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Original Basic Science—Liver

AntagomiR-199a Enhances the Liver Protective Effect of Hypoxia-Preconditioned BM-MSCs in a Rat Model of Reduced-Size Liver Transplantation

Zhang, Weichen MD1; Chen, Qi PhD2; Ye, Yufu PhD1; Zou, Bei MS1; Liu, Yuanxing PhD1; Cheng, Longyu PhD1; Yu, Jun PhD1; Zheng, Shusen MD, PhD1

Author Information
doi: 10.1097/TP.0000000000002928

Abstract

INTRODUCTION

Although multiple approaches have been developed to treat severe liver failure, liver transplantation (LT) remains the only method of treating end-stage liver diseases.1 To combat the shortage of donor livers, both split LT and living donor LT were developed. However, insufficient liver volume would inevitably lead to small-for-size syndrome, which significantly influences the success rate of LT and long-term survival of the recipients.2 There are several theories that attempt to explain small-for-size syndrome, among which ischemia-reperfusion injury (IRI) is believed to be the major mechanism responsible for the deterioration of liver function after LT.3 Therefore, the identification of a method that reduces graft IRI would be of great value to increase the success rate of LT and long-term survival of LT patients.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a group of multipotent stem cells that can differentiate into various mesenchymal cells (eg, osteocytes, adipose cells, and hepatocytes).4,5 In addition to their multipoetency, BM-MSCs exhibit a paracrine capacity to secrete various types of soluble chemokines into the extracellular environment.6 Recently, studies have confirmed that BM-MSCs might reduce liver injury7 and fibrosis8,9 as well as promote liver regeneration.10 However, the protective effect of BM-MSCs varies substantially between different models; the efficacy of BM-MSC therapy remains to be investigated in a model of reduced-size LT.

To optimize the efficacy of MSC therapy, several methods have been developed. Among the available solutions, hypoxia preconditioning is believed to be the most convenient and feasible approach.11 Hypoxia preconditioned MSCs have been proven to be protective in preventing lung fibrosis,12 brain injury,13 cardiac IRI,14 and renal injury.15 Our previous study demonstrated that hypoxia preconditioned BM-MSCs (H-MSCs) promoted liver regeneration in a massive hepatectomy model.16 However, the efficiency of hypoxia preconditioned MSCs remains limited. Thus, novel methods are desperately required to optimize H-MSC therapy.

Vascular endothelial growth factor (VEGF) is one of the most important cytokines involved in liver regeneration. Recently, VEGF has been shown to play an indispensable role in reducing liver injury, ameliorating liver IRI, and maintaining the liver microstructure. Several studies have demonstrated that MSC infusion promotes liver repair via VEGF induction.17 Moreover, it is believed that hypoxia-preconditioning triggers the expression of hypoxia induction factor 1α (Hif-1α), which further promotes VEGF transcription. However, additional evidence is still required to better elucidate the role of the Hif-1α/VEGF axis in H-MSC therapy.

MiRNAs are a group of non-coding RNA which exerts their function through posttranscriptional modulation. MiR-199a has recently been found to be involved in multiple angiogenic cellular processes.18 Additionally, miR-199a inhibits tumor angioneogenesis through the inhibition of Hif-1α and VEGF secretion.19 Whether the downregulation of miR-199a increases the production of VEGF and consequently maximizes the protective effect of H-MSCs remains unknown.

This study aimed to confirm whether the downregulation of miR-199a increases the protective effect of H-MSC in reduced-size LT and discover the underlying mechanism.

MATERIALS AND METHODS

Ethical Approval of the Study Protocol

The study protocol was approved by the Animal Ethics Review Committee of Zhejiang University (Zhejiang, China). All in vivo experiments strictly followed the guidelines for animal care established by Zhejiang University.

