M. Elizabeth Hartnett

Three-year follow-up of a randomized trial comparing focal/grid photocoagulation and intravitreal triamcinolone for diabetic macular edema

OBJECTIVE To report 3-year outcomes of patients who participated in a randomized trial evaluating 1-mg and 4-mg doses of preservative-free intravitreal triamcinolone compared with focal/grid photocoagulation for treatment of diabetic macular edema. METHODS Eyes with diabetic macular edema and visual acuities of 20/40 to 20/320 were randomly assigned to focal/grid photocoagulation or 1 mg or 4 mg of triamcinolone. At the conclusion of the trial, 3-year follow-up data were available in 306 eyes. RESULTS Between 2 years (time of the primary outcome) and 3 years, more eyes improved than worsened in all 3 treatment groups. Change in visual acuity letter score from baseline to 3 years was +5 in the laser group and 0 in each triamcinolone group. The cumulative probability of cataract surgery by 3 years was 31%, 46%, and 83% in the laser and 1-mg and 4-mg triamcinolone groups, respectively. Intraocular pressure increased by more than 10 mm Hg at any visit in 4%, 18%, and 33% of eyes, respectively. CONCLUSIONS Results in a subset of randomized subjects who completed the 3-year follow-up are consistent with previously published 2-year results and do not indicate a long-term benefit of intravitreal triamcinolone relative to focal/grid photocoagulation in patients with diabetic macular edema similar to those studied in this clinical trial. Most eyes receiving 4 mg of triamcinolone as given in this study are likely to require cataract surgery. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00367133.

Plasma Complement Components and Activation Fragments: Associations with Age-Related Macular Degeneration Genotypes and Phenotypes

PURPOSE Several genes encoding complement system components and fragments are associated with age-related macular degeneration (AMD). This study was conducted to determine whether alterations in circulating levels of these markers of complement activation and regulation are also independently associated with advanced AMD and whether they are related to AMD genotypes. METHODS Plasma and DNA samples were selected from individuals in our AMD registry who had progressed to or developed the advanced stages of AMD, including 58 with geographic atrophy and 62 with neovascular disease. Subjects of similar age and sex, but without AMD, and who did not progress were included as controls (n = 60). Plasma complement components (C3, CFB, CFI, CFH, and factor D) and activation fragments (Bb, C3a, C5a, iC3b, and SC5b-9) were analyzed. DNA samples were genotyped for seven single-nucleotide polymorphisms in six genes previously shown to be associated with AMD: CFB, CFH, C2, C3, and CFI and the LOC387715/ARMS2 gene region. The association between AMD and each complement biomarker was assessed by using logistic regression, controlling for age, sex, and proinflammatory risk factors: smoking and body mass index (BMI). Functional genomic analyses were performed to assess the relationship between the complement markers and genotypes. Concordance, or C, statistics were calculated to assess the effect of complement components and activation fragments in an AMD gene-environment prediction model. RESULTS The highest quartiles of Bb and C5a were significantly associated with advanced AMD, when compared with the lowest quartiles. In multivariate models without genetic variants, the odds ratio (OR) for Bb was 3.3 (95% confidence interval [CI] = 1.3-8.6), and the OR for C5a was 3.6 (95% CI = 1.2-10.3). With adjustment for genetic variants, these ORs were substantially higher. The alternative pathway regulator CFH was inversely associated with AMD in the model without genotypes (OR = 0.3; P = 0.01). Positive associations were found between BMI and plasma C3, CFB, CFH, iC3b, and C3a. There were also significant associations between C5a fragment and LOC387715/ARMS2 and C3 genotypes (P for trend = 0.02, 0.04), respectively. C statistics for models with behavioral and genetic factors increased to 0.94 +/- 0.20 with the addition of C3a, Bb, and C5a. CONCLUSIONS Increased levels of activation fragments Bb and C5a are independently associated with AMD. Higher BMI is related to increased levels of complement components. C5a is associated with AMD genotypes. C statistics are stronger with the addition of C3a, Bb, and C5a in predictive models. Results implicate ongoing activation of the alternative complement pathway in AMD pathogenesis.

