Dig Dis Sci DOI 10.1007/s10620-014-3423-9
ORIGINAL ARTICLE
Bone Marrow Stromal Cells Inhibit the Activation of Liver Cirrhotic Fat-Storing Cells via Adrenomedullin Secretion Xiaodong Wang • Wei Zhao • Jinghua Wang • Kexin Shi Xufu Qin • Qingfei Kong • Guangyou Wang • Lili Mu • Hulun Li • Bo Sun • Lijun Shi
•
Received: 25 September 2013 / Accepted: 30 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Cirrhosis, or liver fibrosis, which is mainly triggered by cirrhosis fat-storing cells (CFSCs) activation, has traditionally been considered an irreversible disease. However, recent observations indicate that even advanced fibrosis is still reversible by removing the causative agents. Anti-fibrotic effects of bone marrow-derived stromal cells (BMSCs) have been demonstrated by inhibiting CFSCs via cytokines secretion; however, the mechanisms are still unclear. Aims The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated CFSCs. Methods After the co-culture of CFSCs with BMSCs supernatants with or without the addition of recombinant
rat adrenomedullin (AM)/AM-specific siRNA, western blot analysis was mainly used to detect the differences of relative protein expression on CFSCs. Results BMSC-secreted adrenomedullin (AM) effectively inhibited the proliferation and activation of CFSCs by suppressing the expression of Ang II and its binding receptor, AT1, which resulted in a reduction of p47-phox formation. Conclusions Our data suggested that BMSCs inhibited CFSC activation in vitro via the AM-Ang II-p47-phox signaling pathway, and since CFSC activation is an essential part of hepatic fibrosis process, this inhibition by BMSCs implies us new insights into the potential treatment of hepatic fibrosis via BMSCs.
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3423-9) contains supplementary material, which is available to authorized users.
Keywords Bone marrow stromal cells, BMSCs Cirrhotic fat-storing cells, CFSCs Adrenomedullin, AM a-Smooth muscle actin, a-SMA Collagen protein type I, Collagen I Angiotensin II, Ang II
X. Wang Emergency Department, The 2nd Affiliated Hospital of Harbin Medical University, 148 Bao Jian Road, Nangang District, Harbin 150086, China
X. Qin The Heilongjiang Province Hospital of Nangang Distinct, The 2nd Ward of Digestion Department, 405 Gogol Street, Harbin 150001, China
W. Zhao J. Wang Q. Kong G. Wang L. Mu H. Li B. Sun (&) Department of Neurobiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Neurobiology, 157 Bao Jian Road, Nangang District, Harbin 150081, China e-mail: [email protected]
H. Li B. Sun Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education, Harbin, China
W. Zhao Department of Cell Biology, Harbin Medical University, 157 Bao Jian Road, Nangang District, Harbin 150081, China
L. Shi (&) Department of Gastroenterology, The 1st Affiliated Hospital of Harbin Medical University, 23 You-Zheng Street, Nangang District, Harbin 150001, China e-mail: [email protected]
K. Shi Heilongjiang Province Land Reclamation Headquarters General Hostpital, Digestive Department, 235 Hashuang Road, Nangang District, Harbin 150088, China
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Introduction Cirrhosis, a chronic degenerative disease in which normal liver cells are damaged and replaced by fibrous scar tissue and regenerative nodules, has been considered an irreversible disease that can eventually lead to liver cancer. So far, end-stage disease due to liver cirrhosis is an important cause of death worldwide, and its treatment is extremely difficult. Among the 1.4 million liver disease-related deaths each year worldwide, over 55 % are directly attributable to cirrhosis [1]. Currently, the only effective treatment is liver transplantation; however, donor supply, surgical invasiveness, and costs continue to be problems. More than 10 % of patients die while waiting for a liver transplant. Among the fortunate patients who receive liver transplants, the survival rate is 94 % at 3 months, 88 % at 1 year, and 79 % at 3 years [2]. Thus, it is of great interest to search for an effective alternative to treat this type of life-threatening disease. Recently, stem cell-based therapy has shown promise in animal models and some clinical patients. This novel organ regeneration method can enhance liver repair through differentiation potential and paracrine effects and reduce the risk of immune rejection due to tissue compatibility. And the nonhematopoietic part of the bone marrow, referred to as bone marrow-derived marrow stromal cells (BMSCs), is of interest because of their large in vitro and in vivo differentiation potential. A key advantage of using BMSCs is their immunological properties by secreting TGF-b, hepatocyte growth factor (HGF), nerve growth factor (NGF), and many other cytokines. Therefore, the therapeutic potential of bone marrow stromal cell (BMSC) therapy on liver fibrosis has become an area of great interest, and BMSCs have been widely used in pre-clinical and clinical trials. Cirrhosis fat-storing cells (CFSCs), also known as hepatic stellate cell line (HSCs), are the major source of fibril proteins. These cells play an important role in the progression of hepatic fibrogenesis. They are normally quiescent and reside in the space of Disse (or perisinusoidal space), where they modulate blood flow in the liver [2–5]. However, when the liver is damaged by viral infection, alcoholic hepatitis, or hepatosteatosis, there is a common pathologic mechanism that leads to fibrosis: the generation and proliferation of smooth muscle a-actin (a-SMA)-positive myofibroblasts of periportal and perisinusoidal origin which arise as a consequence of the activation of CFSCs [6, 7], and the occurrence of fibrogenesis. The activated CFSCs are a rich source of type I and III fibrillar collagen, secrete high levels of tissue inhibitors of metalloproteinase (TIMPs) and also express high levels of plateletderived growth factor receptor type b (PDGFRb), which binds to PDGF for the maintenance of CFSC proliferations and migrations [8]. Activated CFSCs are generated in the extracellular matrix (ECM) of the principal cells during liver fibrosis. ECM production is the primary reason for the
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excessive fibrous tissue formation that eventually leads to cirrhosis. Although it has been postulated that activated CFSCs constrict sinusoids and, thereby, worsen portal hypertension [9, 10], the mechanism by which activated CFSCs augment contraction is not fully understood. Recent studies have also shown that BMSCs have therapeutic effects on cirrhotic rats, by modulating CFSCs in vitro via the TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secretion of hepatocyte growth factor (HGF) [11]. CFSC growth can be irreversibly inhibited by BMSCs, followed by cell apoptosis. This apoptotic effect possibly occurs via activation of the JNK pathway by BMSC-secreted NGF and HGF. Apoptosis is enhanced through inhibition of TGF-b [12]. Evidence indicates that BMSCs might have anti-fibrotic effects by inhibiting CFSC activation. In this study, we examined the efficacy of BMSCs in regulating the activation of CFSCs and identified possible mechanisms via in vitro tests. Hopefully, our results might provide new insights into the potential treatment on CFSC fibrogenesis with BMSCs.