LT and Rat Groupings

Syngeneic male Lewis rats (180–200 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). The rats were prepared for 50% reduced-size orthotopic LT. Rats were fastened 1 day before LT. Phenobarbital sodium (50 mg/kg) was intraperitoneally injected to achieve an adequate depth of anesthesia. The liver was harvested and stored in cold 0.9% saline. The caudate lobes and left lateral lobe of the donor liver were resected, with the right lobe and median lobe spared to obtain a 50% liver graft. Kamada’s 2-cuff methods were used during 50% reduced-size orthotopic LT. Briefly, we sutured the donor and recipient suprahepatic vena cava and infrahepatic vena cava with an 8-0 microscopic vascular suture. The 2-cuff method was utilized to reconstruct the common bile duct and portal vain, without reconstruction of the hepatic artery. The operations were performed by an experienced laboratory assistant. The procedure took ~40 min, and both the warm and cold ischemia time was documented in Table S1 (SDC, http://links.lww.com/TP/B801). Hypoxia-preconditioned BM-MSCs (H-MSCs) were prepared and numbered, and a density of 5 × 106 cells were intraportally injected during the surgery. All rats were carefully observed until 6 h postsurgery, and >90% of the rats survived the liver transplant surgery.

The rats that survived the LT were randomly divided into 4 groups and injected with either agomiR-199a or antagomiR-199a accordingly through the tail vein once a day: (1) control (injected saline during and after operation); (2) H-MSCs (injected H-MSCs during operation and saline injection after operation); (3) agomiR-199a (injected H-MSCs during operation and agomiR-199a 80 mg/kg after operation); and (4) antagomiR-199a (injected H-MSCs during operation and antagomiR-199a 80 mg/kg after operation). Each group consisted of at least 6 pairs of liver transplants. On Days 1, 2, 3, and 7 post-LT, the rats were mercifully sacrificed, and the serum and liver samples were harvested simultaneously for further analysis.

To study the mechanism the rats were further divided into 4 groups: (1) Control (saline injection during and after operation); (2) neutralizing VEGF antibody (injected VEGF-ab 50 ng after operation); (3) antagomiR-199a + H-MSC (injected H-MSC during operation and antagomiR-199a 80 mg/kg after operation); and (4) VEGF-ab + antagomiR-199a + H-MSCs (injected H-MSC during operation and VEGF-ab 50 ng plus antaomiR-199a 80 mg/kg after operation). Saline, antagomiR-199a, and the VEGF-ab were injected via the tail vein. AntagomiR-199a was administered once per day following LT. The VEGF-ab was administered every 3 days after LT. Each group consisted of at least 4 pairs of liver transplants. The rats were sacrificed and both the serum and liver samples were collected as described above.

Isolation and Verification of BM-MSCs

Femoral bone marrow samples from the Lewis rats were prepared for BM-MSC isolation. The marrow pellet was washed twice with Hank’s balanced salt solution. The samples were centrifuged at 1000 rpm centrifugation for 10 minutes and the cells were resuspended in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco). The isolated cells were cultured in an incubator at 37°C with 5% CO2. The culture medium was replaced every 3 days to eliminate any nonadherent cells. Antibodies against CD29, CD90, CD31, CD34, and CD45 (Caltag Laboratories, San Francisco, CA) were cocultured with the isolated cells. Flow cytometry was performed according to the manufacturer’s instructions.

BM-MSC osteogenic and adipogenic differentiation medium were purchased from Cyagen Corporation. The MSCs and culture medium were prepared according to the manufacturer’s instructions. Two weeks after induction, the cells were fixed in formalin. Alizarin red and Sudan III solutions were used to stain the calcified bodies and adipose vacuoles. The osteogenic and adipogenic differentiation potential of the BM-MSCs were verified via light microscopy.

Hypoxia-Preconditioniong of BM-MSCs

The isolated BM-MSCs at passages 3–5 were suspended in 0.25% trypsin and plated into a 6-well plate with 10% fetal bovine serum DMEM. After 24 h, the culture medium was replaced, the 6-well plate was further stored in an atmosphere containing 1% oxygen, 5% CO2, and 94% nitrogen for another 24 h. The cells that received hypoxia-preconditioning were harvested and diluted in saline at a concentration of 5 × 106/mL for further intra-portal injection.

Chemicals and Reagents

AgomiR-199a and antagomiR-199a were purchased from Ribobio (Guangzhou, China). The VEGF-ab was purchased from R&D. All primary and secondary antibodies used for the Western blot, including Bax, Bcl-xl, GAPDH, Hif-1α, VEGF, TNF-α, and IL-6 were obtained from Cell Signaling Technology (Danvers, MA).

