D. Scott McLeod

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.

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.

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.

Retrolental fibroplasia : Evidence for a role of the prostaglandin cascade in the pathogenesis of oxygen-induced retinopathy in the newborn beagle

Summary: Aspirin administration, at a dosage producing plasma levels within the human therepeutic range, caused marked inhibition of production of both vascular prostacyclin (a vasodilator) and platelet thromboxane (a vasoconstrictor) in beagle puppies. In addition, aspirin-treated, oxygen-exposed puppies developed retinopathy of significantly greater severity than their unmedicated, oxygen-exposed littermates. Direct ophthalmoscopic observations indicated that whereas sustained oxygen breathing produced retinal vasoconstriction in unmedicated puppies, retinal vessels of aspirintreated littermates became more dilated or remained unchanged. It is postulated that retinal vasoconstriction may be a normal physiologic mechanism to protect the immature retina from damaging effects of high blood oxygen levels; i.e., it may be a protective rather than a pathologic process in response to hyperoxia.Many vascular anomalies which characterize the human disease were present in the retinas of the puppies. Several of the most severely affected puppies treated with aspirin even displayed grade III cicatricial retinopathy (falciform retinal fold). Thus, a major criticism of the retrolental fibroplasia animal model has been addressed by producing cicatricial retrolental fibroplasia in puppies, and the confidence with which results from experimental animal studies might be extrapolated to the clinical situation is thereby strengthened.Speculation: It is conceivable that the susceptibility of any eye to oxygen-associated retinopathy at birth depends upon the extent to which the vasoconstriction protective response is functional as well as upon the degree of retinal vascular maturity attained. In utero, the arterial PO2 is low, and blood flow to the immature retina is high. When the PO2 rises at birth, retinal blood flow does not change, although arterial blood pressure rises; this could be explained by the occurrence of some degree of vasoconstriction in response to elevated arterial PO2 or reduced PO2 which establishes the clinically normal retinal vasotonia. In light of this, those cases of “spontaneous” retrolental fibroplasia reported in premature infants never administered oxygen or in full-term infants who were administered oxygen might simply be examples of individuals with inadequate retinal vasotonia for protection of structurally immature vessels from the effects of elevated arterial PO2, and/or excessively high transmural pressure.

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.

The Initial Fetal Human Retinal Vasculature Develops by Vasculogenesis

There is increasing evidence that the hemangioblast, a common progenitor for hematopoietic cells and endothelial cells, participates in embryonic and extra‐embryonic vasculogenesis in some organs. Whether resident angioblasts or endothelial progenitor cells (EPCs) contribute to human retinal vasculogenesis is still a matter of controversy. To address this controversy, fetal human retinas of 6–23 weeks gestation (WG) were examined using immunohistochemistry and a panel of antibodies against endothelial cell markers (CD34, CD31), a marker for retinal angioblasts and endothelium (CD39/ecto‐ADPase), and a marker for precursors and hemangioblasts (CXCR4). Confocal microscopic spectral analysis and double labeling with Ki67 was used to identify the proliferating cell types. In the inner neuroblastic layer of the 6–8 WG retina and in the putative ganglion cell layer in avascular regions of older eyes (14 WG–20 WG), scattered CD39+ angioblasts were well in advance of forming vasculature. There was a layer of CXCR4+ cells in the inner retina that was reduced in size with development. As blood vessels formed, CD39+ cells were always well in advance of the vascular front and they expressed CXCR4. This demonstrates that a pool of resident angioblasts express CD39 and CXCR4 as they differentiate and participate in vasculogenesis in the fetal human. They retain expression of CD39 as endothelial cells in the newly formed retinal vasculature but they down‐regulate CXCR4 expression. Developmental Dynamics 235:3336–3347, 2006.

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruchs membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.

Intrachoroidal Microvascular Abnormality: A Previously Unrecognized form of Choroidal Neovascularization

PURPOSE To evaluate the histopathologic and histochemical characteristics of intrachoroidal microvascular abnormality. METHODS Forty eyes obtained at autopsy from human donors ranging in age from 20 to 91 years (25 diabetics, 15 nondiabetics) were analyzed. Choroids were processed for alkaline phosphatase flat-embedding. Vascular patterns were examined and analyzed before embedding and serial sectioning. RESULTS Intrachoroidal microvascular abnormality had the most prominent alkaline phosphatase reaction product of choroidal vessels. These formations appeared as ameboid or cobweb-like vascular networks (area, 0.05 to 4.6 mm2) in the choroidal stroma external to the choriocapillaris. They appeared as both single or groups of formations in the posterior pole and equatorial regions in all subjects. Intrachoroidal microvascular abnormality was found in five subjects with diabetes: four with type I diabetes mellitus, and one with type II diabetes mellitus. One subject had proliferative diabetic retinopathy, three had background retinopathy, and one had no retinopathy. Intrachoroidal microvascular abnormality was connected with all levels of choroidal vasculature. Microaneurysms were observed within intrachoroidal microvascular abnormality formations in most subjects but not in other choroidal vessels. CONCLUSIONS Intrachoroidal microvascular abnormality is a form of intrachoroidal neovascularization. This neovascularization has features similar to intraretinal microvascular abnormalities found in diabetic subjects but seems to form independently of the status of retinopathy. The presence of microaneurysms in intrachoroidal microvascular abnormalities and not other choroidal vessels supports the view that aneurysms may be aborted attempts at neovascularization.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague-Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the betaA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate betaA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.