Materials and Methods Animals Healthy Sprague–Dawley SD rats (110–120 g) were purchased from the Animal Institute of the Second Affiliated Hospital of Harbin Medical University and maintained under specific pathogen-free conditions at 20 ± 2 °C and 45 ± 5 % humidity. All animal handling and experimental procedures were performed in accordance with the Guidelines of the Care and Use of Laboratory Animals published by the China National Institute of Health. Cell Preparation For BMSC isolation, whole bone marrow (BM) aspirates from the limbs of normal SD rats were obtained as previously described [13] (See supplementary material). Different passages of BMSCs were cultured in DMEM containing 10 % certified fetal bovine serum qualified for mesenchymal cells (Bioind, Israel), and siRNA knockdown was used to genetically inhibit AM gene expression on BMSCs. BMSCs were divided into several culture dishes containing 2.5 ml culture medium and incubated for 48 h before the supernatants were collected. Cirrhosis fat-storing cells, a rat hepatic stellate cell strain, were donated by the Department of Neurobiology of Harbin Medical University. Cells were sustained on uncoated plastic tissue culture dishes and cultured in DMEM culture medium (Hyclone, USA) supplemented
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with 10 % fetal calf serum (FCS) and digested with 0.25 % trypsin plus 0.02 % EDTA. And for the experiments, CFSCs in good condition were seeded into six-well plates with BMSC culture supernatants prepared as mentioned above and incubated for 48 h before collection, and 2 9 10-7 mol/L recombinant AM (Phoenix Biotech co. LTD) was added as a positive control. Losartan (10-7 M, Santacruz, USA), an AT1 receptor antagonist, was also used. CCK-8 Assay Single CFSC suspensions were adjusted to a concentration of 2 9 105/ml. Four 100 ll aliquots of cell suspensions, each containing 2 9 104 cells, were placed into 96-well flat-bottom microtiter plates (Nunc, Copenhagen, Denmark) in DMEM culture medium, supplemented with 10 % heat-inactivated FCS. Cells were incubated at 37 °C in 5 % CO2 for 24 h until a monolayer formed. The medium was aspirated and followed by 100 ll of supernatant from different passages (P1, P3, P5, and P8) of BMSCs or the supernatant from BMSCs transfected with AM-specific siRNA addition to each well; recombinant AM was added as positive control, and fresh BMSC culture medium was used as a negative control. After 48-h incubation, 10 ll CCK-8 was added to each well. The cells were incubated at 37 °C for another 1–4 h until the cells presented an orange-colored formazan dye. Absorbance was read at 450 nm using a 3550-UV microplate reader (BioRad). All experiments were performed at least three separate times. Results were expressed as the mean optical density (OD) value ± standard deviation (SD). Determination of AM and Ang II levels by Radioimmunoassay Quantitative analysis of adrenomedullin (AM) and angiotensin II (Ang II) levels was performed by AM and Ang IIspecific radioimmunoassay kits (Beijing Sino-UK Institute of Biological Technology, China). Measurements of AM were made on the culture medium of different passages (P1–P8) of BMSCs, and fresh BMSC culture medium was used as a control; for the Ang II detection, CFSCs were cultured in passage 3 BMSC culture supernatant with or without siRNA reagent or recombinant AM. The cell supernatants were prepared after 48-h incubation for the RIA assay, and Ang II levels were detected. Results were expressed as the mean cytokine concentration (ng/ml) ± SD. Immunoblotting Whole-cell lysates were prepared from cultured CFSCs with different supernatants or recombinant AM. Immunoblotting
was performed, as described previously [14]. Briefly, proteins were fractionated by SDS-PAGE (Invitrogen), transferred to polyvinylidene difluoride membrane (Invitrogen), and immunoblotted with primary antibodies against a-SMA (1:400, Sigma-Aldrich), collagen I (1:200, Bios, China), angiotensin type I receptor (AT1), angiotensin type II receptor (AT2) (1:500, Millipore, USA), p47-phox (1:100, Santa Cruz Biotechnology Inc., USA), PDGFRb (1:200, Bios, China), TIMP1 (1:200, Bios, China), and GAPDH (1:2,000, Santa Cruz Biotechnology Inc.) overnight at 4 °C. The blots were then washed with TBST and incubated with goat-anti-rabbit or goat-anti-mouse secondary antibodies (1:1,000) for 2 h at room temperature (RT). Detection of GAPDH severed as controls. The membranes were washed and protein bands were visualized and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences). Immunofluoresence Immunofluorescence staining was performed on CFSCs, which were plated on round coverslips, and cultured in sixwell plates containing P3 BMSC supernatant with or without AM-specific siRNA or the recombinant AM until the confluence reached about 80 % for 48 h. P0 (fresh BMSC culture medium) was used as a control. After 48 h incubation, culture medium was removed, and cells were fixed in 4 % paraformaldehyde for 15 min, then permeabilized with 0.1 % Triton X-100/1 9 PBS for 30 min at room temperature, and blocked by 0.1 % BSA for 30 min, every step was followed by 3 times, extensively for 3–5 min PBS washings. Cells were then incubated with primary antibodies (fluorescein-labeled-rabbit-anti-ratCLR, RAMP2, and RAMP3 were used for AM receptors detection, and rabbit-anti-rat-Collagen I and a-SMA were performed for the observation of CFSC activation, all these primary antibodies were purchased from Bios, Beijing, China) for 1 h at 37 °C, followed by an incubation with polyclonal secondary FITC-conjugated antibodies (SigmaAldrich, USA) for 1 h at RT. The cells were stained with DAPI for nuclei (Sigma-Aldrich, USA) for 5 min and then mounted with glycerol in PBS and photographed with a confocal laser scanning microscope (Olympus, Japan). Gene Knockdown Studies BMSCs were cultured overnight at density of 5 9 105 cells per well in 24-well tissue culture plate (Corning, Denmark). Pre-designed siRNA were used for AM gene knockdown (Santa Cruz, USA). Transfection was performed using siRNA Transfection System (GenePharma Company, China) according to manufacturer instructions. Final siRNA concentration was 50 nM. Transfection was performed for 6 h. After 6 h, medium containing siRNA
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Fig. 1 Adrenomedullin (AM) expression and inhibitory effects of different passages of BMSCs. a, d AM secretion by BMSCs was detected and quantified by RIA (in ng/ml). b, e Inhibitory effects of BMSC paracrine factors on CFSCs by adding BMSC supernatants to
the CFSCs were evaluated by CCK-8 assay. c AM-specific siRNA was used for gene knockdown. Data are shown as mean ± SD, *P \ 0.05; **P \ 0.01
complex was removed and replaced by DMEM supplemented with 10 % FCS. Cells were left undisturbed for 48 h after transfection. One set of cells was also transfected with negative control-scrambled siRNA to rule out the effects on BMSCs because of transfection process. Gene knockdown was confirmed after 48 h with real-time PCR. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Corp., Carlsbad, California, USA) according to suppliers’ instructions. CDNA synthesis and PCR were performed as described previously [14]. The primer pair for AM detection was forward, 50 -CAGGACA AGCAGAGCACGTC-30 , and reverse, 50 -TCTGGCGGTA GCGTTTGAC-30 . MRNA expression levels were normalized to 18 S expressions (forward, 50 -AGTCCCTGCCCTT TGTACACA-30 , reverse, 50 -CGATCCGAGGGCCTCACT A-30 ) and relative fold differences were calculated. The lowest experimental value was set to 1.
Results
Statistical Analysis Differences between groups were analyzed by ANOVA. A P value of less than 0.05 was considered statistically significant.
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AM Secretion and Its Inhibitory Effects on CFSCs by Different Passages of BMSCs To verify the secretion of AM by BMSCs, cell supernatants from different passages (P1–P8) of BMSCs were collected for the detection of AM levels by RIA. The P0 BMSCs (fresh medium without any treatment) contained background levels of AM, whereas higher levels of AM were found in the supernatants of P1–P6 BMSCs. This secretion decreased by P7 and P8 BMSCs (Fig. 1a). These results indicated that P1–P6 BMSCs were suitable for the subsequent experiments because of higher levels of AM secretion. Additionally, to verify the influence of AM secreted by BMSCs, AM-specific siRNA was selected to knockdown the expression of AM by BMSCs at gene level, as shown in Fig. 1c, d; compared with control groups, we detected lower level of AM after AM-specific siRNA treatment at both gene level (Fig. 1c) and protein level (Fig. 1d), implying the expression of AM by BMSCs. After verifying the secretion of AM by BMSCs, we then detected the inhibitory effects of BMSC supernatants on
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Fig. 2 CLR/RAMP2 and CLR/RAMP3 co-expression on CFSCs. Multiple staining with DAPI (blue), CLR (green), and RAMP2/ RAMP3 (red) was manipulated on CFSCs. a, e DAPI, b, f CLR (green), c RAMP2 (red, up), d RAMP3 (red, down), d, h Merge
pictures, yellow color were overlapped by green and red colors, implying the co-expression of CLR and RAMP2 (AMR1), as well as CLR and RAMP3 (AMR2)
CFSC proliferation compared with control group (Fig. 1b). The P3 BMSC supernatant presented the most obvious inhibitory effect on CFSC proliferation, which was correlated with its higher level of AM secretion as measured by RIA. Besides, the inhibitory effect of P3 BMSC supernatant on CFSC proliferation was reversed by AM-specific siRNA treatment (Fig. 1e), which implying the proliferation of CFSCs was inhibited by the secretion of AM secreted by BMSCs. Therefore, we chose the P3 BMSC culture supernatant for the remaining experiments in this paper.
was formed by CLR (green) and RAMP2 (red) overlapping, indicated the AM1 receptor expression. Also, RAMP3 and CLR co-expression was also observed; as illustrated in Fig. 2e–h, both CLR and RAMP3 were distributed in the nucleus and the cytoplasm (Fig. 2h, yellow color).
