Publications by authors named "Tsutomu Nakahara"

148 Publications

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity.

J Pharmacol Sci 2021 Aug 7;146(4):233-243. Epub 2021 May 7.

Department of Molecular Pharmacology, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

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 β-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.
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http://dx.doi.org/10.1016/j.jphs.2021.04.008DOI Listing
August 2021

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

Int J Mol Sci 2021 Apr 23;22(9). Epub 2021 Apr 23.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo 108-8641, Japan.

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 -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.
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http://dx.doi.org/10.3390/ijms22094439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123037PMC
April 2021

L-Citrulline ameliorates the attenuation of acetylcholine-induced vasodilation of retinal arterioles in diabetic rats.

Heliyon 2021 Mar 21;7(3):e06532. Epub 2021 Mar 21.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

In our previous study, we found that the vasodilation of retinal arterioles induced by acetylcholine and BMS-191011, a large-conductance Ca-activated K (BK) channel opener, were diminished in diabetic rats. Currently, few agents ameliorate the impaired vasodilator responses of retinal blood vessels. Our recent finding that the intravenous infusion of L-citrulline dilated retinal arterioles, suggests that L-citrulline could be a potential therapeutic agent for circulatory disorders of the retina. In this study, we determined the effect of an oral L-citrulline treatment on impaired acetylcholine- and BMS-191011-induced vasodilation in the retinal arterioles of diabetic rats. To induce diabetes, rats were administered an intravenous dose of streptozotocin (65 mg/kg) and a 5% D-glucose solution as drinking water. The L-citrulline (2 g/kg/day) and L-arginine (2 g/kg/day) treatments commenced either 15 days before or just after the streptozotocin injection and continued throughout the experimental period. A 29-day treatment with L-citrulline, but not L-arginine, significantly ameliorated the impaired acetylcholine- and BMS-191011-induced retinal vasodilation in diabetic rats without affecting their plasma glucose levels. The 2-week L-citrulline treatment tended to ameliorate the dysfunction of the acetylcholine-induced retinal vasodilation in diabetic rats. In conclusion, these results showed that the retinal blood vessel dysfunction induced by diabetes mellitus could be prevented by the long-term administration of L-citrulline and suggest that the latter could play a potentially prophylactic role in diabetic retinopathy.
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http://dx.doi.org/10.1016/j.heliyon.2021.e06532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020426PMC
March 2021

Role of Epoxyeicosatrienoic Acids in Acetylcholine-Induced Dilation of Rat Retinal Arterioles in Vivo.

Biol Pharm Bull 2021 ;44(1):82-87

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

CYP epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent hyperpolarization (EDH)-related dilation in multiple vascular beds. The present study aimed to determine the role of EETs in the acetylcholine (ACh)-induced dilation of retinal arterioles in rats in vivo. The vasodilator responses were assessed by determining the change in diameter of the retinal arterioles on images of the ocular fundus. The intravitreal injection of 17-octadecynoic acid (1.4 nmol/eye), an inhibitor of CYP epoxygenase, and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E; 2 nmol/eye), an antagonist of EETs, reduced the ACh (0.3-10 µg/kg/min)-induced dilation of the retinal arterioles. The EET antagonist attenuated the vasodilator response to ACh under blockade of nitric oxide (NO) synthases and cyclooxygenases with N-nitro-L-arginine methyl ester (30 mg/kg) plus indomethacin (5 mg/kg). Intravitreal injection of 14,15-EET (0.5 nmol/eye) dilated retinal arterioles and the response was prevented by iberiotoxin, an inhibitor of large-conductance Ca-activated K (BK) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BK channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.
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http://dx.doi.org/10.1248/bpb.b20-00635DOI Listing
January 2021

Pharmacological depletion of retinal neurons prevents vertical angiogenic sprouting without affecting the superficial vascular plexus.

Dev Dyn 2021 Apr 2;250(4):497-512. Epub 2020 Nov 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

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.
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http://dx.doi.org/10.1002/dvdy.263DOI Listing
April 2021

The process of revascularization in the neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Cell Tissue Res 2020 Dec 8;382(3):529-549. Epub 2020 Sep 8.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

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.
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http://dx.doi.org/10.1007/s00441-020-03276-9DOI Listing
December 2020

4-Aminopyridine, a Voltage-Gated K Channel Inhibitor, Attenuates Nitric Oxide-Mediated Vasodilation of Retinal Arterioles in Rats.

