty acids. Results are average values ± SEM, n = 10; •••P < 0.001 for ethanol versus control; ND, not detected (B). Ultrastructural analysis depicting microvesicular and macrovesicular steatosis ([RIGHTWARDS ARROW]), vacuolization (V), and electron-dense mitochondria (M) in KCethanol versus KCcontrol (C, top), and similar buoyancy or lipid droplets (LD) but more dilated ER (▸), lysosomes (L), and electron sense mitochondria (M) in HSCethanol versus HSCcontrol (C, bottom).

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General Methodology.

Endotoxin-free AA, to avoid cellular activation, was added in the presence of bovine serum albumin as a carrier. Cell viability was determined by the (3-(4,5-Dimethylthiazol-2-yl)-2,5-phenyltetrazolium bromide (MTT) assay. Cell proliferation was estimated by methyl[3H]-thymidine incorporation into HSC DNA.9 Apoptosis was measured by flow cytometry with a caspase 3/7 Kit (Invitrogen, Carlsbad, CA). Intracellular and extracellular TNF-α were evaluated by flow cytometry with anti-rat TNF-α phycoerythrin-conjugated antibody (Ab) (BD Pharmingen, San Jose, CA) and by enzyme-linked immunosorbent assay (Biosource, Camarillo, CA), respectively. Intracellular lipid peroxidation was determined by cell sorting using cis-parinaric acid (Invitrogen). Thiobarbituric acid reactive substances were assayed as described.32 Intracellular hydroperoxides (mostly H2O2) and O2· were assessed by flow cytometry using the fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) and dihydroethidium, respectively (Invitrogen). Extracellular hydroperoxides (mainly H2O2) were measured by the ferrous oxidation–xylenol orange assay.33 Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity was determined using lucigenin.34 GSH and GSSG were determined by the recycling method of Tietze35 and Anderson,36 respectively.

Western Blot Analysis.

Anti-collagen type I Ab was provided by Dr. Schuppan (Harvard Medical School, Boston, MA).37 Collagen type I was detected as several high-molecular-weight chains of pro-collagen α1(I) and α2(I) and N-terminally processed pCα1(I) and pCα2(I). For intracellular collagen I, the α1 chain is 139 kDa, and the α2 chain is 129 kDa. In the culture media, intact pro-collagen I predominated along with fully processed collagen I.9, 10, 37 The quantification under the blots refers to the sum of bands from all collagen I isoforms. Alpha-smooth muscle antigen (α-SMA) and β-tubulin Abs were from Sigma (St. Louis, MO).

Statistical Analysis.

Data were analyzed by a two-factor analysis of variance, and results are expressed as means ± standard error of the mean (SEM) (at least n > 3).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chronic Alcohol Feeding Model.

Primary HSC and KC were isolated from rats fed with either control or ethanol Lieber-DeCarli diets. Hematoxylin and eosin staining showed microvesicular and macrovesicular steatosis in livers from ethanol-fed rats (Fig. 1A, bottom versus top). Transaminases and nonesterified fatty acids were elevated twofold and sixfold in the ethanol-fed rats, respectively, and plasma ethanol was 120 mg/dL (Fig. 1B). Ultrastructural analysis depicted microvesicular and macrovesicular steatosis ([RIGHTWARDS ARROW]), vacuolization (V), and some electron-dense mitochondria (M) in KCethanol versus KCcontrol (Fig. 1C, top). More dilated endoplasmic reticulum (▸) and electron-dense mitochondria (M) were observed in HSCethanol versus HSCcontrol, whereas analogous content of lipid droplets (LD) was observed in both (Fig. 1C, bottom). Immediately after perfusion (t = 0 days), collagen I and α-SMA, markers of HSC activation, as well as HSC counts, were higher for HSCethanol compared with HSCcontrol (not shown).

AA and Ethanol Induce Proliferation Rates and Activate KC.