AM Receptor Expression on CFSCs AM exerts its biological effects by interacting with a functional receptor formed by the combination of the calcitonin receptor-like receptor (CLR) with receptor activitymodifying protein-2 and protein-3 (CLR/RAMP2 and CLR/RAMP3, known as AM1 and AM2 receptors, respectively.) [15]. To study the effects of AM secreted by BMSCs on CFSC activation, we then confirmed the presence of AM receptors on CFSCs. Thus, RAMP2, RAMP3, and CLR cellular presence and localization were evaluated in CFSCs by immunofluorescence staining. We evaluated the relative expression of RAMP2 and CLR, the two proteins forming the functional AM1 receptor. As shown in Fig. 2a–d, both CLR and RAMP2 were distributed in the nucleus and the cytoplasm, and the yellow color, which
BMSCs Inhibit the Activation of CFSCs by Interfering with the Expression of a-SMA, Collagen I, TIMP1, and PGDFRb Since BMSCs have benefits to liver fibrosis patients, we test whether BMSCs also have the effect to inhibit the activation of CFSCs in vitro by co-culturing the CFSCs with BMSC supernatants. In our test, a-SMA and collagen I expressions was preferably detected by immunofluoresence. The cells were subjected to fluorescence detection by confocal microscopy. Compared to the control group (Fig. 3a, e), the number of positive a-SMA and collagen I expression-cells were remarkably reduced by the addition of P3 BMSC supernatant, together with weakened fluorescence intensity (Fig. 3b, f), while this inhibitory effect was abolished by AM-specific siRNA treatment (Fig. 3c, g). And in positive control group, the a-SMA and collagen I expression level evaluated by fluorescence-positive cell number and fluorescence intensity was markedly decreased by the addition of recombinant rat AM (Fig. 3d, h).
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Fig. 3 a-SMA and Collagen I expression by CFSCs. a–h Double staining with FITC-conjugated anti-a-SMA/Collagen I (green) and DAPI (blue). Cells were treated with P0 BMSC fresh medium (a, e),
Fig. 4 a-SMA, Collagen I, TIMP1 and PDGFRb expressions of CFSCs were suppressed by BMSCs. Western blot analysis of the CFSC activation markers. a a-SMA, b Collagen I, c TIMP1 and PDGFRb was done under the indicated conditions. Cells in those four panels were treated as follows: 1st panel-fresh BMSC culture medium, 2nd panel-P3 BMSC supernatant, 3rd panelsiRNA treatment, 4th panel-rat recombinant AM addition. Expression levels were normalized to GAPDH. *P \ 0.05
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P3 BMSC culture supernatant (b, f), and P3 BMSCs-treated with AM-specific siRNA culture supernatant (c, g), respectively. Rat recombinant AM was added as positive control (d, h)
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Fig. 5 Alteration of Ang II expression by CFSCs. Ang II secretion by CFSCs treated with different supernatants was detected and quantified by RIA (in pg/ml). *P \ 0.05
Additionally, we quantified the expression of a-SMA and collagen I in the CFSCs with different treatments by Western blot (Fig. 4a, b upper). Compared with the control group (CFSCs only), the ratios of a-SMA/GAPDH and collagen I/GAPDH (Fig. 4a, b lower graphs) were obviously reduced in CFSCs which received P3 BMSC supernatant (P \ 0.05). This reduction was at least partially neutralized by the AM-specific siRNA addition (P \ 0.05), indicating that BMSCs might inhibit a-SMA and collagen I expression by AM secretion. Additionally, recombinant rat AM treatment displayed a similar trend as BMSC supernatant treatment (Fig. 4a, b last panels). Similarly, TIMP1 (Fig. 4c, the 1st line) and PDGFRb (Fig. 4c, the 2nd line) expressions in protein levels were also downregulated by BMSCs and reversed by siRNA treatment. These data showed us that the AM secreted by BMSCs could inhibit the a-SMA, collagen I, TIMP1 and PDGFRb expression, which are known as markers of CFSC activation [16]. Expression of Ang II We quantified the level of Ang II secretion by CFSCs with different treatments via RIA. Results showed that Ang II was inhibited by the addition of the P3 BMSC supernatant, and this inhibition was reversed by the addition of the AMspecific siRNA (P \ 0.05, Fig. 5). This finding confirmed that activated CFSCs highly expressed Ang II, which could induce the phosphorylation of p47 [17]. And AM, the inhibitory cytokine secreted by BMSCs, could effectively suppress Ang II expression in CFSCs. P47-Phox Expression by CFSCs To confirm whether the p47 phosphorylation process was involved in the inhibition of CFSCs, the phosphorylation of p47 in CFSCs was detected via Western blot and compared
Fig. 6 Alteration of P47-phox expression in CFSCs. a Western blot analysis of P47-phox expression with the indicated treatments. Expression levels were normalized to GAPDH. Data are shown as mean ± SD, *P \ 0.05
with a control group (CFSCs only). The p47-phox/GAPDH ratio was clearly diminished in cells with P3 BMSC supernatant (P \ 0.05, Fig. 6). This reduction was canceled by AM-specific siRNA addition (P \ 0.05), implying that AM expression in the BMSC supernatant may inhibit p47-phox expression.
Expression of AT1 and AT2 Ang II has been reported to induce p47-phox phosphorylation and reactive oxygen species (ROS) production by interacting with angiotensin type I receptor (AT1), thereby promoting liver fibrosis. However, the interaction of Ang II with angiotensin type II receptor (AT2) results in a completely anti-fibrotic function. In the above experiment, we confirmed that the activated CFSCs highly expressed Ang II and p47-phox, which were synchronously inhibited by BMSC supernatant addition, and reversed by siRNA treatment, but whether these proteins associated with AT1 and AT2 was still unknown. Therefore, we performed Western blots for the detection of AT1 and AT2 in CFSCs treated with P3 BMSC supernatant with or without siRNA. GAPDH was selected as a control. As shown, P3 BMSC supernatant inhibited AT1 receptor expression, whereas the addition of AM-specific siRNA neutralized this inhibition (Fig. 7a, the first line). However, the P3 BMSC supernatant caused a markedly increase in AT2 expression, and the addition of AM-specific siRNA reversed this effect (Fig. 7a, the 2nd line).
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Fig. 7 Changes of AT1 and AT2 expression by CFSCs. a Western blot analysis for AT1 (the first line) and AT2 (the second line) expression levels in the indicated treatments was done. b Losartan
was added to block AT1 passway with or without recombinant rat AM as listed. GAPDH was set as control. Data are shown as mean ± SD, *P \ 0.05; **P \ 0.01
Additionally, we used another agent losartan, the AT1 receptor antagonist, not the AT2 receptor antagonist, to observe the alternations of AT1. Data had shown that AM addition declined the AT1 level, and losartan addition markedly blocked AT1 receptor expression. Combination of losartan and recombinant rat AM had a similar trend as losartan only (Fig. 7b), these results indicated that AM played a role in the blockage of AT1 receptor.
