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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

Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been considered as an alternative therapy, replacing liver transplantation in clinical trials, to treat liver cirrhosis, an irreversible disease that may eventually lead to liver cancer development. However, low survival rate of the BM-MSCs leading to unsatisfactory efficacy remains a major concern. Gender differences have been suggested in BM-MSCs therapeutic application, but the effect of the androgen receptor (AR), a key factor in male sexual phenotype, in this application is not clear. Using two liver cirrhosis mouse models induced by CCl4 or thioacetamide, we showed that targeting AR in the BM-MSCs improved their self-renewal and migration potentials and increased paracrine effects to exert anti-inflammatory and anti-fibrotic actions to enhance liver repair. Mechanism dissection studies suggested that knocking out AR in BM-MSCs led to improved self-renewal and migration by alteration of the signaling of epidermal growth factor receptor and matrix metalloproteinase 9 and resulted in suppression of infiltrating macrophages and hepatic stellate cell activation through modulation of interleukin (IL)1R/IL1Ra signaling. Therapeutic approaches using either AR/small interfering RNA or the AR degradation enhancer, ASC-J9®, to target AR in BM-MSCs all led to increased efficacy for liver repair. Conclusion: Targeting AR, a key factor in male sexual phenotype, in BM-MSCs improves transplantation therapeutic efficacy for treating liver fibrosis. (HEPATOLOGY 2013;57:1550–1563)

Chronic liver disease (CLD) with cirrhosis is the twelfth leading cause of death, with 27,555 deaths each year in the United States,1 and the 10-year mortality rate is 32%-66%,2 depending on the cause of the cirrhosis. Many factors may contribute to liver cirrhosis, including hepatitis B, hepatitis C, alcoholic liver disease, hepatotoxic drugs, and toxins. Among those factors, chronic alcoholism and hepatitis C are the most common causes to induce cirrhosis in the western world. Because patients with liver cirrhosis may also develop hepatocellular carcinomas (HCCs),3 early treatment of liver cirrhosis with proper therapy will not only improve cirrhotic symptoms, but also prevent HCC incidence.

Current treatment for liver cirrhosis is to prevent further damage of functional hepatocytes, and liver transplantation (LT) remains the standard treatment for advanced liver cirrhosis. However, the efficacy of LT is limited by the availability of donor organs and several adverse effects, such as graft-versus-host disease, mental changes, and complications resulting from perioperation,4, 5 which all may complicate this therapy. Therefore, alternative therapeutic approaches, such as transplantation of hepatocytes, hematopoietic stem cells, endothelial progenitor cells, and bone marrow mesenchymal stem cells (BM-MSCs),6-10 may become other options.

BM-MSC transplantation has been extensively studied in clinical trials. Clinical outcomes displayed short-term relief in liver function. But, unfortunately, long-term observations showed failure with recurring symptoms,11 and only low numbers of BM-MSCs finally migrated to the target liver, which could be the result of high apoptotic rates of BM-MSCs from microischemia.12 Therefore, improving self-renewal and survival of BM-MSCs may become a key step to improve the efficacy of using BM-MSCs to treat cirrhotic livers.

Gender differences have been suggested in BM-MSC transplantation therapy of myocardial infarction, because female BM-MSCs have better therapeutic potential than male BM-MSCs.13 It also has been shown that expression of the androgen receptor (AR), a key factor in male sexual phenotype, was increased as embryonic stem cells proceeded to differentiation, a process initiated after cell proliferation stopped,14 implicating that interruption of androgen/AR signaling may lead to increased stem cell proliferation. Here, we found that targeting AR in BM-MSCs significantly enhanced the self-renewal of BM-MSCs. BM-MSCs treatment using either AR/small interfering RNA (siRNA) or ASC-J9®, an AR degradation enhancer, both lead to better therapeutic efficacy to treat liver cirrhosis.

Materials and Methods

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

Liver Cirrhosis Mouse Models.

CCl4 (Sigma-Aldrich, diluted 1:1 in olive oil; Sigma-Aldrich, St. Louis, MO) or vehicle (olive oil) was administered by intraperitoneal (IP) injection at a dose of 1 mL/kg of body weight twice per week to induce liver cirrhosis. Transplantation of BM-MSCs of wild type (WT) and AR knockout (ARKO) was performed by tail vein injection 1 day after the eighth CCl4 treatment. For knocking down BM-MSCs AR with siRNA, BM-MSCs were infected with lentivirus, which contained either scramble control or AR-siRNA, and green fluorescence protein (GFP). BM-MSCs containing more than 90% GFP-positive signals were prepared to perform transplantation. For ASC-J9®-treated BM-MSCs, BM-MSCs were cultured and treated with either dimethyl sulfoxide (DMSO) or 10 μM of ASC-J9® for 1 week before transplantation. After 8 weeks of treatments (total 16 treatments) with CCl4, mice were sacrificed with pentobarbital, mouse livers were removed to examine for fibrosis, and mouse sera were isolated to assay for liver functions. Thioacetamide (TAA) was administered at a concentration of 300 mg/L in drinking water for 8 weeks to induce liver cirrhosis. Similarly, BM-MSCs transplantation was performed after 4-week treatment, and mice were sacrificed after an additional 4 weeks of TAA challenge. Other materials and methods, including cells, animals, reagents, isolation of BM-MSCs, histologic analysis of liver tissues, liver functional assay, colony-forming unit-fibroblast (CFU-f) assay, cell-growth assay, flow cytometry, western blotting analysis, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), lentivirus production, migration, and statistical analysis, are described in the Supporting Materials.