Biol Pharm Bull 2020 ;43(7):1123-1127

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Nitric oxide (NO) is an important regulator of the retinal blood flow. The present study aimed to determine the role of voltage-gated K (K) channels and ATP-sensitive K (K) channels in NO-mediated vasodilation of retinal arterioles in rats. In vivo, the retinal vasodilator responses were assessed by measuring changes in the diameter of retinal arterioles from ocular fundus images. Intravitreal injection of 4-aminopyridine (a K channel inhibitor), but not glibenclamide (a K channel blocker), significantly attenuated the retinal vasodilator response to the NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Intravitreal injection of indomethacin (a non-selective cyclooxygenase inhibitor) also reduced the NOR3-induced retinal vasodilator response. The combination of 4-aminopyridine and indomethacin produced a greater reduction in the NOR3-induced response than either agent alone. 4-Aminopyridine had no significant effect on pinacidil (a K channel opener)-induced response. These results suggest that the vasodilatory effects of NO are mediated, at least in part, through the activation of 4-aminopyridine-sensitive K channels in the retinal arterioles of rats. NO exerts its dilatory effect on the retinal vasculature of rats through at least two mechanisms, activation of the K channels and enhancement of prostaglandin production.
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http://dx.doi.org/10.1248/bpb.b20-00220DOI Listing
December 2020

Involvement of Gi protein-dependent BK channel activation in β-adrenoceptor-mediated dilation of retinal arterioles in rats.

Naunyn Schmiedebergs Arch Pharmacol 2020 11 5;393(11):2043-2052. Epub 2020 Jun 5.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Circulating catecholamines contribute to the regulation of retinal vascular tone. Our previous studies have demonstrated that the activation of large-conductance Ca-activated K (BK) channels is involved in the β-adrenoceptor-mediated dilation of retinal arterioles in rats. The present study aimed to examine the role of Gi protein in the β-adrenoceptor-mediated activation of BK channels in the retinal arterioles. Images of in vivo rat ocular fundi were captured, and the diameters of retinal arterioles were measured. Systemic blood pressure and heart rate were recorded continuously. Intravenous infusion of formoterol (0.01-0.3 μg/kg/min), a β-adrenoceptor agonist, increased the diameter of retinal arterioles but decreased mean arterial pressure in a dose-dependent manner. Intravitreal injection of iberiotoxin (20 pmol/eye), an inhibitor of BK channels, significantly attenuated the formoterol-induced dilation of retinal arterioles. Similar results were obtained when salbutamol (0.03-3 μg/kg/min), another β-adrenoceptor agonist, was used instead of formoterol. However, iberiotoxin had no significant effect on retinal vasodilator responses to intravenous infusion of denopamine (1-30 μg/kg/min; a β-adrenoceptor agonist), CL316243 (0.3-10 μg/kg/min; a β-adrenoceptor agonist), prostaglandin I (0.03-10 μg/kg/min; a prostanoid IP receptor agonist), and forskolin (1-10 μg/kg/min; an adenylyl cyclase activator). Intravitreal injection of pertussis toxin (66 ng/eye; a Gi protein inhibitor) significantly attenuated the dilation of retinal arterioles induced by formoterol but not by denopamine and CL316243. In the presence of pertussis toxin, iberiotoxin had no inhibitory effect on formoterol-induced dilation of retinal arterioles. These results suggest that stimulation of β-adrenoceptors dilates retinal arterioles through pertussis toxin-sensitive Gi protein-dependent activation of BK channels in rats in vivo.
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http://dx.doi.org/10.1007/s00210-020-01895-1DOI Listing
November 2020

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Biol Pharm Bull 2020 ;43(5):859-863

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

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.
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http://dx.doi.org/10.1248/bpb.b19-01065DOI Listing
January 2021

[Expression changes in microRNA in the retina of retinal degenerative diseases].

Nihon Yakurigaku Zasshi 2020 ;155(2):81-86

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

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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http://dx.doi.org/10.1254/fpj.19121DOI Listing
March 2020

Role of transient receptor potential vanilloid subtype 4 in the regulation of azoymethane/dextran sulphate sodium-induced colitis-associated cancer in mice.

Eur J Pharmacol 2020 Jan 10;867:172853. Epub 2019 Dec 10.

Division of Pathological Sciences, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, 6078414, Japan.

Ca-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.
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http://dx.doi.org/10.1016/j.ejphar.2019.172853DOI Listing
January 2020

Changes in components of the neurovascular unit in the retina in a rat model of retinopathy of prematurity.

