Tsuneaki Omae

Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study.

PURPOSE To evaluate how optical coherence tomography (OCT) angiography depicts clinical fundus findings in patients with diabetic retinopathy (DR). DESIGN Prospective study evaluating imaging technology. METHODS Forty-seven eyes of 25 patients with DR were scanned using a high-speed 840-nm-wavelength spectral-domain optical coherence tomography instrument (RTVue XR Avanti; Optovue, Inc, Fremont, California, USA). Blood flow was detected using the split-spectrum amplitude-decorrelation angiography algorithm. Fluorescein angiography (FA) images were also obtained in all eyes and the ability to visualize microaneurysms, retinal nonperfused areas, and neovascularization was compared with that of the en face OCT angiograms. RESULTS In 42 eyes, microaneurysms detected by FA near the macula appeared as focally dilated saccular or fusiform capillaries on OCT angiograms of the superficial and/or deep capillary plexus. Retinal nonperfused areas visualized by FA appeared as lesions with no or sparse capillaries on OCT angiograms. Area measurement of retinal nonperfusion near the macula in 7 eyes revealed a difference between the extent of nonperfused areas in superficial and deep plexuses. In 4 eyes, the vascular structures of neovascularization at the optic disc were clearly visualized on OCT angiograms. Decreases and re-increases of flow in new vessels were quantified in an eye treated with anti-vascular endothelial growth factor. CONCLUSIONS OCT angiography can clearly visualize microaneurysms and retinal nonperfused areas and enables closer observation of each layer of the retinal capillaries. Quantitative information on new vessels can also be obtained. OCT angiography may be clinically useful to evaluate the microvascular status and therapeutic effect of treatments for DR.

Pioglitazone, a Peroxisome Proliferator–Activated Receptor-γ Agonist, Induces Dilation of Isolated Porcine Retinal Arterioles: Role of Nitric Oxide and Potassium Channels

PURPOSE Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has anti-inflammatory and atheroprotective effects on vascular tissue and may reduce cardiovascular risk in patients with diabetes. The effect of pioglitazone on the retinal microvascular diameter was examined, and it was determined whether the effect depends on the endothelium and/or potassium channels in smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. METHODS Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded diametric responses to pioglitazone. RESULTS The retinal arterioles dilated in a concentration-dependent (10 nM-10 μM) manner in response to pioglitazone and decreased by 60% after endothelium removal. The nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) inhibited pioglitazone-induced vasodilation comparable to denudation. Inhibition of soluble guanylyl cyclase (1H-1,2,4-oxadiazolo[4,3-a]quinoxalin-1-one), blockade of phosphatidylinositol (PI) 3-kinase (wortmannin), and pretreatment with compound C, an AMP-activated protein kinase (AMPK) inhibitor, were comparable to l-NAME. Pioglitazone-induced vasodilation also was inhibited by a nonselective K(+) channel blocker, tetraethylammonium, and a voltage-gated K(+) (Kv) inhibitor, 4-aminopyridine (4-AP). Treatment with intraluminal and extraluminal GW9662, a PPAR-γ antagonist, similarly inhibited pioglitazone-induced vasodilation. Co-administration of l-NAME and 4-AP almost eliminated pioglitazone-induced vasodilation. CONCLUSIONS Pioglitazone elicits endothelium-dependent and -independent dilation of retinal arterioles mediated by NO release and Kv channel activation, respectively. The NO-mediated dilation pathway probably occurs via activation of guanylyl cyclase, PI3-kinase/Akt, and AMPK signaling. Understanding the effect of pioglitazone on retinal vasculature may provide new insights into therapeutic advances for treating diabetic retinopathy.

Adiponectin-induced dilation of isolated porcine retinal arterioles via production of nitric oxide from endothelial cells.

