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Resveratrol, a natural polyphenolic compound, reportedly possesses numerous biological activities, including anti-inflammatory and antioxidant effects. In the current study, we examined (1) the dilator effects of resveratrol on retinal arterioles, (2) the protective effects of resveratrol against excitotoxic retinal injury, and (3) whether these effects are mediated by the AMP-activated kinase (AMPK)-dependent pathway in rats. Male Wistar rats (7 to 10 weeks old) were used in this study. The diameters of the retinal arterioles, mean arterial pressure, and heart rate were measured in vivo. The retinal injury was assessed by histological examination. Intravenous injection of resveratrol (3 mg/kg) increased the diameter of the retinal arterioles without affecting the mean arterial pressure and heart rate. The AMPK inhibitor, compound C (5 mg/kg, intravenously), significantly attenuated the retinal vasodilator response to resveratrol. Seven days after intravitreal injection of N-methyl-d-aspartic acid (NMDA; 25, 50, and 100 nmol/eye), the number of cells located in the ganglion cell layer (GCL) was reduced, along with thinning of the inner plexiform layer. Intravitreal resveratrol injection (100 nmol/eye) reduced the NMDA (25 and 50 nmol/eye)-induced cell loss in the GCL. The neuroprotective effect of resveratrol was significantly but not completely reversed by compound C (10 nmol/eye). These results suggest that resveratrol dilates retinal arterioles and protects against NMDA-induced retinal neurodegeneration via an AMPK-dependent pathway in rats. Resveratrol may have the potential to slow the onset and progression of diseases associated with retinal ischemia by improving impaired retinal circulation and protecting retinal neuronal cells.

Methylglyoxal, a highly reactive dicarbonyl compound, is increased and accumulated in patients with diabetic mellitus. Methylglyoxal forms advanced glycation end products (AGE), contributing to the pathogenesis of diabetic complications, including diabetic retinopathy. Recent studies have shown that methylglyoxal induces diabetic retinopathy-like abnormalities in retinal vasculature. In this study, we investigated the processes and mechanisms of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes) were assessed in the retinas 2, 7, and 14 days after intravitreal injection of methylglyoxal. Intravitreal methylglyoxal injection induced retinal capillary endothelial cell degeneration in a dose- and time-dependent manner. Changes in the shape and distribution of pericytes occurred before the initiation of capillary regression in the retinas of methylglyoxal-injected eyes. The receptor for AGEs (RAGEs) antagonist FPS-ZM1, and the matrix metalloproteinase (MMP) inhibitor GM6001 significantly attenuated methylglyoxal-induced capillary endothelial cell degeneration. FPS-ZM1 failed to prevent pathological changes in pericytes in methylglyoxal-injected eyes. In situ zymography revealed that MMP activity was enhanced at sites of blood vessels with reduced pericyte coverage in methylglyoxal-injected eyes. These results suggest that intravitreal methylglyoxal injection induces pathological changes in pericytes before the initiation of capillary endothelial cell degeneration via an AGE-RAGE-independent pathway. The capillary endothelial cell degeneration is mediated by activating the AGE-RAGE pathway and increasing MMP activity in endothelial cells by impairing pericyte function in the retina.

Background In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses. Results Neuronal cell loss induced by an intravitreal injection of N-methyl-d-aspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer. Conclusions Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer.

Because visual information accounts for 80-90% of sensory information that we get from our circumstance, loss of vision seriously diminishes our quality of life. According to a recent epidemiological study, glaucoma is the first, and retinitis pigmentosa (RP) is the second leading causes of acquired blindness in Japan. Degeneration of the retinal ganglion cells (RGC) and photoreceptor cells causes glaucoma and RP, respectively. Intraocular pressure-lowering therapy is an only effective treatment for glaucoma, and the agents that protect RGC directly against glaucomatous injury have not been available yet. In addition, there is no effective treatment for RP at present. microRNAs are a class of small, endogenous, non-coding RNAs comprised of approximately 20 nucleotides. It has been clarified that microRNAs reduces the stability of the target mRNAs and/or repress the translation of the target genes. A single microRNA can affect the transcription of multiple mRNAs, and almost 30% of human genes are thought to be regulated by microRNAs. Therefore, it has been considered that the expression changes of microRNAs are possible to cause various diseases, such as cancer and neurodegenerative diseases. Recently, the expression changes in microRNAs have been reported in the retina of experimental model animals for glaucoma and RP. The expressional changes of microRNAs are suggested to be related with development and progression of glaucoma and RP. Here, we will discuss about the relationship between the expressional changes of microRNAs and neuronal cell death in glaucoma and RP.