CFSCs are the major source of fibrillar matrix proteins. Studies have shown that CFSC activation is a fundamental feature of liver fibrosis, and hepatic fibrogenesis is mainly triggered by CFSC activation. In vivo, CFSCs undergo a pleiotropic response termed ‘‘activation’’ that also occurs in culture models in vitro and ultimately leads to the transition of CFSCs from resident quiescent CFSCs into myofibroblasts expressing a-SMA [3]. Besides, the onset of CFSC activation coincides with the induction of PDGFRb expression, while PDGF is the most potent mitogen for culture-activated CFSCs [8]. And as reported, pathophysiologic fibrosis is essentially an excessive accumulation of ECM components, particularly type I collagen, together with declined expression and activity of matrix metalloproteinases (MMPs) [21]. However, CFSCs are responsible for the excess production of ECM components. Therefore, in our tests, a-SMA, collagen I, TIMP1 (a natural inhibitor of MMPs), and PDGFR-beta were chose as markers of CFSC activation. In this report, we firstly identified the secretion of AM by different passages of BMSCs via RIA, and this secretion could be decreased by AM-specific siRNA treatment, and data are displayed in Fig. 1a, c. Then, we found that supernatants of P1, P3, P5 passages of AM-expressing BMSCs inhibited the proliferation of CFSCs since their high levels of AM content (Fig. 1b, d), thus P3 passage of BMSC supernatant was selected for the following experiments because of its more effectively function. As known, AM has been reported to act as an anti-fibrotic factor,
Discussion In this study, we found that BMSCs inhibited CFSC proliferation and activation via AM secretions. This inhibition was exerted by decreasing Ang II expression and its receptor AT1 but not AT2, resulting in a reduction of p47phox formation. Traditionally, hepatic cirrhosis has been thought to be irreversible. However, recent evidence from animal studies and human clinical trials has indicated that even advanced fibrosis is reversible [18, 19]. Any intervention that can effectively remove the causative agents could potentially reverse the liver fibrosis process. Hence, the inhibition of the CFSC proliferation and activation, alteration of ECM production, and related subsequent events are crucial goals for intervention in the hepatic fibrogenesis cascade. CFSCs are fibrogenic cells that express p47-phox and produce ROS. They were firstly observed in 1876 by von Kupffer and scarcely studied for almost 100 years [20].
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protecting cells from hypoxic pulmonary damage by inhibiting cell proliferation, collagen synthesis, and TGFb1 production [22, 23]. Besides, we had also confirmed the expression of AM receptors (both AMR1 and AMR2) on CFSCs by immunofluorescence in Fig. 2. Therefore, we tried to verify whether the activation of CFSCs was affected by BMSC supernatant by detecting a-SMA, collagen I, TIMP1, and PDGFRb expression alternations. Compared with the control group, the levels of these four markers of activated CFSCs were all decreased when the cells were incubated in P3 BMSC culture supernatant, but these decreases were remarkably reversed by the siRNA treatment (Figs. 3, 4), and rat recombinant AM was added as a positive control. Based on these four activation markers changes, we concluded that BMSCs could effectively inhibit CFSC activation, at least partially by their secretion of AM. Next, we investigated the potential mechanisms of this regression of CFSC activation. Hepatic fibrinogenesis can be caused by ROS production by different liver cells after various injuries [24, 25]. ROS can stimulate the production of collagen I and may act as an intracellular signaling mediator of the fibrogenic action of TGF-b. NADPH oxidase (NOX) is a multicomponent enzyme complex that generates ROS in response to various stimuli. The NOX complex has been increasingly recognized as a key element of intracellular signaling of hepatic fibrogenesis. Both phagocytic and nonphagocytic NOX components in CFSCs mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy [14]. The molecular components and signaling pathways of various NOX homologues that are critical for fibrotic activity in CFSCs need to be more clearly identified. P47-phox, with its different domains and several phosphorylated sites, is a main regulator of NOX activation by organizing the complex. p47phox-/- mice have been widely used as a genetic model to study NOX inhibition. Bataller et al. [14] demonstrated that p47phox-/- mice showed attenuated liver injury and fibrosis compared with wild-type counterparts after bile duct ligation. To determine whether the p47-phox phosphorylation process was involved in this CFSC inhibition, the phosphorylation of p47-phox was detected by Western blot. As expected, p47-phox was downregulated by BMSC supernatant, but upregulated by the addition of AM-specific siRNA (Fig. 6). These data implied that secretion of AM inhibited CFSCs by interfering with the p47-phox pathway. Better understanding of the different mechanisms and transduction pathways involved in p47-phox phosphorylation and its interactions would be necessary to design new therapeutic agents to downregulate ROS hyperproduction in inflammatory diseases, without decreasing the normal response to bacterial-derived stimuli [26].
Ang II, a pro-oxidant and fibrogenic cytokine, induces p47-phox phosphorylation and ROS production through its co-expression with AT1. However, the interaction of Ang II with AT2 presents a completely anti-fibrotic function. Inhibition of Ang II synthesis or AT1 has been shown to decrease inflammation and hepatic fibrosis in experimental models of chronic liver injury. The fibrogenic response caused by hepatic collagen accumulation was shown to be lower in AT1 knockout mice. Several experiments have demonstrated that Ang II is able to induce ROS production in activated human CFSCs. Ang II-induced ROS formation in CFSCs was shown to be inhibited when CFSCs were treated with either a NOX inhibitor or with the AT1 antagonist. We detected a decreased Ang II expression in CFSCs when P3 BMSC supernatant was added. The addition of AM-specific siRNA reversed this effect (Fig. 5), and these results were consistent with previous reports that AM inhibited Ang II-induced oxidative Stress [27]. To determine whether Ang II expression was associated with AT1 and AT2 levels, we performed Western blot analysis of AT1 and AT2 in CFSCs treated with P3 BMSC supernatant with or without siRNA. The P3 BMSC supernatant dramatically inhibited AT1 expression, and siRNA addition neutralized this inhibition (Fig. 7). The AT2 expression was markedly increased by the P3 BMSC supernatant, but decreased by siRNA treatment (Fig. 7). These findings confirmed that activated CFSCs highly expressed Ang II, which could induce the phosphorylation of p47 by interacting with AT1 but not AT2. Moreover, they also indicated that AM, the inhibitory cytokine secreted by BMSCs, could effectively act on this pathway. BMSCs are multipotent stem cells with the potential to differentiate into liver cells. They play an important role in liver fibrosis treatment without allograft rejection. In this study, we observed that the induction of the AM-Ang II-p47phox signaling pathway could dramatically repress the activation of CFSCs co-cultured with BMSC supernatant in vitro, which might potentially result in the regression of CFSCs fibrogenesis. However, whether BMSCs can inhibit CFSCs in vivo through the AM-Ang II-p47-phox signaling pathway is still unclear. Further studies are needed to clarify the mechanisms in vivo to improve the quality of life of patients and prolong their survival time via BMSC transplantation. Acknowledgments The study is financially supported by Heilongjiang Postdoctoral Science-Research Foundation (LBH-Q11034), Provincial department of international research projects (WB07C04), and Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education (KF201202). Natural Science Foundation of Heilongjiang Province of China (C201441). Conflict of interest There are no financial interests other than the basic academic employment that the authors have, which could create a conflict of interests or appearance of interests with regard to this work.