Cell Tissue Res 2020 Mar 2;379(3):473-486. Epub 2019 Dec 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

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.
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http://dx.doi.org/10.1007/s00441-019-03112-9DOI Listing
March 2020

Cellular Mechanisms of Angiogenesis in Neonatal Rat Models of Retinal Neurodegeneration.

Int J Mol Sci 2019 Sep 25;20(19). Epub 2019 Sep 25.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

Νeuronal and glial cells play an important role in the development of vasculature in the retina. In this study, we investigated whether re-vascularization occurs in retinal neurodegenerative injury models. To induce retinal injury, -methyl-D-aspartic acid (NMDA, 200 nmol) or kainic acid (KA, 20 nmol) was injected into the vitreous chamber of the eye on postnatal day (P)7. Morphological changes in retinal neurons and vasculature were assessed on P14, P21, and P35. Prevention of vascular growth and regression of some capillaries were observed on P14 in retinas of NMDA- and KA-treated eyes. However, vascular growth and re-vascularization started on P21, and the retinal vascular network was established by P35 in retinas with neurodegenerative injuries. The re-vascularization was suppressed by a two-day treatment with KRN633, an inhibitor of VEGF receptor tyrosine kinase, on P21 and P22. Astrocytes and Müller cells expressed vascular endothelial growth factor (VEGF), and the distribution pattern of VEGF was almost the same between the control and the NMDA-induced retinal neurodegenerative injury model, except for the difference in the thickness of the inner retinal layer. During re-vascularization, angiogenic sprouts from pre-existing blood vessels were present along the network of fibronectins formed by astrocytes. These results suggest that glial cells contribute to angiogenesis in neonatal rat models of retinal neurodegeneration.
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http://dx.doi.org/10.3390/ijms20194759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801463PMC
September 2019

Activation of transient receptor potential vanilloid 4 channels dilates rat retinal arterioles through nitric oxide- and BK channel-dependent mechanisms in vivo.

Naunyn Schmiedebergs Arch Pharmacol 2020 01 8;393(1):35-41. Epub 2019 Aug 8.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Transient receptor potential vanilloid 4 (TRPV4) channel, a cation channel expressed in nearly all cell types, plays an important role in the regulation of vascular tone. In the present study, we examined the effect of GSK1016790A, an activator of TRPV4 channels, on the diameter of retinal blood vessels in rats and the underlying mechanisms. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo and diameters of retinal blood vessels were measured. Intravenous infusion of GSK1016790A (0.2-2 μg kg min) increased retinal arteriolar diameter in a dose-dependent manner. The higher dose of GSK1016790A (2 μg kg min) slightly decreased blood pressure. These responses to GSK1016790A were significantly attenuated by intravenous injection of GSK2193874 (0.3 mg/kg), an antagonist of TRPV4 channels. Intravitreal injection of N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase or iberiotoxin, an inhibitor of large-conductance Ca-activated K (BK) channel, significantly attenuated the GSK1016790A-induced increases in retinal arteriolar diameter. These results suggest that activation of TRPV4 channels dilates rat retinal arterioles through NO- and BK channel-dependent mechanisms in vivo.
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http://dx.doi.org/10.1007/s00210-019-01707-1DOI Listing
January 2020

Attenuation of Retinal Endothelial Vasodilator Function in a Rat Model of Retinopathy of Prematurity.

Curr Eye Res 2019 12 18;44(12):1360-1368. Epub 2019 Jul 18.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

: 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.: 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.: 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.: 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.
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http://dx.doi.org/10.1080/02713683.2019.1641825DOI Listing
December 2019

Role of Neuron⁻Glia Signaling in Regulation of Retinal Vascular Tone in Rats.

Int J Mol Sci 2019 Apr 20;20(8). Epub 2019 Apr 20.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

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-glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.
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http://dx.doi.org/10.3390/ijms20081952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514555PMC
April 2019

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina.

Exp Eye Res 2019 05 16;182:101-108. Epub 2019 Mar 16.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.exer.2019.03.005DOI Listing
May 2019

Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization.

Sci Rep 2019 02 7;9(1):1560. Epub 2019 Feb 7.

Department of Physiology, Aichi Medical University, Nagakute, Japan.

