Mammalian target of rapamycin mediates the angiogenic effects of ...

Am J Physiol Gastrointest Liver Physiol 301: G210–G219, 2011. First published January 20, 2011; doi:10.1152/ajpgi.00047.2010.

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells Sara Aleffi,1* Nadia Navari,1* Wanda Delogu,1 Sara Galastri,1 Erica Novo,3 Krista Rombouts,1 Massimo Pinzani,1,2 Maurizio Parola,3 and Fabio Marra1,2 1

Dipartimento di Medicina Interna, 2Center of Research, Transfer and Higher Education DenoTHE, University of Florence, Florence; and 3Dipartimento di Medicina e Oncologia Sperimentali, University of Turin, Italy Submitted 9 February 2010; accepted in final form 16 January 2011

Aleffi S, Navari N, Delogu W, Galastri S, Novo E, Rombouts K, Pinzani M, Parola M, Marra F. Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 301: G210 –G219, 2011. First published January 20, 2011; doi:10.1152/ajpgi.00047.2010.—Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1␣ (HIF-1␣) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1␣ and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1␣ abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1␣ and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPHoxidase. Induction of HIF-1␣ requires NADP(H) oxidase but not mTOR activation. hepatic fibrosis; hypoxia-inducible factor; NADP(H) oxidase; platelet-derived growth factor

tissue-specific counterpart of the “woundhealing” response, a process whereby hepatocellular damage triggers a series of events aimed to limit damage and to preserve tissue function and integrity (22). As in other tissues, the liver wound-healing response triggers inflammation, followed by recruitment and activation of myofibroblast-like cells, which lead to deposition and remodeling of fibrillar extracellular matrix and alterations in the microvascular structure of the tissue (47). Activation of hepatic stellate cells (HSC) represents a major pathway leading to myofibroblast accumulation in different conditions of chronic liver injury, including alcoholic and nonalcoholic steatohepatitis (27). In

LIVER FIBROSIS IS A

* S. Aleffi and N. Navari contributed equally to the present study. Address for reprint requests and other correspondence: F. Marra, Dipartimento di Medicina Interna, Viale Morgagni, 85, I-50134 Florence, Italy (e-mail: [email protected]). G210

response to liver injury, vitamin A-rich stellate cells undergo a process referred to as “activation,” characterized by loss of retinoid stores, and acquisition of a profibrogenic and proinflammatory phenotype, becoming responsive to several soluble mediators (22). More recently, HSC have also been shown to be tightly involved in hepatic neoangiogenesis during chronic liver disease (47). This process contributes to physiological and pathological changes in vascular structure (14) and plays an important role in the development of liver fibrosis and hepatocarcinogenesis (47, 52). Angiogenesis is regulated by the net balance between proangiogenic factors and angiogenic inhibitors. Expression of vascular endothelial growth factor (VEGF) plays a major role as an angiogenic stimulus during fibrogenesis related to chronic liver disease (47). Several cell types have been shown to contribute to VEGF expression within the liver, including activated HSC, which express VEGF both in culture, in response to fibrogenic cytokines, and in vivo, in conditions of experimental liver fibrogenesis (3, 32, 47). Leptin has recently emerged as an important profibrogenic factor. Leptin is primarily produced by adipocytes of the white adipose tissue and regulates food intake and fat metabolism through actions on several tissues, including the central nervous system (2). Accumulating evidence indicates a critical role of leptin in hepatic inflammation and fibrogenesis, based on the observation that defects in leptin production or in leptin receptor signaling result in a marked reduction of liver fibrosis in different models of chronic liver injury (reviewed in Ref. 29). The profibrogenic actions of leptin are based, at least in part, on a direct action on HSC, which express functional leptin receptors, including the “long” form, ObRb (29). We have previously shown that leptin upregulates the expression of VEGF by human HSC and activates hypoxia-inducible factor 1␣ (HIF-1␣), a central transcription factor involved in vascular remodeling, in an oxygen-independent fashion (3). Little information is available on angiogenic signals transduced by ObR in leptin-responsive cells, and in particular no data have been reported in HSC, where regulation of angiogenesis occurs in close synergy with the modulation of fibrosis. The mammalian target of rapamycin (mTOR) is a highly conserved serinethreonine kinase that plays a central role in modulating cell growth and proliferation (21). mTOR activation has been previously involved in the biology of HSC, as a mediator of collagen expression and cell proliferation. However, the possible contribution of mTOR to intracellular signaling downstream of the leptin receptor and upstream of the expression of angiogenic factors has not been adequately explored. In this study, we provide evidence for a role of the mTOR pathway as a novel target of leptin receptor signaling, playing a relevant role in the induction of the angiogenic properties of HSC.