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15. De Martin S, Paliuri G, Belloni A et al. Expression and distribution of the adrenomedullin system in newborn human thymus. PLoS One. 2014;9:e97592. 16. Anan A, Baskin-Bey ES, Bronk SF, Werneburg NW, Shah VH, Gores GJ. Proteasome inhibition induces hepatic stellate cell apoptosis. Hepatology. 2006;43:335–344. 17. Pendyala S, Usatyuk PV, Gorshkova IA, Garcia JG, Natarajan V. Regulation of NADPH oxidase in vascular endothelium: the role of phospholipases, protein kinases, and cytoskeletal proteins. Antioxid Redox Signal. 2009;11:841–860. 18. Arthur MJ. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C. Gastroenterology. 2002;122:1525–1528. 19. Issa R, Zhou X, Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology. 2004;126:1795–1808. 20. Brandao DF, Ramalho LN, Ramalho FS, Zucoloto S, Martinelli Ade L, Silva Ode C. Liver cirrhosis and hepatic stellate cells. Acta Cir Bras. 2006;21(Suppl 1):54–57. 21. Williams EJ, Benyon RC, Trim N, Hadwin R, et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut. 2001;49:577–583. 22. Wang Y, Zhang JS, Qian J, Huang GC, Chen Q. Adrenomedullin regulates expressions of transforming growth factor-beta1 and beta1-induced matrix metalloproteinase-2 in hepatic stellate cells. Int J Exp Pathol. 2006;87:177–184. 23. Hao SL, Yu ZH, Qi BS, Luo JZ, Wang WP. The antifibrosis effect of adrenomedullin in human lung fibroblasts. Exp Lung Res. 2011;37:615–626. 24. Albanis E, Friedman SL. Hepatic fibrosis. Pathogenesis and principles of therapy. Clin Liver Dis. 2001;5:315–334 (v–vi). 25. Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis. J Hepatol. 2001;35:297–306. 26. El-Benna J, Dang PM, Gougerot-Pocidalo MA, Marie JC, BrautBoucher F. p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases. Exp Mol Med. 2009;41:217–225. 27. Yoshimoto T, Gochou N, Fukai N, Sugiyama T, Shichiri M, Hirata Y. Adrenomedullin inhibits angiotensin II-induced oxidative stress and gene expression in rat endothelial cells. Hypertens Res. 2005;28:165–172.
ORIGINAL ARTICLE
Bone Marrow Stromal Cells Inhibit the Activation of Liver Cirrhotic Fat-Storing Cells via Adrenomedullin Secretion Xiaodong Wang • Wei Zhao • Jinghua Wang • Kexin Shi Xufu Qin • Qingfei Kong • Guangyou Wang • Lili Mu • Hulun Li • Bo Sun • Lijun Shi
•
Received: 25 September 2013 / Accepted: 30 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Cirrhosis, or liver fibrosis, which is mainly triggered by cirrhosis fat-storing cells (CFSCs) activation, has traditionally been considered an irreversible disease. However, recent observations indicate that even advanced fibrosis is still reversible by removing the causative agents. Anti-fibrotic effects of bone marrow-derived stromal cells (BMSCs) have been demonstrated by inhibiting CFSCs via cytokines secretion; however, the mechanisms are still unclear. Aims The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated CFSCs. Methods After the co-culture of CFSCs with BMSCs supernatants with or without the addition of recombinant
rat adrenomedullin (AM)/AM-specific siRNA, western blot analysis was mainly used to detect the differences of relative protein expression on CFSCs. Results BMSC-secreted adrenomedullin (AM) effectively inhibited the proliferation and activation of CFSCs by suppressing the expression of Ang II and its binding receptor, AT1, which resulted in a reduction of p47-phox formation. Conclusions Our data suggested that BMSCs inhibited CFSC activation in vitro via the AM-Ang II-p47-phox signaling pathway, and since CFSC activation is an essential part of hepatic fibrosis process, this inhibition by BMSCs implies us new insights into the potential treatment of hepatic fibrosis via BMSCs.
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3423-9) contains supplementary material, which is available to authorized users.
Keywords Bone marrow stromal cells, BMSCs Cirrhotic fat-storing cells, CFSCs Adrenomedullin, AM a-Smooth muscle actin, a-SMA Collagen protein type I, Collagen I Angiotensin II, Ang II
X. Wang Emergency Department, The 2nd Affiliated Hospital of Harbin Medical University, 148 Bao Jian Road, Nangang District, Harbin 150086, China
X. Qin The Heilongjiang Province Hospital of Nangang Distinct, The 2nd Ward of Digestion Department, 405 Gogol Street, Harbin 150001, China
W. Zhao J. Wang Q. Kong G. Wang L. Mu H. Li B. Sun (&) Department of Neurobiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Neurobiology, 157 Bao Jian Road, Nangang District, Harbin 150081, China e-mail: [email protected]
H. Li B. Sun Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education, Harbin, China
W. Zhao Department of Cell Biology, Harbin Medical University, 157 Bao Jian Road, Nangang District, Harbin 150081, China
L. Shi (&) Department of Gastroenterology, The 1st Affiliated Hospital of Harbin Medical University, 23 You-Zheng Street, Nangang District, Harbin 150001, China e-mail: [email protected]
K. Shi Heilongjiang Province Land Reclamation Headquarters General Hostpital, Digestive Department, 235 Hashuang Road, Nangang District, Harbin 150088, China
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Introduction Cirrhosis, a chronic degenerative disease in which normal liver cells are damaged and replaced by fibrous scar tissue and regenerative nodules, has been considered an irreversible disease that can eventually lead to liver cancer. So far, end-stage disease due to liver cirrhosis is an important cause of death worldwide, and its treatment is extremely difficult. Among the 1.4 million liver disease-related deaths each year worldwide, over 55 % are directly attributable to cirrhosis [1]. Currently, the only effective treatment is liver transplantation; however, donor supply, surgical invasiveness, and costs continue to be problems. More than 10 % of patients die while waiting for a liver transplant. Among the fortunate patients who receive liver transplants, the survival rate is 94 % at 3 months, 88 % at 1 year, and 79 % at 3 years [2]. Thus, it is of great interest to search for an effective alternative to treat this type of life-threatening disease. Recently, stem cell-based therapy has shown promise in animal models and some clinical patients. This novel organ regeneration method can enhance liver repair through differentiation potential and paracrine effects and reduce the risk of immune rejection due to tissue compatibility. And the nonhematopoietic part of the bone marrow, referred to as bone marrow-derived marrow stromal cells (BMSCs), is of interest because of their large in vitro and in vivo differentiation potential. A key advantage of using BMSCs is their immunological properties by secreting TGF-b, hepatocyte growth factor (HGF), nerve growth factor (NGF), and many other cytokines. Therefore, the therapeutic potential of bone marrow stromal cell (BMSC) therapy on liver fibrosis has become an area of great interest, and BMSCs have been widely used in pre-clinical and clinical trials. Cirrhosis fat-storing cells (CFSCs), also known as hepatic stellate cell line (HSCs), are the major source of fibril proteins. These cells play an important role in the progression of hepatic fibrogenesis. They are normally quiescent and reside in the space of Disse (or perisinusoidal space), where they modulate blood flow in the liver [2–5]. However, when the liver is damaged by viral infection, alcoholic hepatitis, or hepatosteatosis, there is a common pathologic mechanism that leads to fibrosis: the generation and proliferation of smooth muscle a-actin (a-SMA)-positive myofibroblasts of periportal and perisinusoidal origin which arise as a consequence of the activation of CFSCs [6, 7], and the occurrence of fibrogenesis. The activated CFSCs are a rich source of type I and III fibrillar collagen, secrete high levels of tissue inhibitors of metalloproteinase (TIMPs) and also express high levels of plateletderived growth factor receptor type b (PDGFRb), which binds to PDGF for the maintenance of CFSC proliferations and migrations [8]. Activated CFSCs are generated in the extracellular matrix (ECM) of the principal cells during liver fibrosis. ECM production is the primary reason for the
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excessive fibrous tissue formation that eventually leads to cirrhosis. Although it has been postulated that activated CFSCs constrict sinusoids and, thereby, worsen portal hypertension [9, 10], the mechanism by which activated CFSCs augment contraction is not fully understood. Recent studies have also shown that BMSCs have therapeutic effects on cirrhotic rats, by modulating CFSCs in vitro via the TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secretion of hepatocyte growth factor (HGF) [11]. CFSC growth can be irreversibly inhibited by BMSCs, followed by cell apoptosis. This apoptotic effect possibly occurs via activation of the JNK pathway by BMSC-secreted NGF and HGF. Apoptosis is enhanced through inhibition of TGF-b [12]. Evidence indicates that BMSCs might have anti-fibrotic effects by inhibiting CFSC activation. In this study, we examined the efficacy of BMSCs in regulating the activation of CFSCs and identified possible mechanisms via in vitro tests. Hopefully, our results might provide new insights into the potential treatment on CFSC fibrogenesis with BMSCs.
