Gerard A. Lutty

Understanding age-related macular degeneration (AMD): Relationships between the photoreceptor/retinal pigment epithelium/Bruch’s membrane/choriocapillaris complex

There is a mutualistic symbiotic relationship between the components of the photoreceptor/retinal pigment epithelium (RPE)/Bruchs membrane (BrMb)/choriocapillaris (CC) complex that is lost in AMD. Which component in the photoreceptor/RPE/BrMb/CC complex is affected first appears to depend on the type of AMD. In atrophic AMD (~85-90% of cases), it appears that large confluent drusen formation and hyperpigmentation (presumably dysfunction in RPE) are the initial insult and the resorption of these drusen and loss of RPE (hypopigmentation) can be predictive for progression of geographic atrophy (GA). The death and dysfunction of photoreceptors and CC appear to be secondary events to loss in RPE. In neovascular AMD (~10-15% of cases), the loss of choroidal vasculature may be the initial insult to the complex. Loss of CC with an intact RPE monolayer in wet AMD has been observed. This may be due to reduction in blood supply because of large vessel stenosis. Furthermore, the environment of the CC, basement membrane and intercapillary septa, is a proinflammatory milieu with accumulation of complement components as well as proinflammatory molecules like CRP during AMD. In this toxic milieu, CC die or become dysfunction making adjacent RPE hypoxic. These hypoxic cells then produce angiogenic substances like VEGF that stimulate growth of new vessels from CC, resulting in choroidal neovascularization (CNV). The loss of CC might also be a stimulus for drusen formation since the disposal system for retinal debris and exocytosed material from RPE would be limited. Ultimately, the photoreceptors die of lack of nutrients, leakage of serum components from the neovascularization, and scar formation. Therefore, the mutualistic symbiotic relationship within the photoreceptor/RPE/BrMb/CC complex is lost in both forms of AMD. Loss of this functionally integrated relationship results in death and dysfunction of all of the components in the complex.

Relationship between RPE and choriocapillaris in age-related macular degeneration

PURPOSE The purpose of this study was to examine the relationships between choriocapillaris (CC) and retinal pigment epithelial changes in age-related macular degeneration (AMD). Morphologic changes in the retinal pigment epithelium (RPE)/choriocapillaris complex were quantified in dry and wet forms of AMD, and the results were compared with those in aged control eyes without maculopathy. METHODS Postmortem choroids from three aged control subjects, five subjects with geographic atrophy (GA), and three subjects with wet AMD were analyzed using a semiquantitative computer-assisted morphometric technique developed to measure the percentages of retinal pigment epithelial and CC areas in choroidal wholemounts incubated for alkaline phosphatase activity. The tissues were subsequently embedded in methacrylate and were sectioned so that structural changes could be examined. RESULTS There was a linear relationship between the loss of RPE and CC in GA. A 50% reduction in vascular area was found in regions of complete retinal pigment epithelial atrophy. Extreme constriction of remaining viable capillaries was found in areas devoid of RPE. Adjacent to active choroidal neovascularization (CNV) in wet AMD, CC dropout was evident in the absence of retinal pigment epithelial atrophy, resulting in a 50% decrease in vascular area. Lumenal diameters of the remaining capillaries in wet AMD eyes were similar to those in control eyes. CONCLUSIONS The primary insult in GA appears to be at the level of the RPE, and there is an intimate relationship between retinal pigment epithelial atrophy and secondary CC degeneration. CC degeneration occurs in the presence of viable RPE in wet AMD. The RPE in regions of vascular dropout are presumably hypoxic, which may result in an increase in VEGF production by the RPE and stimulation of CNV.

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate

PURPOSE Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN Experimental animal study. METHODS Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.

