Lloyd Paul Aiello

Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders

BACKGROUND Retinal ischemia induces intraocular neovascularization, which often leads to glaucoma, vitreous hemorrhage, and retinal detachment, presumably by stimulating the release of angiogenic molecules. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific angiogenic factor whose production is increased by hypoxia. METHODS We measured the concentration of VEGF in 210 specimens of ocular fluid obtained from 164 patients undergoing intraocular surgery, using both radioimmuno-assays and radioreceptor assays. Vitreous proliferative potential was measured with in vitro assays of the growth of retinal endothelial cells and with VEGF-neutralizing antibody. RESULTS VEGF was detected in 69 of 136 ocular-fluid samples from patients with diabetic retinopathy, 29 of 38 samples from patients with neovascularization of the iris, and 3 of 4 samples from patients with ischemic occlusion of the central retinal vein, as compared with 2 of 31 samples from patients with no neovascular disorders (P < 0.001, P < 0.001, and P = 0.006, respectively). The mean (+/- SD) VEGF concentration in 70 samples of ocular fluid from patients with active proliferative diabetic retinopathy (3.6 +/- 6.3 ng per milliliter) was higher than that in 25 samples from patients with nonproliferative diabetic retinopathy (0.1 +/- 0.1 ng per milliliter, P = 0.008), 41 samples from patients with quiescent proliferative diabetic retinopathy (0.2 +/- 0.6 ng per milliliter, P < 0.001), or 31 samples from nondiabetic patients (0.1 +/- 0.2 ng per milliliter, P = 0.003). Concentrations of VEGF in vitreous fluid (8.8 +/- 9.9 ng per milliliter) were higher than those in aqueous fluid (5.6 +/- 8.6 ng per milliliter, P = 0.033) in all 10 pairs of samples obtained simultaneously from the same patient; VEGF concentrations in vitreous fluid declined after successful laser photocoagulation. VEGF stimulated the growth of retinal endothelial cells in vitro, as did vitreous fluid containing measurable VEGF. Stimulation was inhibited by VEGF-neutralizing antibodies. CONCLUSIONS Our data suggest that VEGF plays a major part in mediating active intraocular neovascularization in patients with ischemic retinal diseases, such as diabetic retinopathy and retinal-vein occlusion.

Randomized Trial Evaluating Ranibizumab Plus Prompt or Deferred Laser or Triamcinolone Plus Prompt Laser for Diabetic Macular Edema

OBJECTIVE Evaluate intravitreal 0.5 mg ranibizumab or 4 mg triamcinolone combined with focal/grid laser compared with focal/grid laser alone for treatment of diabetic macular edema (DME). DESIGN Multicenter, randomized clinical trial. PARTICIPANTS A total of 854 study eyes of 691 participants with visual acuity (approximate Snellen equivalent) of 20/32 to 20/320 and DME involving the fovea. METHODS Eyes were randomized to sham injection + prompt laser (n=293), 0.5 mg ranibizumab + prompt laser (n=187), 0.5 mg ranibizumab + deferred (> or =24 weeks) laser (n=188), or 4 mg triamcinolone + prompt laser (n=186). Retreatment followed an algorithm facilitated by a web-based, real-time data-entry system. MAIN OUTCOME MEASURES Best-corrected visual acuity and safety at 1 year. RESULTS The 1-year mean change (+/-standard deviation) in the visual acuity letter score from baseline was significantly greater in the ranibizumab + prompt laser group (+9+/-11, P<0.001) and ranibizumab + deferred laser group (+9+/-12, P<0.001) but not in the triamcinolone + prompt laser group (+4+/-13, P=0.31) compared with the sham + prompt laser group (+3+/-13). Reduction in mean central subfield thickness in the triamcinolone + prompt laser group was similar to both ranibizumab groups and greater than in the sham + prompt laser group. In the subset of pseudophakic eyes at baseline (n=273), visual acuity improvement in the triamcinolone + prompt laser group appeared comparable to that in the ranibizumab groups. No systemic events attributable to study treatment were apparent. Three eyes (0.8%) had injection-related endophthalmitis in the ranibizumab groups, whereas elevated intraocular pressure and cataract surgery were more frequent in the triamcinolone + prompt laser group. Two-year visual acuity outcomes were similar to 1-year outcomes. CONCLUSIONS Intravitreal ranibizumab with prompt or deferred laser is more effective through at least 1 year compared with prompt laser alone for the treatment of DME involving the central macula. Ranibizumab as applied in this study, although uncommonly associated with endophthalmitis, should be considered for patients with DME and characteristics similar to those in this clinical trial. In pseudophakic eyes, intravitreal triamcinolone + prompt laser seems more effective than laser alone but frequently increases the risk of intraocular pressure elevation.