Choroidal neovascularization (CNV) is associated with age-related macular degeneration (AMD), a major cause of vision loss among elderly people. Vascular endothelial cell growth factor (VEGF) is essential for the development and progression of AMD, and VEGF signaling molecules are effective targets for the treatment of AMD. We recently reported that activator of G-protein signaling 8 (AGS8), a receptor-independent Gβγ regulator, is involved in VEGF-induced angiogenesis in cultured endothelial cells (EC); however, the role of AGS8 in CNV is not yet understood. This study aimed to explore the role of AGS8 in CNV in cultured cells, explanted choroid tissue, and laser-induced CNV in a mouse AMD model. AGS8 knockdown in cultured choroidal EC inhibited VEGF-induced VEGFR-2 phosphorylation, cell proliferation, and migration. AGS8 knockdown also downregulated cell sprouting from mouse choroidal tissue in ex vivo culture. A mouse model of laser-induced CNV, created to analyze the roles of AGS8 in vivo, demonstrated that AGS8 mRNA was significantly upregulated in choroidal lesions and AGS8 was specifically expressed in the neovasculature. Local AGS8 knockdown in intravitreal tissue significantly inhibited laser-induced AGS8 upregulation and suppressed CNV, suggesting that AGS8 knockdown in the choroid has therapeutic potential for AMD. Together, these results demonstrate that AGS8 plays critical roles in VEGF-induced CNV.
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http://dx.doi.org/10.1038/s41598-018-38067-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367328PMC
February 2019

Probucol Slows the Progression of Cataracts in Streptozotocin-Induced Hyperglycemic Rats.

Pharmacology 2019 5;103(3-4):212-219. Epub 2019 Feb 5.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan,

We examined the effect of probucol, an antihyperlipidemic drug with potent antioxidant properties, on cataract formation in streptozotocin (STZ)-induced hyperglycemic rats that were given 5% D-glucose as drinking water. Probucol treatment was initiated immediately after the induction of hyperglycemia was confirmed. Using full horizontal-plane lens images captured with an original digital camera system, the opacity of central region of lens was assessed by measuring the opaque area in the region. Central opacities were detected after 3 weeks of hyperglycemia, and progressed in a time-dependent manner. The majority of STZ-induced hyperglycemic rats developed severe cataracts after 9 weeks of hyperglycemia. Probucol slowed the progression of cataracts in a dose-dependent manner. Levels of sorbitol and protein carbonyls in lenses of STZ-induced hyperglycemic rats were higher than those of control rats. Probucol suppressed the increase in protein carbonyls, but not of sorbitol, in lenses of STZ-induced hyperglycemic rats. Probucol had no significant effect on increases in plasma concentrations of glucose, total cholesterol, and triglyceride observed in STZ-induced hyperglycemic rats. These results suggest that probucol slows the progression of sugar cataracts, independent of its lipid-lowering effects. The beneficial effect of probucol on cataracts is partially attributable to the attenuation of oxidative damage to lens proteins.
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http://dx.doi.org/10.1159/000496055DOI Listing
March 2019

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

J Pharmacol Sci 2018 Nov 14;138(3):203-208. Epub 2018 Oct 14.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jphs.2018.10.004DOI Listing
November 2018

Anti-cataract Effect of Resveratrol in High-Glucose-Treated Streptozotocin-Induced Diabetic Rats.

Biol Pharm Bull 2018 ;41(10):1586-1592

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Resveratrol, which is a polyphenol found in grapes, peanuts, and other plants, has health benefits for various chronic diseases. The aim of the present study was to examine the effect of resveratrol on cataract formation in diabetic rats. Male Wistar rats (7-week-old) were treated with streptozotocin, and the streptozotocin-treated animals were administered 5% D-glucose in drinking water to promote the formation of cataracts by inducing severe hyperglycemia. Resveratrol supplementation (10 or 30 mg/kg/d) in drinking water was initiated immediately after induction of diabetes was confirmed. The full lens images of the horizontal plane were captured with the digital camera system which we developed. Cataract formation was assessed by an observer-based scoring method and by quantitative analysis of digital images of the lens. Cataracts at the peripheral region of the lens were detected 2 weeks after induction of hyperglycemia and progressed depending on the length of the diabetic period. The majority of them developed severe cataracts after 9 weeks of hyperglycemia. Resveratrol did not prevent the appearance of diabetic cataracts but significantly delayed the progression of cataracts compared with controls. The contents of sorbitol and protein carbonyls in lenses of diabetic rats were higher than those of control rats. Resveratrol suppressed the increase in protein carbonyls, but not of sorbitol, in diabetic lenses. These results suggest that resveratrol delays the progression of diabetic cataracts partially through attenuation of oxidative damage to lens proteins. Resveratrol may be beneficial in preventing the progression of diabetic cataracts.
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http://dx.doi.org/10.1248/bpb.b18-00328DOI Listing
January 2019

[Selective neuronal cell death in retinal degenerative diseases].

