Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

Multiple preferred escape trajectories are explained by a geometric model incorporating prey's turn and predator attack endpoint.

The escape trajectory (ET) of prey - measured as the angle relative to the predator's approach path - plays a major role in avoiding predation. Previous geometric models predict a single ET; however, many species show highly variable ETs with multiple preferred directions. Although such a high ET variability may confer unpredictability to avoid predation, the reasons why animals prefer specific multiple ETs remain unclear. Here, we constructed a novel geometric model that incorporates the time required for prey to turn and the predator's position at the end of its attack. The optimal ET was determined by maximizing the time difference of arrival at the edge of the safety zone between the prey and predator. By fitting the model to the experimental data of fish Pagrus major, we show that the model can clearly explain the observed multiple preferred ETs. By changing the parameters of the same model within a realistic range, we were able to produce various patterns of ETs empirically observed in other species (e.g., insects and frogs): a single preferred ET and multiple preferred ETs at small (20-50°) and large (150-180°) angles from the predator. Our results open new avenues of investigation for understanding how animals choose their ETs from behavioral and neurosensory perspectives.

When a prey spots a predator about to pounce, it turns swiftly and accelerates away to avoid being captured. The initial direction the prey chooses to take ­ known as its escape trajectory ­ can greatly impact their chance of survival. Previous models were able to predict the optimal direction an animal should take to maximize its chances of evading the predator. However, experimental data suggest that prey actually tend to escape via multiple specific directions, although why animals use this approach has not been clarified. To investigate this puzzle, Kawabata et al. built a new mathematical model that better represents how prey and predators interact with one another in the real world. Unlike past models, Kawabata et al. incorporated the time required for prey to change direction and only allowed the predators to move toward the prey for a limited distance. By including these two factors, they were able to reproduce the escape trajectories of real animals, including a species of fish, as well as species from other taxa such as frogs and insects. The new model suggests that prey escape along one of two directions: either by moving directly away from the predator in order to outrun its attack, or by dodging sideways to avoid being captured. Which strategy the prey chooses has some elements of unpredictability, which makes it more difficult for predators to adjust their capturing method. These findings shed light on why escaping in multiple specific directions makes prey harder to catch. The model could also be extended to test the escape trajectories of a wider variety of predator and prey species, which may avoid capture via different routes. This could help researchers better understand how predators and prey interact with one another. The findings could also reveal how sensory information (such as sound and sight) associated with the threat of an approaching predator is processed and stimulates the muscle activity required to escape in multiple different directions.

A novel model of chronic limb ischemia to therapeutically evaluate the angiogenic effects of drug candidates.

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates.NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

ASP1126, a Novel Sphingosine-1-Phosphate-Selective Agonist With a Favorable Safety Profile, Prolongs Allograft Survival in Rats and Nonhuman Primates in Combination With Tacrolimus With a Broad Safety Margin for Bradycardia.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of 5 G protein-coupled receptors (S1P1 to S1P5). Among these, S1P1 is a major regulator of lymphocyte trafficking. Fingolimod, whose active metabolite, fingolimod phosphate, acts as a nonselective S1P-receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating lymphocyte trafficking via downregulation of S1P1 expression on lymphocytes. Here, we describe the pharmacologic profile of a novel S1P1 agonist, ASP1126. ASP1126 preferentially activated S1P1 compared to S1P3 in rat and human guanosine-5'-(γ-thio)-triphosphate (GTPγS) assays. Oral single administration of ASP1126 decreased the number of peripheral lymphocytes and repeated dosing showed a cumulative effect on lymphopenia in both rats and monkeys. ASP1126 prolonged allograft survival in a rat heterotopic heart transplantation model in combination with a subtherapeutic dose of tacrolimus that was independent of drug-drug interactions. In addition, in nonhuman primate (NHP) renal transplantation, pretreatment with ASP1126 reduced not only the number of naive T cells and central memory T cells but also effector memory T cells in the peripheral blood, all of which could contribute to acute graft rejection and prolonged allograft survival in combination with tacrolimus. Further, we confirmed that ASP1126 has a broad ranging safety margin with respect to its effect on lung weight in rats and bradycardia in NHPs, which were the adverse events found in clinical studies of fingolimod. ASP1126 with improved safety profile has the potential to be an adjunct therapy in combination with tacrolimus in clinical transplantation.

Development of microsatellites in Machilus thunbergii (Lauraceae), a warm-temperate coastal tree species in Japan.

PREMISE OF THE STUDY: Microsatellite markers were developed and characterized in a typically coastal, widespread, and dominant tree species of the evergreen broadleaf forests, Machilus thunbergii, for comparison of the genetic diversity and structure of inland populations surrounding the ancient Lake Biwa and coastal populations in Japan. METHODS AND RESULTS: Eighteen polymorphic microsatellites of this species were isolated using an improved technique for isolating codominant compound microsatellite markers. These isolated loci provided compound simple sequence repeat (SSR) markers with polymorphisms of three to 19 alleles per locus, with an average of 10.9. The expected and observed within-population heterozygosities ranged from 0.16 to 0.86 and from 0.13 to 0.72, respectively. CONCLUSIONS: These markers may be useful tools for further investigation of the population genetic structure and biogeographic history of M. thunbergii in the warm-temperate zone of East Asia.