Materials and Methods Animals Healthy Sprague–Dawley SD rats (110–120 g) were purchased from the Animal Institute of the Second Affiliated Hospital of Harbin Medical University and maintained under specific pathogen-free conditions at 20 ± 2 °C and 45 ± 5 % humidity. All animal handling and experimental procedures were performed in accordance with the Guidelines of the Care and Use of Laboratory Animals published by the China National Institute of Health. Cell Preparation For BMSC isolation, whole bone marrow (BM) aspirates from the limbs of normal SD rats were obtained as previously described [13] (See supplementary material). Different passages of BMSCs were cultured in DMEM containing 10 % certified fetal bovine serum qualified for mesenchymal cells (Bioind, Israel), and siRNA knockdown was used to genetically inhibit AM gene expression on BMSCs. BMSCs were divided into several culture dishes containing 2.5 ml culture medium and incubated for 48 h before the supernatants were collected. Cirrhosis fat-storing cells, a rat hepatic stellate cell strain, were donated by the Department of Neurobiology of Harbin Medical University. Cells were sustained on uncoated plastic tissue culture dishes and cultured in DMEM culture medium (Hyclone, USA) supplemented
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with 10 % fetal calf serum (FCS) and digested with 0.25 % trypsin plus 0.02 % EDTA. And for the experiments, CFSCs in good condition were seeded into six-well plates with BMSC culture supernatants prepared as mentioned above and incubated for 48 h before collection, and 2 9 10-7 mol/L recombinant AM (Phoenix Biotech co. LTD) was added as a positive control. Losartan (10-7 M, Santacruz, USA), an AT1 receptor antagonist, was also used. CCK-8 Assay Single CFSC suspensions were adjusted to a concentration of 2 9 105/ml. Four 100 ll aliquots of cell suspensions, each containing 2 9 104 cells, were placed into 96-well flat-bottom microtiter plates (Nunc, Copenhagen, Denmark) in DMEM culture medium, supplemented with 10 % heat-inactivated FCS. Cells were incubated at 37 °C in 5 % CO2 for 24 h until a monolayer formed. The medium was aspirated and followed by 100 ll of supernatant from different passages (P1, P3, P5, and P8) of BMSCs or the supernatant from BMSCs transfected with AM-specific siRNA addition to each well; recombinant AM was added as positive control, and fresh BMSC culture medium was used as a negative control. After 48-h incubation, 10 ll CCK-8 was added to each well. The cells were incubated at 37 °C for another 1–4 h until the cells presented an orange-colored formazan dye. Absorbance was read at 450 nm using a 3550-UV microplate reader (BioRad). All experiments were performed at least three separate times. Results were expressed as the mean optical density (OD) value ± standard deviation (SD). Determination of AM and Ang II levels by Radioimmunoassay Quantitative analysis of adrenomedullin (AM) and angiotensin II (Ang II) levels was performed by AM and Ang IIspecific radioimmunoassay kits (Beijing Sino-UK Institute of Biological Technology, China). Measurements of AM were made on the culture medium of different passages (P1–P8) of BMSCs, and fresh BMSC culture medium was used as a control; for the Ang II detection, CFSCs were cultured in passage 3 BMSC culture supernatant with or without siRNA reagent or recombinant AM. The cell supernatants were prepared after 48-h incubation for the RIA assay, and Ang II levels were detected. Results were expressed as the mean cytokine concentration (ng/ml) ± SD. Immunoblotting Whole-cell lysates were prepared from cultured CFSCs with different supernatants or recombinant AM. Immunoblotting
was performed, as described previously [14]. Briefly, proteins were fractionated by SDS-PAGE (Invitrogen), transferred to polyvinylidene difluoride membrane (Invitrogen), and immunoblotted with primary antibodies against a-SMA (1:400, Sigma-Aldrich), collagen I (1:200, Bios, China), angiotensin type I receptor (AT1), angiotensin type II receptor (AT2) (1:500, Millipore, USA), p47-phox (1:100, Santa Cruz Biotechnology Inc., USA), PDGFRb (1:200, Bios, China), TIMP1 (1:200, Bios, China), and GAPDH (1:2,000, Santa Cruz Biotechnology Inc.) overnight at 4 °C. The blots were then washed with TBST and incubated with goat-anti-rabbit or goat-anti-mouse secondary antibodies (1:1,000) for 2 h at room temperature (RT). Detection of GAPDH severed as controls. The membranes were washed and protein bands were visualized and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences). Immunofluoresence Immunofluorescence staining was performed on CFSCs, which were plated on round coverslips, and cultured in sixwell plates containing P3 BMSC supernatant with or without AM-specific siRNA or the recombinant AM until the confluence reached about 80 % for 48 h. P0 (fresh BMSC culture medium) was used as a control. After 48 h incubation, culture medium was removed, and cells were fixed in 4 % paraformaldehyde for 15 min, then permeabilized with 0.1 % Triton X-100/1 9 PBS for 30 min at room temperature, and blocked by 0.1 % BSA for 30 min, every step was followed by 3 times, extensively for 3–5 min PBS washings. Cells were then incubated with primary antibodies (fluorescein-labeled-rabbit-anti-ratCLR, RAMP2, and RAMP3 were used for AM receptors detection, and rabbit-anti-rat-Collagen I and a-SMA were performed for the observation of CFSC activation, all these primary antibodies were purchased from Bios, Beijing, China) for 1 h at 37 °C, followed by an incubation with polyclonal secondary FITC-conjugated antibodies (SigmaAldrich, USA) for 1 h at RT. The cells were stained with DAPI for nuclei (Sigma-Aldrich, USA) for 5 min and then mounted with glycerol in PBS and photographed with a confocal laser scanning microscope (Olympus, Japan). Gene Knockdown Studies BMSCs were cultured overnight at density of 5 9 105 cells per well in 24-well tissue culture plate (Corning, Denmark). Pre-designed siRNA were used for AM gene knockdown (Santa Cruz, USA). Transfection was performed using siRNA Transfection System (GenePharma Company, China) according to manufacturer instructions. Final siRNA concentration was 50 nM. Transfection was performed for 6 h. After 6 h, medium containing siRNA
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Fig. 1 Adrenomedullin (AM) expression and inhibitory effects of different passages of BMSCs. a, d AM secretion by BMSCs was detected and quantified by RIA (in ng/ml). b, e Inhibitory effects of BMSC paracrine factors on CFSCs by adding BMSC supernatants to
the CFSCs were evaluated by CCK-8 assay. c AM-specific siRNA was used for gene knockdown. Data are shown as mean ± SD, *P \ 0.05; **P \ 0.01
complex was removed and replaced by DMEM supplemented with 10 % FCS. Cells were left undisturbed for 48 h after transfection. One set of cells was also transfected with negative control-scrambled siRNA to rule out the effects on BMSCs because of transfection process. Gene knockdown was confirmed after 48 h with real-time PCR. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Corp., Carlsbad, California, USA) according to suppliers’ instructions. CDNA synthesis and PCR were performed as described previously [14]. The primer pair for AM detection was forward, 50 -CAGGACA AGCAGAGCACGTC-30 , and reverse, 50 -TCTGGCGGTA GCGTTTGAC-30 . MRNA expression levels were normalized to 18 S expressions (forward, 50 -AGTCCCTGCCCTT TGTACACA-30 , reverse, 50 -CGATCCGAGGGCCTCACT A-30 ) and relative fold differences were calculated. The lowest experimental value was set to 1.
Results
Statistical Analysis Differences between groups were analyzed by ANOVA. A P value of less than 0.05 was considered statistically significant.