Real-Time Polymerase Chain Reaction

TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract the total mRNA of each frozen liver sample. A Reverse Transcription Reagent kit with gDNA Eraser (TaKaRa Bio, Shiga, Japan) was used to perform reverse-transcription. Real-time polymerase chain reaction (PCR) was performed using an ABI 7,500 Sequence Detection system (Applied Biosystems, Foster City, CA) with SYBR-green I (TaKaRa Bio). The relative gene-expression profiles were determined by normalizing the level of expression to that of the housekeeping gene (U-6) using the 2−ΔCt method.

Immunohistochemistry Analyses

Liver samples harvested following LT were fixed in formalin and paraffin-embedded. Ki-67 (Cell Signaling Technology) immunohistochemistry was performed for each slide to evaluate the regenerative potential of the liver samples. A total of 20 high-power fields were randomly selected from each slide and the number of Ki-67-positive cells were quantified under light microscopy.

TUNEL Assay

A colorimetric TUNEL Apoptosis Assay Kit (Beyotime Tech) was purchased to evaluate apoptosis. A TUNEL assay was performed according to the manufacturer’s protocol. A light microscope was used to detected TUNEL-positive cells.

Luciferase Reporter Gene System

The 3′-UTR sequences of wild or mutated Hif-1α mRNA were cloned into the luciferase vector, phRL-TK. The constructed plasmids were then co-transfected into BM-MSCs with an miR-199a mimic (Ribobio, Guangzhou, China). The cells were harvested 48 h after co-transfection. The level of luciferase activity was examined using a Renilla Luciferase Assay System (Promega, Sunnyvale, CA).

Nitric Oxide Secretion Detection

A nitric oxide (NO) detection kit was purchased from Beyotime Tech. To detect NO, the cell medium for each treatment group was harvested, and 50 μL of each sample was transferred into a 96-well plate. Griess Reagent was added as indicated in the instructions. The plate was further observed using a spectrophotometer at 540 nm.

Statistical Analysis

The results were analyzed using SPSS v11.0 (IBM, Armonk, NY) and the data were presented as the mean ± SD. All in vitro experiments were repeated at least 3 times. A one-way ANOVA was used to compare the group variables, followed by least-significant difference post hoc test, if required. A P < 0.05 was considered significant.

RESULTS

Verification of BM-MSC Isolation

BM-MSCs were isolated using the methods described above (Figure S1A, SDC, http://links.lww.com/TP/B801). To verify the multi-potency of the isolated BM-MSCs, we cultured the cells in osteogenic and adipogenic differentiation medium. Alizarin red and Sudan III staining were performed to detect mineral bodies (Figure S1B, SDC, http://links.lww.com/TP/B801) and fatty vacuoles (Figure S1C, SDC, http://links.lww.com/TP/B801) in the differentiated MSCs as previously described.

We further examined the expression of clusters of differentiation molecules. As shown in Figure S2 (SDC, http://links.lww.com/TP/B801), the isolated BM-MSCs were positive for CD29 and CD90 but negative for CD31, CD34, and CD45, which correspond to BM-MSCs characteristics.

Downregulation of miR-199a Increased the Liver Protective Effect of H-MSCs In Vivo

To verify the efficiency of the transfection with agomiR-199a and antagomiR-199a, we cocultured the isolated MSCs with agomiR-199a and antagomiR-199a in vitro. RT-PCR was used to evaluate the transfection efficiency. As shown in Figure S3A and S3B (SDC, http://links.lww.com/TP/B801), miR-199a overexpression and inhibition was successfully achieved. No significant differences were observed between the transfected cell lines and control group in the results of the CCK-8 cell counting assay, which indicated that transfection with agomiR-199a and antagomiR-199a did not affect the viability of the isolated BM-MSCs (Figure S3C, SDC, http://links.lww.com/TP/B801).