Vascular Endothelial Growth Factor–Induced Retinal Permeability Is Mediated by Protein Kinase C In Vivo and Suppressed by an Orally Effective β-Isoform–Selective Inhibitor

Increased vascular permeability and excessive neovas-cularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms α, βII, and δ and > threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the β-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC β-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC β-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-asso-ciated ocular disorders such as diabetic retinopathy.

Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGFs mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.

Photocoagulation Treatment of Proliferative Diabetic Retinopathy: The Second Report of Diabetic Retinopathy Study Findings

Data from the Diabetic Retinopathy Study (DRS) show that photocoagulad inhibited the progression of retinopathy. These beneficial effects were noted to some degree in all those stages of diabetic retinopathy which were included in the Study. Some deleterious effects of treatment were also found, including losses of visual acuity and constriction of peripheral visual field. The risk of these harmful effects was considered acceptable in eyes with retinopathy in the moderate or severe retinopathy in the moderate or severe proliferative stage when the risk of severe visual loss without treatment was great. In early proliferative or severe nonproliferative retinopathy, when the risk of severe visual loss without treatment was less, the risks of harmful treatment effects assumed greater importance. In these earlier stages, DRS findings have not led to a clear choice between prompt treatment and deferral of treatment unless and until progression to a more severe stage occurs.

Aflibercept, bevacizumab, or ranibizumab for diabetic macular edema.

BACKGROUND The relative efficacy and safety of intravitreous aflibercept, bevacizumab, and ranibizumab in the treatment of diabetic macular edema are unknown. METHODS At 89 clinical sites, we randomly assigned 660 adults (mean age, 61±10 years) with diabetic macular edema involving the macular center to receive intravitreous aflibercept at a dose of 2.0 mg (224 participants), bevacizumab at a dose of 1.25 mg (218 participants), or ranibizumab at a dose of 0.3 mg (218 participants). The study drugs were administered as often as every 4 weeks, according to a protocol-specified algorithm. The primary outcome was the mean change in visual acuity at 1 year. RESULTS From baseline to 1 year, the mean visual-acuity letter score (range, 0 to 100, with higher scores indicating better visual acuity; a score of 85 is approximately 20/20) improved by 13.3 with aflibercept, by 9.7 with bevacizumab, and by 11.2 with ranibizumab. Although the improvement was greater with aflibercept than with the other two drugs (P<0.001 for aflibercept vs. bevacizumab and P=0.03 for aflibercept vs. ranibizumab), it was not clinically meaningful, because the difference was driven by the eyes with worse visual acuity at baseline (P<0.001 for interaction). When the initial visual-acuity letter score was 78 to 69 (equivalent to approximately 20/32 to 20/40) (51% of participants), the mean improvement was 8.0 with aflibercept, 7.5 with bevacizumab, and 8.3 with ranibizumab (P>0.50 for each pairwise comparison). When the initial letter score was less than 69 (approximately 20/50 or worse), the mean improvement was 18.9 with aflibercept, 11.8 with bevacizumab, and 14.2 with ranibizumab (P<0.001 for aflibercept vs. bevacizumab, P=0.003 for aflibercept vs. ranibizumab, and P=0.21 for ranibizumab vs. bevacizumab). There were no significant differences among the study groups in the rates of serious adverse events (P=0.40), hospitalization (P=0.51), death (P=0.72), or major cardiovascular events (P=0.56). CONCLUSIONS Intravitreous aflibercept, bevacizumab, or ranibizumab improved vision in eyes with center-involved diabetic macular edema, but the relative effect depended on baseline visual acuity. When the initial visual-acuity loss was mild, there were no apparent differences, on average, among study groups. At worse levels of initial visual acuity, aflibercept was more effective at improving vision. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01627249.).

Risks of intravitreous injection: a comprehensive review.