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Classification of retinal pigment epithelial detachments associated with drusen

To understand the natural history of retinal pigment epithelial (RPE) detachments (PEDs) associated with drusen, we retrospectively reviewed 125 PEDs that had not undergone laser treatment over an average follow-up period of 28 months. We identified six types of RPE detachments: pseudo-vitelliform, confluent drusen, serous, vascular, and hemorrhagic lesions as well as PEDs associated with a retinal vascular abnormality. We correlated the type of detachment with visual acuity and anatomic outcome. The poorest results were obtained for PEDs associated with neovascularization (vascular, hemorrhagic, and retinal vascular abnormality types). Eyes presenting with pseudo-vitelliform lesions or confluent drusen had the best visual prognosis and anatomic results. Obvious subretinal new vessels developed in 16 (34%) of the serous PEDs over an average follow-up period of 25 months. Subretinal neovascularization was rare in the pseudo-vitelliform and confluent drusen groups. The proposed classification system may be useful in predicting the natural course of PEDs.

Deep Retinal Vascular Anomalous Complexes in Advanced Age-related Macular Degeneration

PURPOSE The authors describe the clinical characteristics of a group of patients with age-related macular degeneration (AMD), deep retinal vascular anomalous complexes (RVACs), advanced Bruch membrane changes, and severe visual loss. Based on clinical evaluation and imaging studies, the authors hypothesize the cause of such retinal vascular formations. PATIENTS AND METHODS The authors quantified an initial case series of 6 patients and expanded it to 11 patients (14 eyes) with AMD and RVACs diagnosed by fluorescein angiography or slit-lamp examination. Associated pigment epithelial detachments (PEDs) of 13 eyes are described. In addition to the clinical and fluorescein angiography descriptions, infrared imaging and indocyanine green angiography were used to characterize more recently described RVACs and fellow eyes. RESULTS Each study eye had a clearly defined anastomosis connecting the retinal circulation to a vascular complex in the deep retina. The RVACs associated with PEDs assumed a more central location than did typical choroidal neovascularization associated with PEDs. In seven eyes with RVACs, there were clinically recognizable retinovascular findings: intraretinal hemorrhages, telangiectasia, or microaneurysms. Legal blindness occurred in 9 of 11 patients. CONCLUSION These results indicate that retinovascular changes can be associated with nondisciform AMD. The authors speculate that neurodegenerative changes and hypoxia may lead to such changes, the RVAC being a more advanced finding. Closure of an RVAC with photocoagulation is difficult, perhaps because of its higher blood flow. The visual outcome is poor, not only because of the advanced state of the underlying AMD, but also because of the exudative nature of the RVAC.

Pathophysiology and Mechanisms of Severe Retinopathy of Prematurity

Retinopathy of prematurity (ROP) affects only premature infants, but as premature births increase in many areas of the world, ROP has become a leading cause of childhood blindness. Blindness can occur from aberrant developmental angiogenesis that leads to fibrovascular retinal detachment. To treat severe ROP, it is important to study normal developmental angiogenesis and the stresses that activate pathologic signaling events and aberrant angiogenesis in ROP. Vascular endothelial growth factor (VEGF) signaling is important in both physiologic and pathologic developmental angiogenesis. Based on studies in animal models of oxygen-induced retinopathy (OIR), exogenous factors such as oxygen levels, oxidative stress, inflammation, and nutritional capacity have been linked to severe ROP through dysregulated signaling pathways involving hypoxia-inducible factors and angiogenic factors like VEGF, oxidative species, and neuroprotective growth factors to cause phases of ROP. This translational science review focuses on studies performed in animal models of OIR representative of human ROP and highlights several areas: mechanisms for aberrant growth of blood vessels into the vitreous rather than into the retina through over-activation of VEGF receptor 2 signaling, the importance of targeting different cells in the retina to inhibit aberrant angiogenesis and promote physiologic retinal vascular development, toxicity from broad and targeted inhibition of VEGF bioactivity, and the role of VEGF in neuroprotection in retinal development. Several future translational treatments are discussed, including considerations for targeted inhibition of VEGF signaling instead of broad intravitreal anti-VEGF treatment.

Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism

PURPOSE To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE). METHODS Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF(165) was added to cocultures of RPE and EC and the TER was measured. RESULTS RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0.05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0.03). CONCLUSIONS Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC-RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruchs membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.

Chloride Intracellular Channel Protein-4 Functions in Angiogenesis by Supporting Acidification of Vacuoles Along the Intracellular Tubulogenic Pathway

Endothelial cells form capillary tubes through the process of intracellular tubulogenesis. Chloride intracellular channel (CLIC) family proteins have been previously implicated in intracellular tubulogenesis, but their specific role has not been defined. In this study, we show that disruption of the Clic4 gene in mice results in defective angiogenesis in vivo as reflected in a Matrigel plug angiogenesis assay. An angiogenesis defect is also apparent in the retina, both in the decreased spontaneous development of retinal vasculature of unstressed mice and in the dramatically decreased angiogenic response of retinal vessels to an oxygen toxicity challenge. We found that endothelial cells derived from Clic4(-/-) mice demonstrated impaired tubulogenesis in three-dimensional fibrin gels compared with cells derived from wild-type mice. Furthermore, we found that tubulogenesis of wild-type cells in culture was inhibited by both an inhibitor of CLICs and an inhibitor of the vacuolar proton ATPase. Finally, we showed that vacuoles along the endothelial tubulogenesis pathway are acidic in wild-type cells, and that vacuolar acidification is impaired in Clic4(-/-) cells while lysosomal acidification is intact. We conclude that CLIC4 plays a critical role in angiogenesis by supporting acidification of vacuoles along the cell-hollowing tubulogenic pathway.

Activated NAD(P)H Oxidase from Supplemental Oxygen Induces Neovascularization Independent of VEGF in Retinopathy of Prematurity Model

PURPOSE To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity. METHODS Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity. RESULTS Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air. CONCLUSIONS Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.

Characteristics of Exudative Age-related Macular Degeneration Determined In Vivo with Confocal and Indirect Infrared Imaging

PURPOSE To evaluate the current and future interventions in age-related macular degeneration (AMD), it is essential to delineate the early clinical features associated with later visual loss. The authors describe the retinal pigment epithelium (RPE)/Bruch membrane region in ten patients with advance exudative AMD using current angiographic techniques and a noninvasive method: infrared (IR) imaging with the scanning laser ophthalmoscope. METHODS Ten patients with exudative AMD, evidence by choroidal neovascularization (CNV), fibrovascular scar formation, pigment epithelial detachment, or serous subretinal fluid,were examined using IR imaging, fluorescein angiography, indocyanine green angiography, and stereoscopic viewing of fundus slides. The authors determined the number and size of drusen and subretinal deposits and the topographic character of the RPE/Bruch membrane area and of CNV. RESULTS In all patients, IR imaging yielded the greatest number of drusen and subretinal deposits. Sheets of subretinal material, but few lesions consistent with soft drusen, were seen. Infrared imaging provided topographic information of evolving CNV. Choroidal neovascularization appeared as a complex with a dark central core, an enveloping reflective structure which created a halo-like appearance in the plane of focus, and outer retinal/subretinal striae. CONCLUSIONS Infrared imaging provides a noninvasive, in vivo method to image early changes in the RPE/Bruch membrane. It offers advantages over current imaging techniques by minimizing light scatter through cloudy media and enhancing the ability to image through small pupils, retinal hyperpigmentation, blood, heavy exudation, or subretinal fluid. It provides additional information regarding early CNV, and the character of drusen and subretinal deposits.