Results

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

Knockout of AR in BM-MSCs Improves Their Therapeutic Potential in Liver Cirrhosis.

We first characterized the purity of primary isolated BM-MSCs from mice by checking their surface marker expressions and found they were highly positive for CD44 (99.0%) and CD29 (98.5%) and moderately positive for CD117 (29.4%) and CD106 (32.5%), but only had little expression of CD34 (0.59%) and CD45 (1.28%) (as shown in Supporting Fig. 1). These results were consistent with previous reports.15 We also compared surface marker expressions between WT BM-MSCs and ARKO BM-MSCs to see whether knockout (KO) of AR changes BM-MSCs subtypes. The results showed no significant difference between WT and ARKO BM-MSCs (Supporting Fig. 2). We then compared ARKO BM-MSCs versus WT BM-MSCs for their transplantation therapeutic efficacy in CCl4-induced liver cirrhotic mice. Because BM-MSCs express Cre recombinase (Cre) and GFP, we therefore used these two markers to monitor transplantation efficacy. First, we checked whether BM-MSCs were able to migrate to the liver and found both Cre expressions and GFP signals were detected in BM-MSCs-transplanted mouse liver tissues, but not in liver tissues from healthy control or untransplanted CCl4-treated mice (Supporting Fig. 3), indicating that transplanted BM-MSCs indeed migrated to the cirrhotic liver.

Histological analysis revealed that WT BM-MSCs-transplanted livers showed lower levels of collagen (shown by Picro Sirus Red staining; Fig. 1A-a), fibronectin (Fig. 1A-b), and alpha smooth muscle actin (α-SMA; Fig. 1A-c), compared to untransplanted mice. Importantly, we found that ARKO BM-MSCs-transplanted liver showed even much lower fibrotic marker expressions than WT BM-MSC-transplanted liver (Fig. 1A-a-c), suggesting that WT BM-MSCs transplantation improved liver cirrhosis and that ARKO BM-MSCs further enhanced this transplantation therapeutic efficiency. Liver functional assays, performed by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT), and albumin (Fig. 1B-d-f), all showed consistent results in that BM-MSCs-transplanted mice had improvement in liver cirrhosis and that KO of AR in BM-MSCs increased transplantation therapeutic efficacy.

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Figure 1. ARKO BM-MSCs showed better efficacy to suppress liver cirrhosis than WT BM-MSCs in the CCl4- and TAA-induced liver cirrhosis mouse models. (A) (a) Picro Sirius Red staining for collagen deposition, (b) immunohistochemistry staining of fibronectin, and (c) IF staining of α-SMA to detect fibrosis area in healthy mice (N) and mice treated with CCl4, CCl4 plus transplantation of WT-MSCs, and CCl4 plus transplantation of ARKO-MSCs. Graphs on the right side of images are quantification results of images; n = 4∼8. (B) (d) AST and (e) ALT assays were performed to monitor liver function with sera extracted from peripheral blood; n = 8∼12. (f) Albumin was also used to monitor liver function; N = 8∼12. (C) Picro Sirius Red staining was used to stain collagen deposition in the TAA-induced cirrhotic mouse model. Quantifications of relative fibrotic area are shown on the right side of the representative images. (D) (g) AST and (h) ALT assays results obtained from sera collected from TAA-induced cirrhotic mouse models. *P < 0.05; **P < 0.001; ***P < 0.0001. P values were generated with the Student t test.

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We further confirmed these findings in the second liver cirrhosis mouse model in TAA-induced liver cirrhosis mice with consistent results (Fig. 1C,D-g,h), showing better therapeutic outcome in ARKO BM-MSCs-transplanted mice than WT BM-MSCs-transplanted mice, compared to untransplanted CCl4-treated mice.

ARKO in BM-MSCs Demonstrates Improved Therapeutic Effects by Antifibrotic Action in Cirrhotic Livers.

BM-MSCs transplantation has been shown to improve autoimmune disease, sepsis, and myocardial infarction through anti-inflammatory effects. Pro-inflammatory and pro-fibrogenic signals have been linked to liver fibrosis16 and showed that BM-MSCs improved liver cirrhosis through antifibrosis by down-regulating transforming growth factor beta 1 (TGFβ1).17 We examined the expression of the fibrotic marker, TGFβ1, in mice liver tissues and found that ARKO BM-MSCs-transplanted livers showed lower expressions of TGFβ1 and TGFβ receptor 2, compared with WT BM-MSCs-transplanted mice (Fig. 2A-a-c and Supporting Fig. 4A).

