Epoxide-Mediated Trans-Thioglycosylation and Application to the Synthesis of Oligosaccharides Related to the Capsular Polysaccharides of C. jejuni HS:4.

The enzyme-resistant thioglycosides are highly valuable immunogens because of their enhanced metabolic stability. We report the first synthesis of a family of thiooligosaccharides related to the capsular polysaccharides (CPS) of Campylobacter jejuni HS:4 for potential use in conjugate vaccines. The native CPS structures of the pathogen consist of a challenging repeating disaccharide formed with ß(1→4)-linked 6-deoxy-ß-D-ido-heptopyranoside and N-acetyl-D-glucosamine; the rare 6-deoxy-ido-heptopyranosyl backbone and ß-anomeric configuration of the former monosaccharide makes the synthesis of this family of antigens very challenging. So far, no synthesis of the thioanalogs of the CPS antigens have been reported. The unprecedented synthesis presented in this work is built on an elegant approach by using ß-glycosylthiolate as a glycosyl donor to open the 2,3-epoxide functionality of pre-designed 6-deoxy-ß-D-talo-heptopyranosides. Our results illustrated that this key trans-thioglycosylation can be designed in a modular and regio and stereo-selective manner. Built on the success of this novel approach, we succeeded the synthesis of a family of thiooligosaccharides including a thiohexasaccharide which is considered to be the desired antigen length and complexity for immunizations. We also report the first direct conversion of base-stable but acid-labile 2-trimethylsilylethyl glycosides to glycosyl-1-thioacetates in a one-pot manner.

Synthesis and Inclusion Properties of a ß-Cyclodextrin Heptaphosphoramidate.

In this study, we report a novel per-6-substituted ß-cyclodextrin (4) featuring seven phosphoramidate moieties as an innovative host for inclusion. This structurally well-defined host has remarkable water solubility and was isolated in pure form. Analytical techniques such as NMR and ITC were used to probe the molecular interactions with different drug molecules. Our investigations revealed that host 4 can form 2:1 inclusion complexes with various drugs. Further studies showed that the inclusions of drugs by ß-CD host (4) are mostly enthalpy driven, highlighting the potential roles played by the phosphoramidate functionalities of the host. Comparatively, a per-O2, O3-acetylated analog (6) of compound 4 was also obtained, which also shows unusual water solubility but diminished inclusion capability.

The extracellular matrix as modifier of neuroinflammation and recovery in ischemic stroke and intracerebral hemorrhage.

Stroke is the second leading cause of death worldwide and has two major subtypes: ischemic stroke and hemorrhagic stroke. Neuroinflammation is a pathological hallmark of ischemic stroke and intracerebral hemorrhage (ICH), contributing to the extent of brain injury but also in its repair. Neuroinflammation is intricately linked to the extracellular matrix (ECM), which is profoundly altered after brain injury and in aging. In the early stages after ischemic stroke and ICH, immune cells are involved in the deposition and remodeling of the ECM thereby affecting processes such as blood-brain barrier and cellular integrity. ECM components regulate leukocyte infiltration into the central nervous system, activate a variety of immune cells, and induce the elevation of matrix metalloproteinases (MMPs) after stroke. In turn, excessive MMPs may degrade ECM into components that are pro-inflammatory and injurious. Conversely, in the later stages after stroke, several ECM molecules may contribute to tissue recovery. For example, thrombospondin-1 and biglycan may promote activity of regulatory T cells, inhibit the synthesis of proinflammatory cytokines, and aid regenerative processes. We highlight these roles of the ECM in ischemic stroke and ICH and discuss their potential cellular and molecular mechanisms. Finally, we discuss therapeutics that could be considered to normalize the ECM in stroke. Our goal is to spur research on the ECM in order to improve the prognosis of ischemic stroke and ICH.

Gadolinium(III) complex formation with a ß-cyclodextrin ligand: an XAS study of a potential MRI contrast agent.

