Yuko Watanabe

Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line.

Background Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.

Localization of mechanosensitive channel TRPV4 in mouse skin.

A transient receptor potential (TRP) family, TRPV4, is a calcium-permeable swell-activated channel, playing a role in cutaneous mechanosensation. To elucidate the localization in the mechanosensitive endings, we found with immunohistochemistry in mice that TRPV4 was expressed both by small (low threshold) and large (high threshold) dorsal root ganglia neurons. In addition to free nerve endings, TRPV4 was specifically located at cutaneous mechanosensory terminals co-localized with neurofilament 200, including Meissner, Merkel, penicillate and intraepidermal terminals but not including hair follicle palisades. The distribution suggests that the sensation of pressure by mechanosensitive TRPV4 channel is transmitted through A- as well as C-fiber.

Characteristics of urinary and serum soluble Klotho protein in patients with different degrees of chronic kidney disease.

BackgroundKlotho is a single-pass transmembrane protein, which appears to be implicated in aging. The purpose of the present study was to characterize the relationship between the soluble Klotho level and renal function in patients with various degrees of chronic kidney disease (CKD).MethodsThe levels of soluble Klotho in the serum and urine obtained from one hundred thirty-one CKD patients were determined by a sandwich enzyme-linked immunosorbent assay system.ResultsThe amount of urinary excreted Klotho during the 24 hr period ranged from 1.6 to 5178 ng/day (median 427 ng/day; interquartile range [IR] 56.8-1293.1), and the serum Klotho concentration ranged from 163.9 to 2123.7 pg/ml (median 759.7 pg/ml; IR 579.5-1069.1). The estimated glomerular filtration rate (eGFR) was significantly correlated with the log-transformed values of the amount of 24 hr urinary excreted Klotho (r = 0.407, p < 0.01) and the serum Klotho levels (r = 0.232, p < 0.01). However, a stepwise multiple regression analysis identified eGFR to be a variable independently associated only with the log-transformed value of the amount of 24-hr urinary excreted Klotho but not with the log-transformed serum Klotho concentration. Despite the strong correlation between random urine protein-to-creatinine ratio and the 24 hr urinary protein excretion (r = 0.834, p < 0.01), a moderate linear association was observed between the log-transformed value of the amount of 24 hr urinary excreted Klotho and that of the urinary Klotho-to-creatinine ratio (Klotho/Cr) in random urine specimens (r = 0.726, p < 0.01).ConclusionsThe amount of urinary Klotho, rather than the serum Klotho levels, should be linked to the magnitude of the functioning nephrons in CKD patients. The use of random urine Klotho/Cr as a surrogate for the amount of 24-hr urinary excreted Klotho needs to be evaluated more carefully.

The Impact of Nephrectomy and Renal Transplantation on Serum Levels of Soluble Klotho Protein

BACKGROUND Klotho, a single-pass transmembrane protein primarily expressed in the kidneys, parathyroid glands, and choroid plexus of the brain, has a short cytoplasmic tail and a long extracellular domain, which can be cleaved and released as a soluble form. However, information regarding the origins and kinetics of soluble serum Klotho remains poorly understood. We evaluated serial changes in serum Klotho levels among living donors before and after retroperitoneoscopic nephrectomy as well as in their renal transplant recipients. METHODS The levels of soluble Klotho in serum obtained from 10 living donors and their renal transplant recipients were determined using a sandwich enzyme-linked immunosorbent assay system. RESULTS Serum soluble Klotho was detectable in all subjects. The baseline serum Klotho concentrations in the living donors ranged from 726.4 to 1417.1 pg/mL (median, 909.8 pg/mL; interquartile ranges [IR], 754.8-1132.4), whereas that in the concomitant renal transplant recipients ranged from 397.5 to 1047.2 pg/mL (median, 613.0 pg/mL; IR, 445.9-750.8; P = .003). The levels of soluble serum Klotho measured 5 days after retroperitoneoscopic nephrectomy (median, 619.0 pg/mL; IR, 544.6-688.5; P = .001) were significantly lower than the baseline values. Among the renal transplant recipients, no significant changes in serum Klotho levels were observed during the observation period. CONCLUSION Our data regarding soluble serum Klotho levels obtained from living donors support the idea that the kidneys are a major source of soluble serum Klotho in human subjects without a deterioration of renal function. In recipients, concomitant acute kidney injuries and immunosuppressive protocols might modulate the release of soluble Klotho from the grafts into the circulation.

Febuxostat for Hyperuricemia in Patients with Advanced Chronic Kidney Disease

Febuxostat is a nonpurine xanthine oxidase (XO) inhibitor, which recently received marketing approval. However, information regarding the experience with this agent among advanced chronic kidney disease (CKD) patients is limited. In the current study, we investigated the effects of oral febuxostat in patients with advanced CKD with asymptomatic hyperuricemia. We demonstrated, for the first time, that not only the serum levels of uric acid (UA) but also those of 8-hydroxydeoxyguanosine, an oxidative stress marker, were significantly reduced after six months of febuxostat treatment, with no adverse events. These results encouraged us to pursue further investigations regarding the clinical impact of lowering the serum UA levels with febuxostat in advanced CKD patients in terms of concomitantly reducing oxidative stress via the blockade of XO. More detailed studies with a larger number of subjects and assessments of the effects of multiple factors affecting hyperuricemia, such as age, sex, and dietary habits, would shed light on the therapeutic challenges of treating asymptomatic hyperuricemia in patients with various stages of CKD.