Intracellular concentrations of AA in humans are in the μM range and are approximately 10% of total fatty acids in rat plasma,38 depending on metabolic and nutritional status. Both n-3 and n-6 series PUFAs are effective in increasing collagen I in co-cultures of mouse HSC with pooled hepatocytes, KC, and endothelial cells.11 We chose AA to compare its pro-fibrogenic effects in our model with those described by us in co-cultures of rat HSC with hepatocytes.9 Initially, KCcontrol and KCethanol were incubated with increasing doses of AA up to 10 μM and showed dose-dependent cell activation and ROS production (not shown). KC viability was similar in the presence of 0 to 10 μM AA at 24 hours (Suppl. Fig. 1A). Proliferation rates at 24 hours were slightly increased in KCethanol compared with KCcontrol, and AA further elevated cell proliferation (Suppl. Fig. 1B). Moreover, light micrographs indicated that AA induced phenotypic changes in KC such as stretching and generation of cytoplasmic processes and filopodia, suggestive of cell activation (Suppl. Fig. 1C, right versus left panels). These effects were more apparent in KCethanol than in KCcontrol (Suppl. Fig. 1C, bottom versus top panels).

Ethanol and AA Synergize to Increase Oxidant Stress in KC.

Intracellular lipid peroxidation end products, hydroperoxides (mostly H2O2), and O2·, as well as extracellular thiobarbituric acid reactive substances and hydroperoxides, which could impact HSC, were assessed to determine whether the effects observed in KCcontrol and in KCethanol under AA treatment were synergistic to those mediated by chronic alcohol feeding. There was a 2.4-fold and an approximately 12% increase in intracellular and extracellular lipid peroxidation-derived products in KCethanol versus KCcontrol(Fig. 2A, B, white bars). AA further enhanced twofold lipid peroxidation. Levels were greater in KCethanol (Fig. 2A, B, black versus white bars). Moreover, intracellular and extracellular hydroperoxides appeared induced 2.2-fold and approximately 40% in KCethanol versus KCcontrol(Fig. 2C, D, white bars). Likewise, addition of AA elevated hydroperoxides. Values were significantly higher in KCethanol than in KCcontrol(Fig. 2C, D, black versus white bars). Lastly, intracellular O2· was elevated twofold in KCethanol versus KCcontrol(Fig. 2E, white bars) and were enhanced by 2.2-fold by AA in both (Fig. 2E, black versus white bars). Therefore, synergism between ethanol and AA in activating KC and generating ROS appeared likely.

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Figure 2. AA Induces Oxidant Stress Mostly in KCethanol. KCcontrol and KCethanol were incubated with 0-10 μM AA for 24 hours. Intracellular lipid peroxidation assessed by cis-parinaric acid fluorescence (A). Lipid peroxidation end products secreted to the culture medium are shown as nmol thiobarbituric acid reactive substances (B). Intracellular hydroperoxides (mostly H2O2) were measured by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (C). Levels of hydroperoxides (mainly H2O2) released to the culture medium were evaluated by the FOX method (D). Intracellular O2· was determined by flow cytometry using dihydroethidium (E). In all panels, results are expressed as means ± SEM of n > 3. *P < 0.05, **P < 0.01, and ***P < 0.001 for AA-treated versus nontreated, and •P < 0.05, ••P < 0.01, and •••P < 0.001 for ethanol versus control.

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Ethanol and AA Increase NADPH Oxidase Activity, TNF-α, and Apoptosis in KC.

To determine the source of ROS, we next explored possible events occurring in KC that may play a role in regulating the HSC fibrogenic response. KCcontrol and KCethanol were incubated with 0 to 10 μM AA for 24 hours and NADPH oxidase activity —a source of ROS— intracellular TNF-α —an anti-fibrogenic agent— and caspase 3/7 —a marker for apoptosis— were evaluated. KCethanol showed elevated NADPH oxidase activity over that of KCcontrol (Fig. 3A, white bars) and AA further induced it by twofold (Fig. 3A, black versus white bars). Cytochrome P450 2E1 and xanthine oxidase remained similar under either ethanol or AA treatment (not shown). TNF-α was twofold increased in KCethanol versus KCcontrol(Fig. 3B, white bars). Moreover, there was a twofold up-regulation in intracellular TNF-α in KCcontroland a 2.5-fold increase in KCethanoltreated with AA (Fig. 3B, black versus white bars). Lastly, the 6.5-fold increase in caspase 3/7 activity in KCethanol versus KCcontrol (Fig. 3C, white bars) was further enhanced by AA in KCcontrol and in KCethanol (Fig. 3C, black versus white bars). A summary of the main effects on KC can be found in Fig. 3D.