0193-1857/11 Copyright © 2011 the American Physiological Society

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mTOR AND ANGIOGENESIS IN STELLATE CELLS MATERIALS AND METHODS

Materials. Monoclonal antibodies against ␣-smooth muscle actin (␣-SMA) (clone 1A4), vinculin, and ␤-actin were purchased from Sigma Chemical (St. Louis, MO). Monoclonal antibodies against VEGF and polyclonal antibodies against hypoxia-inducible factor (HIF)-1␣ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphorylation-specific, polyclonal antibodies against tuberin/TSC2 (Thr1462), p70 S6 kinase (p70S6k) (Thr389), 4E-binding protein 1 (4E-BP1) (Ser65), AMP-activated protein kinase (AMPK), and LKB1; polyclonal antibodies against AMPK; and rapamycin were purchased from Cell Signaling Technology (Beverly, MA). 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), VEGF, recombinant human leptin, and platelet-derived growth factor (PDGF)-BB were from Peprotech (Rocky Hill, NJ). Diphenyleneiodonium (DPI), rotenone, apocynin, 2=,7=-dichlorodihydrofluorescein diacetate (DCFH-DA), and AICAR were from Sigma Chemical (St. Louis, MO). All other reagents were of analytical grade. Cell culture. Human HSC were isolated from wedge sections of liver tissue unsuitable for transplantation by collagenase/pronase digestion and centrifugation on Stractan gradients as previously described (15). Cells were cultured on uncoated plastic dishes and used for all of the experiments after complete transition toward an activated myofibroblast-like phenotype. The hepatocellular carcinoma line Huh-7 and the hepatoblastoma cell line HepG2 were cultured as previously described (34). Human endothelial cells (HUVEC) were isolated from umbilical cord vein by collagenase treatment as described (10), and used at passage 1– 4. Cells were grown on gelatin-coated plastic, in medium M199 (Sigma-Aldrich) supplemented with 20% heat-inactivated FCS, penicillin (100 units/ml), streptomycin (50 mg/ml), heparin (25 mg/ ml), and bovine brain extract (100 mg/ml) (Life Technologies, Milan, Italy). Western blot analysis. Confluent, serum-starved HSC were treated with the appropriate conditions, quickly placed on ice, and washed with ice-cold phosphate-buffered saline. The monolayer was lysed in RIPA buffer [20 mmol/l Tris·HCl, pH 7.4, 150 mmol/l NaCl, 5 mmol/l

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ethylenediaminetetraacetic acid, 1% Nonidet P-40, 1 mmol/l Na3VO4, 1 mmol/l phenyl methyl sulfonyl fluoride, and 0.05% (wt/vol) aprotinin] and transferred in microcentrifuge tubes. Insoluble proteins were discarded by a 10-min centrifugation at 12,000 rpm at 4°C. Protein concentration was measured in triplicate via a commercially available assay. Equal amounts of proteins were separated by SDSPAGE and analyzed by Western blotting as previously described (8). Gene silencing. Silencing of Raptor in HSC was performed by transfection of small interfering RNA (siRNA) by nucleofection, as previously reported in detail (11). Control, nontargeting siRNA, and siRNA targeting Raptor (as a Dharmacon Smart Pool) were obtained from Thermo Fisher (Lafayette, CO). Detection of intracellular levels of ROS. Intracellular levels of reactive oxygen species (ROS) were detected by means of the semiquantitative DCFH-DA fluorescence technique, as previously described (12), in cells exposed to different stimuli for 15 min. At the end of the incubation, cells positive for DCFH-DA were counted. In vitro angiogenesis assay. To assess the induction of a direct angiogenic effect of HSC treated with different stimuli, subconfluent HSC were serum deprived overnight and then left untreated or incubated with 200 ng/ml leptin or 10 ng/ml PDGF-BB for 2 h. Next, cells were washed with PBS, medium was replaced with fresh serumfree medium, and incubation continued for 24 additional hours. Conditioned media were aliquoted and stored at ⫺80°C until further analyzed. The angiogenic effects of different culture media were assayed by evaluating tube formation in HUVEC, as previously described (28), with minor modifications. Briefly, 1 ml of collagen was added to cultures plates (3-mm petri dishes) and allowed to gel at 37°C for 30 min. HUVEC (105 cells/dish) were gently added to each dish and allowed to adhere to the coating gel for 1 h at 37°C. After 24 h the medium was replaced with the conditions to be tested, including fresh medium (control), medium M199 containing VEGF 100 ng/ml (positive control), or HSC-conditioned media. Conditioned media were diluted 1:1 in M199 with 10% FCS and added to HUVEC cultures for

Fig. 1. Leptin and PDGF activate the mammalian target of rapamycin (mTOR) pathway in hepatic stellate cells (HSC). A: serum-deprived HSC were exposed to 200 ng/ml leptin for the indicated time points. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated forms of p70 S6 kinase (p70S6k), 4E-binding protein 1 (4E-BP1), and AMP-activated protein kinase (AMPK). The membrane was stripped and reprobed for ␤-actin to ensure equal loading. B: serum-deprived HSC were left untreated (Cnt) or incubated with 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) for 30 min or with leptin for 10 or 30 min, as indicated. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated form of LKB1. The membrane was stripped and reprobed for ␤-actin to ensure equal loading. C: the experiment was performed essentially as described for A. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated form of tuberous sclerosis complex (TSC)2 (Thr1462) and then reprobed with anti-vinculin antibodies to ensure equal loading. D: serum-deprived HSC were incubated in the absence or presence of 10 ng/ml PDGF for the indicated time points. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated forms of p70S6k, 4E-BP1, and AMPK. The membrane was stripped and reprobed for ␤-actin to ensure equal loading. In all panels, migration of molecular weight markers is indicated at left, and fold increase over basal, measured by densitometry, is shown at bottom. AJP-Gastrointest Liver Physiol • VOL