Increased nadh oxidase activity in the retina of the BBZ/WOR diabetic rat

This morphological study demonstrates a role for endothelial cells in generating reactive oxygen species in early stages of retinopathy in the BBZ/Wor rat, an obese, noninsulin dependent model of diabetes. Hyperglycemia induced pseudohypoxia results in an imbalance in cytosolic NADH/NAD+. In the oxygen-rich environment of the retina, NADH oxidase generates superoxide radical which is dismutated to hydrogen peroxide. Localization of hydrogen peroxide by the cerium NADH oxidase enzyme activity cytochemical localization technique shows a statistically significant increase of peroxide localization in the central retina of diabetic rats as compared to age-matched, nondiabetic controls. Endothelial cell dysfunction, indicated by leakage of endogenous serum albumin, coincided with areas of NADH oxidase activity localization. In diabetic rats there are increased levels of fibronectin in areas of hydrogen peroxide localization. This in vivo, morphological study is the first demonstration of oxidative injury and endothelial cell dysfunction in the retina of a spontaneous, noninsulin dependent model of diabetes.

Visualization of a developing vasculature

The events involved in vasculogenesis still remain obscure. One difficulty has been the techniques employed to visualize angioblasts, i.e., vascular precursors, during the genesis of blood vessels. The retina provides a unique model for studying these events since it is not completely vascularized in some mammals at birth. Using a previously published magnesium-dependent ATPase technique to visualize the developing retinal vasculature and its precursors, and embedding this tissue in JB-4 methacrylate for serial sectioning, has permitted examination of the retinal vasculogenic processes in dual perspective. The technique has permitted observation of the stages in angioblast differentiation and the apparent importance of glycosaminoglycan-rich cell-free spaces in this process. Perhaps the most important observation is that initial vessel formation occurs by coalescence of angioblasts after differentiation in situ.

Effects of diabetes on the eye.

Hyperglycemia has toxic effects on almost all cells in the body. Ophthalmic complications of hyperglycemia are most profound in cornea and retina. Seventy percent of diabetics suffer from corneal complications, collectively called diabetic keratopathy, which includes include recurrent erosions, delayed wound healing, ulcers, and edema. Confocal microscopy has permitted in vivo imaging of corneal nerves, which are also affected in diabetic subjects. Gene therapies upregulating MNNG HOS transforming gene (cMet) and/or downregulating MMP10 and cathepsin S are potential future therapies for diabetic keratopathy. Diabetic retinopathy (DR) is the most common cause of blindness in people over the age of 50. There is accumulating evidence that DR is an inflammatory disease. The initial events in animal models of DR are increased vascular permeability and leukostasis. This binding of leukocytes to the endothelium results from an increase in intracellular adhesion molecule-1 (ICAM-1) on the retinal capillary endothelium (EC) and expression of CD11/CD18 on the surface of the activated leukocyte. We have observed polymorphonuclear leukocytes (PMNs) at sites of EC vascular dysfunction in diabetic retinas as well as choroid. Anti-inflammatory drugs like etanercept, aspirin, or meloxicam reduce leukostasis and EC death. Future therapies may include repopulation of the acellular capillaries after EC and pericyte death with vascular progenitors made from the patients own blood cells.

Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium

Chitosan, PCEP (poly{[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate}), and magnetic nanoparticles (MNPs) were evaluated for the safe delivery of genes in the eye. Rabbits were injected with nanoparticles either intravitreally (IV) or subretinally (SR) and sacrificed 7 days later. Eyes were grossly evaluated for retinal pigment epithelium abnormalities, retinal degeneration, and inflammation. All eyes were cryopreserved and sectioned for analysis of toxicity and expression of either enhanced green or red fluorescent proteins. All of the nanoparticles were able to transfect cells in vitro and in vivo. IV chitosan showed inflammation in 12/13 eyes, whereas IV PCEP and IV MNPs were not inflammatory and did not induce retinal pathology. SR PCEP was nontoxic in the majority of cases but yielded poor transfection, whereas SR MNPs were nontoxic and yielded good transfection. Therefore, we conclude that the best nanoparticle evaluated in vivo was the least toxic nanoparticle tested, the MNP.