PURPOSE Adiponectin, an important adipocytokine secreted by adipocytes, has anti-inflammatory and atheroprotective effects on vascular tissue via the adiponectin receptor (adipoR). However, the action of adiponectin in the retinal microcirculation is unknown. We examined the direct effect and underlying mechanism of the vasomotor action of adiponectin in porcine retinal arterioles. METHODS Porcine retinal arterioles (internal diameter, 60-90 μm) were isolated, cannulated, and pressurized (55 cmH2O) without flow in this in vitro study. Videomicroscopic techniques were used to record changes in diameter in response to adiponectin. RESULTS The retinal arterioles dilated in a dose-dependent (0.125-7.5 μg/mL) manner in response to adiponectin. The vasodilation decreased significantly after removal of the endothelium. N(G)-nitro-L-arginine methyl ester (a nitric oxide [NO] synthase inhibitor), 1H-1,2,4-oxadia-zolo[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor), but not wortmannin (a phosphatidylinositol 3-kinase inhibitor) inhibited the effect of adiponectin-induced vasodilation comparable with that of denudation. Pretreatment with compound C, an activated protein kinase (AMPK) inhibitor, partially but significantly reduced vasodilation. Incubation with GW6471, a peroxisome proliferator-activated receptor blocker, did not significantly inhibit vasodilation by adiponectin. AdipoR1 and adipoR2 immunoreactions were observed in the endothelium of retinal arterioles. CONCLUSIONS Adiponectin elicits mainly endothelium-dependent dilation of the retinal arterioles. Endothelium-dependent vasodilation likely induced by adiponectin results from NO via activation of guanylyl cyclase that is partially dependent on AMPK activity. Understanding the effect of adiponectin on the retinal vasculature may help improve potential therapies for retinal vascular disorders, especially diabetic retinopathy in patients with type 2 diabetes mellitus.

Characteristics of Retinal Neovascularization in Proliferative Diabetic Retinopathy Imaged by Optical Coherence Tomography Angiography

Purpose To characterize the morphology of neovascularization at the disc (NVD) and neovascularization elsewhere (NVE) in treatment-naïve or previously treated proliferative diabetic retinopathy (PDR) patients using optical coherence tomography (OCT) angiography. Methods En face OCT angiograms of NVD/NVE in 40 eyes of 33 patients with PDR were acquired using RTVue XR Avanti OCT. The morphology of NVD/NVE on OCT angiograms was evaluated, and the activity was determined by biomicroscopy and fluorescein angiography (FA). In 12 eyes that were treated or treatment-naïve, changes in the morphology and vessel area of NVD/NVE before and after panretinal photocoagulation (PRP) were investigated. Results Twenty eyes had treatment-naïve PDR, whereas 20 eyes were previously treated with PRP. All treatment-naïve NVD/NVE had remarkable (i.e., active) leakage in early-phase FA. Ninety-five percent of treatment-naïve NVD/NVE observed by OCT angiography had exuberant vascular proliferation (EVP), identified as irregular proliferation of fine (smaller-caliber) new vessels; whereas, the presence of EVP in previously treated eyes (13/20) was significantly less than in treatment-naïve eyes (65% vs. 95%, P = 0.043). The remaining seven treated eyes had pruned NVD/NVE without EVP, observed as fibrotic changes or faint (inactive) leakage in FA. The vessel areas of NVD/NVE significantly decreased following PRP (n = 12, P = 0.019), and NVD/NVE morphology showed pruning and decreased EVP. Conclusions Exuberant vascular proliferation on OCT angiograms should be considered as an active sign of neovascularization; therefore, morphologic evaluation of neovascularization using OCT angiography may be useful to estimate the activity of each neovascularization in eyes with PDR.

Dilation of porcine retinal arterioles to cilostazol: roles of eNOS phosphorylation via cAMP/protein kinase A and AMP-activated protein kinase and potassium channels.

PURPOSE Cilostazol, a selective inhibitor of phosphodiesterase 3, has antiplatelet aggregation and peripheral vasodilation effects. We examined the effects of cilostazol on the retinal microvascular diameter to determine its dependence on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. METHODS Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded the diametric responses to cilostazol. RESULTS The retinal arterioles dilated in response to cilostazol in a dose-dependent (100 pM-10 μM) manner; the dilation decreased by 60% after endothelial removal. The nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited cilostazol-induced vasodilation comparable to denudation. Inhibition of soluble guanylyl cyclase and blockade of protein kinase A (PKA) were comparable to L-NAME. Compound C, an AMP-activated protein kinase (AMPK) inhibitor, partially inhibited cilostazol-induced vasodilation, which exhibited a weaker inhibitory effect on cilostazol-induced vasodilation than blockade of PKA. The large-conductance Ca²⁺-activated K channel (BK(Ca) channel) blocker, iberiotoxin, also inhibited cilostazol-induced vasodilation. The residual vasodilation decreased further with co-administration of L-NAME and iberiotoxin. CONCLUSIONS Cilostazol elicits endothelium-dependent and -independent dilation of the retinal arterioles mediated by NO release and BK(Ca) channel activation, respectively. Endothelial nitric oxide synthase (eNOS) phosphorylation via the cAMP/PKA and AMPK pathways and consequent activation of the soluble guanylyl cyclase/cyclic guanosine monophosphate pathway might play an important role in cilostazol-induced vasodilation of the retinal arterioles.

