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Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia–reperfusion injury in rats

Sun, Cheuk-Kwan MD, PhD; Chang, Chia-Lo MD; Lin, Yu-Chun PhD; Kao, Ying-Hsien PhD; Chang, Li-Teh PhD; Yen, Chia-Hung PhD; Shao, Pei-Lin PhD; Chen, Chih-Hung MD; Leu, Steve PhD; Yip, Hon-Kan MD

doi: 10.1097/CCM.0b013e31823dae23
Laboratory Investigations

Objectives: Mesenchymal stem cells have previously been shown to offer significant therapeutic benefit in ischemic organ injuries. This study aimed at investigating the therapeutic role of adipose tissue-derived mesenchymal stem cells in hepatic ischemia–reperfusion injury and the underlying mechanisms.

Design: Adult male Fisher rats (n = 30) were equally divided into three groups (group 1: Sham-operated normal controls; group 2: Ischemia-reperfusion injury with intravenous fresh culture medium; group 3: Ischemia-reperfusion injury with intravenous adipose tissue-derived mesenchymal stem cells). Ischemia-reperfusion injury was induced by occluding the vascular supplies of left lobe liver for 60 minutes followed by reperfusion for 72 hrs. Adipose tissue-derived mesenchymal stem cells (1.2 × 106) were administered through tail vein immediately after reperfusion and at 6 hrs and 24 hrs after reperfusion in group 3. All animals were sacrificed 72 hrs after reperfusion.

Setting: Animal laboratory at a medical institute.

Measurements and Main Results: Histologic features, plasma aspartate aminotransferase, hepatic cytokine profile, oxidative stress, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling were analyzed. Seventy-two hrs after reperfusion, plasma aspartate aminotransferase, hepatic oxidative stress, messenger RNA expressions of tumor necrosis factor-a, transforming growth factor-b, interleukin-1b, interleukin-6, endothelin-1, matrix metalloproteinase-9, plasminogen activator inhibitor-1, Bax and caspase-3, protein expression of intercellular adhesion molecule as well as the number of apoptotic nuclei were significantly increased in group 2 compared with group 3, whereas messenger RNA expressions of endothelial nitric oxide synthase, Bcl-2, interleukin-10, protein expressions of reduced nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1, and heme oxygenase-1 were lower in group 2 than group 3.

Conclusions: The results showed that systemic adipose tissue-derived mesenchymal stem cell administration significantly preserved hepatocyte integrity and suppressed inflammatory responses, oxidative stress, and apoptosis in a rodent model of hepatic ischemia–reperfusion injury. (Crit Care Med 2012; 40:–1290)

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Drs. Leu and Yip contributed equally to this work.

The authors have not disclosed any potential conflicts of interest.

From the Departments of Emergency Medicine (C-KS) and Medical Research (Y-CL, Y-HK), E-Da Hospital, I-Shou University, Kaohsiung, Taiwan; the Division of Colorectal Surgery (C-LC), Department of Surgery, the Division of Cardiology (SL, H-KY), the Division of General Medicine (C-HC), Department of Internal Medicine, and the Center for Translational Research in Biomedical Sciences (SL, H-KY), Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan; Basic Science (L-TC), Nursing Department, Meiho University, Pingtung, Taiwan; the Department of Life Science (C-HY), National Pingtung University of Science and Technology, Pingtung, Taiwan; and the Graduate Institute of Medicine (P-LS), College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

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Ethics. All experimental animal procedures were approved by the Institute of Animal Care and Use Committee at our hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23, Washington, DC, National Academy Press, revised 1996).

Animal Grouping and Experiment Protocol. Pathogen-free, adult male Fisher rats weighing 250–300 g (n = 30) (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan) were randomly divided into three groups; group 1: Sham-operated group receiving laparotomy only plus systemic venous administration of fresh culture medium immediately and at 6 hrs and 24 hrs after operation; group 2: The left liver lobe undergoing 60-min ischemia followed by reperfusion for 72 hrs plus systemic venous administration of fresh culture medium immediately, 6 hrs, and 24 hrs after reperfusion; and group 3: The left liver lobe undergoing 60-min ischemia followed by reperfusion for 72 hrs plus systemic venous administration of 1.2 × 106 ADMSCs immediately, 6 hrs, and 24 hrs after reperfusion. All animals were euthanized 72 hrs after operation. Blood samples were collected before and after the experiments and plasma specimens were used for aspartate aminotransferase analysis. Liver specimens were harvested and then stored at –80°C for analyses of messenger RNA (mRNA) and protein expressions. Tissues were also embedded in optimal cutting temperature compound or 4% buffered formaldehyde for cryosectioning and paraffin sectioning, respectively.

