A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

Pure Laparoscopic Versus Open Right Hepatectomy in Live Liver Donors: A Propensity Score-matched Analysis.

OBJECTIVE: The aim of the study was to present the safety and feasibility of pure laparoscopic donor right hepatectomy (PLDRH) in comparison with those of conventional donor right hepatectomy. SUMMARY BACKGROUND DATA: Although the use of PLDRH is gradually spreading worldwide, its outcomes, including the long-term outcomes in both donors and recipients, have not yet been evaluated in a large comparative study. METHODS: We retrospectively reviewed the medical records of 894 donors who underwent living donor liver transplantation between January 2010 and September 2018 at Seoul National University Hospital. We performed 1:1 propensity score matching between the PLDRH and conventional donor right hepatectomy groups. Subsequently, 198 donor-recipient pairs were included in each group. RESULTS: The total operation time (P < 0.001), time to remove the liver (P < 0.001), and warm ischemic time (P < 0.001) were longer in the PLDRH group. None of the donors required intraoperative transfusion or experienced any irreversible disabilities or mortalities. The length of postoperative hospital stay was significantly shorter in the PLDRH group (P < 0.001). The rate of complications in donors was similar between the 2 groups. Although other complication rates in recipients were, however, similar, the rates of early (P = 0.019) and late (P < 0.001) biliary complications in recipients were higher in the PLDRH group. There was no significant difference in overall survival and graft survival between the 2 groups. CONCLUSIONS: PLDRH is feasible when performed at an experienced living donor liver transplantation center. Further studies on long-term recipient outcomes including biliary complications are needed to confirm the safety.

Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation.

[Figure: see text].

Optimal Intervention for Initial Treatment of Anastomotic Biliary Complications After Right Lobe Living Donor Liver Transplantation.

Background: This study evaluated endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic biliary drainage (PTBD) as interventions for patients with anastomotic biliary complications (ABC) after living donor liver transplantation (LDLT). Methods: Prospectively collected data of patients who were diagnosed with ABC after LDLT between January 2013 and June 2017 were retrospectively reviewed. Results: There were 57 patients who underwent LDLT with a right liver graft using duct-to-duct biliary reconstruction and experienced ABC. Among the patients with RAD involvement, there were no significant differences in the intervention success (p = 0.271) and patency rates (p = 0.267) between ERCP and PTBD. Similarly, among the patients with RPD involvement, there were no significant differences in the intervention success (p = 0.148) and patency rates (p = 0.754) between the two procedures. Graft bile duct variation (p = 0.013) and a large angle between the recipient and graft bile duct (R-G angle) (p = 0.012) significantly increased the likelihood of failure of ERCP in the RAD. When the R-G angle was greater than 47.5°, the likelihood of ERCP failure increased. Conclusion: We recommend PTBD when graft bile duct variation is presented in patients with RAD involvement and/or when the R-G angle is greater than 47.5°.

Short-term therapy with anti-ICAM-1 monoclonal antibody induced long-term liver allograft survival in nonhuman primates.

Tolerance induction remains challenging following liver transplantation and the long-term use of immunosuppressants, especially calcineurin inhibitors, leads to serious complications. We aimed to test an alternative immunosuppressant, a chimeric anti-ICAM-1 monoclonal antibody, MD-3, for improving the outcomes of liver transplantation. We used a rhesus macaque liver transplantation model and monkeys were divided into three groups: no immunosuppression (n = 2), conventional immunosuppression (n = 4), and MD-3 (n = 5). Without immunosuppression, liver allografts failed within a week by acute rejection. Sixteen-week-long conventional immunosuppression that consisted of prednisolone, tacrolimus, and an mTOR inhibitor prolonged liver allograft survival; however, recipients died of acute T cell-mediated rejection (day 52), chronic rejection (days 62 and 66), or adverse effects of mTOR inhibitor (day 32). In contrast, 12-week-long MD-3 therapy with transient conventional immunosuppression in the MD-3 group significantly prolonged the survival of liver allograft recipients (5, 96, 216, 412, 730 days; p = .0483). MD-3 effectively suppressed intragraft inflammatory cell infiltration, anti-donor T cell responses, and donor-specific antibody with intact anti-cytomegalovirus antibody responses. However, this regimen ended in chronic rejection. In conclusion, short-term therapy with MD-3 markedly improved liver allograft survival to 2 years without maintenance of immunosuppressant. MD-3 is therefore a promising immune-modulating agent for liver transplantation.

Demarcating the Exact Midplane of the Liver Using Indocyanine Green Near-Infrared Fluorescence Imaging During Laparoscopic Donor Hepatectomy.