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Figure 2. ARKO-MSCs exert improved therapeutic effects by anti-fibrotic and anti-inflammatory actions. (A) Anti-fibrosis effects were shown by reduced expression of fibrosis markers examined with (a) immunohistochemical (IHC) staining of TGFβ1, a key factor in liver fibrosis, in healthy mouse livers and cirrhotic mouse livers treated with CCl4, WT BM-MSCs, and ARKO BM-MSCs. (b) Quantification of TGFβ1 IHC staining; n = 4∼9. (c) TGFβ1 mRNA expression in mouse liver tissues of the same group; n = 9∼12. B. (d) PCNA and α-SMA IF staining. PCNA/SMA double-positive cells (merged image) represent activated HSCs. (e) Quantification results of (d); n = 5∼8. (f) TIMP-2 expression was shown using qRT-PCR; n = 9∼12. Anti-inflammation was confirmed with inhibition of macrophage infiltration. (C) (g) F4/80 staining for macrophage. Arrowheads indicate F4/80-positive signals. (h) Quantification of F4/80-positive area; n = 4∼8. (i) qRT-PCR result of MCP-1 in liver tissues; n = 9∼12. D. (j) IL1Ra, a key player in BM-MSCs-mediated anti-fibrosis and anti-inflammation, was determined by IHC. (k) Quantification of IL1RA-positive area; n = 4∼7. (l) qRT-PCR of IL1RA; N = 9∼12. *P < 0.05; **P < 0.001; ***P < 0.0001. P values were generated with the Student t test.

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We then examined the proliferation of myofibroblasts with double immunofluorescence (IF) staining in liver tissues using antibodies (Abs) of α-SMA and proliferating cell nuclear antigen (PCNA) and found decreased numbers of double stained cells (indicating less proliferating myofibroblasts) in liver tissue of ARKO BM-MSCs-transplanted mice, compared with those transplanted with WT BM-MSCs (Fig. 2B-d,e), suggesting that ARKO BM-MSC transplantation in mice inhibited fibrosis more significantly.

Tissue inhibitor of metalloproteinase 2 (TIMP-2) has been shown to possess antiapoptotic effects on hepatic stellate cells (HSCs) and plays an important role in promoting liver cirrhosis.18 We found that BM-MSCs-transplanted livers have decreased TIMP-2 expression, compared to livers without transplantation. More important, it was shown that ARKO BM-MSCs-transplanted livers showed even lower TIMP-2 expression level, compared with WT BM-MSCs-transplanted mice (Fig. 2B-f), suggesting that HSCs in BM-MSCs-transplanted mice have higher apoptotic potential than untransplanted mice and that knockout in BM-MSCs enhanced this potential.

Depletion of AR in BM-MSCs Improves Its Therapeutic Effects by Anti-inflammatory Action.

Clinically, it has been shown that patients with liver cirrhosis have higher circulating cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha, than healthy patients.19 The increased circulating cytokines could then elevate the circulating monocytes that lead to enhance monocyte/macrophage infiltration in damaged livers.19, 20 We observed lower numbers of F4/80 positively stained cells (indicating infiltrating macrophages) in BM-MSCs-transplanted mice livers, compared to untransplanted mice, and even lower numbers of infiltrated macrophages were detected in ARKO BM-MSCs-treated mice (Fig. 2C-g,h). We also found that ARKO BM-MSCs-treated livers have significantly reduced expression of monocyte chemotactic protein-1 (MCP-1; an indicator of anti-inflammatory action in liver tissues) (Fig. 2C-i), suggesting that transplantation of ARKO BM-MSCs does exert potent anti-inflammation effects in fibrotic livers.

To further confirm anti-inflammation effects, we also examined expressions of IL-1 receptor antagonist (IL1Ra), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) that have been linked to BM-MSC-mediated anti-inflammation/anti-fibrosis therapies in myocardial infarction and liver cirrhosis.7, 21-23 We found significantly increased IL1Ra expressions, at both messenger RNA (mRNA) and protein levels, in ARKO BM-MSCs-transplanted livers, as compared with those transplanted with WT BM-MSCs (Fig. 2D-j-l), and transplanted BM-MSCs are the major cells secreting IL1Ra (Supporting Fig. 5A,B). However, we detected no significant difference in HGF, VEGFB, and VEGFC expressions (Supporting Fig. 4C-E). Surprisingly, we observed significantly reduced MMP-2 and -9 expressions upon BM-MSC transplantation, and ARKO BM-MSCs showed better reduction than WT BM-MSCs (Supporting Fig. 4F,G), implying that BM-MSCs transplantation inhibited inflammatory response. These results are consistent with the clinical observation showing MMP-2 and -9 were elevated in chronic and inflammatory liver disease patients,24, 25 but opposite to the report proposing BM-MSCs therapeutic effects through elevating MMPs.23

AR Suppresses Self-Renewal and Migration Potentials of BM-MSCs.