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Fig. 2. Leptin increases AMPK phosphorylation in hepatocytic cells. A: cultured Huh-7 cells were serum deprived overnight and exposed to 200 ng/ml leptin for the indicated time points. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated forms of AMPK. The membrane was stripped and reprobed for ␤-actin to ensure equal loading. B: cultured HepG2 cells were serum deprived overnight and exposed to 200 ng/ml leptin for the indicated time points. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated forms of AMPK. The membrane was stripped and reprobed for total AMPK to ensure equal loading. Migration of molecular weight markers is indicated at left, and fold increase over basal, measured by densitometry, is shown at bottom.

increased LKB1 phosphorylation (Fig. 1B). However, leptin did not modify the phosphorylation status of LKB1 at time points that preceded the decrease in AMPK phosphorylation. PI3K, ERK, and AMPK converge on the molecular crossroad represented by the tumor suppressor molecules, TSC1 and TSC2 (25). TSC1 and TSC2 form a physical and functional complex, where mutation of either protein is sufficient to release mTOR from negative regulation. Thus inhibition of the formation of the dimeric complex, and/or negative posttranscriptional regulation, such as phosphorylation of TSC2 on Thr1462 residues, provokes dissociation of the complex and results in mTOR activation. In response to leptin concentrations that are effective in activating mTOR, HSC showed enhanced phosphorylation of TSC2 on Thr1462 residues, indicating that dissociation of the tuberous sclerosis complex (TSC) by leptin precedes mTOR activation (Fig. 1C). PDGF is a potent stimulus for several biological actions of HSC and signals through activation of a tyrosine kinasecoupled receptor. We compared the signals induced by leptin with those generated by PDGF, as an additional cytokine that upregulates VEGF by oxygen-independent pathways (50). Similar to the effects of leptin, phosphorylation of both S6K

24 h. The plates were monitored after 24 h and photographed with a contrast phase microscope. Data presentation and statistical analysis. Autoradiograms and autoluminograms are representative of at least three experiments with comparable results. In selected experiments, densitometry data from at least three independent experiments were shown in barograms as means ⫾ SD. Statistical analysis was performed by Student’s t-test. P levels ⱕ0.05 were considered significant. RESULTS

We and others have recently demonstrated that leptin activates ERK and phosphatidylinositol 3-kinase (PI3K) in rodent and human HSC, and signals downstream of these pathways are known to activate mTOR (3, 36). We first analyzed whether leptin could affect the pathways converging on mTOR, which integrates signals from mitogenic growth factors, nutrients, stress, and cellular energy (41). To evaluate this possible action of leptin, we examined the phosphorylation status of two molecules downstream of mTOR, namely p70S6k and the eukaryotic translation initiation factor 4E-BP1. Leptin rapidly and markedly induced phosphorylation of 4E-BP1 and p70S6k on residues that are known to be target of mTOR’s activity (Fig. 1A). AMPK is another upstream regulator of mTOR (11), providing negative signals that counteract those generated by ERK and PI3K. Phosphorylation of AMPK on activationspecific residues was detectable in unstimulated, serum-starved HSC. Exposure to leptin for prolonged periods of time resulted in a progressive reduction of AMPK phosphorylation, suggesting inhibition of this pathway (Fig. 1A). To better understand the possible mechanisms of this action, we analyzed the effects of leptin on the phosphorylation status of LKB1, an upstream kinase that is responsible for AMPK phosphorylation (26). As expected, AICAR, which mimics a low intracellular AMP-to-ATP ratio, AJP-Gastrointest Liver Physiol • VOL

Fig. 3. Effects of mTOR inhibition on angiogenic pathways in response to leptin. A: serum-deprived HSC were left untreated or exposed for 1 h to 200 ng/ml leptin alone or after preincubation with 10 nM rapamycin. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated form of p70S6k. B: the experiment was performed as in A. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated form of 4E-BP1. C: HSC were left untreated or incubated with 200 ng/ml leptin for 4 h, alone or after preincubation with 10 nM rapamycin. Total cell lysates were analyzed by immunoblotting with antiVEGF antibodies. D: HSC were left untreated or incubated with 200 ng/ml leptin for 1 h, alone or after preincubation with 10 nM rapamycin. Total cell lysates were analyzed by immunoblotting with anti-hypoxia inducible factor 1␣ (HIF-1␣) antibodies. In all panels, the blots were stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left. E: data from Western blottings as in A–D were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, leptin; cross-hatched bars, leptin and rapamycin. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone. 301 • AUGUST 2011 •