In the search for improved and safer gadolinium-based magnetic resonance imaging (MRI) contrast agents, macrocyclic cyclodextrins (CDs) attract great interest. Our group previously synthesized a cyclodextrin-based ligand with 1,2,3-triazolmethyl residues conjugated to ß-CD, called ß-CD(A), which efficiently chelates Gd(III) ions. To probe the local structure around the Gd(III) ion in the 1:1 Gd(III): ß-CD(A) complex in aqueous solution (pH 5.5), we used extended X-ray absorption fine structure (EXAFS) spectroscopy. Least-squares curve fitting of the Gd L3-edge EXAFS spectrum revealed 5 Gd-O (4 COO- and 1 H2O) and 4 Gd-N (from two imino and two 1,2,3-triazole groups) bonds around the Gd(III) ion with average distances 2.36 and 2.56 ± 0.02 Å, respectively. A similar EXAFS spectrum was obtained from an aqueous solution of the clinically used MRI contrast agent Na[Gd(DOTA)(H2O)], also 9-coordinated in its first shell. Careful analysis revealed that the mean Gd-N distance is shorter in the Gd(III): ß-CD(A) (1:1) complex, indicating stronger Gd-N bonding and stronger Gd(III) complex formation than with the DOTA4- ligand. This is consistent with the lower free Gd3+ concentration found previously for the Gd(III): ß-CD(A) (1:1) complex than for the [Gd(DOTA)(H2O)]- complex, and shows its potential as an MRI probe. EXAFS spectroscopy revealed a similar Gd(III) 9-coordination although slightly stronger for a modified ß-cyclodextrin: Gd(III) 1:1 complex, [Gd(LH4)]7-, in aqueous solution than for the clinically used MRI contrast agent Na[Gd(DOTA)(H2O)].

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

Regio- and stereo-controlled synthesis of 6-deoxy-ß-D-ido-heptopyranosides related to Campylobacter jejuni HS:4.

Campylobacter jejuni is a bacterial pathogen that causes hundreds of millions of cases of food-borne gastroenteritis worldwide annually. The infection caused by this bacterium is also associated with several forms of post-infectious autoimmune sequelae that can be very serious, including the life-threatening Guillain-Barré syndrome. The capsular polysaccharides (CPS) of C. jejuni HS:4 consist of a very unique repeating disaccharide unit that is characterized by a ß-1,4-linked 6-deoxy-ß-D-ido-heptopyranose and an N-acetyl-ß-D-glucosamine. Eliciting carbohydrate-specific antibodies against the CPS structures of C. jejuni HS:4 is an attractive strategy. The 6-deoxy-ido-configuration of the heptose combined with its ß-anomeric configuration makes the chemical synthesis of the disaccharide very challenging. Here, we report an efficient synthesis to obtain the key repeating disaccharide and its analog in reverse order plus a trisaccharide. Our synthesis features a highly efficient, one-step stereo- and regioselective conversion of ß-D-galacto-heptopyranosides to 6-deoxy-ß-D-ido-heptopyranosides via the intermediate 2,3-anhydro-ß-D-talo-heptopyranosides.

KDM5A silencing transcriptionally suppresses the FXYD3-PI3K/AKT axis to inhibit angiogenesis in hepatocellular cancer via miR-433 up-regulation.

Hepatocellular cancer (HCC) has been reported to belong to one of the highly vascularized solid tumours accompanied with angiogenesis of human umbilical vein endothelial cells (HUVECs). KDM5A, an attractive drug target, plays a critical role in diverse physiological processes. Thus, this study aims to investigate its role in angiogenesis and underlying mechanisms in HCC. ChIP-qPCR was utilized to validate enrichment of H3K4me3 and KDM5A on the promotor region of miR-433, while dual luciferase assay was carried out to confirm the targeting relationship between miR-433 and FXYD3. Scratch assay, transwell assay, Edu assay, pseudo-tube formation assay and mice with xenografted tumours were conducted to investigate the physiological function of KDM5A-miR-433-FXYD3-PI3K-AKT axis in the progression of HCC after loss- and gain-function assays. KDM5A p-p85 and p-AKT were highly expressed but miR-433 was down-regulated in HCC tissues and cell lines. Depletion of KDM5A led to reduced migrative, invasive and proliferative capacities in HCC cells, including growth and a lowered HUVEC angiogenic capacity in vitro. Furthermore, KDM5A suppressed the expression of miR-433 by demethylating H3K4me3 on its promoterregion. miR-433 negatively targeted FXYD3. Depleting miR-433 or re-expressing FXYD3 restores the reduced migrative, invasive and proliferative capacities, and lowers the HUVEC angiogenic capacity caused by silencing KDM5A. Therefore, KDM5A silencing significantly suppresses HCC tumorigenesis in vivo, accompanied with down-regulated miR-433 and up-regulated FXYD3-PI3K-AKT axis in tumour tissues. Lastly, KDM5A activates the FXYD3-PI3K-AKT axis to enhance angiogenesis in HCC by suppressing miR-433.