To dissect the potential mechanisms by which knockout of AR in BM-MSCs could lead to better transplantation efficacy through anti-inflammation/anti-fibrosis signals, we investigated the self-renewal and migration potentials of BM-MSCs that have been shown to improve therapeutic outcomes on myocardial infarction and liver cirrhosis through anti-inflammatory and anti-fibrotic actions.26-28 We found higher self-renewal potential in ARKO BM-MSCs than WT BM-MSCs using the CUF-f assay29 (Fig. 3A-a). Western blotting analysis also showed higher PCNA expression in ARKO BM-MSCs than WT BM-MSCs (Fig. 3A-b).

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Figure 3. (legend on page 1556)Loss of AR in BM-MSCs leads to suppressed HSC activation by enhancing their self-renewal and migration potentials. (A) (a) CFU-f assays were performed to examine the self-renewal potential of WT and ARKO BM-MSCs; n = 7. (b) PCNA was detected using western blotting in WT BM-MSCs (WT1 and WT2) and ARKO BM-MSCs (ARKO1 and ARKO2). α-Tubulin served as the loading control. Numbers below the blot are the quantitative results of PCNA relative expression. (c) p-Erk1/2, Erk1/2, pAkt, Akt, pEGFR, EGFR, and AR were detected in the primary isolated WT BM-MSCs and ARKO BM-MSCs. (d) EGFR expressions in BM-MSCs isolated from WT and ARKO mice at ages of 8, 12, and 16 weeks (n = 4∼12) were measured using the qRT-PCR. (B) To assay mobilization ability, WT BM-MSCs and ARKO BM-MSCs were placed in the upper chamber, and (e) medium alone and (f) hepatocytes were placed in the lower chamber for migration assay. Migration of BM-MSCs was determined by counting BM-MSCs that migrated to the lower side of the membrane. To determine AR influence on BM-MSC migration, MMP-9 mRNA expression was measured using qRT-PCR in (g) BM-MSCs isolated from WT and ARKO mice at 8, 12, and 16 weeks of age, and (h) C3H10T1/2 (mesenchymal stem cells) infected with lentivirus packed with either scramble control or siRNA-AR. (i) Zymography was performed to measure MMP-9 activity of WT and ARKO MSCs. Numbers in (i) indicate individual mice. (j) MMP-9 inhibitor was applied to pretreat WT and ARKO MSCs for 72 hours to mask AR effects on migration. (C) (k) In vivo colocalization of GFP and Ki67 was performed in CCl4-treated mouse livers, transplanted with WT BM-MSC expressing GFP or ARKO BM-MSC expressing GFP, to determine the proliferation rate of BM-MSCs. (l) Ratio of quantification result of colocalization in (k) of GFP and Ki67 to GFP alone. (m) Only GFP-positive cells. (D) Macrophage migration assay. Upper chambers were seeded with macrophages, and lower chambers were seeded with HSC-T6 cells, which were treated with 10 ng/mL of IL-1β in the presence of CM from either low-number WT BM-MSCs (Lo WT CM) or high-number WT BM-MSCs (Hi WT CM) 1 day before being cocultured with macrophage. Twenty-four hours later, migrated macrophages would be counted to evaluate CM's masking effects on IL-1β-stimulated HSCs to secreted chemoattractant proteins to recruit macrophages infiltration. (E) HSC-T6 growth assays were measured using cell counting. HSC-T6 cells were treated with 10 ng/mL of IL-1β and 100 pg/mL of TGFβ1 in the presence of either Lo WT CM or Hi WT CM. HSC-T6 was counted at day 6 to observe masking effects of CM on IL-1β- and TGFβ1-induced HSC-T6 proliferation. *P < 0.05; **P < 0.001; ***P < 0.0001. P values were generated with the Student t test.

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We then dissected the mechanisms by which ARKO BM-MSCs have higher self-renewal ability, and found that knocking out AR in BM-MSCs led to activation of extracellular signal-related kinase 1 and 2 (Erk1/2) and protein kinase B (Akt) signals and their upstream signal, endothelial growth factor/endothelial growth factor receptor (EGF/EGFR; Fig. 3A-c,d), suggesting that AR in BM-MSCs might be able to promote the self-renewal potential through modulation of EGF-Erk1/2 and EGF-Akt signals.

It is interesting to know whether human MSCs (hMSCs) also express AR and whether knockdown of AR in hMSCs results in the similar mechanistic regulation as observed in mouse models. We demonstrated that hMSCs have detectable AR expression (Supporting Fig. 6A). Knockdown of AR in hMSCs enhanced EGFR expression to result in activation of Akt and Erk1/2 (Supporting Fig. 6B,E,F).

We then examined AR knockout effect in BM-MSCs on cell migration using Boyden chamber assays, and found that ARKO BM-MSCs have higher migration ability than WT BM-MSCs, as demonstrated by positively stained migrated cells (Fig. 3B-e,f).

We then dissected the mechanisms by which the ARKO BM-MSCs have higher migration ability, and found that ARKO BM-MSCs have higher MMP-9 expression than WT BM-MSCs (Fig. 3B-g). Similar results were also obtained in the established MSCs cell line (C3H10T1/2 cells) manipulated with AR-siRNA (Fig. 3B-h).