Isolation and Cultivation of ADMSCs. Seven days before hepatic IR induction, adult male Fisher rats (group 3, n = 10) weighing 250–300 g were anesthetized with chloral hydrate (35 mg/kg intraperitoneally). Adipose tissue surrounding the epididymis was carefully dissected and excised. Details on ADMSC isolation can be found in one of our recent studies (13). The isolated ADMSCs were cultured in a 100-mm diameter dish with 10 mL Dulbecco’s minimal essential medium culture medium containing 10% fetal bovine serum for 7 days. Flow cytometric analysis was performed for identification of cellular characteristics after cell labeling with appropriate antibodies.

Phenotypic Characterization of ADMSCs by Flow Cytometry. A sample of cultured cells was used to analyze the cell phenotype by flow cytometry. Briefly, 3 × 105 cells were labeled at 4°C for 20 mins in the dark with the following antibodies: PE-conjugated antibodies against CD90 (BD Pharmingen), CD26 (BD Pharmingen), and CD34 (BD Pharmingen); fluorescein isothiocyanate-conjugated antibodies against CD45 (BioLegend) and c-kit (BD Pharmingen); monoclonal antibodies against CD31 (Abcam), vascular endothelial growth factor (Abcam), and CD21 (Abcam); polyclonal antibodies against von Willebrand factor (Millipore) and KDR (Thermo); and biotin-conjugated antibodies against CD29 (BD Pharmingen). Flow cytometric analysis was performed using a fluorescence-activated cell sorter (FACSCalibur system; Beckmen).

Induction of IR Injury of the Liver. IR injury of the left lobe rat liver was induced as previously described (6). Briefly, the left lobe liver was dissected free from the surrounding ligaments. Hepatic ischemia was induced by obstructing the vessels by placing a 4-0 silk loop around the hilar region of the left liver lobe in group 2 and group 3 rats, whereas group 1 animals received only laparotomy without undergoing hepatic ischemia. Reperfusion was started 60 mins later when the hilar occlusion was released.

Differential Labeling and Systemic Venous Infusion of ADMSCs. Group 3 animals (i.e., the ADMSC-treated group) were briefly anesthetized with inhalational isofluorane (3%). The prepared ADMSCs with quantity of 1.2 × 106 were labeled with CM-DiI (Vybrant DiI cell-labeling solution; Molecular Probes, Inc.) (50 μg/mL) added to the culture medium 30 mins before intravenous administration at three different time points (i.e., immediately after release of occlusion, and then 6 hrs, and 24 hrs later). Group 1 and group 2 animals received equal volume of fresh culture medium at the three identical time points as group 2. The engrafted cells infused at three different time points were identified microscopically by the distinctive red fluorescence emitted by the cells on ultraviolet excitation of the liver section.

Isolation of Mitochondria. The IR-treated livers were excised and washed with buffer A (100 mM Tris-HCl, 70 mM sucrose, 10 mM EDTA, and 210 mM mannitol, pH 7.4). After homogenization and centrifugation (14), the mitochondria-rich pellets were collected and stored at –70°C.

Western Blot Analysis. For quantifying the hepatic protein expression of intercellular adhesion molecule-1, reduced nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1, heme oxygenase-1, and cytochrome c, equal amounts (10–30 μg) of protein extracts from the livers with or without ADMSC administration and those from sham-operated animals were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 8% to 10% acrylamide gradients. The procedures of electrophoresis and electrophoretical transfer were as previously described (13). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) and exposure to Biomax L film (Kodak, Rochester, NY). For quantification, digitized enhanced chemiluminescence signals were analyzed using Labworks UVP software (UVP Labworks, Upland, CA).

RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction Analysis. The mRNA expressions of endothelial nitric oxide synthase, endothelin-1, interleukin (IL)-1β, IL-6, IL-10, transforming growth factor (TGF)- β, plasminogen activator inhibitor (PAI)-1, matrix metalloproteinase (MMP)-9, tumor necrosis factor (TNF)- β, Bax, Bcl-2, and caspase-3 of the three groups of animals were analyzed with real-time quantitative polymerase chain reaction and compared (Table 1). Technical details were according to those previously described (13).

Hepatic Oxidative Stress Analysis. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150, Billerica, MA). The procedure was according to that of our previous study (15). Expressions of two anti-oxidative proteins, nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1 and heme oxygenase-1, were also analyzed and compared among the three groups of animals.

Immunohistochemical Staining for Smooth Muscle Actin, Myeloperoxidase, and Immunofluorescent Staining for Connexin43. Because activated HSCs around hepatic sinusoids express smooth muscle actin (a-SMA), immunohistochemical staining of a-SMA (Sigma) was performed to determine the degree of HSC activation according to the manufacturer’s instructions. To exclude nonspecific positivity from hepatic vasculatures, a-SMA-positive cells lining vascular structure were not counted. Hepatic expression of myeloperoxidase (MPO), which is a marker of activated neutrophil, was also semiquantitatively analyzed using immunohistochemistry with respective antibodies (Sigma, St. Louis, MO). Connexin43 (Cx43), a component of gap junctions, has recently been found to have a role to play in apoptotic signaling (16). The expression of fluorescence-labeled Cx43 was semiquantitatively assessed using Image Tool 3 (IT3) image analysis software (Image Tool for Windows, Version 3.0; University of Texas, Health Science Center, San Antonio, TX) on confocal microscopy-harvested images. For analyses of a-SMA, MPO, and Cx43, three liver sections from each rat were analyzed and three randomly selected high-power fields (1003) were chosen in each section. The mean number per high-power field for a-SMA and MPO and the mean area for Cx43 for each animal was then determined by summation of all numbers divided by 9.

Histologic Analysis of Liver Injury. After hematoxylin and eosin staining, the degree of liver injury was assessed with liver injury score defined as follows: 0 = no notable hepatocyte integrity impairment or sinusoidal distortion; 1 = mild hepatic injury with <25% of section involved; 2 = moderate hepatic injury with 25% to 50% of section involved; and 3 = severe hepatic injury with >50% involved. For each rat, three liver sections were examined and three randomly selected high-power fields (100×) were analyzed in each section. The mean score for each animal was then determined by summation of all numbers divided by 9.

Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End Labeling Assay for Apoptotic Nuclei. For each rat, six sections (three longitudinal and three transverse sections of each liver specimen) were analyzed by an in situ Cell Death Detection Kit, AP (Roche, Basel, Switzerland), according to the manufacturer’s guidelines. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells were examined in three randomly chosen high-power fields (×400). The mean percentage of apoptotic nuclei per high-power field for each animal was then determined by summation of all numbers divided by 18.

Immunofluorescent Staining for Identification of CD31/von Willebrand Factor+ Cells. Immunofluorescent staining was performed for the examinations of CD31+ and von Willebrand factor+ cells using respective primary antibodies. Irrelevant antibodies were used as controls in the present study.

Statistical Analysis. Quantitative data are expressed as means ± SD. Statistical analysis was adequately performed by analysis of variance followed by Bonferroni multiple-comparison post hoc test. Statistical analysis was performed using SAS statistical software for Windows version 8.2 (SAS Institute, Cary, NC). A probability value < .05 was considered statistically significant.

Table 1

Table 1

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Flow Cytometric Analysis. After 7-day culture, the cultivated cells exhibited typical spindle-shaped morphology characteristic of MSCs (Fig. 1, left upper panel). In vitro characterization of isolated ADMSCs was achieved through flow cytometry that showed predominant cellular expressions of CD-90 and CD-29 typical of MSCs (Fig. 1).

Figure 1

Figure 1

Identification of Engrafted ADMSCs. In this study, the infused ADMSCs were identified by the characteristic red fluorescence from CM-DiI, which was confirmed by fluorescent microscopy at the end of the experiment (Fig. 1, right lower panel).

