Role of monocarboxylate transporters in AMPK-mediated protection against excitotoxic injury in the rat retina.

Activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway protects against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal injury. AMPK activation enhances fatty acid metabolism and ketone body synthesis. Ketone bodies are transported into neurons by monocarboxylate transporters (MCTs) and exert neuroprotective effects. In this study, we examined the distribution and expression levels of MCT1 and MCT2 in the retina and analyzed the effects of pharmacological inhibition of MCTs on the protective effects of metformin and 5-aminoimidazole-4-carboxamide (AICAR), activators of AMPK, against NMDA-induced retinal injury in rats. MCT1 was expressed in the blood vessels, processes of astrocytes and Müller cells, and inner segments of photoreceptors in the rat retina, whereas MCT2 was expressed in neuronal cells in the ganglion cell layer (GCL) and in astrocyte processes. The expression levels of MCT2, but not MCT1, decreased one day after intravitreal injection of NMDA (200 nmol). Intravitreal injection of NMDA decreased the number of cells in the GCL compared to the vehicle seven days after injection. Simultaneous injection of metformin (20 nmol) or AICAR (50 nmol) with NMDA attenuated NMDA-induced cell loss in the GCL, and these protective effects were attenuated by AR-C155858 (1 pmol), an inhibitor of MCTs. AR-C155858 alone had no significant effect on the retinal structure. These results suggest that AMPK-activating compounds protect against NMDA-induced excitotoxic retinal injury via mechanisms involving MCTs in rats. NMDA-induced neurotoxicity may be associated with retinal neurodegenerative changes in glaucoma and diabetic retinopathy. Therefore, AMPK-activating compounds may be effective in managing these retinal diseases.

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis).

PURPOSE: Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). METHODS: OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. RESULTS: Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. CONCLUSIONS: These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

The Role of microRNAs Related to Apoptosis for N-Methyl-d-Aspartic Acid-Induced Neuronal Cell Death in the Murine Retina.

Glaucoma is one of the leading causes of acquired blindness and characterized by retinal ganglion cell (RGC) death. MicroRNAs are small noncoding RNAs that degrade their target mRNAs. Apoptosis is one of the common mechanisms leading to neuronal death in many neurodegenerative diseases, including glaucoma. In the present study, we identified microRNAs that modulate RGC death caused by the intravitreal injection of N-methyl-d-aspartic acid (NMDA). We found an upregulation of miR-29b and downregulation of miR-124 in the retina of the NMDA-injected eyes. The intravitreal injection of an miR-29b inhibitor 18 h before NMDA injection reduced RGC death and the downregulation of myeloid cell leukemia 1 (MCL-1), an anti-apoptotic factor, induced by intravitreal NMDA. The intravitreal injection of an miR-124 mimic 18 h before NMDA injection also reduced RGC death and the upregulation of B-cell/chronic lymphocytic leukemia lymphoma 2 (bcl-2)-associated X protein (Bax) and bcl-2 interacting protein (Bim), pro-apoptotic factors, induced by intravitreal NMDA. These data suggest that expressional changes in microRNA are involved in the excitotoxicity of RGCs, and that complement and/or inhibition of microRNA may be a potential therapeutic approach for the diseases related to the excitotoxicity of RGCs, such as glaucoma and retinal central artery occlusion.

Role of mammalian target of rapamycin in the formation and progression of retinopathy of prematurity-like vascular abnormalities in neonatal rats.

Retinopathy of prematurity (ROP), a retinal disease that can occur in premature infants, can lead to severe visual impairment. In this study, we examined the preventive and therapeutic effects of mammalian target of rapamycin complex 1 (mTORC1) inhibition on abnormal retinal blood vessels in a rat model of ROP. To induce ROP-like vascular abnormalities, rats were subcutaneously treated with KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on postnatal day 7 (P7) and P8. KRN633-treated (ROP) rats were treated subcutaneously with the mTORC1 inhibitor rapamycin according to preventive and therapeutic protocols, i.e., from P11 to P13 (P11-P13) and from P14 to P20 (P14-P20), respectively. To compare with the effects of VEGF inhibition, KRN633 was administered according to similar protocols. Changes in retinal vasculature, phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, and the proliferative status of vascular cells were evaluated at P14 and P21 using immunohistochemistry. Rapamycin treatment from P11 to P13 prevented increases in arteriolar tortuosity, capillary density, and the number of proliferating vascular cells, and eliminated pS6 immunoreactivity in ROP rats. KRN633 treatment at P11 and P12 (P11/P12) also prevented the appearance of ROP-like retinal blood vessels. Rapamycin treatment from P14 to P20 failed to attenuate arteriolar tortuosity but prevented increases in capillary density and proliferating vascular cell number at the vascular front, but not at the central zone. KRN633 treatment from P14 to P20 significantly reduced abnormalities in the retinal vasculature; however, the effects were inferior to those of KRN633 treatment on P11/P12. These results suggest that activation of the mTORC1 pathway in proliferating endothelial cells contributes to the appearance and progression of ROP-like retinal blood vessels. Therefore, inhibition of mTORC1 may be a promising approach for selectively targeting abnormal retinal blood vessels in ROP.