To investigate the role of miR-199a in hypoxia-preconditioned BM-MSCs (H-MSC), reduced-size LT was performed in rats, H-MSCs were injected into the portal vein during the operation; saline, agomiR-199a, and antagomiR-199a were injected via the tail vein. The rats were mercifully sacrificed on Days 1, 2, 3, and 7 after LT, at which time liver and serum samples were collected. As shown in Figure 1A and B, H-MSCs were associated with decreased levels of serum ALT and AST compared with the control at 3 days after LT. AntagomiR-199a amplified the protective effect of H-MSCs by further decreasing the level of ALT and AST. In contrast, the combination of agomiR-199a and H-MSCs exhibited no differences regarding the level of ALT and AST compared with the control group. The liver grafts were weighed and documented before and after the operation, and the net increase in the graft weight was calculated. As shown in Figure 1C, treatment with antagomiR-199a was associated with an increase in the net graft weight after LT. The graft:body weight ratio was also calculated and is displayed in Figure 1D. Treatment with agomiR-199a displayed a decrease in the liver:body ratio after LT compared with the control. Treatment with antagomiR-199a + H-MSCs and H-MSCs alone was associated with a moderate increase in the liver:body ratio; however, the results were not significant. To further discuss H-MSCs and antagomiR-199a in ameliorating liver IRI, a liver H&E staining was performed for each rat. As shown in Figure 1E, agomiR-199a plus H-MSCs showed a slightly increased level of infiltrated neutrophils. Besides, antagomiR-199a plus H-MSCs showed limited hepatic necrosis than other treatment.

FIGURE 1
FIGURE 1:
AntagomiR-199a enhanced the protective effect of the H-MSC infusion. The serum and liver samples of the LT rats were collected on Day 3 after LT. ALT (A) and AST (B) levels were measured via ELISA. AntagomiR-199a treatment had the lowest level of ALT and AST among all groups. Treatment with agomiR-199a increased the level of ALT and AST. (N = 6 *P < 0.05; **P < 0.01). First, we weighed the liver graft before the LT operation. After sacrificing the rats, the liver grafts were resected and weighed once again. The net increment of the liver weight (C) was defined as the difference between the 2 weights mentioned above. D, The ratio of liver:body weight was measured and calculated. E, An H&E staining was performed, representative H&E staining image are displayed for each group. H-MSC, hypoxia-preconditioned MSC; LT, liver transplantation; MSC, mesenchymal stem cells.

We also explored whether antagomiR-199a alone injection showed a significantly protective effect independent of H-MSCs. As shown in Figure S4A (SDC, http://links.lww.com/TP/B801), antagomiR-199a showed an insignificant decreased ALT level at 3 days after LT. Besides, no differences in AST, net liver weight increasement, liver:body weight ratio were noticed among agomiR, antagomiR and control group (Figure S4B–D, SDC, http://links.lww.com/TP/B801). On the other hand, agomiR and antagomiR-199a alone showed a limited effect in regulating apoptosis and inflammation (Figure S4E, SDC, http://links.lww.com/TP/B801).

The Downregulation of miR-199a Aided H-MSCs in Inhibiting Apoptosis and Ameliorating Liver IRI

To evaluate the antiapoptotic effect of antagomiR-199a and H-MSCs, the liver samples were subjected to analysis by RT-PCR and Western blot. Figure 2A shows that treatment with antagomiR-199a + H-MSCs significantly decreased the level of transcription of the pro-apoptotic protein, Bax. In contrast, the level of Bcl-xl mRNA was elevated following antagomiR-199a treatment, which correlated with its anti-apoptotic effect (Figure 2B). A Western blot was performed to evaluate the level of Bcl-xl and Bax protein expression (Figure 2C). To further evaluate the level of apoptosis present in the liver grafts, a TUNEL assay was performed. As shown in Figure 2D, multiple TUNEL-positive cells were noted in the control group. H-MSCs treatment displayed fewer TUNEL-positive cells than the control group. Moreover, antagomiR-199a plus H-MSCs exhibited the lowest number of TUNEL-positive cells among all of the groups. A total of 20 high-power fields were randomly selected, and the TUNEL-positive cells under each field were counted. The percentage of TUNEL positive cells were calculated and displayed in Figure 2E.