To further dissect how AR regulates MMP-9 at the transcriptional level, we constructed an MMP-9 promoter (ranged from +2 to −2629) hooked with a luciferase vector to test whether AR could negatively regulate MMP-9 promoter transactivation activity, and found that AR could suppress MMP-9 expression in promoter regulation (Supporting Fig. 7A-C). We also applied the zymography assay to detect MMP-9 activity, and found higher MMP-9 proteolytic activity in ARKO BM-MSCs, compared with WT BM-MSCs (Fig. 3B-i). This was also confirmed in studies using hMSCs manipulated with AR-siRNA (Supporting Fig. 6C,F).

To test whether ARKO-mediated enhanced migration is MMP-9 dependent, we pretreated ARKO BM-MSCs with an MMP-9 inhibitor and performed the migration assay, and results showed that the addition of the MMP-9 inhibitor indeed masked ARKO-mediated enhanced migration ability (Fig. 3B-j), suggesting that AR needs to go through MMP-9 to exert its influence on BM-MSC migration.

Together, results from three different types of assays all proved that MMP-9 is a critical molecule to mediate the enhanced migration ability of ARKO BM-MSCs.

Finally, we confirmed the above-described findings showing KO of AR in BM-MSCs increased self-renewal potential and migration capacity in CCl4-induced liver cirrhotic mice. Consistently, ARKO BM-MSCs-transplanted liver showed higher Ki67/GFP double-positive stained cells (representing proliferating transplanted BM-MSCs) than WT BM-MSCs (Fig. 3C-k-m).

Enhanced Self-Renewal and Migration Potentials of BM-MSCs Leads to Better Anti-fibrotic and Anti-inflammatory Actions.

To correlate the increased self-renewal and migration potentials of ARKO BM-MSCs improvement in anti-fibrosis and anti-inflammatory actions, we used conditioned medium (CM) of BM-MSCs to test their effects on macrophage migration and HSCs proliferation. Results showed that BM-MSCs-inhibited macrophage migration (anti-inflammatory effects) and HSCs proliferation (anti-fibrotic actions) were BM-MSCs-number dependent (Fig. 3D,E), suggesting that KO of AR-increased self-renewal and migration of BM-MSCs resulted in more BM-MSCs to exert better anti-inflammation and anti-fibrotic actions. Together, results (from Fig. 3A-E) concluded that KO of AR in BM-MSCs led to increased self-renewal and migration potentials of BM-MSCs and these resulted in better transplantation therapeutic efficacy to treat liver cirrhosis by exerting better anti-fibrotic and anti-inflammatory effects. These phenotypes were involved in the modulation of EGF-Erk1/2/Akt signals, as well as MMP-9 signals.

IL1Ra, Secreted From BM-MSCs, Is the Key Molecule That Mediates ARKO BM-MSCs-Exerted Anti-inflammatory and Anti-fibrotic Effects.

All above-described results demonstrated that higher numbers of BM-MSCs migrating into the cirrhotic liver led to better transplantation therapeutic efficacy with higher anti-inflammatory and anti-fibrotic effects (Fig. 3D,E). We were interested to know whether there are any secreted paracrine factors influenced by knockout of AR in BM-MSCs to contribute to anti-inflammatory and -fibrotic actions. To test this possibility, we investigated expressions of HGF, VEGFB, VEGFC, TIMP-2, bone morphogenetic protein, activin membrane-bound inhibitor (BAMBI), and IL1Ra in ARKO and WT BM-MSCs, and found that ARKO BM-MSCs expressed much higher levels of IL1Ra (but not HGF, VEGFB, VEGFC, TIMP-2, and BAMBI) than WT BM-MSCs (Fig. 4A-a and Supporting Fig. 8A-E). Similar results were observed in hMSCs manipulated with AR-siRNA (Supporting Fig. 6D,F). Molecular mechanism dissection revealed that AR acts on the promoter region to inhibit IL1Ra transcription (Supporting Fig. 9A-C).