The acute intravenous toxicity of biological stains, dyes, and other fluorescent substances

Abstract Sodium fluorescein and indocyanine green are currently used as indicators for angiography of the retinal and choroidal vasculatures. This study was conducted to suggest additional indicator substances for this technique. Fifty-three dyes and biological stains were evaluated by the following criteria: intravenous LD50, histopathological damage from intravenous administration, binding properties in blood, in vivo metabolism of the substances, fluorescent and absorption spectra, and impurities detected with thin-layer chromatography. Male Swiss-Webster mice were used for the intravenous toxicity studies and for the metabolism and LD50 determinations. Several cyanines were modified by recrystallization with sodium iodide and an oxazine was sulfonated to improve their solubility and biocompatibility. Both techniques were successful in improving the intravenous toxicity and the solubility of the dyes in water. Iodination was not successful on the merocyanines. Patent blue VF and 8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium salt were promising since both had impurities yet their LD50s exceeded 1000 mg/kg. Fluorones with two salt sites (NaO and COONa) were soluble at 10% or more in water. Nonhalogenated fluorones were excreted totally in the urine, while those with eight halogen atoms were removed via the biliary system. Those fluorones with six or less halogen atoms were excreted by both systems. Twenty-three of the fifty-three dyes were tolerated well enough for use in angiography of the ocular fundus. The majority of the substances had at least one impurity. In general, the cationic dyes were poorly tolerated; only 2 of the 23 acceptable substances were cationic. Those dyes that were anionic or dipolar were the best tolerated, with the dipolar molecules being cleared, at least partially, by the liver.

Beyond the definitions of the phenotypic complications of sickle cell disease: an update on management.

The sickle hemoglobin is an abnormal hemoglobin due to point mutation (GAG → GTG) in exon 1 of the β globin gene resulting in the substitution of glutamic acid by valine at position 6 of the β globin polypeptide chain. Although the molecular lesion is a single-point mutation, the sickle gene is pleiotropic in nature causing multiple phenotypic expressions that constitute the various complications of sickle cell disease in general and sickle cell anemia in particular. The disease itself is chronic in nature but many of its complications are acute such as the recurrent acute painful crises (its hallmark), acute chest syndrome, and priapism. These complications vary considerably among patients, in the same patient with time, among countries and with age and sex. To date, there is no well-established consensus among providers on the management of the complications of sickle cell disease due in part to lack of evidence and in part to differences in the experience of providers. It is the aim of this paper to review available current approaches to manage the major complications of sickle cell disease. We hope that this will establish another preliminary forum among providers that may eventually lead the way to better outcomes.

C-reactive protein and complement factor H in aged human eyes and eyes with age-related macular degeneration

Background There is increasing evidence that inflammation and immune-mediated processes (complement activation) play an important role in age-related macular degeneration (AMD) pathogenesis. A genetic variation in the gene encoding complement factor H (CFH) and plasma levels of C-reactive protein (CRP), a systemic marker of subclinical inflammation, have consistently been shown to be associated with an increased risk for AMD. In the present study, we examined the immunolocalisation of CRP and CFH in aged control human donor eyes (n=10; mean age 79 years) and eyes with AMD (n=18; mean age 83 years). Methods Alkaline phosphatase immunohistochemistry was performed using polyclonal antibodies against CRP and CFH on cryopreserved tissue sections from disc/macular blocks. Three independent masked observers scored the reaction product (0–8). Results In aged control eyes, the retinal pigment epithelium/Bruchs membrane/choriocapillaris (RPE/BrM/CC) complex including intercapillary septa (ICS) had the most prominent immunostaining for CRP and CFH. CRP was significantly higher than controls in BrM/CC/ICS and choroidal stroma in early and wet AMD eyes (p<0.05). In contrast, CFH was significantly lower in BrM/CC/ICS complex of AMD choroids than in controls (p<0.05). Interestingly, CRP and CFH were significantly reduced in BrM/CC/ICS complex in atrophic area of macula in geographical atrophy (p<0.05). Drusen and basal laminar deposits were intensely positive for CRP and CFH. Conclusion These immunohistochemical findings show that changes in distribution and relative levels of CRP and CFH were evident in early and late AMD eyes. This suggests that high levels of CRP and insufficient CFH at the retina/choroid interface may lead to uncontrolled complement activation with associated cell and tissue damage. This study supports the hypothesis that inflammation and immune-mediated mechanisms are involved in the pathogenesis of AMD.