Qinlong Liu

Sphingosine kinase-2 inhibition improves mitochondrial function and survival after hepatic ischemia–reperfusion

BACKGROUND & AIMS The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR. METHODS Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion. RESULTS Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25- and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54-91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration, all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/re-oxygenation resulted in increases of SK2 mRNA, S1P levels, and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes. CONCLUSIONS SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death, and survival after hepatic IR and represents a new target for the treatment of IR injury.

Acute Ethanol Causes Hepatic Mitochondrial Depolarization in Mice: Role of Ethanol Metabolism

Background/Aims An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Methods Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg). Results Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼70% and ∼20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Conclusions Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde.

Inhibition of Sphingosine Kinase-2 Suppresses Inflammation and Attenuates Graft Injury after Liver Transplantation in Rats

Inflammation mediates/promotes graft injury after liver transplantation (LT). This study investigated the roles of sphingosine kinase-2 (SK2) in inflammation after LT. Liver grafts were stored in UW solution with and without ABC294640 (100 µM), a selective inhibitor of SK2, before implantation. Hepatic sphingosine-1-phosphate (S1P) levels increased ∼4-fold after LT, which was blunted by 40% by ABC294640. Hepatic toll-like receptor-4 (TLR4) expression and nuclear factor-κB (NF-κB) p65 subunit phosphorylation elevated substantially after transplantation. The pro-inflammatory cytokines/chemokines tumor necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10 mRNAs increased 5.9-fold, 6.1-fold and 16-fold, respectively following transplantation, while intrahepatic adhesion molecule-1 increased 5.7-fold and monocytes/macrophage and neutrophil infiltration and expansion of residential macrophage population increased 7.8–13.4 fold, indicating enhanced inflammation. CD4+ T cell infiltration and interferon-γ production also increased. ABC294640 blunted TLR4 expression by 60%, NF-κB activation by 84%, proinflammatory cytokine/chemokine production by 45–72%, adhesion molecule expression by 54% and infiltration of monocytes/macrophages and neutrophils by 62–67%. ABC294640 also largely blocked CD4+ T cell infiltration and interferon-γ production. Focal necrosis and apoptosis occurred after transplantation with serum alanine aminotransferase (ALT) reaching ∼6000 U/L and serum total bilirubin elevating to ∼1.5 mg/dL. Inhibition of SK2 by ABC294640 blunted necrosis by 57%, apoptosis by 74%, ALT release by ∼68%, and hyperbilirubinemia by 74%. Most importantly, ABC294640 also increased survival from ∼25% to ∼85%. In conclusion, SK2 plays an important role in hepatic inflammation responses and graft injury after cold storage/transplantation and represents a new therapeutic target for liver graft failure.

Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers.

This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20 h after gavaging with ethanol (5 g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5 μM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.

Amphiregulin Stimulates Liver Regeneration After Small-for-Size Mouse Liver Transplantation

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small‐for‐size liver transplantation. Livers of male C57BL/6 mice were reduced to ∼50% and ∼30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5‐Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 μg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small‐for‐size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

Small-for-Size Liver Transplantation Increases Pulmonary Injury in Rats: Prevention by NIM811

Pulmonary complications after liver transplantation (LT) often cause mortality. This study investigated whether small-for-size LT increases acute pulmonary injury and whether NIM811 which improves small-for-size liver graft survival attenuates LT-associated lung injury. Rat livers were reduced to 50% of original size, stored in UW-solution with and without NIM811 (5 μM) for 6 h, and implanted into recipients of the same or about twice the donor weight, resulting in half-size (HSG) and quarter-size grafts (QSG), respectively. Liver injury increased and regeneration was suppressed after QSG transplantation as expected. NIM811 blunted these alterations >75%. Pulmonary histological alterations were minimal at 5–18 h after LT. At 38 h, neutrophils and monocytes/macrophage infiltration, alveolar space exudation, alveolar septal thickening, oxidative/nitrosative protein adduct formation, and alveolar epithelial cell/capillary endothelial apoptosis became overt in the lungs of QSG recipients, but these alterations were mild in full-size and HSG recipients. Liver pretreatment with NIM811 markedly decreased pulmonary injury in QSG recipients. Hepatic TNFα and IL-1β mRNAs and pulmonary ICAM-1 expression were markedly higher after QSG transplantation, which were all decreased by NIM811. Together, dysfunctional small-for-size grafts produce toxic cytokines, leading to lung inflammation and injury. NIM811 decreased toxic cytokine formation, thus attenuating pulmonary injury after small-for-size LT.

Improvement of liver injury and survival by JNK2 and iNOS deficiency in liver transplants from cardiac death mice

BACKGROUND & AIMS Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. METHODS Livers explanted from wild-type, inducible nitric oxide synthase knockout (iNOS(-/-)), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. RESULTS After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS deficiency. JNK inhibition and JNK2 deficiency, but not JNK1 deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS deficiency, JNK inhibition and JNK2 deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. CONCLUSION The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization, and is an attractive target to improve liver function and survival in CDD liver transplantation recipients.

