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.
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.
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.
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.
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.
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.
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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