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Figure 3. Ethanol and AA increase NADPH oxidase, TNF-α, and Caspase 3/7. NADPH oxidase activity in KCcontrol and in KCethanol incubated with 0-10 μM AA for 24 hours (A). Intracellular TNF-α (B) and caspase 3/7 activities (C) evaluated by flow cytometry. In all panels, results are average values ± SEM of n = 4, *P < 0.05, **P < 0.01, ***P < 0.001 for AA-treated versus nontreated and •P < 0.05, ••P < 0.01, and •••P < 0.001 for ethanol versus control. Summary of the effects of chronic ethanol consumption and AA on KC; [UPWARDS ARROW]: up-regulation by ethanol; equation image : up-regulated by AA (D).

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KC Promote HSC Activation, Proliferation, and Collagen I Deposition in the Presence of AA.

We previously demonstrated the ability of KC to induce the HSC pro-fibrogenic behavior via ROS-dependent mechanisms.2 To further our knowledge, we questioned whether KC could enhance the pro-fibrogenic behavior of HSC in the presence of AA as well. Using the co-culture model (Fig. 4A),2 phenotypical changes in HSC co-cultured with KC, such as stretching and generation of cytosolic processes establishing contacts among cells, were more apparent in HSC co-cultured with KC than in HSC cultured alone (Fig. 4B, HSC/KC versus HSC). AA enhanced the activated phenotype in the co-cultured HSC (Fig. 4B, HSC/KC + AA versus HSC/KC). Consistent with the light micrographs, there was a fourfold increase in proliferation of HSC in co-culture compared with HSC cultured alone (Fig. 4C, solid lines). Addition of AA enhanced 1.7-fold HSC proliferation rates only in the co-culture (Fig. 4C, top dashed line versus solid line). Likewise, the already elevated ROS levels in the co-culture2 were synergistically enhanced by AA (not shown).

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Figure 4. KC promote HSC activation, proliferation, and collagen I deposition in the presence of AA. Co-culture model: After liver perfusion, elutriation, and cell sorting with CD163-fluorescein isothiocyanate (inset), 5 × 105 KC were plated on cell culture inserts and 1.5 × 105 HSC were seeded on the bottom plates in 4 mL Dulbecco's minimum essential medium F12 containing 10% fetal bovine serum. One hour later, the KC medium was replaced to remove any remaining endothelial cells that do not adhere at short times after plating while KC do. Twenty-four hours after, the medium was discarded and either empty inserts (co-culture controls) or inserts containing KC were transferred onto the HSC plates. Fresh serum-free medium (4 mL) was added and samples of HSC lysates or culture medium were collected at selected times (A). Light micrographs of primary HSC cultured alone or with KC in the presence of AA (original magnification ×200) (B). Methyl[3H]-thymidine incorporation into the DNA of HSC cultured alone or with KC in the presence of AA (C). Cells were co-cultured up to 7 days in the presence of AA and samples of cell lysate and culture medium were analyzed for intracellular and extracellular collagen I and β-tubulin (loading control) by western blot. Results are in arbitrary densitometry units under the blots, and the quantification of the signal corrected by that of β-tubulin was referred to the nontreated HSC, which was assigned a value of 1 (D). In (C) and (D), results are average values ± SEM of n = 4, *P < 0.05 and ***P < 0.001 for AA-treated versus nontreated, ••P < 0.01 and •••P < 0.001 for co-culture versus control. Primary HSC cultured alone or with KC were incubated with vitamin E (25 μM) —an antioxidant— and DPI (1 nM) —an inhibitor of NADPH oxidase— 2 hours before addition of AA and intracellular collagen I and β-tubulin (loading control) were evaluated by western blot. Results are in arbitrary densitometry units under the blots and are average values of n = 3 (SEM not shown) (E).