Indocyanine green (ICG) near-infrared fluoroscopy has been recently implemented in pure laparoscopic donor hepatectomy (PLDH). This study aims to quantitatively evaluate the effectiveness of ICG fluoroscopy during liver midplane dissection in PLDH and to demonstrate that a single injection of ICG is adequate for both midplane dissection and bile duct division. Retrospective analysis was done with images acquired from recordings of PLDH performed without ICG (pre-ICG group) from November 2015 to May 2016 and with ICG (post-ICG group) from June 2016 to May 2017. 30 donors from the pre-ICG group were compared with 46 donors from the post-ICG group. The operation time was shorter (P = 0.002) and postoperative peak aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were lower (P = 0.031 and P = 0.019, respectively) in the post-ICG group than the pre-ICG group. Within the post-ICG group, the color intensity differences between the clamped versus nonclamped regions in the natural, black-and-white, and fluorescent modes were 39.7 ± 36.2, 89.6 ± 46.9, and 19.1 ± 36.8 (mean ± SD, P < 0.001), respectively. The luminosity differences were 37.2 ± 34.5, 93.8 ± 32.1, and 26.7 ± 25.7 (P < 0.001), respectively. Meanwhile, the time from when ICG was injected to when the near-infrared camera was turned on for bile duct visualization was 85.6 ± 25.8 minutes. All grafts received from the 46 donors were successfully transplanted. In conclusion, ICG fluoroscopy helps to reduce operation time and lower postoperative AST/ALT levels. ICG injection visualized with black-and-white imaging is most effective for demarcating the liver midplane during PLDH. A single intravenous injection of ICG is sufficient for midplane dissection as well as bile duct division.

Dextroplantation of Left Liver Graft in Infants.

The position of the left side liver graft is important, and it could lead to complications of the hepatic vein (HV) and portal vein (PV), especially in a small child using a variant left lateral section (vLLS) graft. The purpose of this study was to evaluate the outcome of a novel technique for the implantation of a vLLS graft to the right side (dextroplantation) in infants. For 3 years, 10 consecutive infants underwent dextroplantation using a vLLS graft (group D). The graft was implanted to the right side of the recipient after 90° counterclockwise rotation; the left HV graft was anastomosed to inferior vena cava using the extended right and middle HV stump, and PV was reconstructed using oblique anastomosis without angulation. Surgical outcomes were compared with the historical control group (n = 17, group C) who underwent conventional liver transplantation using a vLLS during infancy. Group D recipients were smaller than group C (body weight <6 kg: 50.0% versus 11.8%; P = 0.03). The rate of graft-to-recipient weight ratio >4% was higher in group D (60.0%) than C (11.8%; P = 0.01). Surgical drains were removed earlier in group D than in group C (15 versus 18 postoperative days [PODs]; P = 0.048). Each group had 1 PV complication (10.0% versus 5.9%); no HV complication occurred in group D, but 3 HV complications (17.6%) occurred in group C (P > 0.05). Hospital stay was shorter in group D than in group C (20 versus 31 PODs; P = 0.02). Dextroplantation of a vLLS graft, even a large-for-size one, was successful in small infants without compromising venous outcomes, compared with conventional vLLS transplantation. We could remove the surgical drains earlier and reduce hospital stays in cases of dextroplantation.

Complications of polytetrafluoroethylene graft use in middle hepatic vein reconstruction in living donor liver transplantation: a retrospective, single-centre, long-term, real-world experience.

In living donor liver transplantation (LDLT) of the right lobe, polytetrafluoroethylene (PTFE) grafts may be used for anterior drainage. This study aimed to determine the risk factors of PTFE graft-associated complications. Data from patients who underwent LDLT of the right lobe with middle hepatic vein reconstruction using PTFE grafts between January 2005 and December 2012 were retrospectively reviewed. Among 360 patients, PTFE graft-associated complications occurred in 17 patients (group B) (4.7%); recipients without these complications comprised group A (95.3%). The 1-, 6- and 12-month patency rates were significantly lower in group B (P < 0.001, P = 0.002 and P = 0.007). In group B, eight patients (47.1%) required surgical intervention, three patients (17.6%) suffered from infectious complications, and 14 patients (82.4%) experienced PTFE graft migration into the adjacent organs, namely the common bile duct (n = 3, 17.6%), stomach (n = 1, 5.9%), duodenum (n = 5, 29.4%) and jejunum (n = 5, 29.4%). The proportion of recipients who underwent hepaticojejunostomy, had abdominal adhesions and received interventions in/around the liver after LDLT was higher in group B (P < 0.001). Although the incidence of PTFE graft-associated complication is low, close long-term follow-up is needed, especially in patients with risk factors.

Donor wound satisfaction after living-donor liver transplantation in the era of pure laparoscopic donor hepatectomy.

