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PURPOSE To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. METHODS To observe the effect of fasudil,mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury,CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. RESULTS The incidence of CNV was significantly lower after treatment with 100 μM and 300 μM fasudil than with PBS,especially with 100 μM fasudil. Meanwhile,the incidences of corneal epithelial defects was lower (n=15,all ptextless0.01). After treatment with 100 μM fasudil,the intensity of DHE fluorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5,all ptextless0.01),and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF,TNF-a,MMP-8,and MMP-9 in the 100 μM fasudil group and the PBS group (n=8,all ptextless0.05). The production of HO-1 protein in the 100 μM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample,ptextless0.05). CONCLUSIONS 100 μM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration,reduction of ROS,and upregulation of HO-1 protein after fasudil treatment. View Publication

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion,stress fiber formation,and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study,we investigated the antiangiogenic effect of fasudil,one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration,viability,and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation,focal adhesion assembly,and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore,fasudil inhibited VEGF-induced phosphorylation of myosin light chain,which is one of the main substrates of ROCK. Therefore,the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis,which resulted in the decrease of cell viability. Moreover,fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore,fasudil might be useful for the treatment of angiogenesis-related diseases,especially cancer. View Publication

BACKGROUND Glioma is the most common form of primary malignant brain tumor in adults,with approximately 4 cases per 100 000 people each year. Gliomas,like many tumors,are thought to primarily metabolize glucose for energy production; however,the reliance upon glycolysis has recently been called into question. In this study,we aimed to identify the metabolic fuel requirements of human glioma cells. METHODS We used database searches and tissue culture resources to evaluate genotype and protein expression,tracked oxygen consumption rates to study metabolic responses to various substrates,performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates,and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. RESULTS We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition,we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover,inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. CONCLUSIONS Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition,the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. View Publication

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However,whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis,here we combined bioinformatic analysis of the FASN-related immune cell landscape,real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout ( FASN KO ) cell models,and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression,cytolytic activity signatures,and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity,accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy. Subject terms: Cancer metabolism,Oncogenesis View Publication

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus,developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein,we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time,we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally,CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets,supporting the role of FA metabolism for Treg function. Furthermore,we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively,our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor,thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo. View Publication

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase,which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms,each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis,including Notch proteins,which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition,a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells,resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β,thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably,concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition. View Publication

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96,TIGIT,and CD226 are receptors that bind to a communal ligand,CD155,and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established,whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies,we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus,soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy." View Publication

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB,it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study,we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly,fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation,suggesting that although BCR and FcγRIIB can both recruit SHIP,this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation,but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP,whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk,as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP,but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. View Publication

UNLABELLED This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs,(b) the delivery of cEPI-NCSCs into the spinal cord,and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study,we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord,may promote angiogenesis and tissue remodeling,and elicit a tissue response that may be beneficial in patients with spinal cord injury. SIGNIFICANCE It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities,clinically and pathologically,to the syndrome in people. For this reason,dogs provide a readily accessible,clinically realistic,spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI. View Publication

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are a promising source of cells for regenerating myocardium. However,several issues,especially the large-scale preparation of hiPS-CMs and elimination of undifferentiated iPS cells,must be resolved before hiPS cells can be used clinically. The cell-sheet technique is one of the useful methods for transplanting large numbers of cells. We hypothesized that hiPS-CM-sheet transplantation would be feasible,safe,and therapeutically effective for the treatment of ischemic cardiomyopathy.backslashnbackslashnMETHODS AND RESULTS: Human iPS cells were established by infecting human dermal fibroblasts with a retrovirus carrying Oct3/4,Sox2,Klf4,and c-Myc. Cardiomyogenic differentiation was induced by WNT signaling molecules,yielding hiPS-CMs that were almost 90% positive for α-actinin,Nkx2.5,and cardiac troponin T. hiPS-CM sheets were created using thermoresponsive dishes and transplanted over the myocardial infarcts in a porcine model of ischemic cardiomyopathy induced by ameroid constriction of the left anterior descending coronary artery (n=6 for the iPS group receiving sheet transplantation and the sham-operated group; both groups received tacrolimus daily). Transplantation significantly improved cardiac performance and attenuated left ventricular remodeling. hiPS-CMs were detectable 8 weeks after transplantation,but very few survived long term. No teratoma formation was observed in animals that received hiPS-CM sheets.backslashnbackslashnCONCLUSIONS: The culture system used yields a large number of highly pure hiPS-CMs,and hiPS-CM sheets could improve cardiac function after ischemic cardiomyopathy. This newly developed culture system and the hiPS-CM sheets may provide a basis for the clinical use of hiPS cells in cardiac regeneration therapy. View Publication

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner,representing a unique model to study reprogramming and an important step toward establishing a safe,clinical grade of cells for therapeutic use. In this study,we provide a detailed protocol for isolation,preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in textless1 week,with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks,respectively. View Publication

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However,most of the studies used skin fibroblasts as the starting population for reprogramming,which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore,iPS cells can be readily derived from adult hASCs in a feeder-free condition,thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion,are easy to maintain in culture,and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells. View Publication