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and 4EBP-1 was markedly increased by PDGF (Fig. 1D), together with increased phosphorylation of TSC2 (data not shown). Additionally, the phosphorylation of AMPK observed in unstimulated cells was markedly reduced, in a time-dependent fashion. These experiments show that both leptin and PDGF activate mTOR via upstream signals that include downregulation of AMPK activation. In different cell types, including hepatocytes, leptin has been shown to increase the activation of AMPK, which contributes to the metabolic effects of this adipokine (30). To confirm that leptin-induced decrease in AMPK phosphorylation is cell specific, we assessed the effects of leptin in two hepatocytic cell lines, Huh-7 and HepG2 (Fig. 2). In both cell types, leptin resulted in a two- to threefold increase in AMPK phosphorylation. To evaluate the biological significance of mTOR activation by leptin in HSC, we employed a pharmacological inhibition strategy using rapamycin, an immunosuppressive drug that targets the activity of mTOR through the target of rapamycin complex 1 (mTORC1) molecular complex (35). Rapamycin treatment of HSC resulted in a significant inhibition of phosphorylation of both p70S6k and 4E-BP1 with respect to cells only treated with leptin, indicating the efficacy of this inhibitor on the mTOR pathway (Fig. 3, A, B, and E). To establish a link between mTOR activation and angiogenic pathways, we evaluated leptin-induced expression of VEGF in the presence or absence of rapamycin. Leptin markedly increased intracellular levels of VEGF, whereas in the presence of rapamycin this action was significantly inhibited (Fig. 3, C and E). To further investigate the mechanisms underlying the upregulation of VEGF by leptin, we analyzed the effects of rapamycin on the protein levels of HIF-1␣, a key regulator of the expression of

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several molecules involved in the angiogenic process, including VEGF (39). As previously shown (3), leptin significantly increased HIF-1 ␣ levels, but no inhibitory effects were observed in cells exposed to rapamycin (Fig. 3, D and E). These data provide evidence for a regulatory role of the mTOR pathway in mediating the angiogenic effects of leptin and indicate that the effects of mTOR on VEGF are largely independent of changes in HIF-1␣ expression. We extended these analyses to signals induced by PDGF. Rapamycin markedly and significantly reduced activation of mTOR, as shown by its ability to block PDGF-induced phosphorylation of p70S6k and 4E-BP1 (Fig. 4A). Similarly to leptin, PDGF increased the expression of both VEGF and HIF-1␣ (Fig. 4, B and C). However, whereas rapamycin inhibited VEGF expression, virtually no effects were observed on the abundance of HIF-1␣ (Fig. 4, B–D). To provide additional evidence for the role of mTOR in leptin-mediated signaling in HSC, we investigated the effects of knockdown of Raptor, a component of the mTORC1 complex (5), by gene silencing (Fig. 5). Treatment with Raptor siRNA reduced the levels of the protein of ⬃50%. This was associated with a reduced ability of leptin to phosphorylate p70S6k and 4E-BP1 (Fig. 5A). Moreover, the ability of leptin to significantly increase VEGF expression was lost in cells treated with Raptor siRNA (Fig. 5, B and C). Similar data were obtained when PDGF was used as an agonist (data not shown). These data confirm that mTOR signaling via the mTORC1 complex is necessary to mediate the stimulating effects of leptin on VEGF in human HSC. We next evaluated whether the mTOR pathway was implicated in the regulation of other biological actions of leptin in

Fig. 4. Effects of mTOR inhibition on angiogenic pathways in response to PDGF. A: serum-deprived HSC were left untreated, or exposed for 1 h to 10 ng/ml PDGF alone or after preincubation with 10 nM rapamycin. Total cell lysates were analyzed by immunoblotting with antibodies directed against the phosphorylated form of 70S6K or 4EBP1. B: the experiment was conducted as described in A, but cells were treated with PDGF with or without rapamycin for 24 h and membranes were blotted with antibodies against HIF-1␣. C: the experiment was conducted as described in B, but membranes were blotted with antibodies against VEGF. In all panels, the blots were stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left. D: data from Western blottings as in A–C were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, PDGF; cross-hatched bars, PDGF and rapamycin. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. PDGF alone.

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Fig. 5. Raptor silencing inhibits leptin-mediated increase in VEGF. A: HSC were transfected with control small interfering RNA (siRNA) or with siRNA directed against Raptor by nucleofection, as described in MATERIALS AND METHODS. Cells were then deprived of serum and incubated in the presence or absence of 200 ng/ml leptin for 1 h. Total cell lysates were analyzed by immunoblotting with antibodies directed against Raptor or the phosphorylated forms of 4E-BP1 or p70S6k. The membrane was stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left, and fold increase over basal, measured by densitometry, is shown at bottom. Images for all lanes were captured from the same gel. A space has been inserted to indicate deletion of one or more lanes. B: the experiment was conducted as described in A, but incubation with leptin was conducted for 24 h, and membranes were blotted with antibodies against VEGF. Migration of molecular weight markers is indicated at left. Images for all lanes were captured from the same gel. A space has been inserted to indicate deletion of one or more lanes. C: data from Western blottings as in B were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). *P ⱕ 0.05 vs. control.