Histologic Derangement, Hepatocyte Integrity, and Neutrophil Activities. Compared with the animals after IR injury without cell therapy (group 2), which had the highest mean liver injury score, significant improvement was noted in the ADMSC-treated group (group 3) (Fig. 2A–D). Plasma aspartate aminotransferase activities of all the animals are shown in Figure 2E. The activities were significantly elevated after IR injury in group 2 and group 3 compared with group 1 (i.e., the normal controls). Furthermore, the activities were remarkably higher in group 2 than in group 3. Consistently, the number of MPO-positive cells, an indicator of activated neutrophils, was significantly elevated after IR injury (Fig. 3A–D). The number was notably higher in group 2 than in group 3 animals. These findings suggest a protective action of ADMSC infusion against hepatic IR-induced impairment in hepatocyte integrity and cell-mediated inflammatory responses. Furthermore, the number of α-SMA-positive cells, a marker of activated HSCs, was significantly increased after IR injury of the liver and was higher in group 2 compared with that in group 3 (Fig. 3E–H). The result implies that HSCs were activated after hepatic IR but were significantly alleviated after ADMSC administration.

Figure 2

Figure 2

Figure 3

Figure 3

Cytokine Profile Analysis. Real-time polymerase chain reaction analysis of liver homogenate revealed significant elevations in cytokine expressions after IR injury, including those of TNF- α, MMP-9, PAI-1, IL-1 β, IL-6, IL-10, and TGF- β, implying IR-elicited inflammatory responses and HSC activation (Fig. 4). The mRNA expressions of TNF- α, MMP-9, PAI-1, IL-1 β, IL-6, and TGF- β were significantly suppressed, whereas IL-10 expression was notably augmented after administration of ADMSCs. The results suggest a role of ADMSC in anti-inflammation and HSC stabilization in this experimental setting. The expressions of endothelial nitric oxide synthase and endothelin-1, two indicators of vasoactivity and microcirculation, showed an opposite trend after induction of hepatic IR (Fig. 4H–I). The former showed a decrease in expression, whereas the latter exhibited an increase. After IR, the expression of endothelial nitric oxide synthase was markedly preserved and that of endothelin-1 was significantly reduced in the animals with ADMSC treatment compared with those without. The findings may reflect a relatively well-preserved hepatic perfusion after hepatic IR injury.

Figure 4

Figure 4

Western Blotting on Intercellular Adhesion Molecule Expression and Oxidative Stress. The protein expression of intercellular adhesion molecule was notably enhanced after IR of the liver without treatment (group 2) but the upregulation was significantly suppressed after ADMSC treatment (group 3) to a level comparable to that of the normal controls (group 1) (Fig. 5A). On the other hand, the protein expression of nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1, a biomarker of antioxidation, was significantly elevated after IR injury of the liver in group 3 but not in group 2 (Fig. 5B). Furthermore, the protein expression of heme oxygenase-1, another antioxidative index, was notably increased only in the ADMSC-treated animals (group 3) after IR (Fig. 5C). Furthermore, oxyblot analysis showed elevated oxidative stress in group 2 but not in group 3 (Fig. 5D). The results suggest an anti-inflammatory and an antioxidative action of ADMSC against hepatic IR injury.

Figure 5

Figure 5

Expressions of Cx43, Cytochrome C, Apoptotic Markers, and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End Labeling. Immunofluorescent staining of Cx43 showed a remarkable increase after hepatic IR injury, which was significantly reduced after ADMSC treatment (Fig. 6A–D). On the other hand, the protein expression of mitochondrial cytochrome C was significantly suppressed after hepatic IR injury without treatment (group 2) but significantly preserved after ADMSC administration (group 3) to a level comparable to that of the sham-operated animals (group 1). The cytosolic expression of cytochrome C, however, exhibited an opposite trend (Fig. 6E–F). The increased release of mitochondrial cytochrome C, a proapoptotic factor, and its suppression after ADMSC treatment may imply an antiapoptotic role of ADMSC in this experimental setting.