Exosome‐mediated extracellular release of polyadenylate‐binding protein 1 in human metastatic duodenal cancer cells

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis‐ and endocytosis‐like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ‐521) and metastatic (AZ‐P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC‐MS/MS and Western blot analyses, polyadenylate‐binding protein 1 (PABP1) was found to be predominantly abundant in AZ‐P7a exosomes. The amount of exosomal PABP1 in AZ‐P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin‐proteasome pathway (MG‐132 and PYR‐41). Treatment of AZ‐P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1‐immunoreactive products cleaved via a proteolysis‐like process. Taken together, these results suggest that AZ‐P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome‐mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.

Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy

To examine whether and how heart ANG II influences the coordination between cardiomyocyte hypertrophy and coronary angiogenesis and contributes to the pathogenesis of diabetic cardiomyopathy, we used Spontaneously Diabetic Torii (SDT) rats treated without and with olmesartan medoxomil (an ANG II receptor blocker). In SDT rats, left ventricular (LV) ANG II, but not circulating ANG II, increased at 8 and 16 wk after diabetes onset. SDT rats developed LV hypertrophy and diastolic dysfunction at 8 wk, followed by LV systolic dysfunction at 16 wk, without hypertension. The SDT rat LV exhibited cardiomyocyte hypertrophy and increased hypoxia-inducible factor-1α expression at 8 wk and to a greater degree at 16 wk and interstitial fibrosis at 16 wk only. In SDT rats, coronary angiogenesis increased with enhanced capillary proliferation and upregulation of the angiogenic factor VEGF at 8 wk but decreased VEGF with enhanced capillary apoptosis and suppressed capillary proliferation despite the upregulation of VEGF at 16 wk. In SDT rats, the phosphorylation of VEGF receptor-2 increased at 8 wk alone, whereas the expression of the antiangiogenic factor thrombospondin-1 increased at 16 wk alone. All these events, except for hyperglycemia or blood pressure, were reversed by olmesartan medoxomil. These results suggest that LV ANG II in SDT rats at 8 and 16 wk induces cardiomyocyte hypertrophy without affecting hyperglycemia or blood pressure, which promotes and suppresses coronary angiogenesis, respectively, via VEGF and thrombospondin-1 produced from hypertrophied cardiomyocytes under chronic hypoxia. Thrombospondin-1 may play an important role in the progression of diabetic cardiomyopathy in this model.

Next generation sequencing approach for detecting 491 fusion genes from human cancer.

Next-generation DNA sequencing (NGS) of the genomes of cancer cells is contributing to new discoveries that illuminate the mechanisms of tumorigenesis. To this end, the International Cancer Genome Consortium and The Cancer Genome Atlas are investigating novel alterations of genes that will define the pathways and mechanisms of the development and growth of cancers. These efforts contribute to the development of innovative pharmaceuticals as well as to the introduction of genome sequencing as a component of personalized medicine. In particular, chromosomal translocations that fuse coding sequences serve as important pharmaceutical targets and diagnostic markers given their association with tumorigenesis. Although increasing numbers of fusion genes are being discovered using NGS, the methodology used to identify such fusion genes is complicated, expensive, and requires relatively large samples. Here, to address these problems, we describe the design and development of a panel of 491 fusion genes that performed well in the analysis of cultured human cancer cell lines and 600 clinical tumor specimens.

Enhancement of anti-STLV-1/HTLV-1 immune responses through multimodal effects of anti-CCR4 antibody

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory diseases. Because anti-HTLV-1 immune responses are critical for suppressing infected cells, enhancing cellular immunity is beneficial for the treatment of HTLV-1-associated diseases. Using simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaques, we analyzed the immune responses to viral antigens and the dynamics of virus-infected cells. The chemokine receptor CCR4 is expressed on STLV-1 infected cells, and administration of humanized monoclonal antibody to CCR4, mogamulizumab, dramatically decreased the number of STLV-1-infected cells in vivo. Concurrently, mogamulizumab treatment enhanced STLV-1 specific CD4+ and CD8+ T cell responses by simultaneously targeting CCR4+ effector regulatory T (Treg) cells and infected cells. Mogamulizumab promoted the phagocytosis of CCR4+ infected cells by macrophages, which likely enhanced antigen presentation. Vaccination with recombinant vaccinia virus (rVV) expressing viral antigens suppressed the proviral load and the number of Tax-expressing cells. Enhanced T-cell responses were also observed in some ATL patients who were treated with mogamulizumab. This study shows that mogamulizumab works not only by killing CCR4+ infected cells directly, but also by enhancing T cell responses by increasing the phagocytosis of infected cells by antigen-presenting cells and suppressing CCR4+ effector Treg cells.

Association between Serum Soluble Klotho Levels and Mortality in Chronic Hemodialysis Patients

Klotho is a single-pass transmembrane protein predominantly expressed in the kidney. The extracellular domain of Klotho is subject to ectodomain shedding and is released into the circulation as a soluble form. Soluble Klotho is also generated from alternative splicing of the Klotho gene. In mice, defects in Klotho expression lead to complex phenotypes resembling those observed in dialysis patients. However, the relationship between the level of serum soluble Klotho and overall survival in hemodialysis patients, who exhibit a state of Klotho deficiency, remains to be delineated. Here we prospectively followed a cohort of 63 patients with a mean duration of chronic hemodialysis of 6.7 ± 5.4 years for a median of 65 months. Serum soluble Klotho was detectable in all patients (median 371 pg/mL, interquartile range 309–449). Patients with serum soluble Klotho levels below the lower quartile (<309 pg/mL) had significantly higher cardiovascular and all-cause mortality rates. Furthermore, the higher all-cause mortality persisted even after adjustment for confounders (hazard ratio 4.14, confidence interval 1.29–13.48). We conclude that there may be a threshold for the serum soluble Klotho level associated with a higher risk of mortality.