Akane Morita

KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase, Induces Intrauterine Growth Restriction in Mice

We previously reported that treatment with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, during mid-pregnancy caused intrauterine growth restriction resulting from impairment of blood vessel growth in the labyrinthine zone of the placenta and fetal organs. However, the relative sensitivities of blood vessels in the placenta and fetal organs to vascular endothelial growth factor (VEGF) inhibitors have not been determined. In this study, we aimed to examine the effects of KRN633 on the vasculatures of organs in mother mice and their newborn pups by immunohistochemical analysis. Pregnant mice were treated daily with KRN633 (5 mg/kg) either from embryonic day 13.5 (E13.5) to E17.5 or from E13.5 to the day of delivery. The weights of the pups of KRN633-treated mice were lower than those of the pups of vehicle-treated mothers. However, no significant difference in body weight was observed between the vehicle- and KRN633-treated mice. The vascular development in the organs (the pancreas, kidney, and intestine) and intestinal lymphatic formation of the pups of KRN633-treated mothers was markedly impaired. In contrast, the KRN633 treatment showed no significant effect on the vascular beds in the organs, including the labyrinthine zone of the placenta, of the mother mice. These results suggest that blood vessels in fetal organs are likely to be more sensitive to reduced VEGF signaling than those in the mother. A partial loss of VEGF function during pregnancy could suppress vascular growth in the fetus without affecting the vasculature in the mother mouse, thereby increasing the risk of intrauterine growth restriction.

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.

Treatment of Mid-Pregnant Mice with KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase, Induces Abnormal Retinal Vascular Patterning in Their Newborn Pups

We previously reported that treatment of mid-pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid-pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633-treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633-treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633-treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network.

Anti-angiogenic effects of valproic acid in a mouse model of oxygen-induced retinopathy

Pathological retinal angiogenesis contributes to the pathogenesis of several ocular diseases. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). Herein, we investigated the effects of valproic acid and vorinostat, a HDAC inhibitor, on pathological retinal angiogenesis in mice with oxygen-induced retinopathy (OIR). OIR was induced in neonatal mice by exposure to 80% oxygen from postnatal day (P) 7 to P10 and to atmospheric oxygen from P10 to P15. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from P10 to P14. At P15, retinal neovascular tufts and vascular growth in the central avascular zone were observed in mice with OIR. Additionally, immunoreactivity for phosphorylated ribosomal protein S6 (pS6), an indicator of mammalian target of rapamycin (mTOR) activity, was detected in the neovascular tufts. Both valproic acid and vorinostat reduced the formation of retinal neovascular tuft without affecting vascular growth in the central avascular zone. Valproic acid reduced the pS6 immunoreactivity in neovascular tufts. Given that vascular endothelial growth factor (VEGF) activates mTOR-dependent pathways in proliferating endothelial cells of the neonatal mouse retina, these results suggest that valproic acid suppresses pathological retinal angiogenesis by interrupting VEGF-mTOR pathways.

Establishment of an abnormal vascular patterning model in the mouse retina

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia.

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors: Transient Phenotypic Change in Vascular Cells

Background: A short‐term interruption of vascular development causes structural abnormalities in retinal vasculature. However, the detailed changes in vascular components (endothelial cells, pericytes, and basement membranes) remain to be fully determined. The present study aimed to provide a detailed description of morphological changes in vascular components following a short‐term interruption of retinal vascular development in mice. Results: Two‐day treatment of neonatal mice with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg, subcutaneously) on postnatal day (P)0 and P1 (P0/1) and P4 and P5 (P4/5) induced different degrees and patterns of impairment of retinal vascular development. Three days after completion of the treatment, the delayed radial vascular growth occurred in P0/1 group mice, whereas in P4/5 group mice, revascularization preferentially occurred in the central avascular area, and radial vascular growth remained suppressed by P10. Differences in α‐smooth muscle actin expression in pericytes were noted in the processes between normal vascular formation and vascular regrowth. The changes in vascular cells were associated with the hypoxia‐induced enhancement of VEGF expression in the superficial retinal layer. Conclusions: These findings suggest that the phenotype of vascular cells is altered following a short‐term interruption of vascular development in the retina. Developmental Dynamics 247:699–711, 2018.

Role of Glial Cells in μ-Opioid Receptor-Mediated Vasodilation in the Rat Retina

ABSTRACT Purpose: Our recent study demonstrated that herkinorin, a non-opioid μ-receptor agonist derived from salvinorin A, dilates retinal arterioles through stimulation of μ-opioid receptors in rats. Activation of neuronal nitric oxide (NO) synthase and the presence of ganglion cells in the retina appear to be crucial for inducing μ-opioid receptor-mediated retinal vasodilation. In the present study, we examined the role of the interaction between neurons and glia in the retinal vasodilator mechanism involving μ-opioid receptors in rats. Materials and methods: The localization of μ-opioid receptors and neuronal NO synthase (nNOS) in the rat retina was examined using immunohistochemistry. The retinal vascular responses were evaluated by measuring the diameter of retinal arterioles in in vivo fundus images. Both systemic blood pressure and heart rate were continuously recorded. Results: Immunoreactivity of μ-opioid receptors was found in ganglion cells and astrocytes, while that of nNOS was detected in ganglion cells and amacrine cells. Herkinorin increased retinal arteriolar diameter without significantly changing mean blood pressure and heart rate. The retinal vasodilator response to herkinorin was significantly attenuated by treatment with glial toxins (fluorocitrate and disialoganglioside-GD1b). The glial toxins markedly prevented vasodilation induced by intravitreal injection, but not by intravenous infusion, of NOR3, an NO donor. Conclusion: These results suggest that retinal glial cells play an important role in the μ-opioid receptor-mediated retinal vasodilation in rats. Stimulation of μ-opioid receptors on retinal ganglion cells may affect the activity of glial cells, thereby changing retinal vascular tone.

Mammalian Target of Rapamycin (mTOR) as a Potential Therapeutic Target in Pathological Ocular Angiogenesis

Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.