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Figure 4. IL1Ra is the key factor secreted by ARKO BM-MSCs to mediate anti-inflammatory and anti-fibrotic actions. (A) (a) IL1Ra mRNA expressions were detected in WT and ARKO BM-MSCs isolated from mice 8, 12, and 16 weeks of age; n = 4∼12. Immunoblotting gel showed IL1Ra expressions in WT BM-MSC CM (WT CM) and ARKO BM-MSC CM (ARKO CM). Numbers indicate individual mice. Macrophage migration assays were performed with Raw 264.7 on the upper chamber and HSC on the lower chamber in the presence of either (b) LPS or (c) IL-1β with the combination of either WT or ARKO CM. HSCs were treated with above-mentioned conditions 1 day before coculture with RAW 264.7. After 24-hour incubation, migrated Raw 264.7 cells were counted to evaluate CM-masking effects on IL-1β-stimulated HSCs in secretion of chemoattractant proteins to recruit macrophage infiltration and to further determine anti-inflammatory actions. (d) HSCs growths were examined with the addition of IL-1β in the presence of either WT CM or ARKO CM. (B) (e) PCNA and α-SMA expressions were analyzed to detect HSCs activation after macrophage CM treatment, and (f) WT and ARKO CM were used to block this activation. To confirm the role of IL1Ra in ARKO BM-MSC-suppressed macrophage migration, 10 μg/mL of IL1Ra neutralization Abs were used to mask WT and ARKO CM effects on (g) macrophage migration induced with 10 ng/mL of IL-1β and (h) HSCs activation induced with 10 ng/mL of IL-1β and 100 pg/mL of TGFβ1. (C) (i) CCL2, (j) CXCL1, and (k) CXCL2 expressions of HSCs stimulated with 10 ng/mL of IL1β were measured upon WT and ARKO CM treatment. (D) Anti-IL1Ra neutralization Abs were preincubated with WT and ARKO CM for 4 hours and (l) CCL2, (m) CXCL1, and (n) CXCL2 expressions in HSCs were measured in the WT and ARKO CM treatments in the presence of IL-1β. P values were generated with the Student t test. *P < 0.05; **P < 0.001; ***P < 0.0001.

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Clinical evidence has indicated that IL-1β, the ligand of IL-1 receptor, is elevated in patients with liver cirrhosis and that this elevation is correlated with increased circulating monocytes.19, 20 In addition, IL-1 signals have been suggested to play critical roles in HSCs activation and proliferation and are linked to macrophage infiltration.30, 31 We demonstrated that the addition of one of the most powerful inflammatory stimuli, lipopolysaccharide (LPS), and IL-1β both led to increased macrophage migration into HSCs. We also observed that IL-1β stimulated HSCs growth. When we pretreated HSCs with WT or ARKO BM-MSCs CM, the increased macrophage migration and HSCs growth were suppressed probably the result of the effects of secreted IL1Ra (macrophage migration result is shown in Fig. 4A-b,c and Supporting Fig. 10A; HSCs growth result is shown in Fig. 4A-d and Supporting Fig. 10B,C). ARKO BM-MSC CM showed better suppression than WT BM-MSC CM, suggesting that higher levels of IL1Ra molecule were secreted in ARKO BM-MSCs.

Because macrophages have been shown to be one source of the IL-β in CLDs32 and macrophage CM could enhance HSCs activation and growth,33 we then tested BM-MSCs CM effect on macrophage-induced HSCs growth and activation (Fig. 4B-e), and found that ARKO BM-MSCs showed better suppression than WT BM-MSCs on HSCs growth (Figure 4B-f), indicating that ARKO BM-MSCs could suppress macrophage-induced HSCs growth more significantly through secreting more IL1Ra to block IL1R signaling. Importantly, the addition of the IL1Ra neutralizing Ab did abolish the inhibitory effect significantly (Fig. 4B-g,h), confirming that the effect of ARKO BM-MSCs on anti-inflammatory and -fibrotic actions might need to go through the IL1Ra molecule.

To further explore whether BM-MSCs-secreted IL1Ra is the major source to mediate anti-inflammatory and -fibrotic effects, IL-1β was used to stimulate macrophage (chemoattractant) protein expression and WT and ARKO BM-MSCs CM were used to test whether they could block this enhancement. Because IL-1β showed induction in MCP-1 (chemokine [C-C motif] ligand 2; CCL2), chemokine (C-X-C motif) ligand (CXCL)1, and CXCL2, consistently in two different types of HSCs (Supporting Fig. 11), expressions of CCL2, CXCL1, and CXCL2 were further examined upon BM-MSC CM treatment. Results showed that ARKO BM-MSC CM had higher suppression on CCL2, CXCL1, and CXCL2 expression than did WT BM-MSCs CM (Fig. 4C-i-k). Pretreating BM-MSCs CM with IL1Ra neutralization Ab significantly masked BM-MSCs CM inhibition on CCL2, CXCL1, and CXCL2, suggesting that BM-MSCs exerted anti-inflammatory actions through IL1Ra inhibiting IL1 signaling to abolish the elevation of CCL2, CXCL1, and CXCL2 blocking macrophage infiltration (Fig. 4D-l-n).

Together, results (from Fig. 4) concluded that KO of AR in BM-MSCs led to more secreted IL1Ra that resulted in suppression of macrophage infiltration (anti-inflammation) and HSCs activation (anti-fibrosis) and then yielded better transplantation therapeutic efficacy to treat liver cirrhosis.

Targeting AR in BM-MSCs With AR-siRNA or ASC-J9® Leads to Better Self-Renewal and Migration Potential.