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We previously reported that co-culture with KC increased HSC collagen I expression.2 We now show that AA treatment further elevated both intracellular and extracellular collagen I in the co-culture 2.3-fold (Fig. 4D, lane 4 versus lane 2). To establish a link between the availability of AA-derived ROS (Fig. 2, and not shown), activation of NADPH oxidase (Fig. 3A), and the fibrogenic response (Fig. 4D), HSC cultured alone or with KC were incubated with an antioxidant (vitamin E) or a NADPH oxidase inhibitor [diphenyleneiodonium (DPI)] 2 hours before AA addition. Both treatments prevented the increase in collagen I by AA; however, DPI appeared more effective (Fig. 4E; lanes 8 and 12 versus lane 4).

Chronic Ethanol Feeding Enhances HSC Activation.

Before determining the potential synergistic effects of ethanol and AA on the HSC pro-fibrogenic response, we analyzed the basal effects of chronic ethanol consumption in HSC cultured alone or with KC. Both HSC and KC were isolated from rats fed the control or the ethanol Lieber-DeCarli diets and co-cultured. HSCethanol proliferated faster than HSCcontrol (Fig. 5A, B, dotted lines). KCcontrol and KCethanol elevated HSCcontrol and HSCethanol proliferation rates; however, effects were greater in the presence of KCethanol(Fig. 5A, B, solid versus dashed lanes for the KCethanol effect, and dashed versus dotted lines for the KCcontrol effect).

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Figure 5. KC enhance proliferation rates mostly in HSCethanol. A time-course experiment up to 6 days was carried out with HSCcontrol and HSCethanol co-cultured with empty inserts (control co-cultures) or with inserts containing KCcontrol or KCethanol (therefore, six culture conditions). Light micrographs of primary HSCcontrol and of HSCethanol co-cultured for 6 days with KCcontrol or KCethanol (original magnification ×200). (A) Cell proliferation assessed by the rate of incorporation of methyl[3H]-thymidine into the DNA of HSC. Results are expressed as average values ± SEM of n = 4; the significance is not indicated in the graph because of limited space (B).

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Divergent Response of HSCethanol to the Pro-Fibrogenic Effects Triggered by KC.

To address the potential KC-derived effects by ethanol on the fibrogenic response, intracellular and extracellular collagen I and α-SMA were analyzed and found to be elevated in HSCethanol compared with HSCcontrol cultured alone (Fig. 6A, compare HSCcontrol and HSCethanol at 3 days —lane 4 versus lane 1— and at 6 days —lane 10 versus lane 7—. This is in agreement with the enhanced proliferation rate of HSCethanol (Fig. 5A, B) and with the change in phenotype (Fig. 5A, top panels). Co-culture of HSCcontrol with either KCcontrol or KCethanol increased intracellular and secreted collagen I levels (Fig. 6A, lanes 2 and 3 versus lane 1 at 3 days, and lanes 8 and 9 versus lane 7 at 6 days).2 However, co-culture of HSCethanolwith either type of KC lowered intracellular and secreted collagen I and α-SMA (Fig. 6A, lanes 5 and 6 versus lane 4 at 3 days, and lanes 11 and 12 versus lane 10 at 6 days). Thus, KC had a pro-fibrogenic effect on HSCcontrol but an apparent anti-fibrogenic effect on HSCethanol.2