HSC. Directional cell migration is considered a critical feature leading to deposition of extracellular matrix in discrete areas of the hepatic acinus in relation to the type and extent of damage. Both leptin and PDGF significantly increased the ability of HSC to migrate in Boyden chamber experiments (Fig. 6). Upon preincubation with rapamycin, the ability of leptin or PDGF to induce cell migration was blocked. These data demonstrate that the mTOR pathway is required also for the motogenic actions of leptin and PDGF. Many cytokines, including leptin, induce intracellular generation of ROS that contribute to generate downstream signals relevant for the modulation of cellular biological activities (19). We first established whether leptin induces ROS generation in human HSC, monitoring their production in cells loaded with the fluorescent dye DCFH-DA (Fig. 7). Leptin significantly increased the number of cells positive for DCFHDA, indicating the ability to increase intracellular ROS generation in HSC. These effects were inhibited by different antioxidants, including rotenone and two inhibitors of NADP(H) oxidase, namely DPI and apocynin (Fig. 7). Moreover, exposure of HSC to leptin was associated with a marked increase of the redox-sensitive protein heme oxygenase-1 (Fig. 8A), confirming leptin-induced accumulation of ROS in HSC. AJP-Gastrointest Liver Physiol • VOL

We next explored the contribution of ROS to leptin’s angiogenic signals using N-acetyl-cysteine (NAC), which blocks intracellular and extracellular ROS generation. Leptin-induced increase in VEGF expression was significantly inhibited by NAC, which also reduced leptin-mediated increase in HIF-1␣ (Fig. 8, B–E). These data indicate the relevance of oxidative stress-related products in the pathway leading from the leptin receptor to VEGF induction. Activation of NADP(H) oxidase has been recently reported in HSC exposed to leptin, but the contribution of this pathway to leptin-mediated increase in VEGF has not been explored (19). We employed DPI, a specific inhibitor of different NAPD(H) isoforms, to test the role of this molecule in leptininduced actions. DPI blocked the increase in VEGF induced by leptin (Fig. 9, A and B). In addition, DPI limited leptinmediated increase in HIF-1␣ abundance, in a similar fashion to cells incubated with NAC (Fig. 8, D and F). Thus upregulation of VEGF by leptin requires ROS generation, which is provided, at least in part, by activation of NADP(H) oxidase. Activation of NADP(H) oxidase was required also for the effects of PDGF, since inhibition of NADP(H) oxidase by DPI significantly reduced the expression of VEGF (Fig. 9, C and D). In addition, DPI reduced the ability of both leptin and PDGF to activate mTOR, as indicated by a reduced phosphorylation of p70S6k and 4E-BP1 (data not shown). Taken together, these data indicate that interference with intracellular generation of ROS blocks the ability of different cytokines to activate the mTOR pathway and to increase VEGF expression. Finally, we assessed whether the ability of soluble mediators, such as leptin or PDGF, to upregulate VEGF was accompanied by the ability to induce angiogenesis in a functional assay. We analyzed the ability of conditioned media from cells pulsed with leptin or PDGF to induce tube formation in HUVEC, a well-established model of angiogenesis (28). Compared with nonconditioned media or to media conditioned in the absence of recombinant cytokines, medium from cells treated with leptin or PDGF induced an evident appearance of tubule-like structures, an in vitro counterpart of angiogenesis (Fig. 10). These effects were remarkably similar to those elicited by recombinant VEGF, further indicating a critical role of this factor for the effects mediated by HSC-conditioned media.

Fig. 6. Rapamycin inhibits leptin and PDGF-induced chemotaxis of HSC. Serum-deprived HSC were preincubated for 30 min with 1 ␮M rapamycin or its vehicle, and chemotaxis in response to 200 ng/ml leptin or 10 ng/ml PDGF was assayed in modified Boyden chambers, as described in MATERIALS AND METHODS. At the end of the experiment, migrated cells were counted. Data are means ⫾ SD from 3 independent experiments. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone; ⽧⽧P ⱕ 0.05 vs. PDGF alone. 301 • AUGUST 2011 •

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Fig. 7. Leptin increases oxidative stress in cultured HSC. A: serum-deprived HSC were preloaded with dichlorodihydrofluorescein diacetate (DCFH-DA), a fluorescent dye sensitive to reactive oxygen species (ROS), as described in MATERIALS AND METHODS, and left untreated or incubated with 100 ng/ml leptin, alone or together with 1 ␮M diphenyleneiodonium (DPI), 2.5 ␮M rotenone, or 100 ␮M apocynin for 15 min, as indicated. Photomicrographs were taken by phase contrast (left) or fluorescence microscopy (middle) and overlays are shown at right. B: the number of cells positive for DCFH-DA were counted and shown as barograms (means ⫾ SD, n ⫽ 3). *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone.