Purpose: To evaluate the prevalence of the most common serious adverse events associated with intravitreous (IVT) injection. Methods: A systematic search of the literature via PubMed from 1966 to March 1, 2004, was conducted to identify studies evaluating the safety of IVT injection. Data submitted in New Drug Applications to the U.S. Food and Drug Administration for drugs administered into the vitreous were included where available. Serious adverse events reported in each study were recorded, and risk per eye and risk per injection were calculated for the following serious adverse events: endophthalmitis, retinal detachment, iritis/uveitis, intraocular hemorrhage, ocular hypertension, cataract, and hypotony. Rare complications also were noted. Results: Data from 14,866 IVT injections in 4,382 eyes were analyzed. There were 38 cases of endophthalmitis (including those reported as pseudoendophthalmitis) for a prevalence of 0.3% per injection and 0.9% per eye. Excluding cases reported specifically as pseudoendophthalmitis, the prevalence of endophthalmitis was 0.2% per injection and 0.5% per eye. Retinal detachment, iritis/uveitis, ocular hypertension, cataract, intraocular hemorrhage, and hypotony were generally associated with IVT injection of specific compounds and were infrequently attributed by the investigators to the injection procedure itself. Retinal vascular occlusions were described rarely in patients after IVT injection, and it was unclear in most cases whether these represented true injection-related complications or chance associations. Conclusion: The risk of serious adverse events reported after IVT injection is low. Nevertheless, careful attention to injection technique and appropriate postinjection monitoring are essential because uncommon injection-related complications may be associated with permanent vision loss.

A Randomized Trial Comparing Intravitreal Triamcinolone Acetonide and Focal/Grid Photocoagulation for Diabetic Macular Edema

OBJECTIVE To evaluate the efficacy and safety of 1-mg and 4-mg doses of preservative-free intravitreal triamcinolone in comparison with focal/grid photocoagulation for the treatment of diabetic macular edema (DME). DESIGN Multicenter, randomized clinical trial. PARTICIPANTS Eight hundred forty study eyes of 693 subjects with DME involving the fovea and with visual acuity of 20/40 to 20/320. METHODS Eyes were randomized to focal/grid photocoagulation (n = 330), 1 mg intravitreal triamcinolone (n = 256), or 4 mg intravitreal triamcinolone (n = 254). Retreatment was given for persistent or new edema at 4-month intervals. The primary outcome was evaluated at 2 years. MAIN OUTCOME MEASURES Visual acuity measured with the electronic Early Treatment Diabetic Retinopathy Study method (primary), optical coherence tomography-measured retinal thickness (secondary), and safety. RESULTS At 4 months, mean visual acuity was better in the 4-mg triamcinolone group than in either the laser group (P<0.001) or the 1-mg triamcinolone group (P = 0.001). By 1 year, there were no significant differences among groups in mean visual acuity. At the 16-month visit and extending through the primary outcome visit at 2 years, mean visual acuity was better in the laser group than in the other 2 groups (at 2 years, P = 0.02 comparing the laser and 1-mg groups, P = 0.002 comparing the laser and 4-mg groups, and P = 0.49 comparing the 1-mg and 4-mg groups). Treatment group differences in the visual acuity outcome could not be attributed solely to cataract formation. Optical coherence tomography results generally paralleled the visual acuity results. Intraocular pressure increased from baseline by 10 mmHg or more at any visit in 4%, 16%, and 33% of eyes in the 3 treatment groups, respectively, and cataract surgery was performed in 13%, 23%, and 51% of eyes in the 3 treatment groups, respectively. CONCLUSIONS Over a 2-year period, focal/grid photocoagulation is more effective and has fewer side effects than 1-mg or 4-mg doses of preservative-free intravitreal triamcinolone for most patients with DME who have characteristics similar to the cohort in this clinical trial. The results of this study also support that focal/grid photocoagulation currently should be the benchmark against which other treatments are compared in clinical trials of DME.