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Figure 6. TNF-α and GSH Up-Regulation Plays a Role in Modulating the Fibrogenic Response in HSCethanol. HSCcontrol and HSCethanol co-cultures were evaluated for intracellular and extracellular collagen I, α-SMA, and β-tubulin (loading control) by western blot analysis. The quantification of the signal was referred to that of the non-treated HSC at 3 days, which was assigned a value of 1 (A). Levels of TNF-α in the culture medium at 6 days of co-culture (B). Intracellular HSC GSH (C) and GSSG levels (C, inset). The HSCethanol co-cultures were incubated with a TNF-α neutralizing Ab —to block TNF-α— or with L-buthionine sulfoximine —to deplete GSH—; and intracellular and extracellular collagen I and β-tubulin expression were analyzed by western blot. Results are expressed as arbitrary densitometry units under the blots; the quantification of the signal was referred to that of the nontreated HSC, which was assigned a value of 1 (D). Results are average values ± SEM [SEM not shown in (A) and (D)] n = 4, •P < 0.05, ••P < 0.01, and •••P < 0.001 for co-culture versus control, and **P < 0.01, and ***P < 0.001 for HSCethanol versus HSCcontrol.

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TNF-α and GSH Mediate the Anti-fibrogenic Action of KC on HSCethanol.

Because of the ability of ethanol to induce ROS, ROS-sensitive cytokines, and growth factors, we analyzed the expression of candidate anti-fibrogenic signals that could mediate the down-regulation of collagen I in the HSCethanol co-cultures. Although a minor increase in TNF-α was observed in HSCcontrol co-cultured with KCcontrol or with KCethanol(Fig. 6B, left bars), there was an 11-fold and a 22-fold increase in secreted TNF-α in HSCethanol co-cultured with KCcontrol and with KCethanol, respectively (Fig. 6B, right bars). Although the antioxidant defense in HSC was similar under all culture conditions (not shown), there was a twofold increase in GSH levels in HSCethanol versus HSCcontrol(Fig. 6C, white bars). A modest decrease in HSC GSH was observed by co-culture with either KC type, whereas GSSG remained similar (Fig. 6C, inset). We then evaluated whether through regulating TNF-α and GSH levels, collagen I expression could be restored to that of non–co-cultured HSCethanol. Addition of a TNF-α neutralizing Ab or treatment with L-buthionine sulfoximine, which depletes GSH pools, restored intracellular and extracellular collagen I expression to that found in nontreated and non–co-cultured HSCethanol (Fig. 6D; lanes 5, 6, 8, and 9 versus lane 1), suggesting a role for TNF-α and GSH on collagen I expression in the HSCethanol co-cultures.

AA Induces Collagen I Expression in the HSCethanol Co-Cultures by Lowering HSCethanol GSH and TNF-α and increasing GSSG and TGF-β.

To define the potential effects of AA as a second “hit”, the control and the ethanol co-cultures were incubated with AA and intracellular and extracellular collagen I was analyzed. HSCcontrol were responsive to AA, both when cultured alone or when either KC type was present, increasing collagen I expression (Fig. 7A, lanes 4–6 versus lanes 1–3). Moreover, HSCethanol, which had lower collagen I in the co-culture with either KC type, appeared responsive to AA, restoring or sustaining collagen I induction above values of non—co-cultured HSCethanol (Fig. 7A, lanes 10–12 versus lanes 7–9). To determine whether TNF-α and GSH played a role in the effects mediated by AA in the HSCethanol co-cultures, secreted TNF-α and HSC intracellular GSH were measured. Addition of AA lowered secreted TNF-α slightly in the HSCcontrol and considerably in the HSCethanol co-cultures. Similar results were observed for GSH pools, whereas GSSG increased (Figs. 7B-D, light and dark gray versus white bars). More importantly, AA triggered a twofold and a 10-fold increase in TGF-β in the HSCcontrol and HSCethanolco-cultures, respectively (Fig. 7E, light and dark gray versus white bars). Therefore, GSSG and TGF-β, a well-known pro-fibrogenic factor, appeared to counteract the anti-fibrogenic signals (that is, TNF-α and GSH) coexisting in the HSCethanolco-culture.