Association Between Diabetic Retinopathy and Flow-Mediated Vasodilation in Type 2 DM

Objective: Retinal endothelial dysfunction is a key in the etiogenesis of diabetic retinopathy (DR), in patients with type 2 diabetes mellitus (DM). Brachial artery flow-mediated vasodilation (FMD) is a marker of endothelial function associated with production of endogenous nitric oxide. Using FMD, we investigated the relationship between macrovascular function and DR. Methods: We studied 74 patients with type 2 DM, including non-DR (NDR) (n = 30); mild nonproliferative DR (NPDR) (n = 16); moderate NPDR (n = 10); severe NPDR (n = 10); and proliferative DR (PDR) (n = 8); and 21 age-matched controls. We measured FMD in each group. Retinal blood flow and pulsatility ratios were measured using laser Doppler velocimetry. Results: FMD decreased significantly in patients with DM compared with healthy control subjects. No significant differences were found in FMD among the NDR, mild NPDR, and moderate NPDR groups. FMD decreased significantly in the severe NPDR and PDR groups compared with the NDR group. FMD was significantly and negatively correlated with duration of DM and pulsatility ratio. Conclusion: Systemic endothelial dysfunction appears to be associated with DR and vascular abnormalities in patients with type 2 DM.

Homocysteine inhibition of endothelium-dependent nitric oxide-mediated dilation of porcine retinal arterioles via enhanced superoxide production.

PURPOSE Elevated plasma concentration of homocysteine, a sulfur-containing amino acid, is an emerging risk factor for cardiovascular diseases. Recent epidemiologic studies have confirmed that elevated homocysteine levels are associated with ocular vascular diseases; however, the direct effect of homocysteine on ocular microvascular reactivity remains unknown. We investigated whether homocysteine affects endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and whether oxidative stress and distinct protein kinase signaling pathways are involved in the homocysteine-mediated effect. METHODS Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Diameter changes were recorded using videomicroscopy techniques. RESULTS Intraluminal treatment with homocysteine (1 mM, 180 minutes) significantly attenuated arteriolar dilation in response to the endothelium-dependent NO-mediated agonists bradykinin and A23187 but not in response to the endothelium-independent NO donor sodium nitroprusside. In the presence of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), the nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H oxidase inhibitor apocynin, p38 kinase inhibitor SB203580, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist pioglitazone, the detrimental effect of homocysteine on bradykinin-induced dilation was prevented; however, neither the xanthine oxidase inhibitor allopurinol, the JNK inhibitor SP600125, or pioglitazone with PPAR-γ inhibitor GW9662 had that effect. CONCLUSIONS Homocysteine inhibits endothelium-dependent NO-mediated dilation in the retinal arterioles by producing superoxide from NAD(P)H oxidase, which appears to be linked with p38 kinase. By impairing endothelium-dependent NO-mediated vasoreactivity, homocysteine potentially facilitates development of retinal vascular diseases. In addition, pioglitazone can prevent homocysteine-induced endothelial dysfunction possibly by activating PPAR-γ.