Disrupted Renal Mitochondrial Homeostasis after Liver Transplantation in Rats

Background Suppressed mitochondrial biogenesis (MB) contributes to acute kidney injury (AKI) after many insults. AKI occurs frequently after liver transplantation (LT) and increases mortality. This study investigated whether disrupted mitochondrial homeostasis plays a role in AKI after LT. Methods Livers were explanted from Lewis rats and implanted after 18 h cold storage. Kidney and blood were collected 18 h after LT. Results In the kidney, oxidative phosphorylation (OXPHOS) proteins ATP synthase-β and NADH dehydrogenase-3 decreased 44% and 81%, respectively, with marked reduction in associated mRNAs. Renal PGC-1α, the major regulator of MB, decreased 57% with lower mRNA and increased acetylation, indicating inhibited synthesis and suppressed activation. Mitochondrial transcription factor-A, which controls mtDNA replication and transcription, protein and mRNA decreased 66% and 68%, respectively, which was associated with 64% decreases in mtDNA. Mitochondrial fission proteins Drp-1 and Fis-1 and mitochondrial fusion protein mitofusin-1 all decreased markedly. In contrast, PTEN-induced putative kinase 1 and microtubule-associated protein 1A/1B-light chain 3 increased markedly after LT, indicating enhanced mitophagy. Concurrently, 18- and 13-fold increases in neutrophil gelatinase-associated lipocalin and cleaved caspase-3 occurred in renal tissue. Both serum creatinine and blood urea nitrogen increased >2 fold. Mild to moderate histological changes were observed in the kidney, including loss of brush border, vacuolization of tubular cells in the cortex, cast formation and necrosis in some proximal tubular cells. Finally, myeloperoxidase and ED-1 also increased, indicating inflammation. Conclusion Suppression of MB, inhibition of mitochondrial fission/fusion and enhancement of mitophagy occur in the kidneys of recipients of liver grafts after long cold storage, which may contribute to the occurrence of AKI and increased mortality after LT.

Sa1036 Hepatic Fibrosis After Small-for-Size Liver Transplantation in Rats: Possible Role of TGFβ/SMAD Signaling

this study, we explored whether concomitant medication altered the risk of hepatic fibrosis in NAFLD. Specifically, we sought to ascertain the association between RAS blocking agents (angiotensin converting enzyme blocker or angiotensin II receptor blocker) and severity of hepatic fibrosis in NAFLD patients with hypertension. Methods: This study cohort consisted of 259 hypertensive patients with biopsy proven NAFLD prospectively followed up in hepatology outpatient clinics in Cleveland, OH. Clinical data including demographics, anthropometry, medical history, medication history, biochemical and liver biopsy findings were evaluated. Patients with and without RAS blocker use were compared in relation to liver fibrosis stage. Utilizing electronic medical records, risk factors for advanced fibrosis were explored, in particular, the association between commonly used medications and advanced fibrosis. Independent risk factors for advanced fibrosis were ascertained using binary regression. Results: The mean age of the cohort was 52.2 years and mean BMI was 37.33 kg/m2. Females, diabetics and patients with advanced fibrosis comprised 64.9%, 60.2% and 35.9% of the cohort respectively. Patients treated with RAS blockers had significantly less ballooning (1.05 vs. 1.33, p=0.002) and lower fibrosis stage (1.64 vs. 2.24, p=0.001) on liver biopsy compared to patients not treated with RAS blockers. Compared to patients without advanced fibrosis, patients with advanced fibrosis were older (54.6 vs. 50.9 years, p=0.014), more often diabetic (76.3 vs. 50.9%, p=0.000), had more ballooning on histology (1.57 vs. 0.93, p=0.000), more likely to be on insulin (22.6 vs. 11.5%, p=0.018), and sulfonylureas (29.0 vs. 15.8%, p=0.011) while less likely to be on RAS blockers (49.5 vs. 70.3%, p=0.001). On multivariate analysis, grade of ballooning had an independent positive association with advanced fibrosis (OR 7.539; 95%CI 3.402-16.706, p=0.000) while usage of RAS blockers had a negative association with advanced fibrosis (OR 0.293; 95%CI 0.1090.787, p=0.015). Conclusion: Hypertensive patients with NAFLD treated with RAS blockers had less advanced hepatic fibrosis, suggesting a beneficial role for angiotensin blockers to protect against NASH fibrosis. Prospective clinical trials are warranted to explore this association in greater detail.

Mo1786 8-Pcpt-cGMP Protects Against Mitochondrial Dysfunction and Improves Survival of Steatotic Grafts After Partial Liver Transplantation

A S L D A b st ra ct s (p,0.001), lower LDH releasing in culture medium (p ,0.001) and ROS-production (p,0.01). Finally, co-cultured hepatocytes with cMSC also presented lower ROS (p,0.001) and higher expression of Nrf2 (p,0.01). CONCLUSION: We concluded that cMSC might be able to reduce TAA-induced chronic liver injury through theirs antioxidant activity and hepatoprotective effects, showing curative potential for liver diseases.