Nihon Yakurigaku Zasshi 2018 ;152(2):58-63

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Retinal degenerative diseases, such as glaucoma and retinitis pigmentosa (RP), are the leading causes of blindness in adults. In Japan, glaucoma is a leading cause, and RP is third major cause of acquired blindness. Specific types of neurons are injured in the patients of glaucoma and RP. Retinal ganglion cells (RGC) are specifically degenerated in glaucoma. Excitotoxicity caused by excess glutamate in the retinal extracellular space is thought to be one of the mechanisms of RGC death induced by glaucoma and retinal central artery occlusion. Retinal ischemia-reperfusion, intravitreal NMDA injection, intravitreal NO donor injection and knock out of glutamate aspartate transporter, which are used as the experimental models of glaucoma, are known to induce RGC death. RGCs are vulnerable for excess glutamate and oxidative stress related to NO, and this vulnerability may be involved in pathogenesis of glaucomatous optic neuropathy. RP, which is characterized by progressive photoreceptor-selective degeneration, is caused by mutation of the genes related to the function of photoreceptor and retinal pigment epithelium. It has not been thoroughly clarified how the mutations induce specific photoreceptor death. Tunicamycin is widely known to induce ER stress, and intravitreal tunicamycin cause photoreceptor-specific degeneration. Therefore, ER stress may cause photoreceptor-selective degeneration in RP.
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http://dx.doi.org/10.1254/fpj.152.58DOI Listing
May 2019

Brilliant Blue G protects against photoreceptor injury in a murine endotoxin-induced uveitis model.

Exp Eye Res 2018 12 29;177:45-49. Epub 2018 Jul 29.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, 108-8641, Japan.

We previously reported that P2X7 receptor antagonists prevented the retinal injury caused by N-methyl-d-aspartic acid. It has been reported that activation of P2X7 receptor is involved in the secretion of proinflammatory cytokines by macrophages, monocytes, and microglia. Although retinal inflammation is known to cause photoreceptor cell death, it is unclear whether a noncompetitive antagonist of P2X7 receptor can protect photoreceptor cells against inflammation-induced injury. We examined whether Brilliant Blue G (BBG), a potent non-competitive antagonist of P2X7 receptor, had neuroprotective effects on photoreceptor cell injury in a murine endotoxin-induced uveitis (EIU) model. EIU was evoked by lipopolysaccharides (LPS; 10 mg/kg/day) administered intraperitoneally once a day for 4 days. BBG (50 mg/kg/day) and indomethacin (10 mg/kg) were also injected intraperitoneally just before LPS injection. BBG significantly prevented photoreceptor cell loss and reduction of the amplitudes of dark-adapted and light-adapted flush electroretinograms induced by LPS, whereas indomethacin did not show such protective effects. These results indicated that BBG is protective against photoreceptor cell injury in EIU in the mice in vivo, suggesting that P2X7 receptor antagonists may be good candidates for preventing photoreceptor degeneration induced by inflammation.
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http://dx.doi.org/10.1016/j.exer.2018.07.028DOI Listing
December 2018

Establishment of an abnormal vascular patterning model in the mouse retina.

J Pharmacol Sci 2018 Apr 14;136(4):177-188. Epub 2018 Mar 14.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jphs.2018.03.002DOI Listing
April 2018

GYY4137, an Extended-Release Hydrogen Sulfide Donor, Reduces NMDA-Induced Neuronal Injury in the Murine Retina.