Role of Ca2+-dependent and Ca2+-sensitive mechanisms in sphingosine 1-phosphate-induced constriction of isolated porcine retinal arterioles in vitro

Although sphingosine 1-phosphate (S1P), a bioactive lipid derived from activated platelets, has a variety of physiologic effects on vessels, no reports have described the effect of S1P on the retinal circulation. We examined the effect and underlying mechanism of the vasomotor action of S1P on porcine retinal arterioles. The porcine retinal arterioles were isolated, cannulated, and pressurized without flow for in vitro study. S1P-induced diameter changes were recorded using videomicroscopic techniques. S1P elicited concentration-dependent (1 nM-10 μM) vasoconstriction of the retinal arterioles that was abolished by the S1P receptor 2 (S1PR2) antagonist JTE-013. S1P-induced vasoconstriction was abolished by the Rho kinase (ROCK) inhibitor H-1152 and was inhibited partly by the protein kinase C (PKC) inhibitor Gö-6983. The inhibition of phospholipase C by U73122 and L-type voltage-operated calcium channels (L-VOCCs) by nifedipine inhibited S1P-induced vasoconstriction; a combination of both inhibitors abolished S1P-induced vasoconstriction. Furthermore, inhibition of myosin light chain kinase (MLCK) by ML-9 significantly blocked S1P-induced vasoconstriction; further coadministration of ML-9 with H-1152 or Gö-6983 abolished S1P-induced vasoconstriction. The current data suggest that S1P elicits vasoconstriction of the retinal arterioles via S1PR2 in vascular smooth muscle cells and this vasoconstriction may be mediated by the Ca2+ -sensitive pathway via activation of PKC leading to activation of ROCK and the Ca2+ -dependent pathway via activation of L-VOCCs resulting in activation of MLCK.

Relationship Between Retinal Blood Flow and Serum Adiponectin Concentrations in Patients With Type 2 Diabetes Mellitus

PURPOSE To study the relationship between retinal microcirculation and serum adiponectin, an important adipocytokine secreted by adipocytes, concentrations in patients with type 2 diabetes mellitus. METHODS Using a laser Doppler velocimetry system, we simultaneously measured the retinal blood flow (RBF) values and retinal vessel diameter and blood velocity in 64 consecutive Japanese patients (mean age ± SD, 59.8 ± 10.4 years) with type 2 diabetes with no or mild nonproliferative diabetic retinopathy. We compared the values with the RBF and serum adiponectin concentrations in these patients. The patients were divided into two groups based on sex (33 males, 31 females). RESULTS The plasma adiponectin concentrations were correlated positively with the retinal vessel diameter (r = 0.480; P = 0.005), retinal blood velocity (r = 0.399; P = 0.02), and RBF (r = 0.518; P = 0.002) and correlated negatively with the retinal arterial vascular resistance (r = -0.598; P = 0.0002) in males, but not females, with type 2 diabetes with early-stage diabetic retinopathy. Multiple regression analysis showed that the plasma adiponectin level was independently and positively correlated with RBF and negatively correlated with retinal arterial vascular resistance. CONCLUSIONS Our results indicated that a high concentration of serum adiponectin may be associated with increased RBF, probably via the increased blood velocity and dilated vessel diameter in males with type 2 diabetes with early-phase diabetic retinopathy.

Imaging of the Corneal Subbasal Whorl-like Nerve Plexus: More Accurate Depiction of the Extent of Corneal Nerve Damage in Patients With Diabetes

PURPOSE To show that noninvasive in vivo corneal confocal microscopy (IVCM) can make more accurate imaging of the corneal subbasal nerve plexus possible. This diagnostic technique monitors the status of diabetic peripheral neuropathy. However, it is difficult to accurately confirm the corneal area captured by IVCM, which can induce measurement errors. Because the whorl-like characteristic pattern of the corneal subbasal nerve plexus is in the inferocentral cornea, we evaluated whether IVCM images of the whorl-like patterns can accurately evaluate the corneal nerve fibers in diabetic neuropathy. METHODS Forty-seven patients with diabetes (DM group) and 21 healthy control subjects underwent IVCM examination to compare the characteristics of the corneal subbasal nerve plexus around the central cornea (conventional method) and the whorl-like pattern in the inferocentral cornea (study method). We measured the total corneal nerve fiber and branch length (CNFL). RESULTS The total CNFL were significantly shorter in the DM group than in the control group and tended to decrease with progression of diabetic retinopathy, nephropathy, neuropathy, and decreased corneal sensation. There was a significant positive correlation between the CNFL values obtained with the conventional method and those obtained with the study method. The coefficient of variation of the CNFL values in the study method was significantly smaller than in the conventional method. CONCLUSIONS Our findings indicated that IVCM measurements of the whorl-like patterns may accurately define the extent of corneal nerve damage in order to monitor diabetic peripheral neuropathy.