FIGURE 2
FIGURE 2:
AntagomiR-199a increased the antiapoptotic effect of H-MSCs. Liver samples were collected as previously described, liver homogenates were acquired, and the samples were prepared for mRNA and protein isolation for further study. The level of Bax (A) and Bcl-xl (B) mRNA were measured by q-PCR. C, A Western-blot was performed to observe the level of Bax and Bcl-xl expression. D, An immunohistochemistry TUNEL assay was further conducted to evaluate the apoptotic level of the groups. The apoptotic cells were dyed a brown color using a TUNEL assay. E, Twenty high-power fields were randomly chosen for from each slide. The number of apoptotic cells for each group was counted, and the percentage of apoptotic cells were calculated (*P < 0.05; **P < 0.01; ***P < 0.001). H-MSC, hypoxia-preconditioned MSC; MSC, mesenchymal stem cells.

To evaluate the level of inflammation in the liver grafts, q-PCR was used to quantify the level of TNF-α (Figure 3A) and IL-6 (Figure 3B) mRNA expression. An injection with H-MSCs significantly decreased the level of serum of TNF-α and IL-6 mRNA, and an inhibition of miR-199a further suppressed TNF-α and Il-6 transcription. A Western blot was performed to examine the level of TNF-α and IL-6 expression. TNF-α and IL-6 were significantly inhibited following treatment with antagomiR-199a + H-MSCs (Figure 3C). Ki-67 immunohistochemistry was performed to estimate the liver regeneration potential of H-MSC treatment. Figure 3D shows that H-MSC treatment was associated with a higher number of ki-67-positive cells compared with the control group, and the group that received antagomiR-199a plus H-MSCs displayed the most ki-67-positive cells among all of the groups. The percentage of ki-67-positive cells was calculated and displayed in Figure 3E.

FIGURE 3
FIGURE 3:
AntagomiR-199a increased the anti-inflammatory effect of H-MSCs. Liver samples were prepared as previously described, and mRNA and protein samples were prepared for qPCR and Western blot. The level of TNF-α (A) and IL-6 (B) mRNA was measured by qPCR. C, The level of TNF-α and IL-6 protein expression was determined by Western blot. Taken together, antagomiR-199a H-MSCs displayed a decrease in the level of TNF-α and IL-6 expression. D, Ki-67 immunochemistry was performed to estimate the regeneration potential of the liver grafts. E, Ki-67-positive cells were counted, and the percentage of Ki-67-positive cells was calculated for each group (*P < 0.05; ***P < 0.001). H-MSC, hypoxia-preconditioned MSC; MSC, mesenchymal stem cells.

AntagomiR-199a Enhanced the Liver Protective Effect of H-MSC IRI Through Hif-1α/VEGF Activation

We further examined the level of miR-199a in the liver samples following treatment with H-MSCs and agomiR or antagomiR-199a. H-MSC treatment displayed an insignificant increase of miR-199a expression (Figure 4A). Treatment of agomiR-199a and antagomiR-199a with H-MSCs exhibited similar results as that presented in Figure S3 (SDC, http://links.lww.com/TP/B801). These results imply that agomiR-199a and antagomiR-199a peripherally injection showed strong influences on liver expression of miR-199a.

FIGURE 4
FIGURE 4:
MiR-199a directly inhibited the activation of the Hif-1α/VEGF pathway. Total RNA and protein samples were prepared for each liver graft. A, The level of miR-199a in each group. B, A Western blot was performed to evaluate the expression of Hif-1α and VEGF in vivo. A luciferase gene reporter system was successfully constructed (C). Treatment with agomiR-199a significantly decreased the relative luciferase activity of Hif-1α. However, when Hif-1α was knocked out, the relative luciferase activity remained constant. D, A Western blot was performed to evaluate the expression of Hif-1α and VEGF in vitro. The cells were incubated in a 95% N2 incubator, and the cell culture medium was collected at 1, 6, and 24 h after hypoxia procedure (E). The concentration of nitric oxide was measured by Griess Reagent (*P < 0.05; **P < 0.01). Hif-1α, hypoxia induction factor 1α; H-MSC, hypoxia-preconditioned MSC; MSC, mesenchymal stem cells; NO, nitric oxide; VEGF, vascular endothelial growth factor.

Based on the theory that MSCs protect against liver IRI through the Hif-1α/VEGF axis, a Western blot was performed to analyze the level of Hif-1α and VEGF expression in vivo. As displayed in Figure 4B, both Hif-1α and its downstream effector, VEGF, were successfully induced by the H-MSCs. Treatment with antagomiR-199a significantly enhanced the level of Hif-1α and VEGF expression. In contrast, treatment with agomiR-199a displayed the opposite effect. Taken together, antagomiR-199a facilitated the H-MSC-mediated reversal of liver IRI following reduced-size LTx.