BACKGROUND: Donor safety and cosmetic outcome are the main concerns raised by most living-donors. Pure laparoscopic living-donor hepatectomy (PLLDH) can provide the balance between those concerns. No studies evaluated the donors' satisfaction after PLLDH. The aim of this study is to evaluate the donors' satisfaction after PLLDH compared with donors who underwent open approach. METHODS: We randomly assigned a questionnaire (Donor satisfaction questionnaire) to the donors, operated between 2011 and 2017, during their follow-up visits in the outpatient clinic. Donors who responded to the questionnaire were included in our study. Donors were divided into 3 groups: L group (conventional inverted L incision), M group (midline incision), and PL group (laparoscopic approach). RESULTS: 149 donors were included in our study. L group included 60 donors (40.3%), M group included 39 patients (26.2%), and PL group included 50 patients (33.5%). There were no significant differences between the groups regarding preoperative and perioperative outcomes apart from shorter operation time in PL group and higher wound infection in M group. Body image scale was significantly better in PL group (p = 0.001). Cosmetic scale was significantly higher in PL group (p = 0.001). Regarding self-confidence scale, it was significantly higher in PL group (p = 0.001). There was no significant difference between the groups regarding the sense of dullness or numbness on the scar (p = 0.113). CONCLUSION: PLLDH is safe and feasible for living-donor hepatectomy. Donors operated by pure laparoscopic approach have better satisfaction scores compared to conventional open approach.

Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

BACKGROUND: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS: Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions. RESULTS: Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage. CONCLUSIONS: Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Pure Laparoscopic Versus Open Left Hepatectomy Including the Middle Hepatic Vein for Living Donor Liver Transplantation.

Pure laparoscopic donor hepatectomy (PLDH) has become increasingly accepted in the era of minimally invasive surgeries. However, the outcomes of pure laparoscopic donor left hepatectomy (PLDLH) are relatively less known than for left lateral sectionectomy or right hepatectomy. This study aimed to report our experience with and the outcomes of PLDLH including the middle hepatic vein (MHV) and to compare these outcomes with conventional donor left hepatectomy (CDLH). The medical records of living liver donors between January 2010 and January 2018 at Seoul National University Hospital were retrospectively reviewed. Donors who underwent left hepatectomy including the MHV were included. To minimize selection bias, donors who underwent CDLH after the initiation of the PLDH program were excluded. Finally, there were 18 donors who underwent CDLH and 8 who underwent PLDLH. The median (interquartile range [IQR]) warm ischemia time (11 [10-16] minutes versus 4 [2-7] minutes; P = 0.001) was longer in the PLDLH group than the CDLH group. The total operation time (333 [281-376] minutes versus 265 [255-308] minutes; P = 0.09) and time to remove the liver (245 [196-276] minutes versus 182 [172-205] minutes; P = 0.08) were also longer in PLDLH although not statistically significant. The length of postoperative hospital stay was significantly shorter in the PLDLH group (7 [7-8] days versus 9 [8-10] days; P = 0.01). There were no postoperative complications in the PLDLH group. The rate of complications in recipients was similar in both groups. In conclusion, PLDLH including the MHV appears to be safe and feasible. Further analysis including longterm outcomes is needed.

Repurposing pyridoxamine for therapeutic intervention of intravascular cell-cell interactions in mouse models of sickle cell disease.

Adherent neutrophils on vascular endothelium positively contribute to cell-cell aggregation and vaso-occlusion in sickle cell disease. In the present study, we demonstrated that pyridoxamine, a derivative of vitamin B6, might be a therapeutic agent to alleviate intravascular cell-cell aggregation in sickle cell disease. Using real-time intravital microscopy, we found that one oral administration of pyridoxamine dose-dependently increased the rolling influx of neutrophils and reduced neutrophil adhesion to endothelial cells in cremaster microvessels of sickle cell disease mice challenged with hypoxia-reoxygenation. Short-term treatment also mitigated neutrophil-endothelial cell and neutrophil-platelet interactions in the microvessels and improved the survival of sickle cell disease mice challenged with tumor necrosis factor-α. The inhibitory effects of pyridoxamine on intravascular cell-cell interactions were potentiated by co-treatment with hydroxyurea. We observed that long-term (5.5 months) oral treatment with pyridoxamine significantly diminished the adhesive function of neutrophils and platelets and down-regulated the expression of E-selectin and intercellular adhesion molecule-1 on the vascular endothelium in tumor necrosis factor-α-challenged sickle cell disease mice. Ex vivo studies revealed that the surface amount of αMß2 integrin was significantly decreased in stimulated neutrophils isolated from sickle cell disease mice treated with pyridoxamine-containing water. Studies using platelets and neutrophils from sickle cell disease mice and patients suggested that treatment with pyridoxamine reduced the activation state of platelets and neutrophils. These results suggest that pyridoxamine may be a novel therapeutic and a supplement to hydroxyurea to prevent and treat vaco-occlusion events in sickle cell disease.

YAP Controls Endothelial Activation and Vascular Inflammation Through TRAF6.