Biol Pharm Bull 2018 ;41(4):657-660

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

We previously reported that systemic administration with sodium hydrogen sulfide, a rapid-release donor compound of hydrogen sulfide (HS), protected retinal neurons against N-methyl-D-aspartic acid (NMDA)-induced injury. For clinical application of HS donors for retinal neurodegeneration, topical administration with an extended-release donor compound will be better. In the present study, we histologically investigated whether GYY4137, an extended-release hydrogen sulfide donor, had a protective effect on NMDA-induced retinal injury in the mice in vivo. Male and female B6.Cg-Tg(Thy1-CFP)23Jrs/J and C57BL/6J mice anesthetized with a mixture of ketamine and xylazine were subjected to intravitreal NMDA injection (80 nmol/eye). GYY4137 was intravitreally administered with NMDA simultaneously. Morphometric evaluation was carried out seven days after NMDA injection. Intravitreal NMDA induced retinal ganglion cell loss. GYY4137 (1, 10 and 100 nmol/eye) significantly reduced retinal ganglion cell loss seven days after NMDA injection. GYY4137 (10 nmol/eye) decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells 12 h after NMDA injection. These results suggest that extended release donor compounds of HS protect retinal neurons against excitotoxicity induced by intravitreal NMDA in the mice in vivo through its anti-oxidative activity.
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http://dx.doi.org/10.1248/bpb.b17-01032DOI Listing
September 2018

Iron-chelating agents attenuate NMDA-Induced neuronal injury via reduction of oxidative stress in the rat retina.

Exp Eye Res 2018 06 9;171:30-36. Epub 2018 Mar 9.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo 108-8641, Japan.

Excitoneurotoxicity is regarded as one of the mechanisms of the death of retinal ganglion cells induced by retinal central artery occlusion and glaucoma. Oxidative stress is at least in part involved in excitoneurotoxicity. Fenton reaction, which is catalyzed by Fe, is known to cause formation of hydroxyl radical, one of reactive oxygen species, suggesting that chelation of iron may be protective against excitoneurotoxicity. In the present study, we histologically evaluated whether zinc-deferoxamine (Zn-DFO) and deferasirox (DFX), common iron-chelating agents, were protective against N-methyl-D-aspartate (NMDA)-induced retinal injury in the rat in vivo. Male Sprague-Dawley rats were subjected to intravitreal NMDA injection (200 nmol/eye). Zn-DFO (1, 3, 10, and 30 mg/kg), Zn (0.1, 0.2 and 0.6 mg/kg) and DFX (20 mg/kg) were intraperitoneally administered. Morphometric evaluations using paraffin-embedded retinal sections, and detection of Fe using SiRhoNox-1, a fluorescent probe of labile Fe in the retinal frozen sections were carried out. Intravitreal NMDA resulted in strong positive signals of SiRhoNox-1 in the ganglion cell layer 24 h after NMDA injection, suggesting that intravitreal NMDA caused Fe accumulation in the retinal ganglion cells. Intravitreal NMDA induced retinal ganglion cell loss 7 days after NMDA injection. Zn-DFO (1, 3, 10, and 30 mg/kg), ZnCl (0.2 mg/kg, a corresponding dose of 1 mg/kg Zn-DFO) and DFX (20 mg/kg) prevented the damage of retinal ganglion cells, whereas 0.6 mg/kg ZnCl, which is a corresponding dose of 3 mg/kg Zn-DFO, did not show any protective effects. Zn-DFO (30 mg/kg) significantly decreased the intensity of the fluorescence of SiRhoNox-1 and the transferrin immunofluorescence 24 h after NMDA injection, the number of TUNEL-positive cells 24 h after NMDA injection, that of 8-OHdG-positive cells, and that of 4-hydroxy-2-nonenal-positive cells 12 and 24 h after NMDA injection. These data suggest that iron-chelating agents protected retinal neurons against excitoneurotoxicity via reduction of iron content and oxidative stress in the rats in vivo. We proposed that treatment with iron-chelating agents would be a new strategy for the retinal diseases caused by excitoneurotoxicity.
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http://dx.doi.org/10.1016/j.exer.2018.03.008DOI Listing
June 2018

Methylglyoxal Impairs β-Adrenoceptor-Mediated Vasodilatory Mechanisms in Rat Retinal Arterioles.

Biol Pharm Bull 2018 ;41(2):272-276

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on β-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the β-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca-activated K (BK) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BK channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates β-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the β-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BK channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.
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http://dx.doi.org/10.1248/bpb.b17-00861DOI Listing
August 2018

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

Exp Eye Res 2018 03 2;168:115-127. Epub 2018 Feb 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

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 (10 mg/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.
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http://dx.doi.org/10.1016/j.exer.2017.12.007DOI Listing
March 2018

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Dev Dyn 2018 05 5;247(5):699-711. Epub 2018 Jan 5.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

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.
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http://dx.doi.org/10.1002/dvdy.24614DOI Listing
May 2018

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

Biol Pharm Bull 2017 ;40(12):2045-2049

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

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.
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http://dx.doi.org/10.1248/bpb.b17-00475DOI Listing
July 2018
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