Figure 6

Figure 6

Apoptosis Assay. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining demonstrated a remarkable increase in the number of apoptotic nuclei in the liver after IR injury (group 2). The increase, however, was significantly suppressed by the administration of ADMSCs (group 3) (Fig. 7A–D). Consistently, analyses of mRNA expressions of Bax, caspase-3, and Bcl-2 revealed notably elevated Bax and caspase-3 expressions after IR injury of the liver (Fig. 7E–F). These increments, however, were significantly suppressed after ADMSC treatment. The mRNA expression of Bcl-2, on the other hand, showed an opposite trend (Fig. 7G). In concert with these findings, analyses of the protein expression of Bax showed a significant shift from the cytosolic to mitochondrial compartment after IR and this was significantly reversed after stem cell treatment to a degree comparable to that of the normal control (Fig. 7H–J). Furthermore, the protein expression of cleaved poly (ADP-ribose) polymerase, an index of caspase-3 activation, was markedly increased after IR injury but significantly suppressed after ADMSC treatment. The results, therefore, may signify an antiapoptotic effect of ADMSC treatment in IR injury of the liver.

Figure 7

Figure 7

Prevalence of CD31/von Willebrand Factor-Positive Cells. Semiquantitative analysis of the percentage of CD31-positive cells demonstrated a notable IR-induced reduction, which was significantly restored after ADMSC treatment (Fig. 8A–D). Consistently, the number of cells positive for von Willebrand factor, another endothelial cell marker, substantially dropped after IR injury but was significantly elevated after cell therapy.

Figure 8

Figure 8

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Significance of Hepatic IR Injury and the Findings of This Study. Because IR injury of the liver has now been widely recognized as a significant contributor to clinical morbidity, a myriad of treatment strategies have been proposed as potential solutions. Although recent therapeutic efforts including ischemic preconditioning and pharmacologic interventions have been reported to exert a beneficial effect on hepatic IR injury, there is insufficient evidence to support their clinical use (8). The application of MSCs in the treatment of ischemic organ disorder, on the other hand, has recently caught much attention (17). In addition, the application of stem cell in the treatment of renal (18), cardiac (19), and brain (20) IR injuries in animal models has also been sporadically reported. However, to date, no study has focused on the role of MSC in hepatic IR injury and the underlying mechanisms.

Our previous studies have demonstrated the unique properties of autologous bone marrow-derived endothelial progenitor cell and bone marrow-derived mononuclear cells in the alleviation of dilated cardiomyopathy (15) and pulmonary hypertension (21) in the rat characterized by mitochondrial damage and microvascular damage, respectively. In view of similar disease mechanisms compared with our recent studies (15, 21), we tested the hypothesis that administration of ADMSCs is also beneficial in the treatment of hepatic IR injury through investigating its impact on hepatocyte integrity, cellular activation (i.e., PMN and HSC), oxidative stress, apoptosis, and expressions of key vasoactive as well as pro- and anti-inflammatory molecules in the current study.

Cellular Elements in Hepatic IR Injury. Current concept supports that hepatic IR injury is a biphasic process with different underlying mechanisms (22). The early phase, which occurs 0–4 hrs post-IR, involves Kupffer cell activation that results in morphologic changes (23), priming, the release of reactive oxygen species (24), and proinflammatory cytokines such as TNF- α (25) and IL-1 β (26). The sources of reactive oxygen species have been proposed to be the xanthine oxidase pathway and mitochondria (27). On the other hand, in the late (or subacute) phase of reperfusion (i.e., 5–24 hrs post-IR), PMNs have a pivotal role to play (28, 29). Generation of reactive oxygen species (30) and inflammatory mediators such as leukotriene B4 (31) as well as proteases such as MPO (28) from PMNs are the key events in the subacute phase of IR injury that is also characterized by more severe postischemic injury and gross hepatocyte destruction (28).