Resveratrol dilates arterioles and protects against N-methyl-d-aspartic acid-induced excitotoxicity in the rat retina.

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.

The process of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats.

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.

Tumor necrosis factor-α and matrix metalloproteinase-9 cooperatively exacerbate neurovascular degeneration in the neonatal rat retina.

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.

Pharmacological inhibition of Na+/K+-ATPase induces neurovascular degeneration and glial cell alteration in the rat retina.

Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(-)-2-amino-5-phosphonopentanoic acid, an antagonist of N-methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats.

PURPOSE: Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. METHODS: Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. RESULTS: Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. CONCLUSION: These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload.

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.

LPA2 promotes neuronal differentiation and neurite formation in neocortical development.

Lysophosphatidic acid (LPA) is a bioactive lipid that activates the G protein-coupled receptors, LPA1-6, which are associated with a wide number of cellular responses including proliferation, migration, differentiation, and survival. Although LPA1-6 are expressed in the developing brain, their functions in brain development are not fully understood. In the present study, we analyzed the temporal expression pattern of LPA receptors (LPARs) during neocortical development and found that LPA2 is highly expressed in neural stem/progenitor cells (NS/PCs) in the embryonic neocortex. LPA2 activation on cultured NS/PCs using GRI977143, a selective LPA2 agonist, promoted neuronal differentiation. LPA2-induced neuronal expansion was inhibited by FR180204, an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, suggesting that LPA2 promotes neuronal differentiation via Erk1/2 signaling. In addition, LPA2 activation promotes neurite elongation and branch formation. These results suggest that LPA2 is a critical regulator of neuronal differentiation and development.

Involvement of Gap Junctions in Acetylcholine-Induced Endothelium-Derived Hyperpolarization-Type Dilation of Retinal Arterioles in Rats.

An electrical communication between the endothelial and smooth muscle cells via gap junctions, which provides the signaling pathway known as endothelium-dependent hyperpolarization (EDH), plays a crucial role in controlling the vascular tone. In this study, we investigated the role of gap junctions in the acetylcholine (ACh)-induced EDH-type dilation of rat retinal arterioles in vivo. The dilator response was evaluated by measuring the diameter of retinal arterioles. Intravitreal injection of gap junction blockers (18ß-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18ß-glycyrrhetinic acid under treatment with a combination of NG-nitro-L-arginine methyl ester (a nitric oxide (NO) synthase inhibitor; 30 mg/kg) and indomethacin (a cyclooxygenase inhibitor; 5 mg/kg). The NO- and prostaglandin-independent, EDH-related component of ACh-induced dilation of retinal arterioles was prevented by intravitreal injection of iberiotoxin, which inhibits large-conductance Ca2+-activated K+ channels. Furthermore, the combination of 18ß-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18ß-glycyrrhetinic acid revealed no significant effect on NOR3 (an NO donor)-induced retinal vasodilator response. These results suggest that gap junctions contribute to the ACh-induced, EDH-type dilation of rat retinal arterioles in vivo.

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

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 ß2-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.

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

In our previous study, we found that the vasodilation of retinal arterioles induced by acetylcholine and BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) 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.

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

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.

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

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 NG-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 Ca2+-activated K+ (BKCa) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BKCa channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.

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

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.

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

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.

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

Nitric oxide (NO) is an important regulator of the retinal blood flow. The present study aimed to determine the role of voltage-gated K+ (KV) channels and ATP-sensitive K+ (KATP) 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 KV channel inhibitor), but not glibenclamide (a KATP 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 KATP 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 KV 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 KV channels and enhancement of prostaglandin production.

Involvement of Gi protein-dependent BKCa channel activation in ß2-adrenoceptor-mediated dilation of retinal arterioles in rats.

Circulating catecholamines contribute to the regulation of retinal vascular tone. Our previous studies have demonstrated that the activation of large-conductance Ca2+-activated K+ (BKCa) channels is involved in the ß2-adrenoceptor-mediated dilation of retinal arterioles in rats. The present study aimed to examine the role of Gi protein in the ß2-adrenoceptor-mediated activation of BKCa 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 ß2-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 BKCa channels, significantly attenuated the formoterol-induced dilation of retinal arterioles. Similar results were obtained when salbutamol (0.03-3 µg/kg/min), another ß2-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 ß1-adrenoceptor agonist), CL316243 (0.3-10 µg/kg/min; a ß3-adrenoceptor agonist), prostaglandin I2 (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 ß2-adrenoceptors dilates retinal arterioles through pertussis toxin-sensitive Gi protein-dependent activation of BKCa channels in rats in vivo.