To more effectively demonstrate the interaction between miR-199a and the Hif-1α/VEGF axis, we constructed a luciferase gene reporter system. As shown in Figure 4C, treatment with agomiR-199a significantly decreased the level of luciferase activity. However, when Hif-1α was knocked out, miR-199a did not affect luciferase activity. Taken together, Hif-1α was the direct downstream target of miR-199a. Isolated BM-MSCs transfected with agomiR-199a or antagomiR-199a were stored in a 95% N2 incubator for 24 hours hypoxia process, after which the cell samples were collected to perform a Western blot. Figure 4D shows the hypoxia-induced expression of Hif-1α and downstream VEGF. AntagomiR-199a further triggered the activation of Hif-1α and VEGF; however, agomiR-199a suppressed the Hif-1α/VEGF axis. NO production is believed to be closely associated with VEGF secretion. The cell culture medium was collected at 1, 6, and 24 h after incubating the cells under hypoxic conditions. The NO concentration in each group was measured using Griess Reagent method. As displayed in Figure 4E, hypoxia-induced NO production and treatment with antagomiR-199a further enhanced the production of NO; however, treatment with agomiR-199a decreased H-MSC-mediated NO secretion.

To determine whether the downregulation of miR-199a aided H-MSCs in protecting against liver IRI through Hif-1α/VEGF activation in vivo, we injected VEGF-ab after the operation. The level of ALT and AST was measured on Days 1, 2, 3, and 7 following LT. Treatment with the VEGF-ab alone did not influence the level of ALT (Figure 5A) and AST (Figure 5B); however, the VEGF-ab was able to inhibit the protective effect of H-MSCs pretreated with antagomiR-199a (***P < 0.001). VEGF-ab treatment moderately inhibited liver regeneration, as shown in Figure 5C. The graft weight increment was inhibited on Days 1 and 7 after LT. No significant differences were observed in the liver:body ratio following treatment with the VEGF-ab (Figure 5D). We also performed an H&E staining to observe microstructure of liver samples. As displayed in Figure 5E, VEGF-Ab slightly reversed protective effect of antagomiR-199a plus H-MSCs by increasing level of liver congestion and neutrophil infiltration.

FIGURE 5
FIGURE 5:
Treatment with a VEGF-neutralizing antibody blocked the protective effect of antagomiR-199a + H-MSCs. On Day 3 after LT, the serum and liver samples were harvested accordingly. The level of ALT (A) and AST (B) were measured via an ELISA. Treatment with an anti-VEGF antibody significantly increased the level of serum ALT and AST following treatment with antagomiR-199a + H-MSCs. (N = 4; *P < 0.05; **P < 0.01; ***P < 0.001). The net increment of liver weight (C) and liver:body ratio (D) were analyzed as previously noted. E, Liver sample H&E staining were performed; representative images of each groups were displayed. H-MSC, hypoxia-preconditioned MSC; LT, liver transplantation; MSC, mesenchymal stem cells; VEGF, vascular endothelial growth factor.

VEGF-ab Treatment Blocked the Antiapoptotic and Anti-inflammatory Effect of the Combination of antagomiR-199a + H-MSC

Treatment with the VEGF-ab alone did not influence the expression of Bax (Figure 6A) or Bcl-xl (Figure 6B). However, when treated with antagomiR-199a and H-MSCs, the VEGF-ab significantly increased the level of Bax expression and decreased Bcl-xl expression. A Western blot was performed to further evaluate the level of Bax and Bcl-xl protein expression (Figure 6C). A TUNEL study was conducted to evaluate the level of apoptosis in the liver grafts (Figure 6D). The number of TUNEL-positive cells was counted for each slide and the percentage of apoptotic cells were calculated and presented in Figure 6E. Treatment with VEGF-ab alone displayed no obvious effect on the number of apoptotic cells; however, it substantially increased the number of TUNEL-positive cells following treatment with antagomiR-199a + H-MSCs.