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AM Secretion and Its Inhibitory Effects on CFSCs by Different Passages of BMSCs To verify the secretion of AM by BMSCs, cell supernatants from different passages (P1–P8) of BMSCs were collected for the detection of AM levels by RIA. The P0 BMSCs (fresh medium without any treatment) contained background levels of AM, whereas higher levels of AM were found in the supernatants of P1–P6 BMSCs. This secretion decreased by P7 and P8 BMSCs (Fig. 1a). These results indicated that P1–P6 BMSCs were suitable for the subsequent experiments because of higher levels of AM secretion. Additionally, to verify the influence of AM secreted by BMSCs, AM-specific siRNA was selected to knockdown the expression of AM by BMSCs at gene level, as shown in Fig. 1c, d; compared with control groups, we detected lower level of AM after AM-specific siRNA treatment at both gene level (Fig. 1c) and protein level (Fig. 1d), implying the expression of AM by BMSCs. After verifying the secretion of AM by BMSCs, we then detected the inhibitory effects of BMSC supernatants on
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Fig. 2 CLR/RAMP2 and CLR/RAMP3 co-expression on CFSCs. Multiple staining with DAPI (blue), CLR (green), and RAMP2/ RAMP3 (red) was manipulated on CFSCs. a, e DAPI, b, f CLR (green), c RAMP2 (red, up), d RAMP3 (red, down), d, h Merge
pictures, yellow color were overlapped by green and red colors, implying the co-expression of CLR and RAMP2 (AMR1), as well as CLR and RAMP3 (AMR2)
CFSC proliferation compared with control group (Fig. 1b). The P3 BMSC supernatant presented the most obvious inhibitory effect on CFSC proliferation, which was correlated with its higher level of AM secretion as measured by RIA. Besides, the inhibitory effect of P3 BMSC supernatant on CFSC proliferation was reversed by AM-specific siRNA treatment (Fig. 1e), which implying the proliferation of CFSCs was inhibited by the secretion of AM secreted by BMSCs. Therefore, we chose the P3 BMSC culture supernatant for the remaining experiments in this paper.
was formed by CLR (green) and RAMP2 (red) overlapping, indicated the AM1 receptor expression. Also, RAMP3 and CLR co-expression was also observed; as illustrated in Fig. 2e–h, both CLR and RAMP3 were distributed in the nucleus and the cytoplasm (Fig. 2h, yellow color).
AM Receptor Expression on CFSCs AM exerts its biological effects by interacting with a functional receptor formed by the combination of the calcitonin receptor-like receptor (CLR) with receptor activitymodifying protein-2 and protein-3 (CLR/RAMP2 and CLR/RAMP3, known as AM1 and AM2 receptors, respectively.) [15]. To study the effects of AM secreted by BMSCs on CFSC activation, we then confirmed the presence of AM receptors on CFSCs. Thus, RAMP2, RAMP3, and CLR cellular presence and localization were evaluated in CFSCs by immunofluorescence staining. We evaluated the relative expression of RAMP2 and CLR, the two proteins forming the functional AM1 receptor. As shown in Fig. 2a–d, both CLR and RAMP2 were distributed in the nucleus and the cytoplasm, and the yellow color, which
BMSCs Inhibit the Activation of CFSCs by Interfering with the Expression of a-SMA, Collagen I, TIMP1, and PGDFRb Since BMSCs have benefits to liver fibrosis patients, we test whether BMSCs also have the effect to inhibit the activation of CFSCs in vitro by co-culturing the CFSCs with BMSC supernatants. In our test, a-SMA and collagen I expressions was preferably detected by immunofluoresence. The cells were subjected to fluorescence detection by confocal microscopy. Compared to the control group (Fig. 3a, e), the number of positive a-SMA and collagen I expression-cells were remarkably reduced by the addition of P3 BMSC supernatant, together with weakened fluorescence intensity (Fig. 3b, f), while this inhibitory effect was abolished by AM-specific siRNA treatment (Fig. 3c, g). And in positive control group, the a-SMA and collagen I expression level evaluated by fluorescence-positive cell number and fluorescence intensity was markedly decreased by the addition of recombinant rat AM (Fig. 3d, h).
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Fig. 3 a-SMA and Collagen I expression by CFSCs. a–h Double staining with FITC-conjugated anti-a-SMA/Collagen I (green) and DAPI (blue). Cells were treated with P0 BMSC fresh medium (a, e),
Fig. 4 a-SMA, Collagen I, TIMP1 and PDGFRb expressions of CFSCs were suppressed by BMSCs. Western blot analysis of the CFSC activation markers. a a-SMA, b Collagen I, c TIMP1 and PDGFRb was done under the indicated conditions. Cells in those four panels were treated as follows: 1st panel-fresh BMSC culture medium, 2nd panel-P3 BMSC supernatant, 3rd panelsiRNA treatment, 4th panel-rat recombinant AM addition. Expression levels were normalized to GAPDH. *P \ 0.05
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P3 BMSC culture supernatant (b, f), and P3 BMSCs-treated with AM-specific siRNA culture supernatant (c, g), respectively. Rat recombinant AM was added as positive control (d, h)
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Fig. 5 Alteration of Ang II expression by CFSCs. Ang II secretion by CFSCs treated with different supernatants was detected and quantified by RIA (in pg/ml). *P \ 0.05
Additionally, we quantified the expression of a-SMA and collagen I in the CFSCs with different treatments by Western blot (Fig. 4a, b upper). Compared with the control group (CFSCs only), the ratios of a-SMA/GAPDH and collagen I/GAPDH (Fig. 4a, b lower graphs) were obviously reduced in CFSCs which received P3 BMSC supernatant (P \ 0.05). This reduction was at least partially neutralized by the AM-specific siRNA addition (P \ 0.05), indicating that BMSCs might inhibit a-SMA and collagen I expression by AM secretion. Additionally, recombinant rat AM treatment displayed a similar trend as BMSC supernatant treatment (Fig. 4a, b last panels). Similarly, TIMP1 (Fig. 4c, the 1st line) and PDGFRb (Fig. 4c, the 2nd line) expressions in protein levels were also downregulated by BMSCs and reversed by siRNA treatment. These data showed us that the AM secreted by BMSCs could inhibit the a-SMA, collagen I, TIMP1 and PDGFRb expression, which are known as markers of CFSC activation [16]. Expression of Ang II We quantified the level of Ang II secretion by CFSCs with different treatments via RIA. Results showed that Ang II was inhibited by the addition of the P3 BMSC supernatant, and this inhibition was reversed by the addition of the AMspecific siRNA (P \ 0.05, Fig. 5). This finding confirmed that activated CFSCs highly expressed Ang II, which could induce the phosphorylation of p47 [17]. And AM, the inhibitory cytokine secreted by BMSCs, could effectively suppress Ang II expression in CFSCs. P47-Phox Expression by CFSCs To confirm whether the p47 phosphorylation process was involved in the inhibition of CFSCs, the phosphorylation of p47 in CFSCs was detected via Western blot and compared
Fig. 6 Alteration of P47-phox expression in CFSCs. a Western blot analysis of P47-phox expression with the indicated treatments. Expression levels were normalized to GAPDH. Data are shown as mean ± SD, *P \ 0.05
with a control group (CFSCs only). The p47-phox/GAPDH ratio was clearly diminished in cells with P3 BMSC supernatant (P \ 0.05, Fig. 6). This reduction was canceled by AM-specific siRNA addition (P \ 0.05), implying that AM expression in the BMSC supernatant may inhibit p47-phox expression.
Expression of AT1 and AT2 Ang II has been reported to induce p47-phox phosphorylation and reactive oxygen species (ROS) production by interacting with angiotensin type I receptor (AT1), thereby promoting liver fibrosis. However, the interaction of Ang II with angiotensin type II receptor (AT2) results in a completely anti-fibrotic function. In the above experiment, we confirmed that the activated CFSCs highly expressed Ang II and p47-phox, which were synchronously inhibited by BMSC supernatant addition, and reversed by siRNA treatment, but whether these proteins associated with AT1 and AT2 was still unknown. Therefore, we performed Western blots for the detection of AT1 and AT2 in CFSCs treated with P3 BMSC supernatant with or without siRNA. GAPDH was selected as a control. As shown, P3 BMSC supernatant inhibited AT1 receptor expression, whereas the addition of AM-specific siRNA neutralized this inhibition (Fig. 7a, the first line). However, the P3 BMSC supernatant caused a markedly increase in AT2 expression, and the addition of AM-specific siRNA reversed this effect (Fig. 7a, the 2nd line).
