Hiroko Ushikubo

Expression of UDP-Glucuronosyltransferase 1 (UGT1) and Glucuronidation Activity toward Endogenous Substances in Humanized UGT1 Mouse Brain

Although UDP-glucuronosyltransferases (UGTs) are important phase II drug-metabolizing enzymes, they are also involved in the metabolism of endogenous compounds. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in the human brain has not been fully clarified. Recently, humanized UGT1 mice (hUGT1 mice) in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus have been developed. In the present study, the expression pattern of UGT1As in brains from humans and hUGT1 mice was examined. We found that UGT1A1, 1A3, 1A6, and 1A10 were expressed in human brains. The expression pattern of UGT1As in hUGT1 mouse brains was similar to that in human brains. In addition, we examined the expression of UGT1A1 and 1A6 in the cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in the cerebellum and cerebral cortex of 6-month-old hUGT1 mice were expressed at a significantly higher rate than those of 2-week-old hUGT1 mice. A difference in expression levels between brain regions was also observed. Brain microsomes exhibited glucuronidation activities toward estradiol and serotonin, with mean values of 0.13 and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play an important role in function regulation of endogenous compounds in a region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo.

Preventive Effects of Rapamycin on Inflammation and Capillary Degeneration in a Rat Model of NMDA-Induced Retinal Injury

Inhibitors of the mammalian target of rapamycin (mTOR) have been shown to protect against neuronal injury, but the mechanisms underlying this effect are not fully understood. The present study aimed to examine the effects of rapamycin, an inhibitor of the mTOR pathway, on inflammation and capillary degeneration in a rat model of N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. Inflammation and capillary degeneration were evaluated by counting the numbers of CD45-positive leukocytes and Iba1-positive microglia, and by measuring the length of empty basement membrane sleeves, respectively. Marked increases in the numbers of leukocytes and microglia were observed 1 d after intravitreal injection of NMDA (200 nmol), and significant capillary degeneration was observed after 7 d. These NMDA-induced changes were significantly reduced by the simultaneous injection of rapamycin (20 nmol) with NMDA. These results suggest that rapamycin has preventive effects on inflammation and capillary degeneration during retinal injury.

Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice.

Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca2+ influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp1]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp1]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.

Protective Effects of Everolimus against N-Methyl-D-aspartic Acid-Induced Retinal Damage in Rats

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-D-aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDA-induced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1-positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells.

A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina

Background: Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice. Results: A dose‐dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1–10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density. Conclusions: These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186–200, 2017.

Exposure to High-Concentration Oxygen in the Neonatal Period Induces Abnormal Retinal Vascular Patterning in Mice.

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high-concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration-dependent manner. In retinas of hyperoxia-exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short-term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.

Retinal neuronal cell loss prevents abnormal retinal vascular growth in a rat model of retinopathy of prematurity

ABSTRACT A short‐term blockade of the vascular endothelial growth factor (VEGF)‐mediated pathway in neonatal rats results in formation of severe retinopathy of prematurity (ROP)‐like retinal blood vessels. The present study aimed to examine the role of retinal neurons in the formation of abnormal retinal blood vessels. Newborn rats were treated subcutaneously with the VEGF receptor tyrosine kinase inhibitor, KRN633 (10mg/kg), or its vehicle (0.5% methylcellulose in water) on postnatal day (P) 7 and P8. To induce excitotoxic loss of retinal neurons, N‐methyl‐D‐aspartic acid (NMDA) was injected into the vitreous chamber of the eye on P9. Changes in retinal morphology, blood vessels, and proliferative status of vascular cells were evaluated on P11 and P14. The number of cells in the ganglion cell layer and the thickness of the inner plexiform layer and inner nuclear layer were significantly decreased 2 days (P11) after NMDA treatment. The pattern and degree of NMDA‐induced changes in retinal morphology were similar between vehicle‐treated (control) and KRN633‐treated (ROP) rats. In ROP rats, increases in the density of capillaries, the tortuosity index of arteries, and the proliferating vascular cells were observed on P14. The expansion of the endothelial cell network was prevented, and the capillary density and the number of proliferating cells were reduced in NMDA‐treated retinas of both control and ROP rats. Following NMDA‐induced neuronal cell loss, no ROP‐like blood vessels were observed in the retinas. These results suggest that retinal neurons play an important role in the formation of normal and ROP‐like retinal blood vessels. HIGHLIGHTSShort‐term VEGF inhibition interrupts retinal vascular development in rats.The impaired vascular development induces retinal hypoxia and VEGF expression.The enhanced VEGF‐VEGFR2 pathway induces abnormal vascular formation.Excitotoxic loss of retinal neurons prevents abnormal vascular formation.Retinal neurons play an important role in abnormal retinal vascular formation.

Retinal region-dependent susceptibility of capillaries to high-concentration oxygen exposure and vascular endothelial growth factor receptor inhibition in neonatal mice.

Retinal blood flow insufficiency due to capillary loss induces hypoxia in the retina, leading to an abnormal angiogenesis, relating to ischemic retinopathy. To better understand the mechanism and process of retinal capillary regression, we examined the process of hyperoxia- and vascular endothelial growth factor receptor (VEGFR) inhibitor-induced retinal capillary regression in neonatal mice. We also investigated the effects of Ca(2+) channel blockers, amlodipine and nicardipine, on hyperoxia-induced capillary regression. The regression of capillaries adjacent to arteries began immediately after the mice were exposed to 80% oxygen on postnatal day 7. An apparent avascular zone was established within 24 h after the initiation of oxygen exposure, whereas capillaries in the retinal vascular front were not affected. Axitinib, an inhibitor of VEGFR tyrosine kinase, induced capillary regression throughout the retinal vasculature. High-concentration oxygen exposure affected the capillaries on the arterial side of the retinal circulation more preferentially than axitinib. The Ca(2+) channel blockers significantly delayed hyperoxia-induced capillary regression and changes in the capillaries on the arterial side. These results suggest that the decreased blood flow due to arterial constriction contributes to hyperoxia-induced capillary regression. Compounds that improve the retinal blood flow may prevent ischemia by preventing capillary loss.

Protective effects of PF-4708671 against N-methyl-d-aspartic acid-induced retinal damage in rats.

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N‐methyl‐d‐aspartic acid (NMDA)‐induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF‐4708671, an inhibitor of S6 kinase, protects against NMDA‐induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45‐positive leukocytes and Iba1‐positive microglia. Surprisingly, simultaneous injection of PF‐4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF‐4708671 and rapamycin likely protect against NMDA‐induced retinal damage via distinct pathways. The neuroprotective effect of PF‐4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF‐4708671 is reported to be a S6 kinase inhibitor.