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Figure 7. AA Restores the Pro-Fibrogenic Response in the HSCethanol Co-culture. The co-cultures were treated with AA and intracellular and extracellular collagen I and β-tubulin expression were analyzed by western blot. Results are expressed as arbitrary densitometry units under the blots and are average values of n = 3; the quantification of the signal was referred to that of the nontreated HSCcontrol, which was assigned a value of 1 (A). Under the same experimental conditions as in (A), levels of TNF-α in the culture medium (B), intracellular HSC GSH (C), HSC GSSG (D), and TGF-β (E) are shown. Results are average values ± SEM (SEM not shown in panel A) of n = 4, •P < 0.05, ••P < 0.01, and •••P < 0.001 for co-culture versus control, *P < 0.05, **P < 0.01, and ***P < 0.001 for HSCethanol versus HSCcontrol, and ▪P < 0.05, ▪▪P < 0.01, and ▪▪▪P < 0.001 for AA-treated versus nontreated.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We previously developed a co-culture system to explore the crosstalk between KC and HSC.2 We now focused on delineating potential mechanisms whereby administration of two “hits”, chronic ethanol feeding and a representative PUFA (such as AA), could modulate KC activation and the fibrogenic response in HSC and provide additional evidence on the complex intercellular communication that links both cell types with pathological ECM remodeling (for a summary of the main findings, see Fig. 8).

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Figure 8. Summary of Results and Proposed Model. I. Effects of chronic alcohol consumption and AA on HSC; II. Effects of AA in the control co-culture; the gray box refers to previous findings published in Hepatology 2006;44(6);1487–1501; III. Effects of AA in the co-culture of HSCcontrol with KCethanol; IV. Effects of AA in the co-culture of HSCethanol with KCcontrol or KCethanol. The contribution of AA is indicated in bold face. [UPWARDS ARROW]: up-regulation by ethanol, [DOWNWARDS ARROW]: down-regulation by ethanol, equation image : up-regulated by AA, equation image: down-regulated by AA, ≈: slight change.

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As a first step, we investigated the effects of chronic ethanol feeding and addition of AA on KC function and the release of critical mediators that could impact on ECM remodeling. KCethanol had a more activated phenotype, proliferated faster, and were more apoptotic than KCcontrol, and these effects were increased by AA. We then speculated that ROS would be the likely candidates for the above-described phenotype that could drive downstream effects on HSC. As previously reported by others,39, 40 KCethanol generated more ROS than KCcontrol, and generation of these species was further enhanced by AA. Likewise, NADPH oxidase activity, a key pro-oxidant enzyme well-known to play a role in alcoholic liver disease,19, 41 increased in KCethanol compared with KCcontrol, and AA synergistically up-regulated the activity. Because growth factors are highly ROS-sensitive and, as a corollary, they could regulate ROS production in KC, we evaluated levels of TNF-α, known to be increased in alcoholic liver disease,22, 42 and to contribute to collagen I expression.7 Indeed, KCethanol showed elevated TNF-α, which was up-regulated by AA as well.

Once the basal effects of ethanol and AA on KC were identified, we determined the contribution of AA to the fibrogenic response in HSC, using the control co-culture previously established, and observed that the induced HSC activation, proliferation, ROS generation, and collagen I expression2 was additionally enhanced by AA. Vitamin E, an antioxidant that inhibits the cascade of lipid peroxidation-derived reactions, prevented the increase in collagen I by AA only in the co-culture. Because vitamin E directly affected the response to AA by lowering ROS production in KC cultured alone, its antifibrogenic potential was likely restricted to KC-derived ROS. However, DPI blunted the up-regulation of collagen I by both the co-culture and the AA treatment. DPI inhibits NADPH oxidase not only in KC but also in HSC,39 which could as well play a role, and may account for its maximal effects on collagen I down-regulation.