FIGURE 6
FIGURE 6:
Treatment with a VEGF neutralizing antibody blocked the antiapoptotic effect of antagomiR-199a + H-MSCs. The mRNA and protein samples were prepared as previously mentioned. The level of Bax (A) and Bcl-xl (B) transcription was determined by qPCR, and a Western blot was conducted to further assess the level of Bax and Bcl-xl expression (C). D, An immunohistochemistry TUNEL assay was used to estimate the level of apoptotic cells in all the groups. The brown-stained cells were considered to be apoptotic cells when observed by light microscopy. E, The percentage of apoptotic cells was analyzed (*P < 0.05; **P < 0.01; ***P < 0.001). H-MSC, hypoxia-preconditioned MSC; MSC, mesenchymal stem cells; VEGF, vascular endothelial growth factor.

Treatment with the VEGF-ab did not exhibit any influence on the level of TNF-α (Figure 7A) and IL-6 (Figure 7B) mRNA expression. However, the level of TNF-α and IL-6 expression was significantly increased following treatment with antagomiR-199a + H-MSCs. A Western blot was performed to explore the level of TNF-α and IL-6 protein expression (Figure 7C) and Ki-67 immunohistochemistry was used to estimate the regenerative potential of the liver grafts. As shown in Figure 7D, treatment with the VEGF-ab decreased the number of Ki-67-positive cells in the H-MSCs + antagomiR-199a group. The percentage of Ki-67-positive cells was calculated and presented in Figure 7E. Figure 7F presents the level of NO and shows that treatment with the VEGF neutralizing antibody was associated with a decrease in NO production following treatment with antagomiR-199a + H-MSCs.

Figure 7
Figure 7:
Treatment with a VEGF-neutralization antibody blocked the anti-inflammatory effect of antagomiR-199a + H-MSCs. The level of TNF-α (A) and IL-6 (B) mRNA was measured by qPCR, and the secretion of TNF-α and IL-6 was further determined by Western blot (C). When treated with the VEGF neutralizing antibody, the level of TNF-α and IL-6 expression in the cells that received antagomiR-199a + H-MSCs was significantly elevated. D, Ki-67 immunochemistry was performed to estimate the regeneration potential of the liver grafts. E, Ki-67-positive cells were counted and the percentage of Ki-67-positive cells in each group was calculated. F, Secretion of nitric oxide was determined using Griess Reagent (*P < 0.05; **P < 0.01; ***P < 0.001). H-MSC, hypoxia-preconditioned MSC; MSC, mesenchymal stem cells; NO, nitric oxide; VEGF, vascular endothelial growth factor.

DISCUSSION

In the present study, we found that antagomiR-199a enhanced the liver protective effect of hypoxia-preconditioned BM-MSCs in a rat model of reduced-size LT. Our previous study demonstrated that H-MSCs protected the liver through the induction of VEGF in a rat model of massive hepatectomy.16 Based on these findings, we further hypothesized that H-MSCs could also be applied in a rat reduced-size LT model; however, unlike the effect on the massive hepatectomy model, the liver protective effect of H-MSCs in reduced-size LT has room for improvement. We further speculated whether antagomiR-199a, a specific inhibitor of miR-199a, could enhance the effect of H-MSCs in the reduced-size LT model.

BM-MSCs are a type of multipotent stem cell that can differentiate into multiple types of mesenchymal cells, including adipocytes,20 chondrocytes,21 osteocytes,22 hepatocytes,23 and cardiomyocytes.24 In addition to its multipotency, MSCs secrete an abundance of cytokines,25 exosomes, and embedded miRNAs26 into the extracellular environment, which play a pivotal role in various cellular processes. Based on the above-mentioned characteristics of BM-MSCs, therapies based on BM-MSCs have been widely used in a number of diseases. Clinical trials have found that that MSC transplantation has a protective effect in acute myocardial infarction patients,27,28 and BM-MSC therapy is also believed to be promising for the treatment of idiopathic pulmonary fibrosis.29,30 Recently, the function of BM-MSCs has been well-studied in various liver disease models. Moreover, the intravenous31 or intraportal8 injection of MSCs has been found to ameliorate liver fibrosis in rats; some studies and clinical trials of BM-MSC transplantation have shown inspiring results in treating liver cirrhosis.32-34 In addition, MSC therapy was shown to rescue the liver from IRI in rats.7,35,36 Based on the anti-IRI effect, MSCs have been further utilized in LT models. A study by Du et al found that MSCs rescued the liver graft from IRI in a rat reduced-sized LT model.10 Similarly, Wu et al37 discovered that Heme oxygenase-1 enhanced the anti-inflammatory effect of BM-MSCs in rat LT. Although MSC therapy has proven to be beneficial in LT, the effect has varied substantially among each of the different conditions. Although the intra-portal injection of MSCs during surgery was shown to be beneficial in several liver disease models, repeated MSC injection seems to be infeasible after surgery, which further influences the efficacy of MSC therapy. Hence, new methods are still critically required to further optimize H-MSCs therapy.38