In the present study, IR-induced MPO expression was significantly reduced after ADMSC administration, signifying a decrease in PMN-triggered inflammatory response. On the other hand, oxidative stress was significantly reduced in animals with ADMSC infusion compared with those without. Consistently, the expression of antioxidative proteins including nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1 and heme oxygenase-1 were also notably increased after ADMSC administration. Furthermore, in concert with the results of a previous study demonstrating an enhanced contractility of HSCs after warm hepatic ischemia as a contributor to persistent impairment in hepatic perfusion and mitochondrial respiration during reperfusion (32), the findings of the current study showed an enhanced post-IR hepatic expression of TGF- β, which is a key cytokine for HSC activation. The increase, interestingly, was significantly reduced after ADMSC infusion, suggesting suppressed HSC activation after ADMSC administration. This is further supported by the finding of suppressed α -SMA expression, an indicator of HSC activation, after ADMSC treatment. Besides, the expression of Cx43, which has been shown to be upregulated in HSC during its activation (33), followed the same trend as that of TGF- β and further strengthens the observation. Taken together, the findings suggest that the activation of both PMN and HSC, key effector cells for perpetuating inflammatory responses in the liver, was significantly suppressed after ADMSC treatment.

The Role of Cytokines in Hepatic IR Injury. Cytokines such as TNF- α and IL-1 are generated during hepatic IR (25, 26, 34). There is a body of evidence suggesting that TNF- α is a significant contributor to IR-induced liver injury (35, 36). TNF- α is known to induce cell death through apoptosis (37). On the other hand, studies also demonstrate a role of IL-1 in the induction of hepatic IR injury. Gene transfer of IL-1 receptor antagonist into the rat liver was found to be protective in terms of reduction in proinflammatory cytokines production and improved survival (38). Furthermore, IL-1 receptor I-knockout [IL-1RI(–/–)] mice showed significantly reduced PMN recruitment and nuclear factor-κB activation compared with wild-type mice during IR (39). Another clinical study showed that overproduction of acute reactant cytokines such as IL-6 from the portal system during hepatic IR relates positively with postoperative hepatocyte injury in humans (40). Several theories have been put forward to explain the phenomenon, including upregulation of adhesion molecules such as intercellular adhesion molecule (41, 42) and/ or their receptors (e.g., Mac-1) (42, 43). The correlation between hepatic IR and cytokine release is now so well established that cytokines such as TNF- α, IL-1 β, and IL-6 have been accepted as clinical indicators for the degree of hepatic IR injury (40).

Accordingly, the results of the present study demonstrated a remarkable increase in TNF- α after IR injury of the liver. The elevation, however, was significantly suppressed after ADMSC administration, implying a reduction in hepatic inflammatory response through ADMSC infusions. The finding is consistent with elevated post-IR expressions of proinflammatory cytokines including MMP-9, PAI-1, IL-1 β, and IL-6, which also showed significant reductions after ADMSC treatment. Consistent changes in the protein expression of intercellular adhesion molecule in the present study further reinforce the results.

On the other hand, other interleukins were found to be protective against hepatic IR injury. For example, pretreatment of liver grafts with IL-10 was shown to decrease posttransplant transaminase levels in the pig (44). Also, exogenous IL-10 was found to protect against hepatic IR injury by suppressing nuclear factor-κB activation and subsequent expression of proinflammatory mediators (45, 46). In the current study, IL-10 was demonstrated to increase after hepatic IR injury and further significant elevation was noted after ADMSC administration. The result, therefore, suggests a protective role of ADMSC treatment in this experimental setting.

IR Injury and Hepatic Microcirculation. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm hepatic IR injury (47). The severity of IR-induced microcirculatory disturbances is proportional to the duration of ischemia (47, 48). One of the most obvious findings in the hepatic microvascular bed during IR is the heterogeneous perfusion pattern because cessation of sinusoidal perfusion occurs over certain focal areas and slowing of blood flow happens in other regions (49). A disturbance in the delicate equilibrium among nitric oxide, endothelin-1, and carbon monoxide (50, 51) that leads to sinusoidal narrowing through HSC contraction (50) has been reported to be an important contributor to the microcirculatory derangement. This in turn enhances leukocyte–endothelial contacts and promotes leukostasis that further hampers sinusoidal blood flow, although most sinusoids containing PMN are still conducting flow (48, 52). As a result, the “no-reflow phenomenon” occurs, causing hypoxic cell injury (48, 53). In combination with the release of proinflammatory cytokines and reactive oxygen species, this leads to the ultimate consequence of cell death and parenchymal failure (54).