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Matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF)-α contribute to the pathogenesis of several ocular diseases. Previous studies have shown that MMP-9 activation plays an important role in capillary degeneration in injured retinas. In this study, we aimed to determine the roles of TNF-α in capillary degeneration and MMP-9 activation in the injured retina. In rats, retinal injury was induced by intravitreal injection of N-methyl-D-aspartic acid (NMDA, 200 nmol) at postnatal day 7. We examined (1) the effects of blocking MMP-9 and TNF-α signaling pathway on capillary degeneration, (2) changes in protein levels and distribution of MMP-9 and TNF-α, and (3) the interaction between MMP-9 and TNF-α in regulating the expression level of each protein in retinas of NMDA-injected eyes. Intravitreal injection of GM6001, an MMP inhibitor, or TNF-α neutralizing antibody (anti-TNF-α Ab) attenuated capillary degeneration in retinas of NMDA-injected eyes. Protein levels of TNF-α increased 2 h after NMDA injection, whereas those of MMP-9 increased 4 h after the injection. Anti-TNF-α Ab suppressed activation of MMP-9 in retinas of NMDA-injected eyes, whereas GM6001 diminished the TNF-α protein expression. Incubation of recombinant TNF-α with supernatants of homogenized retina increased protein levels and activity of MMP-9. These results suggest that TNF-α and MMP-9 collaboratively increase their expression levels in the retina following neurodegeneration, thus leading to retinal capillary degeneration. The cooperative interaction between MMP-9 and TNF-α could be involved in the exacerbation of retinal neurovascular degeneration.

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague–Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a β2-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

Misdirected vascular growth frequently occurs in the neovascular diseases in the retina. However, the mechanisms are still not fully understood. In the present study, we created capillary-free zones in the central and peripheral retinas in neonatal mice by pharmacological blockade of vascular endothelial growth factor (VEGF) signaling. Using this model, we investigated the process and mechanisms of revascularization in the central and peripheral avascular areas. After the completion of a 2-day treatment with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5, revascularization started on P8 in the central avascular area where capillaries had been dropped out. The expression levels of VEGF were higher in the peripheral than in the central avascular area. However, the expansion of the vasculature in the peripheral avascular retina remained suppressed until revascularization had been completed in the central avascular area. Additionally, we found disorganized endothelial cell division, misdirected blood vessels with irregular diameters, and abnormal fibronectin networks at the border of the vascular front and the avascular retina. In the central avascular area, a slight amount of fibronectin as non-vascular component re-formed to provide a scaffold for revascularization. Mechanistic analysis revealed that higher levels of VEGF attenuated the migratory response of endothelial cells without decreasing the proliferative activity. These results suggest that the presence of concentration range of VEGF, which enhances both migration and proliferation of the endothelial cells, and the structurally normal fibronectin network contribute to determine the proper direction of angiogenesis.

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. Keywords: Histone deacetylase, Mammalian target of rapamycin, Pathological angiogenesis, Retina, Vascular endothelial growth factor

Accumulated temperature, representing the sum of average daily temperatures, is an objective index for assessment of the maturity of vegetable and fruit crops, including melon. We attempted to manage melon cultivation using time–temperature indicators (TTIs), that reflect temperature history during the cultivation period. The objective of this study was to develop a method for simple prediction and estimation of melon maturity using TTIs based on the Maillard reaction to assist in the decision of when to harvest melons. We examined the performance of TTIs during melon cultivation in greenhouses. Prototype TTIs were prepared using a combination of d-xylose (1.0–2.0 mol/kg) and glycine (2.0–3.5 mol/kg) and the correspondence between changes in color and time–temperature histories of melons during cultivation investigated. The color of TTIs changed from colorless to dark brown, as expected. In some areas of the greenhouse, accumulated temperatures differed by more than 50 °C day, depending on location within the greenhouse, resulting in variation in the color development of TTIs. TTIs could successfully indicate differences in accumulated temperatures, due to temperature irregularities in a greenhouse, as a difference in color. Furthermore, the relationship between accumulated temperature and melon quality at harvest time was investigated. Accumulated temperature was positively correlated with soluble solids content (SSC) and negatively correlated with melon firmness. The prototype TTIs corresponded well with the level of melon maturity, and can indicate SSC and firmness by color development; therefore, they have potential for use in prediction of individual melon maturity during cultivation.

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. © 2017 Wiley Periodicals, Inc.

Background: Astrocytes migrate into the retina through the optic nerve head via the axons of retinal ganglion cells, and spread radially towards 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. This article is protected by copyright. All rights reserved.

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.