Efficacy of novel methylenecyclohexenone derivatives as TrxR inhibitors in suppressing the proliferation and metastasis of human cancer cells.

A series of mono- and di-methylenecyclohexenone derivatives, 3a-f and 4a-f, respectively, were designed and synthesized from piperlongumine (PL) and their in vitro and in vivo pharmacological properties were evaluated. A majority of the compounds exhibited a potent antiproliferative effect on five human cancer cell lines, especially those causing breast cancer. Compound 4f showed the highest antiproliferative potency among all of the compounds, almost a 10-fold higher inhibitory potency against thioredoxin reductase (TrxR) compared with PL in cells causing breast cancer. In addition, 4f was found to increase the levels of reactive oxygen species (ROS), thus leading to more potent antiproliferative effects. More importantly, the suppression assays of migration and invasion revealed that compound 4f could reverse the epithelial-mesenchymal transition induced by the transforming growth factor ß1, and exhibit prominent anti-metastasis effects. Compound 4f also showed strong inhibition potency toward solid tumors of breast cancer in vivo. Our findings show that compound 4f is a promising therapeutic candidate in the treatment of breast cancer, which, however, needs further research to be proved.

Amphiphilic Cyclodextrin-Based Liquid Crystals for Proton Conduction.

Novel cyclodextrin (CD)-based amphiphilic poly(carboxylic acid)s that self-assemble into highly ordered smectic liquid crystalline mesophases were investigated as a novel class of protonic conductors. These structurally well-defined materials are synthesized from nontoxic and environment-friendly CDs, which possess a unique face-to-face pseudosymmetry. By taking advantage of such geometry, a series of flexible tetraethylene glycol groups terminated with a carboxylic acid functionality were introduced to the CD's secondary face, resulting in the formation of long-range 2D hydrogen-bond networks in the smectic mesophases over a wide temperature window. This new material was found to exhibit impressive proton conductivities in solid states, up to 1.4 × 10-2 S cm-1 at 70 °C and 95% humidity. This constitutes the first report of amphiphilic CD-based liquid crystals applied as proton conductive materials.

Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis.

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Dysfunction of pulmonary surfactant mediated by phospholipid oxidation is cholesterol-dependent.

Pulmonary surfactant forms a cohesive film at the alveolar air-lung interface, lowering surface tension, and thus reducing the work of breathing and preventing atelectasis. Surfactant function becomes impaired during inflammation due to degradation of the surfactant lipids and proteins by free radicals. In this study, we examine the role of reactive nitrogen (RNS) and oxygen (ROS) species on surfactant function with and without physiological cholesterol levels (5-10%). Surface activity was assessed in vitro in a captive bubble surfactometer (CBS). Surfactant chemistry, monolayer fluidity and thermodynamic behavior were also recorded before and after oxidation. We report that physiologic amounts of cholesterol combined with oxidation results in severe impairment of surfactant function. We also show that surfactant polyunsaturated phospholipids are the most susceptible to oxidative alteration. Membrane thermodynamic experiments showed significant surfactant film stiffening after free radical exposure in the presence of cholesterol. These results point to a previously unappreciated role for cholesterol in amplifying defects in surface activity caused by oxidation of pulmonary surfactant, a finding that may have implications for treating several lung diseases.

Synthesis and Unprecedented Complexation Properties of ß-Cyclodextrin-Based Ligand for Lanthanide Ions.