To apply these findings in clinical application by targeting AR in BM-MSCs (mimicking genetic ARKO BM-MSC effects in treating liver cirrhosis), we applied the currently available agents, such as AR-siRNA and ASC-J9®, that could degrade AR in selective cells with little side effects.34 We found ARKO with lentiviral AR-siRNA infection in primary WT BM-MSCs (or C3H10T1/2 and D1 cells) led to increased cell migration and proliferation (Fig. 5A-a-c,B). Similar results were also obtained when we replaced AR-siRNA with ASC-J9®. Results showed that ASC-J9® treatment in WT BM-MSCs caused elevated migration into regular media or to hepatocytes (Fig. 5C-d-f). ASC-J9® was also applied to WT BM-MSCs to determine its effect on WT BM-MSC self-renewal and proliferation potential, and results from the bromodeoxyuridine (BrdU) assay proved that ASC-J9® treatment led to enhanced self-renewal and proliferation in WT BM-MSCs (Fig. 5D). Zymographic analysis also showed that AR-siRNA or ASC-J9® treatment increased MMP-9 activity (Fig. 5E,F). Together, results (from Fig. 5A-F) conclude that targeting AR in BM-MSCs with either AR-siRNA or ASC-J9® yielded similar effects, when compared with BM-MSC effects isolated from ARKO mice, showing better anti-inflammation and anti-fibrosis effects.

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Figure 5. (legend on page 1560)Targeting AR by ASC-J9® or AR-siRNA leads to enhanced self-renewal and migration potentials of BM-MSCs in vitro. (A) (a) C3H10T1/2 (C3), (b) D1 (mouse BM MSCs), and (c) WT-MSCs were infected with lentivirus linked either with scramble control or AR-siRNA. After flow cytometry sorting with the GFP signal (lentivirus plasmid expresses GFP), cells were subjected to the Boyden chamber transwell migration assay; n = 3∼5. (B) Cell growth of C3H10T1/2-sc and C3H10T1/2-siAR were measured with the methyl thiazol tetrazolium assay; n = 4. (C) (d) C3 cells were treated with DMSO and 10 μM of ASC-J9® for 72 hours, and then equal cell amounts were seeded on the upper chamber to start the migration assay for 24 hours; n = 4. Primary isolated WT MSCs were also treated with DMSO and ASC-J9® for 72 hours, then equal cell amounts of each treatment were placed in the upper chamber, and (e) regular medium or (f) hepatocytes were placed in the lower chamber for 24-hour migration assay. Cells migrated to another side (lower side) of membrane were counted; n = 4. (D) WT BM-MSCs were treated with DMSO and ASC-J9® for 48 hours and incubated with BrdU for another 24 hours. BrdU-positive ratio was determined from counting BrdU signals to total nuclei; n = 5. (E and F) WT BM-MSCs infected with scramble or AR-siRNA and treated with DMSO or ASC-J9® for 72 hours were placed in six-well plates at the same density with serum-free media. Supernatants were collected to measure MMP-9 activity with the zymography assay. *P < 0.05; **P < 0.001. P values were generated with the Student t test. BrdU, bromodeoxyuridine.

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New Therapy of Targeting AR in BM-MSCs Using AR-siRNA and/or ASC-J9® to Treat Liver Cirrhosis.

With consistent in vitro results obtained (Fig. 5), it was essential to test whether concordant outcomes could be reached in the in vivo mouse liver cirrhosis model. As expected, we found that lentiviral AR-siRNA infected BM-MSCs have better transplantation therapeutic effects in treating liver cirrhotic mice induced with CCl4 or TAA than scramble control BM-MSCs, when demonstrated using collagen deposition staining and expressions of fibronectin and α-SMA (Fig. 6A-a-c and Supporting Fig. 12A,B). This conclusion was further supported in liver functional assays in CCl4-induced liver cirrhosis mice (Fig. 6B-d-f). Similar results were also obtained in the TAA-induced liver cirrhosis mouse model (Supporting Fig. 12C).

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Figure 6. Targeting AR in WT BM-MSCs improves therapeutic potentials in CCl4-induced liver cirrhosis mice. (A) Therapeutic effects of targeting AR in WT BM-MSCs with siRNA-AR on liver cirrhosis were determined using (a) Picro Sirius Red staining, (b) immunohistochemical (IHC) staining of fibronectin, and (c) IF staining of α-SMA in healthy mouse livers and cirrhotic mouse livers with treatment of CCl4 alone, CCl4 plus transplantation of WT BM-MSCs infected with scramble control (WT MSC-sc), and CCl4 plus transplantation of WT BM-MSCs infected with siRNA-AR (WT MSC-siAR). Graphs located in the right side of images are quantification results of images; n = 4∼10. (B) (d) AST, (e) ALT, and (f) albumin were measured in sera from healthy, CCl4 alone, CCl4 plus WT MSC-sc, and CCl4 plus WT MSC-siAR mice; n = 8∼12. (C) Therapeutic effects of targeting AR in WT-MSCs with AR degradation enhancer ASC-J9® on liver cirrhosis were examined using (g) Picro Sirius Red staining, (h) IHC staining of fibronectin, and (i) IF staining of α-SMA in healthy mice and cirrhotic mice treated with CCl4 alone, CCl4 plus transplantation of WT BM-MSCs treated with DMSO, and CCl4 plus transplantation of WT BM-MSCs treated with ASC-J9®. Graphs located on the right side of images are quantification; N = 4∼10. D. (j) AST, (k) ALT, and (l) albumin were measured in sera isolated from the same mice; n = 8∼12. *P < 0.05; **P < 0.001; ***P < 0.0001. P values were generated with the Student t test.