Cost-Effectiveness of Detecting and Treating Diabetic Retinopathy

Diabetic eye disease, which leads to macular edema (maculopathy) and retinal neovascularization (retinopathy), is one of the most common vascular complications of diabetes mellitus and represents the leading cause of new cases of blindness among persons of working age [1-3]. Recently published national, multicenter, prospective trials have shown that properly timed laser photocoagulation substantially reduces the likelihood of blindness in persons with sight-threatening diabetic retinopathy [4-7]. We and others have shown that timely detection and treatment of retinopathy in Americans with insulin-dependent (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM) result in considerable savings for the federal budget and, in some cases, for health payer organizations [8-13]. Preventive eye care programs for diabetic persons have been proposed by the National Eye Institute [14], the Centers for Disease Control and Prevention [15], and the American Academy of Ophthalmology [16]. The importance of ophthalmologic screening for diabetic persons is highlighted by its inclusion in the Health Plan Employer Data and Information Set (HEDIS II; National Committee for Quality Assurance, Washington, D.C.) adopted throughout the managed care industry. Increasingly, a cost-effectiveness analysis based on dollars spent per quality-adjusted life-year (QALY) saved has been advocated as a strategy for societal health care allocation decisions. In this report, we evaluate the potential cost-effectiveness of screening, recruitment, and treatment programs for diabetic eye disease. Unlike our previous analyses, which dealt specifically with cost savings to the federal budget, this analysis is expressed in terms of cost-effectiveness from the perspective of health insurers. Methods Model Structure We used the PROPHET (Prospective Population Health Event Tabulation) Modeling System detailed in previous articles [8-10]. The PROPHET model is an epidemiology-based net-work simulation program designed for modeling the progression of a chronic irreversible disease. The model is based on Monte Carlo simulation and analyzes events and costs incurred during the course of an irreversible chronic disease; it considers each patient individually. Monte Carlo simulation allows a simple, probability-based solution, using random number generation, of complex disease progression processes over time that would otherwise need to be expressed as multivariate differential equations. For instance, if there is a 1% likelihood of a person in the model progressing to proliferative retinopathy over the next time interval, the simulation software draws a random number between 0 and 1. If that number is 0.01 or less, the person in question is recorded as having developed proliferative retinopathy. If the random number is greater than 0.01, that person remains in the baseline state. The simulation begins with a theoretical cohort representing all Americans in a given age group who develop diabetes in a single year. A complete set of clinical assumptions used in the model is shown in the Appendix. Data on incidence of diabetes, treatment with insulin, progression of diabetic eye disease, and vision loss are drawn from cross-sectional and longitudinal studies [17-24]. Outcomes of laser photocoagulation for proliferative retinopathy and macular edema are based on published clinical trial results. In the Diabetic Retinopathy Study, panretinal photocoagulation was initially reported to reduce the likelihood of severe vision loss by 60% as compared with no treatment. However, many patients enrolled in this study had advanced disease and, hence, a worse outcome than patients identified for care today. When the published results of panretinal photocoagulation in the Early Treatment Diabetic Retinopathy Study are compared with the no treatment group of the Diabetic Retinopathy Study, an 84% reduction in progression to severe vision loss is found (1.48% as compared with 8.7%). We believe this to be a conservative finding because reanalysis of the primary data suggests that the treatment effect may be over 90% [25]. In the case of treatment for macular edema, published data have used the clinically relevant end point of doubling the visual angle rather than the end point of legal blindness, which determines eligibility for federally funded insurance and entitlement programs. However, additional data are available from the Early Treatment Diabetic Retinopathy Study, which used a visual acuity end point (that is, the proportion of eyes with vision worse than 20/100) corresponding more exactly to current definitions of legal blindness [26]. The relative benefit of treatment as compared with no treatment is assumed to be permanent, based on two 15-year follow-up studies of patients treated with photocoagulation. In applying these data, we do not assume that successfully treated persons will not lose vision but rather that the reduction in risk for vision loss associated with treatment is constant over time [27, 28]. In our model, each person is assessed for disease progression and mortality by age [29], disease duration, and disease severity [30-33]. Persons being screened, according to the preferred practice pattern for diabetic retinopathy of the American Academy of Ophthalmology [34], are assessed for the presence of proliferative retinopathy and macular edema using published sensitivities for ophthalmic examination [35, 36]. Those persons predicted to have proliferative retinopathy or clinically significant macular edema detected by screening are next assessed for treatment outcome. Treatment