Here, we report the synthesis and detailed studies on the coordination chemistry of a novel chemically modified polyaminocarboxylate (5) based on ß-cyclodextrin (CD) scaffold for lanthanides. The target ligand is prepared in a highly efficient manner (seven total steps) from ß-CD using the readily available iminodiacetic acid as a starting material. A propargyl group is attached to the iminodiacetate via N-alkylation, and the obtained derivative is efficiently conjugated to the ß-CD scaffold via the copper(I)-mediated 1,3-dipolar cycloaddition. The generated 1,2,3-triazolmethyl residues advantageously provide a competent chelating group while displacing the metal coordination center away from the primary rim of ß-CD, to afford the required conformational flexibility. The functional groups from each of the two adjacent glucopyranosyl units of ß-CD complete a uniquely created octavalent coordination sphere for lanthanides while still sparing one site for dynamic water coordination. To help study the coordination chemistry of CD ligand 5, we also design a relevant maltoside ligand 6, which faithfully represents one submetal-binding section of ligand 5. Thanks to HRMS and NMR studies, we successfully elucidate the coordination chemistries of synthesized ligands. The octavalent coordination sphere of ligand 5 shows strong binding affinity to lanthanides. By potentiometric titration experiments, ligand 5 is found to bind gadolinium(III), forming 1:1, 1:2, and 1:3 multinuclear complexes with lanthanides, thus possessing great capacity for catalyzing the dynamic water-exchange. Further NMR studies also reveal that the formed ligand 5/Gd(III) complexes show significantly better abilities to alter T1 relaxivities of coordinated water than DOTA-Gd(III) and also some of the synthetic CD probes reported in the literature.

The role of multilayers in preventing the premature buckling of the pulmonary surfactant.

The pulmonary surfactant is a protein-lipid mixture that spreads into a film at the air-lung interface. The highly-compacted molecules of the film keep the interface from shrinking under the influence of otherwise high surface tension and thus prevent atelectasis. We have previously shown that for the film to withstand a high film pressure without collapsing it needs to assume a specific architecture of a molecular monolayer with islands of stacks of molecular multilayers scattered over the area. Surface activity was assessed in a captive bubble surfactometer (CBS) and the role of cholesterol and oxidation on surfactant function examined. The surfactant film was conceptualized as a plate under pressure. Finite element analysis was used to evaluate the role of the multilayer stacks in preventing buckling of the plate during compression. The model of film topography was constructed from atomic force microscope (AFM) scans of surfactant films and known physical properties of dipalmitoylphosphatidylcholine (DPPC), a major constituent of surfactant, using ANSYS structural-analysis software. We report that multilayer structures increase film stability. In simulation studies, the critical load required to induce surfactant film buckling increased about two-fold in the presence of multilayers. Our in vitro surfactant studies showed that surface topography varied between functional and dysfunctional films. However, the critical factor for film stability was the anchoring of the multilayers. Furthermore, the anchoring of multilayers and mechanical stability of the film was dependent on the presence of hydrophobic surfactant protein-C. The current study expands our understanding of the mechanism of surfactant inactivation in disease.

Receptor-independent, direct membrane binding leads to cell-surface lipid sorting and Syk kinase activation in dendritic cells.

Binding of particulate antigens by antigen-presenting cells is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. By using atomic force microscopy as a tool for real-time single-cell activation analysis, we report that uric acid crystals could directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein-based cellular contacts. Binding of particulate substances activated Syk kinase-dependent signaling in dendritic cells. These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell-surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation, and adjuvanticity.

Total Synthesis of ß-d-ido-Heptopyranosides Related to Capsular Polysaccharides of Campylobacter jejuni HS:4.