DISCUSSION

Accumulating evidence indicates a relevant role of leptin in the fibrogenic process (30). Animals defective in leptin secretion or with mutations in leptin receptors have a marked impairment in hepatic fibrogenesis when induced by intoxication with different chemicals or by experimental steatohepatitis. The molecular mechanisms underlying the profibrogenic actions of leptin are manifold and not completely characterized. It has been demonstrated that leptin upregulates expression of the profibrogenic cytokine transforming growth factor-␤, acting on Kupffer cells and sinusoidal endothelial cells (24, 48). In addition, several groups have demonstrated that leptin directly modulated the biology of HSC, as indicated by the fact that these cells express ObRb, the signaling-competent form of leptin receptor, and by the ability of leptin to exert profibrogenic actions in cultured rodent and human HSC (13, 29). Leptin has been implicated in the regulation of multiple actions of HSC, including proliferation, survival, extracellular matrix production, and secretion of proinflammatory cytokines (29). We recently demonstrated that an additional action of leptin is the upregulation of VEGF, a pivotal cytokine for the induction of neoangiogenesis (3). AJP-Gastrointest Liver Physiol • VOL

The main finding of the present study is the elucidation of the signaling pathways that regulate VEGF expression in response to leptin, namely the mTOR pathway and generation of ROS via NADP(H) oxidase. The ability of leptin to interact with mTOR has not been previously shown in HSC, or in other liver-derived cells. mTOR receives positive and negative inputs from molecules located upstream, especially the TSC1/ TSC2 complex, a tumor-suppressor gene that integrates signals derived from outside and inside the cell (51). TSC1/TSC2 provide inhibitory signals toward the mTOR pathway by inhibiting the guanine-exchange factor Rheb, which is critical for Raptor-associated activation of mTOR. In the presence of nutrient shortage or stressful conditions, activation of TSC1/ TSC2 blocks mitogenic and synthetic pathways and promotes cell autophagy. In contrast, postreceptor mitogenic signals, such as those mediated by Ras-ERK and PI3K-Akt, suppress TSC signals and result in mTOR activation (51). The fact that leptin phosphorylates TSC2 on “inhibitory” residues is in agreement with previous data from our group and other groups, showing that interaction of leptin with the ObRb receptor on HSC leads to activation of ERK 1/2 and of PI3K/Akt, both of which concur to inhibit the TSC complex and hence to activate 301 • AUGUST 2011 •

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Fig. 8. ROS generation by leptin is required for activation of angiogenic pathways. A: HSC were incubated in the presence or absence of 200 ng/ml leptin for 24 h. Total cell lysates were analyzed by immunoblotting with antibodies directed against heme oxygenase-1 (HO-1). The blots was stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left, and fold increase over basal, measured by densitometry, is shown at bottom. B: serum-starved HSC were preincubated with 100 ␮M N-acetyl-cysteine (NAC) for 1 h and then exposed to 200 ng/ml leptin for 1 additional hour. Total cell lysates were analyzed by immunoblotting with antibodies directed against VEGF. The blot was stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left. C: data from Western blottings as in B were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, leptin; cross-hatched bars, leptin and NAC. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone. D: HSC were left untreated or incubated with 200 ng/ml leptin in the presence or absence of 100 ␮M NAC or 20 ␮M DPI, as indicated, for 1 h. Total cell lysates were analyzed by immunoblotting with antibodies directed against HIF-1␣. The blot was stripped and reprobed for ␤-actin. Migration of molecular weight markers is indicated at left. E: data from Western blottings comparing samples as in lanes 1–3 of D were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, leptin; cross-hatched bars, leptin and NAC. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone. F: data from Western blottings comparing samples as in lanes 1, 2, and 4 of D were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, leptin; cross-hatched bars, leptin and DPI. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone.

mTOR (3, 13, 36). The involvement of mTOR in leptin signaling is supported by findings in other cellular systems. In vascular smooth muscle cells, leptin’s ability to induce cell hyperplasia was limited by rapamycin (40). Moreover, leptinmediated increase in mTOR activity in the hypothalamus is critical for the anorectic signals mediated by this hormone (17). The mTOR pathway cross-talks with AMPK, an important regulator of metabolism and multiple cellular functions (42). Leptin requires AMPK activation to suppress hepatic glucose production (4), and in cultured hepatocytic cells leptin was found to upregulate AMPK activation (46). We confirmed these results, since leptin increased phosphorylation of AMPK in both Huh-7 and HepG2. In contrast, leptin reduced AMPK phosphorylation in HSC, acting independently of LKB1 actiAJP-Gastrointest Liver Physiol • VOL