failures are determined, net costs and benefits during the cycle are tabulated, and the cycle repeats itself at 2-month intervals throughout the lifetime of the cohort. Monte Carlo simulation is used for determining outcome of all events in each cycle, including disease progression, disease detection, treatment outcome, and mortality [37]. The remaining assumptions about disease progression and outcome of treatment are unchanged from our previous reports. Incident Cases The total number of incident cases of diabetes mellitus in the U.S. population is derived from previously published age-specific incident rates [17]. When applied to age-specific 1988 U.S. population figures [38], as shown in Table 1, these rates predict that 576 136 persons will develop NIDDM in the United States annually. This estimate is consistent with previous predictions [39, 40]. Table 1. Age-specific Annual Incident Cases of IDDM and NIDDM in the United States* Expenditures Screening and treatment costs are derived from the average Medicare charges in 1990 [41]. Although costs of care may vary among insurers, the Medicare fee schedule is increasingly becoming the basis for computing medical care reimbursement for many indemnity and managed care insurance plans. To the extent that actual costs of care are less than the charges used as inputs in this model, ophthalmologic screening for diabetic persons will be more cost-effective than predicted in our analysis. All costs are expressed in 1990 U.S. dollars using a discount rate of 5%, based on published techniques [42-48]. Some debate remains among health economists about whether investments in health care should be subjected to present value analysis (that is, discounted) just like any other financial investment. The purpose of discounting is to account for the time-value of money or universal preference for funds now rather than funds later. This value is most appropriately measured by common borrowing costs over and above inflation. Public investments are often discounted at 4% to 5% because it is the rate over inflation at which governments typically borrow funds (that is, sell bonds). The justification for discounting an investment such as screening for diabetic eye disease is that the investment value will be realized only over a period of years, whereas alternative investments in health carefor example, prenatal care or childhood vaccinationmay be realized over a shorter term. For this reason, we have discounted person-years of sight and QALYs by 5% (except in Table 3 where comparability to other published cost-effectiveness figures is needed). As can be seen from Table 2 and Table 3, applying discounted values is a more conservative approach because discounting reduces the predicted cost-effectiveness of the proposed intervention. Table 2. Cost-effectiveness of Treating Diabetic Eye Disease* Table 3. Comparative Cost-Utility Ratios* Screening Implementation The estimated present implementation of appropriate screening in patients with NIDDM is derived from analysis of the studies by Klein and coworkers [49] and Witkin and Klein [50], who found that 40% of these patients in Wisconsin were not meeting the suggested guidelines for ophthalmic care. Analysis of the 1989 National Health Interview Survey suggests that 50% of persons with self-reported diabetes reported no dilated eye examination in the past year [51]. Consequently, we have chosen 60% as an estimate for the prevailing implementation of appropriate eye care in patients with NIDDM. We assumed that screening under an ideal program is done according to the preferred practice pattern for diabetic retinopathy published by the American Academy of Ophthalmology [34], which suggests using dilated ophthalmoscopy annually for patients with no retinopathy and examination every 6 months for those with retinopathy. This pattern is consistent with recommendations published by the American College of Physicians and the American Diabetes Association [52]. Assignment of Quality-Adjusted Life-Years to Outcomes In 1989, 74.4% of 12 500 legally blind persons were successfully rehabilitated, and 25.6% were considered poorly adjusted after rehabilitation efforts (Avery C, U.S. Department of Education. Personal communication). Drummond [53] estimated that a year of blindness for well-adjusted and poorly adjusted persons

Evolving guidelines for intravitreous injections.

Intravitreous (IVT) injection is increasingly being incorporated into the management of ocular diseases. While only fomivirsen sodium (Vitravene™) is currently approved by the Food and Drug Administration as an IVT injection, the number of approved IVT injections indications is anticipated to grow on the basis of promising results from ongoing clinical studies. Despite the potential benefits that may be derived from intraocular injections of different agents, no guidelines have been published previously for IVT injection. The purpose of this document is to identify specific strategies for the delivery of IVT injection that may reduce risks and improve outcomes. Consensus was sought among a panel of investigators, surgeons experienced with this technique, and industry representatives. Objective evidence was sought for all guidelines, but consensus was accepted where evidence remains incomplete. In the absence of either evidence or consensus, the current manuscript identifies outstanding issues in need of further investigation. It is anticipated that more complete guidelines will evolve over time, potentially altering some of the guidelines included here, based on new applications of IVT injection, additional clinical experience, and results of clinical trials.