RATIONALE: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. OBJECTIVE: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. METHODS AND RESULTS: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell-specific deletion of YAP. CONCLUSIONS: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.

DREAM plays an important role in platelet activation and thrombogenesis.

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5'-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iß (PI3K-Iß). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iß activation. These results suggest that platelet DREAM regulates PI3K-Iß activity and plays an important role during thrombus formation.

Different prognostic factors and strategies for early and late recurrence after adult living donor liver transplantation for hepatocellular carcinoma.

BACKGROUND: Some patients with hepatocellular carcinoma (HCC) recurrence after LT show good long-term survival. We aimed to determine the prognostic factors affecting survival after recurrence and to suggest treatment strategies. METHODS: Between January 2000 and December 2015, 532 patients underwent adult living donor liver transplantation (LDLT) for HCC. Among these, 92 (17.3%) who experienced recurrence were retrospectively reviewed. RESULTS: The 1-, 3-, and 5-year survival rates after recurrence were 59.5%, 23.0%, and 11.9%, respectively. In multivariate analysis, time to recurrence >6 months and surgical resection after recurrence were related to longer survival after recurrence, while multi-organ involvement at the time of primary recurrence was related to poorer survival. We classified patients into early (≤6 months) and late (>6 months) recurrence groups. In the early recurrence group, tumor size >5 cm in the explant liver, liver as the first detected site of recurrence, and multiple organ involvement at primary recurrence were related to survival on multivariate analysis. In the late recurrence group, mammalian target of rapamycin inhibitor (mTORi) usage and multi-organ involvement were significantly associated with the prognosis on multivariate analysis. CONCLUSIONS: Various therapeutic approaches are needed depending on the period of recurrence after LT and multiplicity of involved organs.

A directional switch of integrin signalling and a new anti-thrombotic strategy.

Integrins have a critical role in thrombosis and haemostasis. Antagonists of the platelet integrin αIIbß3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of causing bleeding. It is therefore desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally. Inside-out signalling activates integrins through a talin-dependent mechanism. Integrin ligation mediates thrombus formation and outside-in signalling, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive but distinct sites within the integrin ß3 cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out signalling and also ligand-induced integrin activation, but is not required for outside-in signalling. Integrin ligation induces transient talin dissociation and Gα13 binding to an EXE motif (in which X denotes any residue), which selectively mediates outside-in signalling and platelet spreading. The second talin-binding wave is associated with clot retraction. An EXE-motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signalling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signalling and, on the basis of this mechanism, designed a potent new anti-thrombotic drug that does not cause bleeding.

The learning curve in pure laparoscopic donor right hepatectomy: a cumulative sum analysis.

BACKGROUND: Although the use of pure laparoscopic donor hepatectomy (PLDH) is increasingly common, it remains limited to a few experienced centers and no data on the learning curve are currently available. The aim of this study is to evaluate the learning curve associated with the use of pure laparoscopic donor right hepatectomy (PLDRH). METHODS: Data from donors undergoing PLDRH performed by a single surgeon between November 2015 and October 2017 were retrospectively reviewed. The learning curve was evaluated using the cumulative sum (CUSUM) method based on duration of surgery. RESULTS: Of 100 donors evaluated, none required transfusion or conversion to open hepatectomy and no irreversible disability or mortality was reported. The mean operative time was 320.7 ± 51.8 min, and all grafts were successfully transplanted. The CUSUM analysis demonstrated a learning curve of approximately 60 cases of PLDRH. Estimated total liver volume > 1400 cm3 and double portal vein orifices were seen to be risk factors for longer surgery time. Having adjusted for case mix with these factors, the risk-adjusted CUSUM analysis demonstrated a learning curve of 65-70 cases of PLDRH. CONCLUSIONS: In conclusion, PLDRH is a feasible and safe procedure with a learning curve of 65-70 cases.

Pure laparoscopic donor right hepatectomy: perspectives in manipulating a flexible scope.

BACKGROUND: Interest in pure laparoscopic donor hepatectomy (PLDH) is increasing worldwide with the donor's cosmetic demands and improvements in surgical techniques. Efficient manipulation of flexible scope is mandatory for successful PLDH, especially in right hepatectomy which requires more mobilization. This study provides guidelines on how to manipulate optimally a flexible scope. METHODS: Data from 158 donors who underwent pure laparoscopic donor right hepatectomy (PLDRH) between November 2015 and December 2017 were retrospectively reviewed. RESULTS: None of the donors required transfusion, conversion to open hepatectomy, or experienced any irreversible disabilities or mortalities. Three types of laparoscopic view provided by the flexible scope, which are bird's eye view, low angle view, and lateral view, were applied to each step of the procedure. CONCLUSIONS: PLDRH can be successfully performed with maximizing visibility given by the tips and pitfalls in manipulating the flexible scope.