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Fig. 7 Changes of AT1 and AT2 expression by CFSCs. a Western blot analysis for AT1 (the first line) and AT2 (the second line) expression levels in the indicated treatments was done. b Losartan
was added to block AT1 passway with or without recombinant rat AM as listed. GAPDH was set as control. Data are shown as mean ± SD, *P \ 0.05; **P \ 0.01
Additionally, we used another agent losartan, the AT1 receptor antagonist, not the AT2 receptor antagonist, to observe the alternations of AT1. Data had shown that AM addition declined the AT1 level, and losartan addition markedly blocked AT1 receptor expression. Combination of losartan and recombinant rat AM had a similar trend as losartan only (Fig. 7b), these results indicated that AM played a role in the blockage of AT1 receptor.
CFSCs are the major source of fibrillar matrix proteins. Studies have shown that CFSC activation is a fundamental feature of liver fibrosis, and hepatic fibrogenesis is mainly triggered by CFSC activation. In vivo, CFSCs undergo a pleiotropic response termed ‘‘activation’’ that also occurs in culture models in vitro and ultimately leads to the transition of CFSCs from resident quiescent CFSCs into myofibroblasts expressing a-SMA [3]. Besides, the onset of CFSC activation coincides with the induction of PDGFRb expression, while PDGF is the most potent mitogen for culture-activated CFSCs [8]. And as reported, pathophysiologic fibrosis is essentially an excessive accumulation of ECM components, particularly type I collagen, together with declined expression and activity of matrix metalloproteinases (MMPs) [21]. However, CFSCs are responsible for the excess production of ECM components. Therefore, in our tests, a-SMA, collagen I, TIMP1 (a natural inhibitor of MMPs), and PDGFR-beta were chose as markers of CFSC activation. In this report, we firstly identified the secretion of AM by different passages of BMSCs via RIA, and this secretion could be decreased by AM-specific siRNA treatment, and data are displayed in Fig. 1a, c. Then, we found that supernatants of P1, P3, P5 passages of AM-expressing BMSCs inhibited the proliferation of CFSCs since their high levels of AM content (Fig. 1b, d), thus P3 passage of BMSC supernatant was selected for the following experiments because of its more effectively function. As known, AM has been reported to act as an anti-fibrotic factor,
Discussion In this study, we found that BMSCs inhibited CFSC proliferation and activation via AM secretions. This inhibition was exerted by decreasing Ang II expression and its receptor AT1 but not AT2, resulting in a reduction of p47phox formation. Traditionally, hepatic cirrhosis has been thought to be irreversible. However, recent evidence from animal studies and human clinical trials has indicated that even advanced fibrosis is reversible [18, 19]. Any intervention that can effectively remove the causative agents could potentially reverse the liver fibrosis process. Hence, the inhibition of the CFSC proliferation and activation, alteration of ECM production, and related subsequent events are crucial goals for intervention in the hepatic fibrogenesis cascade. CFSCs are fibrogenic cells that express p47-phox and produce ROS. They were firstly observed in 1876 by von Kupffer and scarcely studied for almost 100 years [20].
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protecting cells from hypoxic pulmonary damage by inhibiting cell proliferation, collagen synthesis, and TGFb1 production [22, 23]. Besides, we had also confirmed the expression of AM receptors (both AMR1 and AMR2) on CFSCs by immunofluorescence in Fig. 2. Therefore, we tried to verify whether the activation of CFSCs was affected by BMSC supernatant by detecting a-SMA, collagen I, TIMP1, and PDGFRb expression alternations. Compared with the control group, the levels of these four markers of activated CFSCs were all decreased when the cells were incubated in P3 BMSC culture supernatant, but these decreases were remarkably reversed by the siRNA treatment (Figs. 3, 4), and rat recombinant AM was added as a positive control. Based on these four activation markers changes, we concluded that BMSCs could effectively inhibit CFSC activation, at least partially by their secretion of AM. Next, we investigated the potential mechanisms of this regression of CFSC activation. Hepatic fibrinogenesis can be caused by ROS production by different liver cells after various injuries [24, 25]. ROS can stimulate the production of collagen I and may act as an intracellular signaling mediator of the fibrogenic action of TGF-b. NADPH oxidase (NOX) is a multicomponent enzyme complex that generates ROS in response to various stimuli. The NOX complex has been increasingly recognized as a key element of intracellular signaling of hepatic fibrogenesis. Both phagocytic and nonphagocytic NOX components in CFSCs mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy [14]. The molecular components and signaling pathways of various NOX homologues that are critical for fibrotic activity in CFSCs need to be more clearly identified. P47-phox, with its different domains and several phosphorylated sites, is a main regulator of NOX activation by organizing the complex. p47phox-/- mice have been widely used as a genetic model to study NOX inhibition. Bataller et al. [14] demonstrated that p47phox-/- mice showed attenuated liver injury and fibrosis compared with wild-type counterparts after bile duct ligation. To determine whether the p47-phox phosphorylation process was involved in this CFSC inhibition, the phosphorylation of p47-phox was detected by Western blot. As expected, p47-phox was downregulated by BMSC supernatant, but upregulated by the addition of AM-specific siRNA (Fig. 6). These data implied that secretion of AM inhibited CFSCs by interfering with the p47-phox pathway. Better understanding of the different mechanisms and transduction pathways involved in p47-phox phosphorylation and its interactions would be necessary to design new therapeutic agents to downregulate ROS hyperproduction in inflammatory diseases, without decreasing the normal response to bacterial-derived stimuli [26].
Ang II, a pro-oxidant and fibrogenic cytokine, induces p47-phox phosphorylation and ROS production through its co-expression with AT1. However, the interaction of Ang II with AT2 presents a completely anti-fibrotic function. Inhibition of Ang II synthesis or AT1 has been shown to decrease inflammation and hepatic fibrosis in experimental models of chronic liver injury. The fibrogenic response caused by hepatic collagen accumulation was shown to be lower in AT1 knockout mice. Several experiments have demonstrated that Ang II is able to induce ROS production in activated human CFSCs. Ang II-induced ROS formation in CFSCs was shown to be inhibited when CFSCs were treated with either a NOX inhibitor or with the AT1 antagonist. We detected a decreased Ang II expression in CFSCs when P3 BMSC supernatant was added. The addition of AM-specific siRNA reversed this effect (Fig. 5), and these results were consistent with previous reports that AM inhibited Ang II-induced oxidative Stress [27]. To determine whether Ang II expression was associated with AT1 and AT2 levels, we performed Western blot analysis of AT1 and AT2 in CFSCs treated with P3 BMSC supernatant with or without siRNA. The P3 BMSC supernatant dramatically inhibited AT1 expression, and siRNA addition neutralized this inhibition (Fig. 7). The AT2 expression was markedly increased by the P3 BMSC supernatant, but decreased by siRNA treatment (Fig. 7). These findings confirmed that activated CFSCs highly expressed Ang II, which could induce the phosphorylation of p47 by interacting with AT1 but not AT2. Moreover, they also indicated that AM, the inhibitory cytokine secreted by BMSCs, could effectively act on this pathway. BMSCs are multipotent stem cells with the potential to differentiate into liver cells. They play an important role in liver fibrosis treatment without allograft rejection. In this study, we observed that the induction of the AM-Ang II-p47phox signaling pathway could dramatically repress the activation of CFSCs co-cultured with BMSC supernatant in vitro, which might potentially result in the regression of CFSCs fibrogenesis. However, whether BMSCs can inhibit CFSCs in vivo through the AM-Ang II-p47-phox signaling pathway is still unclear. Further studies are needed to clarify the mechanisms in vivo to improve the quality of life of patients and prolong their survival time via BMSC transplantation. Acknowledgments The study is financially supported by Heilongjiang Postdoctoral Science-Research Foundation (LBH-Q11034), Provincial department of international research projects (WB07C04), and Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education (KF201202). Natural Science Foundation of Heilongjiang Province of China (C201441). Conflict of interest There are no financial interests other than the basic academic employment that the authors have, which could create a conflict of interests or appearance of interests with regard to this work.
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