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Consistent therapeutic outcomes in cirrhotic liver mice were obtained from ASC-J9®-treated WT BM-MSCs. Expression of fibrosis markers confirmed that WT BM-MSCs treated with the ASC-J9® suppressed liver cirrhosis better than vehicle-treated WT BM-MSCs (Fig. 6C-g-i and Supporting Fig. 12D,E). Liver functional assays showed similar results in the CCl4 (Fig. 6D-j-l) and TAA models (Supporting Fig. 12F).

Collectively, targeting AR in BM-MSCs with either AR-siRNA or ASC-J9® may represent a new potential therapeutic approach to battle liver cirrhosis.

Discussion

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

Androgen/AR signaling, as a critical determining factor in sexual phenotypes and maturation, is well documented. It is generally agreed that androgen/AR signaling stimulates differentiation of prostate, bone, and muscle.35-37 During the embryonic stem cell differentiation process, AR amount gradually increases.38 In natural development, androgen/AR signaling seems to enhance the differentiation process, except for adipocyte differentiation. This observation triggered us to think about whether this fact exists in disease treatment when BM-MSCs transplantation therapy is performed. Indeed, it has been suggested that gender difference does exist using BM-MSCs transplantation therapy in heart diseases.13

Although gender differences have been suggested in BM-MSC transplantation therapy with the observation that female BM-MSCs have better therapeutic potential than male BM-MSCs,13 effects of AR, a key factor in male sexual phenotype, in BM-MSC therapeutic application remain unclear. In this study, we used a genetic deletion approach to prove that deletion of AR in BM-MSCs increases their self-renewal potential (consistent results were observed in female ARKO mice; data not shown), migration capacity, anti-inflammatory actions, and anti-fibrotic effects to better treat liver cirrhosis. Because loss of BM-MSCs after transplantation cannot be prevented as a result of microischemia, our finding that knockout of AR in BM-MSCs increases functions and numbers of BM-MSCs after transplantation can provide the future basis for improving BM-MSC transplantation in liver cirrhosis as well as other diseases that have currently adopted BM-MSC transplantation therapy in clinical trials.

In therapeutic approaches, we also clearly demonstrated that targeting AR using either AR-siRNA or ASC-J9® in BM-MSCs both led to better therapeutic outcomes in treating liver cirrhosis, suggesting that targeting BM-MSC AR could exert better BM-MSC transplantation therapeutic efficacy in treating cirrhotic liver. The advantage of targeting AR with ASC-J9® is that we can handle AR degradation in in vitro cultured BM-MSCs before transplanting into liver, and several early in vivo mice studies all proved that ASC-J9® has little side effects and mice have normal sexual activity with healthy serum testosterone level.

Our mechanism studies pointed out that targeting AR in BM-MSCs leads to better therapeutic efficiency to treat liver cirrhosis through four major pathways (as illustrated in Fig. 7). Targeting AR in BM-MSCs could (1) increase EGFR to enhance their self-renewal potential (Fig. 3A), (2) elevate the MMP-9 to stimulate their migration (Fig. 3B), and (3) promote IL1Ra production to inhibit macrophage infiltration and HSCs proliferation (Fig. 4A,B). Increased IL1Ra production, in turn, suppress IL-1/IL1R signaling involved in macrophage infiltration and HSCs growth (Fig. 4). The combinational effects from those four pathways all led to improve therapeutic potential in liver cirrhosis.

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Figure 7. Illustration of detailed mechanism by which AR mediates BM-MSCs transplantation therapy in liver cirrhosis. Targeting AR in BM-MSCs (1) increases self-renewal potential through EGFR and (2) enhances migration capacity by elevating MMP-9 production, resulting in more BM-MSCs reaching the target organ. Depletion of AR in BM-MSCs stimulates IL1Ra production to suppress IL1/IL1R signals to inhibit HSCs-secreting chemoattractant proteins, such as CCL2, CXCL1, and CXCL2, (3) leading to less macrophage infiltration, which has been shown to activate HSCs through TGFβ1, causing less HSCs activation to relieve liver fibrosis. (4) Enhanced IL1Ra production also can antagonize IL1/IL1R-exerted effects on HSCs activation directly to suppress liver fibrosis.

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In summary, we describe a process by which targeting AR, a key factor in male sexual phenotype, in BM-MSCs improves transplantation therapeutic efficacy for treating liver fibrosis. This finding might also be helpful in other diseases that have recently adopted BM-MSCs transplantation therapy in clinical trials.40

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 Karen Wolf (University of Rochester Medical Center, Rochester, NY) for help in editing the manuscript for this article. The authors also thank Dr. Haiyan Pang's (University of Rochester Medical Center) help in BM-MSCs transplantation.

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

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