VEGF rescues cigarette smoking-induced human RPE cell death by increasing autophagic flux: implications of the role of autophagy in advanced age-related macular degeneration.

PURPOSE: Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated. METHODS: Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases. RESULTS: Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 µM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk. CONCLUSIONS: Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.

In vivo detection of acute ischemic damages in retinal arterial occlusion with optical coherence tomography: a "prominent middle limiting membrane sign".

PURPOSE: To describe characteristic findings of acute retinal ischemic damage in optical coherence tomography. METHODS: Eighteen cases of acute retinal arterial occlusion with available fundus photography, optical coherence tomography, and/or fluorescein angiography in the early phase (<1 month) with more than 2 months follow-up were reviewed. A site-to-site analysis between optical coherence tomography morphology and correlating fundus images were done on each visit. RESULTS: Retinal opacities at first presentation were vague to mild opacity in four eyes, moderate (affecting visibility of underlying choroidal vessels) in seven, severe (yellow to whitish) in five, and very severe (chalky white) in two. These changes eventually disappear within 1 month (8 of 9 eyes). Inner retinal hyperreflectivity and a "prominent middle limiting membrane" in optical coherence tomography were consistently noticed up to 1 month showing regional correlation with the retinal opaque areas and was readily identified even in areas with vague or disappeared retinal opacities. Later, inner retinal atrophic changes replace these ischemic optical coherence tomography signs. CONCLUSION: A prominent middle limiting membrane sign is a useful indicator of acute ischemic retinal damage, especially in cases showing subtle or resolved retinal opacities before the onset of atrophic changes.

New insights into the pathoanatomy of diabetic macular edema: angiographic patterns and optical coherence tomography.

PURPOSE: To describe the pathoanatomy of diabetic macular edema in optical coherence tomography and its correlation with fluorescein angiography patterns. METHODS: Sixty eyes of 56 patients were analyzed. Diabetic macular edema was classified into typical focal leakage (from microaneurysm), typical diffuse leakage (the capillary plexus), or combined/questionable leakage using fluorescein angiography and retinal thickness profiles. The leakage and pooling patterns in fluorescein angiography were matched to the corresponding optical coherence tomography images and analyzed. RESULTS: Focal leakage shows swelling predominantly in the outer plexiform layer (OPL). Deeply located microaneurysms directly leak into the loose fiber portion of OPL (Henle layer) through the "fluid conductivity barrier" (synaptic portion of OPL). Diffuse leakage caused swelling predominantly in the inner nuclear layer and secondarily in the OPL. The deep capillary plexus is located between the two "fluid barriers" (inner plexiform layer and OPL); thus, diffuse leakage is primarily related with swelling in the inner nuclear layer. In the combined/questionable leakage, partial sections consisting of inner nuclear layer swelling and much larger areas of OPL/outer nuclear layer swelling are noticed. CONCLUSION: Based on the concept of the fluid conductivity barrier, we revealed a correlation between the intraretinal location of the leakage source and where the fluid accumulated within the retinal layers.

Vascular endothelial growth factor as an autocrine survival factor for retinal pigment epithelial cells under oxidative stress via the VEGF-R2/PI3K/Akt.

PURPOSE: Vascular endothelial cell growth factor (VEGF) is strongly induced by oxidative stress in retinal pigment epithelial (RPE) cells, and VEGF-A is a survival factor for various cell types. This study was conducted to determine whether the autocrine VEGF signaling pathway in RPE cells is involved in the mechanism of adaptive response to oxidative stress. METHODS: ARPE-19 cells were treated with hydrogen peroxide, and cell death was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Survival analysis was performed with pretreatment of VEGF-A-neutralizing antibodies, VEGF receptor tyrosine kinase inhibitor (SU5416), or VEGF-A receptor-neutralizing antibodies (anti-VEGF-R1 and anti-VEGF-R2). The expression of VEGF-A, -R1, -R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR or Western blot analysis. Phosphorylation of VEGF-R2 was detected with immunoprecipitation and immunoblot analysis. RESULTS: Hydrogen peroxide-induced cell death was promoted by pretreatment with VEGF-A and anti-VEGF-R2-neutralizing antibodies, but not with anti-VEGF-R1-neutralizing antibody. Phosphorylation of VEGF-R2 in RPE cells was induced by hydrogen peroxide, and pretreatment with anti-VEGF-A-neutralizing antibody inhibited phosphorylation. Phosphorylation of Akt under oxidative stress was abrogated by pretreatment with neutralizing antibodies against either VEGF-A or SU5416. CONCLUSIONS: Autocrine VEGF-A enhanced RPE cell survival under oxidative stress; the autocrine VEGF-A/VEGF-R2/PI3K/Akt pathway is involved. Neutralization of VEGF-A signaling, as in eyes with age-related macular degeneration, may influence RPE cell survival.

Foveal ganglion cell layer damage in ischemic diabetic maculopathy: correlation of optical coherence tomographic and anatomic changes.

