Ichiro Miyoshi

Subventricular Zone-Derived Neural Progenitor Cells Migrate Along a Blood Vessel Scaffold Toward the Post-stroke Striatum

The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ‐derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre‐existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron‐blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ‐derived NPCs in Flk1‐EGFP knock‐in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ‐derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region. STEM CELLS 2010;28:545–554

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high‐grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp–TAg transgenic mouse, which expresses the SV40 large T‐antigen (TAg) under the control of the mouse müllerian‐specific Ovgp‐1 promoter. Histological analysis of the fallopian tubes of mogp–TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal‐appearing p53‐positive epithelium that are similar to ‘p53 signatures’ in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non‐invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp–TAg and wild‐type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low‐grade and high‐grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease. Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/− nor Chk2−/− mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/−Chk2−/− and Chk1+/−Chk2+/− mice have a progressive cancer‐prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra‐S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double‐mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53‐ or Rb‐dependent oncogene‐induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

Deficiency of the tensin2 gene in the ICGN mouse : an animal model for congenital nephrotic syndrome

The ICGN mouse is a model for nephrotic syndrome (NS) which presents with proteinuria, hyperlipidemia, and edema. In this study we attempted to identify the gene(s) responsible for NS. By analyzing albuminuria in 160 (ICGN × MSM)F1 × ICGN backcross progenies, we found that NS in the ICGN mouse is caused by more than one gene. We then performed a quantitative trait locus (QTL) analysis and detected a QTL with a very high LOD score peak in the telomeric region of Chr 15. By analyzing the nucleotide sequence of 22 genes located close to the QTL, we found that the tensin2 gene of the ICGN mouse possessed an 8-nucleotide deletion mutation in exon 18, leading to a frameshift and giving rise to a terminal codon at a premature position. Analyses of in situ hybridization and immunohistochemistry revealed that tensin2 was expressed in podocytes and tubular epithelial cells in normal mice but not in the ICGN mouse. These data raise the possibility that a mutation of the tensin2 gene is responsible for NS of the ICGN mouse and tensin2 is a prerequisite for the normal kidney function.

Immunosuppressive and Prometastatic Functions of Myeloid-Derived Suppressive Cells Rely upon Education from Tumor-Associated B Cells

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfβR1/TgfβR2 signaling. MDSC fully educated in this manner exhibit an increased production of reactive oxygen species and NO and more efficiently suppress CD4(+) and CD8(+) T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfβR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immunosuppressive and prometastatic functions of MDSC.

Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: Possible involvement of immune cells in mesenteric adipose tissue

The anti-diabetic effects of Brazilian propolis were examined using ob/ob mice. Although repeated injection of an ethanol extract of Brazilian propolis (100 mg/kg, ip, twice a week for 12 weeks) did not affect body weight gain and food intake of ob/ob mice, blood glucose and plasma cholesterol levels were significantly attenuated. Moreover, the propolis extract partially restored glucose tolerance and insulin resistance, indicating anti-diabetic properties of the extract. The propolis-treated mice exhibited lower weight gain in mesenteric adipose tissue, while weight gains in inguinal and epididymal adipose tissues were not modulated. Flow cytometric and microscopic analyses suggested that the extract promoted accumulation of eosinophils into mesenteric and epididymal adipose tissues. Alternatively, the ratio of M1-like macrophages to M2-like macrophages in mesenteric adipose tissue was reduced by the propolis injection, coincident with the decrement of the number of interleukin-12A+ cells. Levels of M1 macrophage markers, such as Itgax and Il12b transcripts, were decreased in the vascular stromal fraction of mesenteric adipose tissue, whereas those of pan-macrophage markers Emr1 and Cd68 were not influenced. Microarray and subsequent gene ontology term analyses suggested that propolis attenuated immune activation in mesenteric adipose tissues. Taken together, this indicates that Brazilian propolis improves diabetes in ob/ob mice, presumably through modification of immune cells in mesenteric adipose tissues.

Impairment of neuropsychological behaviors in ganglioside GM3-knockout mice.

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.

Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mϕs in renal crystal formation during hyperoxaluria using CSF-1-deficient mice, which lack M2Mϕs. Compared with wild-type mice, CSF-1-deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1-deficient and wild-type mice. Flow cytometry of sorted renal Mϕs showed that CSF-1 deficiency resulted in a smaller population of CD11b(+)F4/80(+)CD163(+)CD206(hi) cells, which represent M2-like Mϕs. Additionally, transfusion of M2Mϕs into CSF-1-deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2-polarized Mϕs than M1-polarized Mϕs or renal tubular cells. Gene array profiling showed that CSF-1 deficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation.

SCF Fbxo22 -KDM4A targets methylated p53 for degradation and regulates senescence

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCFFbxo22-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCFFbxo22 ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCFFbxo22-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22−/− mice are almost half the size of Fbxo22+/− mice owing to the accumulation of p53. These results indicate that SCFFbxo22-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.