The 6-deoxy-ß-d-ido-heptopyranoside related to the capsular polysaccharides of C. jejuni HS:4 is very remarkable, owing to the unique, multifaceted structural features that have been combined into one molecule, which include (1) the rare ido-configuration, (2) the unusual 7-carbon backbone, and (3) the challenging ß-(1→2)-cis-anomeric configuration. Two distinct strategies toward the total synthesis of this interesting target are reported. The first involved establishment of the ß-d-idopyranosyl configuration from ß-d-galactopyranosides, prior to a C-6-homologation extending the d-hexose to the desired 6-deoxy-d-heptose. However, this approach encountered difficulties due to the significantly reduced reactivity of the 6-position of the ß-d-idopyranosides, so instead a second strategy was employed, which involved first carrying out a 6-homologation on the less flexible d-galactopyranose, followed by a very successful conversion to the desired ß-d-ido-configuration found in the target heptopyranoside (2). This report is the first successful synthesis of the 6-deoxy-ß-d-ido-heptopyranoside, which could possess interesting immunological properties.

CXCL10/CXCR3 signaling mobilized-regulatory T cells promote liver tumor recurrence after transplantation.

BACKGROUND & AIMS: Liver graft injury and tumor recurrence are the major challenges of liver transplantation for the patients with hepatocellular carcinoma (HCC). Here, we aimed to explore the role and mechanism of liver graft injury mobilizing regulatory T cells (Tregs), which lead to late phase tumor recurrence after liver transplantation. METHODS: The correlation among tumor recurrence, liver graft injury and Tregs mobilization were studied in 257 liver transplant recipients with HCC and orthotopic rat liver transplantation models. The direct roles of CXCL10/CXCR3 signaling on Tregs mobilization and tumor recurrence were investigated in CXCL10-/- and CXCR3-/- mice models with hepatic IR injury. RESULTS: Clinically, patients received the graft with graft weight ratio (GWR) <60% had higher HCC recurrence after liver transplantation than the recipients with GWR ⩾60% graft. More circulating Tregs and higher intragraft TLR4/CXCL10/CXCR3 levels were detected in recipients with GWR <60% graft. These results were further validated in rat transplantation model. Foxp3+ cells and expressions of TLR4, CXCL10, TGFß, CTLA-4 and CD274 were increased in rat liver tumor tissues from small-for-size graft group. In mouse model, the mobilization and recruitment of Tregs were decreased in TLR4-/-, CXCL10-/- and CXCR3-/- mice compared to wild-type mice. Moreover, less CXCR3+ Tregs were recruited into liver in CXCL10-/- mice after hepatic IR injury. The knockout of CXCL10 and depletion of Tregs inhibited tumor recurrence after hepatic IR injury. CONCLUSION: CXCL10/CXCR3 signaling upregulated at liver graft injury directly induced the mobilization and intragraft recruitment of Tregs, which further promoted HCC recurrence after transplantation. LAY SUMMARY: There were positive correlation among tumor recurrence, circulating Tregs and liver graft injury after human transplantation for HCC patients. The knockout of CXCL10 decreased hepatic recruitment of CXCR3+ Tregs and late phase tumor recurrence after hepatic IR injury.

Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins­ConA, LcH, and PSA­that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma.

BACKGROUND & AIMS: The roles of alternatively activated (M2) macrophages on pro-tumour phenotypes have been well documented in many cancers except hepatocellular carcinoma (HCC). Considering their close relationship with chronic tissue injuries as well as enhanced tumour invasiveness and growth, we aimed to investigate the direct effects of M2 macrophages on HCC. METHODS: M2 macrophages in 95 HCC clinical specimens were quantified using immunohistochemistry and quantitative PCR. The pro-tumour functions and the underlying molecular mechanisms of M2 macrophages in HCC were investigated in vivo and in an in vitro co-culture system. RESULTS: In the clinical study, high M2-specific CD163 (hazard ratio=2.693; p=0.043) and scavenger receptor A (hazard ratio=3.563; p=0.044) levels indicated poor prognosis and correlated with increased tumour nodules and venous infiltration in HCC patients. In an orthotopic model, the liver tumour volume was increased 3.26-fold (1.27 cm3±0.36) after M2 macrophage injection compared with the control (0.39 cm3±0.05) (p=0.032). An increased rate of lung metastasis was also found in the treatment group. In vitro, co-cultivation with M2 macrophages elevated the number of HCC cells (MHCC97L) and migration events by 1.3-fold and 3.2-fold, respectively (p<0.05). Strongly induced by MHCC97L, M2 macrophage-derived CCL22 was proven to enhance tumour migration capacities and correlate with venous infiltration in HCC patients. Increased epithelial-mesenchymal transition (EMT) via Snail activation in MHCC97L was found to be promoted by M2 macrophages and CCL22. CONCLUSIONS: M2 macrophages contribute to poor prognosis in HCC and promote tumour invasiveness through CCL22-induced EMT.