PURPOSE: To describe the morphologic features of ischemic diabetic maculopathy by high-resolution optical coherence tomography (OCT) and their correlation with the damaged foveal avascular zone (FAZ) on fluorescein angiography (FA). DESIGN: Observational case series. PARTICIPANTS: One hundred twenty-four eyes of 63 patients with diabetic retinopathy and acceptable FA and OCT images were studied. Twenty-three normal fellow eyes of 23 nondiabetic patients with unilateral acute central serous choroidopathy also were studied. METHODS: High-speed Fourier-domain OCT was used with a speckle noise-reduction technique to obtain detailed horizontal and vertical images through the center of the fovea and horizontal raster scans every 100 microm. Foveal ganglion cell layer (GCL) damage was identified on OCT as an evident difference in foveal thickness and contour compared with a normal fovea or as asymmetry within the fovea. Fluorescein angiography was performed by confocal scanning laser ophthalmoscope (HRA 2; Heidelberg Engineering, Heidelberg, Germany), and FAZ damage visible during the FA arterial phase was graded according to the Early Treatment Diabetic Retinopathy Study (ETDRS) FA grading system. Correlations were sought between foveal GCL damage identified on OCT and FA capillary dropout sites. MAIN OUTCOME MEASURES: Foveal GCL damage on OCT, the size of the foveola on OCT (defined as the area of GCL thickness <10 microm), ETDRS grading of FAZ on FA, and visual acuity. RESULTS: Among the 124 eyes with diabetic retinopathy, 62 (50%) had FA evidence of either FAZ damage higher than grade 1 or FAZ capillary loss. In these eyes, damage to the FAZ seen on FA also could be detected on OCT (positive predictive value, 84.5%; negative predictive value, 72.9%), and locations of FAZ damage seen on FA corresponded well with sites of foveal GCL damage on OCT. In nondiabetic, normal eyes, the size of the foveola on OCT matched the size of the FAZ on FA. CONCLUSIONS: Evidence of foveal GCL damage on OCT is a good indicator of macular ischemic damage in eyes with diabetic retinopathy. Although in this study FA was more sensitive than OCT in detecting vascular damage, OCT provides objective results and seems to be a good noninvasive substitute for FA.

Surgical technique for suture fixation of a single-piece hydrophilic acrylic intraocular lens in the absence of capsule support.

We present a new intraocular lens (IOL) fixation technique that reduces the presence of intraocular knots and eliminates the need for scleral flaps while attaining excellent central positioning of the IOL. It also provides rapid visual rehabilitation from the insertion of a foldable IOL through a small corneal incision. This single-loop suture technique is an easy, convenient, and practical procedure, with minimum complications in the treatment of aphakia and the management of cataract surgery in eyes without capsular support.

Problems associated with the 25-gauge transconjunctival sutureless vitrectomy system during and after surgery.

OBJECTIVE: To report the initial experiences with the 25-gauge transconjunctival sutureless vitrectomy (TSV) system, including intraoperative and postoperative problems. METHODS: We retrospectively reviewed the medical records and surgical videotapes of 50 consecutive patients who underwent vitrectomy performed by one surgeon using the TSV system. RESULTS: Intraoperatively, we encountered such problems as difficulty in inserting the microcannula, which led to deformity, instability of the microcannula, self-disconnection of the infusion tip and resultant lens damage, and conversion to 20-gauge conventional vitrectomy. Postoperatively, there were 8 cases with hypotony (IOP < 6 mm Hg) on day 1, 6 cases with elevated IOP, and 3 cases with retinal detachment during follow-up. CONCLUSION: Though certain problems exist during and after surgeries using TSV, this system is both convenient and safe for various vitreoretinal procedures.

A case of inadvertent anterior chamber and corneal stromal injection with antibiotics during cataract operation.

PURPOSE: To report a case of inadvertent anterior chamber and cornea stromal injection with high dose antibiotics and steroids during cataract operation. METHODS: During cataract operation on a 78 year-old female patient, high dose gentamicin (20 mg/0.5 ml) and dexamethasone (2 mg/0.5 ml) were inadvertently injected into the anterior chamber and cornea stroma when making cornea edema for sealing of the incision sites. Anterior chamber irrigation with balanced salt solution (BSS) was immediately administered. On postoperative day one, extensive cornea edema was noted, and best-corrected visual acuity was 0.2. Descemet's membrane folds were observed around the corneal incision sites. Topical 5% NaCl and 1% prednisolone were started. RESULTS: Four weeks postoperatively, corneal edema began to reduce significantly. At four months postoperatively, corneal edema fully resolved, and best-corrected visual acuity was 0.8. However, some Descemet's membrane folds still remained, and a decrease in the number of endothelial cells was noted by specular microscope. CONCLUSIONS: In this case involving anterior chamber and cornea stromal injection with high dose antibiotics and steroids, immediate anterior chamber irrigation with balanced salt solution seemed an appropriate management, and the patient's long-term visual acuity appears good. To prevent such mistakes, precise labeling of all solutions and use of different syringe needles should be considered.