Mir-199a was first found to be closely associated with hepatocellular carcinoma. In a study by Hou et al, the miRNAomes of human liver and HCC samples were compared, and revealed that miR-199a was the third most abundant miRNA in the liver, and the downregulation of miR-199a in HCC patients correlated with poor survival.39 In particular, miR-199a was found to interfere with HCC glucose metabolism, which inhibited HCC development and progression.40 Morita et al41 further found that decreased miR-199a expression was associated with a high rate of HCC recurrence following LT. In contrast, miR-199a inhibition was found to facilitate engraft and hepatic repopulation of embryonic stem cell-derived hepatocytes, which optimized the therapeutic effect of stem cell therapy for the treatment of liver diseases.42 As shown in our previous study, H-MSCs produced more VEGF than unconditioned BM-MSCs.16 Based on hypoxia preconditioning, we found that miR-199a inhibition further increased the secretion of VEGF. Moreover, agomiRs and antagomiRs are recently discovered miRNA derivatives, which displayed an miRNA mimic and inhibitor effect, respectively, without requiring transfection.43 Although a repeated intra-portal injection of MSCs seems impossible, a repeated injection of antagomiR-199a may represent an ideal method by which the protective characteristics of H-MSCs can be maintained. However, to further clarify the role of miR-199a in LT and MSC physiology, additional research is required. Our study was the first to combine antagomiR-199a with H-MSCs and verify its effect in a reduced-size LT model.

Our study still suffered from one major limitation. Although antagomiR-199a was found to significantly increase the liver protective effect of the H-MSC infusion, whether antagomiR-199a specifically inhibited miR-199a in MSCs remains unknown. Several types of liver-resident cells are believed to participate in liver IRI and liver regeneration (eg, hepatocytes,44 sinusoidal endothelial cells,45 and hepatic stellate cells46) which could possibly be the target of antagomiR-199a. In our study, antagomiR-199a was administered via the caudal vein following LT, which made it virtually impossible to directly determine which cell type in the liver was target of antagomiR-199a. Ideally, to determine the interaction between antagomiR-199a and MSCs, the construction of a stably transfected miR-199a-inhibited MSC cell line using a lentivirus transfection system is required.47 Unfortunately, to achieve a qualified stalely transfected cell line, the cells transfected with the lentivirus need to be screened using antibiotics for several passages. However, the multipoetency and cell viability of isolated BM-MSCs will inevitably decline after 5 passages or more.48 Taken together, we are currently unable to directly ascertain whether antagomiR-199a inhibited the level of miR-199a in H-MSCs. Due to this limitation, the only conclusion that can be drawn is that antagomiR-199a co-stimulated the activation of the Hif-1α/VEGF axis in conjunction with H-MSCs. Thus, further evidence is required to better demonstrate the mechanism of antagomiR-199a in our transplantation model.

In conclusion, we found that antagomiR-199a significantly increased the hepatic protective effect of H-MSCs in a rat model of reduced-size LT. In terms of the specific mechanism, VEGF-ab inhibited the protective effect of antagomiR-199a + H-MSCs, which illustrated that the inhibition of miR-199a protected the liver grafts via activation of the Hif-1α/VEGF pathway. Taken together, our findings suggest that antagomiR-199a combined with H-MSCs may ameliorate liver injury and promote liver regeneration following LT.

ACKNOWLEDGMENTS

We thank Dr Chen Wei and Shen Jian for the selfless help and technical assistance.

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