Purpose: Retinopathy of prematurity (ROP) is characterized by morphological abnormalities in retinal blood vessels, but how an episode of ROP affects vascular function remains to be fully elucidated. The purpose of the present study was to assess the distribution of pericyte/smooth muscle in retinal blood vessels and retinal vasodilator responses in a rat model of ROP.Methods: ROP was induced in rats by the subcutaneous injection of the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8. The distribution of pericyte/smooth muscle in retinal blood vessels was examined on P14 and P35 by immunohistochemistry. Retinal vasodilator responses were assessed on P35 by measuring the diameter of retinal arterioles in fundus images.Results: In retinas of KRN633-treated (ROP) rats, progressive angiogenesis, tortuous arteries, enlarged veins, and enhanced expression of α-smooth muscle actin in pericytes on capillaries and veins were observed on P14. These abnormalities in retinal vasculature showed a tendency to normalize by P35. Vasodilation of retinal arterioles induced by acetylcholine, an endothelium-dependent vasodilator, was smaller in P35 ROP rats than age-matched controls, whereas retinal vasodilator responses to the nitric oxide (NO) donor NOR3 were unaltered.Conclusions: Phenotypic changes in pericytes occur in the ROP model rats and endothelium-dependent vasodilatory mechanisms in retinal blood vessels are impaired. The impaired vasodilator function may contribute to the progression and pathogenesis of ROP.

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron&ndash, glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.

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.

Ca2+-permeable ion channels, such as transient receptor channels, are one of the potential therapeutic targets in cancer. Transient receptor potential vanilloid subtype 4 (TRPV4) is a nonselective cation channel associated with cancer progression. This study investigates the roles of TRPV4 in the pathogenesis of colitis-associated cancer (CAC) in mice. The role of TRPV4 was examined in azoxymethane (AOM)/dextran sulphate sodium (DSS)-induced murine CAC model. The formation of colon tumours induced by AOM/DSS treatment was significantly attenuated in TRPV4-deficient mice (TRPV4KO). TRPV4 was co-localised with markers of angiogenesis and macrophages. AOM/DSS treatment upregulated the expression of CD105, vascular endothelial growth factor receptor 2, and TRPV4 in wildtype, but the upregulation of CD105 was significantly attenuated in TRPV4KO. Bone marrow chimera experiments indicated that TRPV4, expressed in both vascular endothelial cells and bone marrow-derived macrophages, played a significant role in colitis-associated tumorigenesis. There was no significant difference in the population of hematopoietic cells, neutrophils, and monocytes between untreated and AOM/DSS-treated WT and TRPV4KO on flow cytometric analysis. TRPV4 activation by a selective agonist induced TNF-α and CXCL2 release in macrophages. Furthermore, TRPV4 activation enhanced the proliferation of human umbilical vein endothelial cells. These results suggest that TRPV4 expressed in neovascular endothelial cells and bone marrow-derived macrophages contributes to the progression of CAC in mice.

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N-methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration.

The chiral guanidine-catalyzed conjugate addition of 5H-oxazol-4-ones to 2-chloroacrylonitrile was developed with high enantiomeric and diastereomeric control. Introduction of an electron-donating 4-methoxyphenyl group as an aromatic substituent (Ar) on 5H-oxazol-4-one was suitable for inhibiting undesirable cyclization and achieving high stereoselectivity in this conjugate addition. The derivatization of an obtained product was also performed to afford a chiral α-siloxyimide, which was subsequently converted to the corresponding α-siloxy-γ-lactam in a few steps.

In this Letter, a chiral guanidine-catalyzed 1,4-addition reaction of 5H-oxazol-4-ones to vinyl ketones is described. The 1,4-addition reaction proceeded with a high enantioselectivity using 5H-oxazol-4-ones, substituted with a 3-chloro-5-methylphenyl group, as pronucleophiles and a newly developed chiral guanidine as a catalyst. The optimized reaction conditions were also effective in achieving excellent enantioselectivity in the 1,6-addition to dienones. Subsequent solvolysis of the 1,4-addition adduct yielded the corresponding α-hydroxy-5-oxocarboxylates.

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.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.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.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.

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.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.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. © 2017 Wiley Periodicals, Inc.

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.

We have previously reported that treatment of newborn mice with KRN633, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, delayed retinal vascularization leading to abnormal retinal vascular growth and patterns. To determine whether similar abnormalities are observed in newborn mice treated with other VEGF receptor tyrosine kinase inhibitors, we administered axitinib to mice on the day of birth and on the following day. When compared with control pups, a significant delay in retinal vascularization was observed in pups treated with axitinib (5 mg/kg). Axitinib-treated pups had a very dense capillary network on postnatal day (P) 6 and fewer central arteries and veins on P8 and P12. Central veins, but not arteries, were significantly enlarged on P8. These abnormalities were similar to those observed in KRN633-treated pups and probably represent a common phenotype induced by short-term treatment with VEGF receptor inhibitors in newborn mice. Therefore, mice treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms of retinal vascular formation and patterning.

A chiral guanidine-catalyzed 1,6-addition reaction of 5H-oxazol-4-ones to α,β,γ,δ-diunsaturated ketones is described in this study. The addition reaction proceeded regiospecifically to yield a 1,6-...

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.