vation. These results may provide an additional mechanism of the profibrogenic section of leptin, since we and others have shown that AMPK negatively modulates the activated status of HSC (1, 11). Interestingly, others have recently reported that leptin downregulates phosphorylation of AMPK in HSC (43), whereas only a very minor activation has been shown by Handy et al. (23). Considering that leptin increases AMPK activation in other cell types, such as endothelial cells (33), different, cell-specific pathways appears to characterize the relation between leptin signaling and AMPK. Activation of mTOR has been previously reported to modulate some aspects of HSC biology, in particular proliferation in response to cytokines (53), and to reduce fibrosis in an in vivo model of chronic liver damage (7). In the present study, we demonstrate that mTOR activation is critical for agonistinduced expression of VEGF, the main angiogenic cytokine released by HSC. In addition, we found that rapamycin blocked the ability of both leptin and PDGF to stimulate directional migration of HSC, a feature that contributes to the development of fibrogenesis. These observations are supported by the inhibitory effects of rapamycin, which interfere with formation of the mTORC1 complex. Moreover, knockdown of Raptor, which specifically contributes to formation of the mTORC1 complex, was associated with the reduced ability of leptin or PDGF to increase VEGF expression, confirming with a molecular approach the relevance of this pathway. Evidence accumulated in the past 5 years has contributed to define the critical role of angiogenesis in the fibrogenic process (reviewed in Ref. 20). On the one hand, angiogenesis closely parallels the development of fibrogenesis, and inhibition of neovessel formation is effective in limiting the deposition of extracellular matrix (31, 45, 52). On the other hand, fibrogenic cells, such as HSC, actively participate in the angiogenic process, releasing soluble mediators such as VEGF and angio-

Fig. 9. NADP(H) oxidase activation mediates upregulation of VEGF expression by leptin or PDGF. HSC were left untreated or incubated with 200 ng/ml leptin in the presence or absence of 20 ␮M DPI for 24 h. Total cell lysates were analyzed by immunoblotting with antibodies directed against VEGF. The blots were stripped and reprobed for ␤-actin to ensure equal loading. Migration of molecular weight markers is indicated at left. B: data from Western blottings as in A were quantified by densitometry and shown as barograms (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, leptin; cross-hatched bars, leptin and DPI. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. leptin alone. C: the experiment was performed as in A, but 10 ng/ml PDGF was used as a stimulus. D: data from Western blottings as in C were quantified by densitometry and shown as a barogram (means ⫾ SD, n ⫽ 3). Open bars, control; solid bars, PDGF; cross-hatched bars, PDGF and DPI. *P ⱕ 0.05 vs. control; ⽧P ⱕ 0.05 vs. PDGF alone. 301 • AUGUST 2011 •

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Fig. 10. Leptin induces production of angiogenic factors in cultured HSC. Human umbilical vein endothelial cells were incubated with nonconditioned medium (a), 100 ng/ml recombinant VEGF (b, c), as a positive control, or medium conditioned (CM) for 24 h by unstimulated HSC (d, e), or by HSC pulsed with leptin (f–i) or PDGF (j–m) as indicated in MATERIALS AND METHODS. Representative phase contrast photomicrographs taken at the end of incubation are shown. Please note tube formation in b–c, f–i, and j–m. Original magnification: ⫻10 (a–g, j–k); ⫻20 (h–i, l–m).

poietin-1 (3, 44, 47). We previously reported that VEGF is a major angiogenic cytokine regulated by leptin, whereas angiopoietin-1 expression was not significantly modified by treatment with this adipokine (3). In the present study we expanded these observations by showing that conditioned media from HSC treated with leptin induce angiogenesis in an in vitro system, as indicated by a tube formation assay using cultured HUVEC. These data reinforce the notion that soluble mediators, including leptin, play a critical role in mediating the accumulation of new vessels that accompanies fibrogenesis. Whereas several profibrogenic actions of leptin have been extensively studied, little is known on the mechanisms that lead to leptin-mediated secretion of VEGF. The involvement of mTOR as a regulator of VEGF secretion in HSC complements the established role of this signaling system in the modulation of vessel formation during chronic wound healing and cancer. Interference with mTOR has been shown to inhibit neoangiogenesis during formation and spreading of solid tumors, including hepatocellular carcinoma, which mostly develops in the context of fibrosis (37). Moreover, administration of rapaAJP-Gastrointest Liver Physiol • VOL

Fig. 11. Model for the regulation of VEGF expression by leptin receptor activation in HSC. TSC, tuberous sclerosis complex. 301 • AUGUST 2011 •