Post-transplant endothelial progenitor cell mobilization via CXCL10/CXCR3 signaling promotes liver tumor growth.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) receiving living donor liver transplantation appear to possess significantly higher tumor recurrence than the recipients receiving deceased donor liver transplantation. The underlying mechanism for HCC recurrence after transplantation remains unclear. Here, we aim to investigate the impact of small-for-size liver graft injury on HCC recurrence after transplantation. METHODS: The correlation between tumor recurrence, liver graft injury, CXCL10 expression and endothelial progenitor cell (EPC) mobilization was studied in 115 liver transplant recipients and rat orthotopic liver transplantation (OLT) models. The direct role of CXCL10/CXCR3 signaling on EPC mobilization was investigated in CXCL10(-/-) mice and CXCR3(-/-) mice. The role of EPCs on tumor growth and angiogenesis was further investigated in an orthotopic liver tumor model. RESULTS: Clinically, patients with small-for-size liver grafts (<60% of standard liver weight, SLW) had significantly higher HCC recurrence (p=0.04), accompanied by more circulating EPCs and higher early-phase intragraft and plasma CXCL10 levels, than the recipients with large grafts (≥60% of SLW), which were further validated in rat OLT models. Circulatory EPC mobilization was reduced after liver injury both in CXCL10(-/-) mice and CXCR3(-/-) mice in comparison to wild-type controls. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners in vitro. Early-phase EPC/CXCL10 injection enhanced orthotopic liver tumor growth, angiogenesis and metastasis in nude mice. CONCLUSIONS: Post-transplant enhanced CXCL10/CXCR3 signaling in small-for-size liver grafts directly induced EPC mobilization, differentiation and neovessel formation, which further promotes tumor growth. Targeting CXCL10/CXCR3 signaling may attenuate early-phase liver graft injury and prevent late-phase tumor recurrence/metastasis after transplantation.

The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response.

OBJECTIVE: We aim to investigate the role of aldose reductase (AR) in hepatic ischemia-reperfusion injury (IRI) of normal and fatty livers and to explore the underlying mechanisms. BACKGROUND: Hepatic IRI is a typical inflammatory response during liver surgery. It contributes to liver graft failure or nonfunction after transplantation. Increasing evidence implicates that AR plays a key role in a number of inflammatory diseases. However, the role of AR in hepatic IRI is still unknown. METHODS: Intragraft AR expression profile and the association with liver graft injury were investigated in both human and rat liver transplantation using normal or fatty graft. The direct role of AR in hepatic IRI was studied in the AR knockout mice IRI model with or without fatty liver. They were further validated by the simulated IRI in vitro model using fatty LO2 cells with or without AR inhibitor zopolrestat and primary peritoneal macrophages isolated from AR knockout and wild-type mice. Gene expression of inflammatory cytokines/chemokines, the infiltration of macrophages/neutrophils, and NF-κB pathway activation were compared among different groups. RESULTS: AR was overexpressed in liver graft after human and rat liver transplantation and correlated with consequent liver injuries. The knockout of AR significantly attenuated hepatic sinusoidal damage and apoptosis in both normal and fatty livers after IRI. The expression of proinflammatory cytokines/chemokines and neutrophil chemoattractants, infiltration of macrophage and neutrophil, and activation of inflammation-associated NF-κB and JNK pathway were downregulated in AR knockout mice. Furthermore, the inhibition of AR effectively suppressed macrophage migration and decreased lipopolysaccharide (LPS)-induced production of proinflammatory cytokines/chemokines in isolated macrophages. CONCLUSIONS: The deficiency of AR attenuated hepatic IRI in both normal and fatty livers by reducing liver inflammatory responses.