In the current study, expression of endothelin-1, which is a key mediator of HSC contraction (55), and enhanced expression of α -SMA and TGF- β, which are markers of HSC activation, were notably increased after IR injury but were suppressed by the administration of ADMSCs. Accordingly, the expression of endothelial nitric oxide synthase, the key enzyme responsible for nitric oxide release in hepatic microvasculature, followed the opposite trend. In addition, the remarkable reduction in the number of cells with endothelial cell markers (i.e., CD31 and von Willebrand factor) after IR suggests an IR-elicited impairment of sinusoidal endothelial integrity, which was significantly restored after ADMSC treatment. The results, therefore, may imply a preservation of sinusoidal perfusion after IR injury through ADMSC administration.

Impact of IR on Hepatocyte Integrity and the Effect of ADMSC Treatment. Consistent with the findings of our previous studies (6, 7), significant microscopic distortion in hepatic architecture was evident after IR injury (Fig. 2). The injury, however, was significantly suppressed after ADMSC treatment. Consistently, IR-induced elevation in plasma aspartate aminotransferase, which reflects the impairment in hepatocyte integrity, was also significantly reduced after ADMSC treatment. The findings suggest a positive therapeutic role of ADMSCs in alleviating hepatic IR-induced microvascular collapse and preserving hepatic microarchitecture.

Impact of IR on Hepatic Apoptosis and the Effect of ADMSC Treatment. In concert with the results from other investigators (5, 56), the present study showed that hepatic IR injury was associated with a remarkable increase in the number of apoptotic nuclei. The increase, however, was significantly reduced after ADMSC treatment in the present study. Consistent trends in IR-induced changes in expressions of Bax, caspase 3, and cleaved poly (ADP-ribose) polymerase were also noted. Furthermore, the release of proapoptotic factor such as cytochrome C from the mitochondrial intermembranous space is known to play an important role in the progression of apoptosis (57). The results of the present study demonstrated not only a significant release of mitochondrial cytochrome C to the cytosolic compartment, but also a notable shift of Bax in the opposite direction after IR insult. In addition, the protein expression of cleaved poly (ADP-ribose) polymerase, an index of caspase-3 activation, was also remarkably increased after IR and significantly repressed after ADMSC administration. These findings, together with the opposite changes in mRNA expression of Bcl-2, suggest that systemic infusion of ADMSCs is beneficial in suppressing hepatic IR-induced apoptotic cell death.

Limitations. Despite the intriguing findings in the current study, there are several limitations. First, the overall positive therapeutic effects from administration of ADMSC at three different time points did not specify the relative beneficial impact of each bolus. The optimal timing for stem cell administration, therefore, remains unclear. The choice of earlier time points for investigation would further improve our understanding of the mechanistic basis of ADMSC treatment in this experimental setting. Second, the present study focused on warm hepatic IR injury in situ to simulate the clinical condition of compromise in hepatic perfusion like in hepatic surgery and shock. The therapeutic potential of ADMSC in the scenario of liver transplantation, which also involves cold preservation of the liver followed by reperfusion, was not assessed. Finally, there was a lack of functional studies in the current investigation so that the question of whether the alleviation in IR-induced injuries at cellular and molecular levels after ADMSC treatment is also accompanied by a functional improvement remains unanswered. Further experimental efforts are required to clarify the issue.

Conclusions. The present study represents the first attempt to address the therapeutic potential of autologous MSCs in treating hepatic IR injury, which is one of the key issues in current surgery. Our results showed that the positive impact from systemic administration of ADMSCs may be attributable to a suppression of cellular activation and a cascade of subcellular mechanisms including reduction of proinflammatory cytokine release, alleviation of IR-induced oxidative stress, preservation of hepatic microcirculation, and decline in apoptosis. The proposed mechanism underlying the positive therapeutic effects of ADMSCs against hepatic IR injury in the rodent model is shown in Figure 9.

Figure 9

Figure 9

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Financial support from a research grant from Chang Gung Memorial Hospital and Chang Gung University (Grant No. CMRPG881151) is gratefully acknowledged.

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