Next, we assessed the involvement of chronic alcohol feeding to the effects mediated by KC on HSC. Chronic ethanol feeding induced activation, cell proliferation, collagen I expression, and GSH levels in HSC cultured alone. The co-cultures established with cells isolated from control and from ethanol-fed rats indicated that HSCcontrol and HSCethanol proliferation rates increase in the co-cultures and are higher in the presence of KCethanol. HSCcontrol responded to KCcontrol and KCethanol signals up-regulating collagen I; however, HSCethanol were resistant. Based on the above-identified mediators released by KC, we measured GSH and TNF-α in the co-cultures as candidate antagonistic signals. Although GSH recycling was considered, GSSG levels remained unchanged; therefore, it is possible that the GSH increase in HSCethanol may derive from up-regulated hepatocyte-generated GSH export, because sinusoidal efflux plays a major role in hepatic GSH homeostasis.43, 44 The HSCethanol co-cultures showed a clear induction of TNF-α and similar GSH levels, suggesting a likely defense mechanism triggered by alcohol in HSC. To confirm this possibility, the HSCethanol co-cultures were incubated in the presence of a TNF-α neutralizing Ab or L-buthionine sulfoximine, which depletes GSH pools. Both treatments restored collagen I protein to that of HSCethanol cultured alone. Because both cell types produce TNF-α and GSH, it is conceivable that the TNF-α neutralization had an effect on both KC and HSCethanol; likewise, L-buthionine sulfoximine most likely depleted GSH levels on KC and HSCethanol as well, promoting oxidative stress, and triggering collagen I up-regulation.

To examine the cellular events that could take place by a potential synergism between ethanol and AA, the HSCcontrol and the HSCethanol co-cultures were incubated in the presence of AA. Collagen I protein was up-regulated over levels found in the HSCcontrol co-cultures. Whereas HSCethanol appeared resistant to the effects mediated by the co-culture with KCcontrol or with KCethanol, collagen I expression was restored to above basal levels when AA was present. We then analyzed whether TNF-α or GSH could play a role in the latter effects. AA significantly decreased TNF-α and GSH levels, whereas in some way it increased GSSG in HSCethanol, likely leaving HSCethanol vulnerable to lipid peroxidation end products and other species, which may have triggered the reestablishment of the profibrogenic response. It is feasible that alcohol elevates GSH in HSC through hepatocyte GSH efflux as a protective mechanism, even though hepatocytes could also release pro-fibrogenic and pro-inflammatory signals,9 but when a second “hit” such as AA enters the picture, HSC GSH stores are depleted, GSSG increases, and the pro-fibrogenic phenotype is reinstated. More notably, AA elicited a striking up-regulation of TGF-β, a powerful profibrogenic factor, which antagonizes TNF-α and favors collagen I induction.4 Indeed, the TNF-α and the TGF-β responsive elements on the COL1A2 promoter overlap,4 and Sp1, a ROS-sensitive transcription factor, is the point of convergence of the TGF-β and TNF-α signaling pathways on the COL1A2 gene.4

In view of the association between ethanol, TNF-α, GSH, and collagen I protein expression in HSC, it is tempting to speculate that HSCethanol may develop a mechanism such that TNF-α and GSH may help the cell cope with the pro-oxidant injury, and surpass the increase in TGF-β. These two molecules may act as anti-fibrogenic signals in early liver injury. Likewise, HSCethanol, which appear more responsive to self-activation, undergo somehow a diversion of phenotype in the presence of KC, so that although some HSC may remain proliferative and perhaps pro-inflammatory, others may have greater potential for a profibrogenic response to KC. Similarly, ethanol feeding may induce HSC-derived mediators such as platelet-derived growth factor BB, which will clearly enhance proliferation whereas other KC-derived factors such as TNF-α or metalloproteinases could act by decreasing collagen I.

Activation of ECM-producing HSC when there is active tissue damage, as occurs in alcoholic liver disease, could reveal chronic activation of the tissue repair process, where, as a consequence of the reiterated damage, accumulation of collagen I may be impaired because of effective remodeling. It is also likely that the profibrogenic role of AA in the co-cultures supersedes the defense pathway started by ethanol. A potential synergistic role for ethanol and AA to the mechanism of KC-mediated ECM remodeling is suggested whereby chronic ethanol consumption may sensitize HSC to up-regulate antagonistic factors (TNF-α and GSH) that may help the cell to cope with injury, whereas AA-derived reactions may operate as a negative feedback by increasing GSSG and TGF-β.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Arthur I. Cederbaum for his helpful insight during this project. We thank Drs. Raquel Urtasun and Laura Conde de la Rosa for their critical review of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_22592_sm_SupFig1.tif2705KSupplemental Figure 1

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