Anti-programmed cell death 1 antibody reduces CD4+PD-1+ T cells and relieves the lupus-like nephritis of NZB/W F1 mice.

Programmed cell death 1 (PD-1) is an immunosuppressive receptor that transduces an inhibitory signal into activated T cells. Although a single nucleotide polymorphism in the gene for PD-1 is associated with susceptibility to systemic lupus erythematosus, the role of PD-1 in systemic lupus erythematosus is still not well understood. In this study, we used NZB/W F1 mice, a model of lupus-like nephritis, to examine the function of PD-1 and its ligands. PD-1 was predominantly expressed on CD4(+) T cells that infiltrated the kidney, and CD4(+)PD-1(high) T cells produced higher levels of IFN-gamma than CD4(+)PD-1(low) or CD4(+)PD-1(-) T cells. Stimulation with PMA/ionomycin caused splenic CD4(+)PD-1(+) T cells to secrete high levels of IFN-gamma, IL-10, low levels of TNF-alpha, faint levels of IL-2, IL-21, and no IL-4, IL-17. In vivo anti-PD-1 mAb treatment reduced the number of CD4(+)PD-1(+) T cells in the kidney of NZB/W F1 mice and significantly reduced their mortality rate (p = 0.03). Conversely, blocking PD-L1 using an anti-PD-L1 mAb increased the number of CD4(+)PD-1(+) T cells in the kidney, enhanced serum IFN-gamma, IL-10, and IgG2a ds-DNA-Ab levels, accelerated the nephritis, and increased the mortality rate. We conclude that CD4(+)PD-1(high) T cells are dysregulated IFN-gamma-producing, proinflammatory cells in NZB/W F1 mice.

PD-1+ memory phenotype CD4+ T cells expressing C/EBPalpha underlie T cell immunodepression in senescence and leukemia.

Although altered T cell function plays a part in immunosenescence, the mechanisms remain uncertain. Here we identify a bona fide age-dependent PD-1(+) memory phenotype (MP) CD4(+) T cell subpopulation that hardly proliferates in response to T cell receptor (TCR) stimulation and produces abundant osteopontin at the cost of typical T cell lymphokines. These T cells demonstrate impaired repopulation in Rag2(-/-) mice, but a homeostatic proliferation in gamma-ray-irradiated mice. These T cells also reveal a unique molecular signature, including a strong expression of C/EBPalpha normally expressed in myeloid-lineage cells, with diminished c-Myc and cyclin D1. Transduction of Cebpa in regular CD4(+) T cells inhibited the TCR-mediated proliferation with c-Myc and cyclin D1 repression and caused a striking activation of Spp1 encoding osteopontin along with concomitant repression of T cell lymphokine genes. Although these T cells gradually increase in number with age and become predominant at the senescent stage in normal mice, the generation is robustly accelerated during leukemia. In both conditions, their predominance is associated with the diminution of specific CD4(+) T cell response. The results suggest that global T cell immunodepression in senescence and leukemia is attributable to the increase in PD-1(+) MP CD4(+) T cells expressing C/EBPalpha.

Dynamics of distribution and performance of ramets constructing genets: a demographic-genetic study in a clonal plant, Convallaria keiskei.

BACKGROUND AND AIMS: In clonal plants producing vegetative offspring, performance at the genet level as well as at the ramet level should be investigated in order to understand the entire picture of the population dynamics and the life history characteristics. In this study, demography, including reproduction and survival, the growth patterns and the spatial distributions of ramets within genets of the clonal herb Convallaria keiskei were explored. METHODS: Vegetative growth, flowering and survival of shoots whose genets were identified using microsatellite markers were monitored in four study plots for 3 years (2003-2005). The size structures of ramets in genets and their temporal shifts were then analysed. Their spatial distributions were also examined. KEY RESULTS: During the census, 274 and 149 ramets were mapped in two 1 x 2 m plots, and 83 and 94 ramets in two 2 x 2 m quadrats. Thirty-eight genotypes were identified from 580 samples. Each plot included 5-18 genets, and most ramets belonged to the predominant genet(s) in each plot. Shoots foliated yearly for several years, but flowering ramets did not have an inflorescence the next year. A considerable number of new clonal offspring persistently appeared, forming a bell-shaped curve of the size structure of ramets in each genet. Comparing the structures modelled by the normal distributions suggested variation among ramets belonging to a single genet and variation among genets. Furthermore, spatial analyses revealed clumped and distant distributions of ramet pairs in a genet, in which the distant patterns corresponded to the linearly elongating clonal growth pattern of this species. CONCLUSION: Characteristics of ramet performances such as flowering and recruitment of clonal offspring, in addition to growth, played a large part in the regulation of genet dynamics and distribution, which were different among the studied genets. These might be characteristics particularly relevant to clonal life histories.

An improved technique for isolating codominant compound microsatellite markers.

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)6(AG)5 or (TC)6(AC)5 and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.