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mycin has been shown to be effective in reducing tumor load and spreading, and rapamycin or its derivatives have been recently proposed as an additional treatment for a number of cancers (49). The observation that rapamycin interferes with expression of VEGF by HSC identifies a possible additional level of interaction that may be relevant in the context of hepatocellular carcinoma. In the past decade, several levels of interaction between ROS and cytokine receptor signaling have been identified (16), and in general, a transient generation of ROS occurs upon interaction between the receptor and its cognate ligand. Similarly, interaction of HSC with leptin has been shown to result in ROS generation, which is necessary for proliferation and survival signals (13). We confirmed that exposure of HSC to leptin induces accumulation of ROS, as determined by using a specific fluorescent indicator, and that these effects were antagonized by inhibition of NADP(H) oxidase. Activation of NADP(H) oxidase contributes to ROS generation and cytokine receptor signaling in many cell types, including HSC (18). NADP(H) oxidase is expressed by phagocytic cells, where it mediates the oxidative burst involved in the innate defense against bacteria, and by nonphagocytic cells, where different isoforms such as p47phox or p91phox are expressed (18). In HSC, activation of nonphagocytic NADP(H) oxidase isoforms has been shown to mediate profibrogenic actions in response to angiotensin II, both in vitro and in vivo (6). Recently, De Minicis et al. (19) have explored the contribution of NADP(H) oxidase to leptin-induced ROS generation in HSC. Diphenylene iodonium reduced ROS generation associated with exposure of HSC to leptin, and blocked activation of ERK and PI3K. Moreover, in HSC isolated from mice deficient in p47phox, leptin’s ability to induce HSC proliferation and secretion of proinflammatory cytokines such as MCP-1 was blunted (19). In the present study, we extend the involvement of NADP(H) oxidase to the control of VEGF secretion in response to leptin and PDGF. Inhibition of NADP(H) oxidase resulted in the inability of leptin to increase VEGF expression, indicating that generation of ROS in response by NADP(H) oxidase activation is a critical step for angiogenic signaling. Inhibition of NADP(H) oxidase was also associated with reduced activation of mTOR, indicating that this kinase lies on a signaling pathway that involves NADP(H) oxidase, PI3K, and ERK. Activation of angiogenic signals, including expression of VEGF, is triggered by cell exposure to low oxygen tension, resulting in upregulation of several hypoxia-sensitive transcription factors, including HIF-1␣ (38). However, HIF-1␣ may be also activated in an oxygen-independent fashion by several cytokines, including leptin and PDGF, in HSC (3). Inhibition of NADP(H) oxidase resulted in a significant reduction of HIF-1␣ activation. This observation is in clear agreement with our previous findings showing that pharmacological inhibition of either ERK or PI3K activation blocks HIF-1␣ activation by leptin (3). Although in other cell types, changes in mTOR activity have been found to modulate HIF-1␣ activation (9), in the present study inhibition of mTOR by rapamycin did not have any major effects on HIF-1␣ activation. Taken together, the available data suggest a model where leptin stimulates VEGF through at least two parallel signaling pathways, one involving HIF-1␣, and one related to mTOR activation (Fig. 11). It is of note that the signaling events elucidated above AJP-Gastrointest Liver Physiol • VOL

for leptin are shared by PDGF, one of the most potent fibrogenic cytokines, and a major mitogen for HSC. This fact adds further value to the observations of the present study, since it suggests that interference with mTOR activation is likely to block VEGF in response to a number of mediators sharing the ability to activate this important pathway. These results should add further impulse to clinical studies investigating drugs that interfere with the mTOR pathway, which have been demonstrated to be potentially effective as antifibrogenic and anticancer agents in preclinical studies. ACKNOWLEDGMENTS We are indebted to Dr. Lucia Napione and Dr. Federico Bussolino (IRCC Candiolo, Turin) for kindly providing HUVEC cultures. GRANTS This study was supported by grants from MIUR (2006063809) and from the University of Florence (to F. Marra) and from Regione Piemonte (to M. Parola). In addition, the research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/20072013) under grant agreement no. HEALTH-F2-2009-241762 for the project FLIP (to F. Marra). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Adachi M, Brenner DA. High molecular weight adiponectin inhibits proliferation of hepatic stellate cells via activation of adenosine monophosphate-activated protein kinase. Hepatology 47: 677–685, 2008. 2. Ahima RS, Osei SY. Leptin signaling. Physiol Behav 81: 223–241, 2004. 3. Aleffi S, Petrai I, Bertolani C, Parola M, Colombatto S, Novo E, Vizzutti F, Anania FA, Milani S, Rombouts K, Laffi G, Pinzani M, Marra F. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology 42: 1339 –1348, 2005. 4. Andreelli F, Foretz M, Knauf C, Cani PD, Perrin C, Iglesias MA, Pillot B, Bado A, Tronche F, Mithieux G, Vaulont S, Burcelin R, Viollet B. Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin. Endocrinology 147: 2432–2441, 2006. 5. Avruch J, Hara K, Lin Y, Liu M, Long X, Ortiz-Vega S, Yonezawa K. Insulin and amino-acid regulation of mTOR signaling and kinase activity through the Rheb GTPase. Oncogene 25: 6361–6372, 2006. 6. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, Qian T, Schoonhoven R, Hagedorn CH, Lemasters JJ, Brenner DA. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest 112: 1383–1394, 2003. 7. Biecker E, De Gottardi A, Neef M, Unternahrer M, Schneider V, Ledermann M, Sagesser H, Shaw S, Reichen J. Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms. J Pharmacol Exp Ther 313: 952–961, 2005. 8. Bonacchi A, Romagnani P, Romanelli RG, Efsen E, Annunziato F, Lasagni L, Francalanci M, Serio M, Laffi G, Pinzani M, Gentilini P, Marra F. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem 276: 9945–9954, 2001. 9. Brugarolas J, Kaelin WG ,Jr. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes. Cancer Cell 6: 7–10, 2004. 10. Bussolino F, Wang JM, Defilippi P, Turrini F, Sanavio F, Edgell CJ, Aglietta M, Arese P, Mantovani A. Granulocyte and granulocytemacrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature 337: 471–473, 1989. 11. Caligiuri A, Bertolani C, Guerra CT, Aleffi S, Galastri S, Trappoliere M, Vizzutti F, Gelmini S, Laffi G, Pinzani M, Marra F. Adenosine 301 • AUGUST 2011 •

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