|Molecular Vision 1999;
Received 24 May 1999 | Accepted 2 November 1999 | Published 3 November 1999
Changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration
Ajit B. Majji,1 Masanobu
Uyama,3 Shin Yoneya4
1Wilmer Eye Institute, Baltimore, MD; 2Scheie Eye Institute, Philadelphia, PA; 3Kansai Medical University, Osaka, Japan; 4Saitama Medical School, Saitama, Japan
Correspondence to: Gerard Lutty, Ph.D., Wilmer Eye Institute, 170 Woods Research Building, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD, 21287-9115; Phone: (410) 955-6750; FAX: (410) 955-3447; email: firstname.lastname@example.org
Retinal pigment epithelial cells (RPE) and the choriocapillaris are on opposite sides of Bruch's membrane and control transport in and out of the retina. In age-related macular degeneration (AMD), they may also be responsible for deposition of material in and on Bruch's membrane and the formation or regression of choroidal neovascularization (CNV). Indocyanine green (ICG) angiography can be used to visualize the choroidal vasculature and CNV. Filling of the choriocapillaris with ICG was delayed in subjects older than 50 years of age, and areas of hypofluorescence were observed in maculas of AMD subjects, often associated with CNV. Laser Doppler flowmetry of the choriocapillaris in the macula demonstrated that choroidal blood flow and volume are reduced in subjects older than 46 years of age and further decreased in subjects with AMD. The human choriocapillaris can be histologically studied in two dimensions by incubating the tissue for alkaline phosphatase activity, flat-embedding it in transparent polymer and sectioning it. Using this technique, choriocapillaris dropout was found to be associated with deposition of material in Bruch's membrane in diabetic subjects. When RPE are removed from Bruch's membrane, the choriocapillaris degenerates; the regeneration of choriocapillaris can be blocked by Genistein, a tyrosine kinase inhibitor. Finally, RPE cells may produce substances that both stimulate the formation and regression of CNV in animal models. These studies suggest that there may be a reduction in choriocapillaris flow in AMD, and this loss of choriocapillaris can be associated with the Bruch's membrane deposits that are hallmarks of AMD. Furthermore, RPE may stimulate the formation and regression of CNV and RPE loss can result in loss of choriocapillaris.
The lesions identified as causing loss of central vision in age-related macular degeneration are detachment of the retinal pigment epithelium (RPE), outer retinal atrophy, and new blood vessel growth between Bruch's membrane and the retina. The accumulation in Bruch's membrane of waste products, presumably derived from RPE, is believed to play a major role in the induction of these lesions. Two theories that have evolved to explain accumulation of material in AMD are: (a) deposits accumulate and cause choroidal vascular dysfunction and loss; (b) choriocapillaris dysfunction or dropout initiates the accumulation of waste associated with Bruch's membrane . It is believed that transport across Bruch's membrane is reduced when deposits accumulate, resulting in a continuum of waste deposition . Dropout of choriocapillaris could also cause RPE to become ischemic and produce growth factors that stimulate the formation of choroidal neovascularization (CNV) in AMD. Changes in choriocapillaris and RPE and their relationship with Bruch's membrane deposits and CNV are explored.
Evidence for possible ICG perfusion defects in patients with AMD
Indocyanine green (ICG) angiography is a useful tool for detecting choroidal neovascularization in patients with AMD. However, few ICG observations have been reported concerning either aging changes of the choroid or choroidal vascular, other than choroidal neovascularization in patients with AMD. Recently, Yoneya et al reported early dye filling patterns in young, healthy volunteers using a video ICG system (modified TCR 50-IA, Topcon, Tokyo, Japan), which uses a 790 nm diode laser as an emission light [3,4]. Based on these observations, aging changes in the choroid were evaluated using this system. They also investigated changes in patients with AMD and the possible role of choroidal vascular changes in the development of AMD.
Video ICG angiography was performed on 35 eyes from 35 healthy, normal volunteers (21-81 years old, average age 50.5 years old) for 30 minutes after ICG injection. A fixed area of 64 x 64 pixels in the macula was used for evaluating the number of arterioles in the image of the early arterial phase with IMAGEnet (Topcon). The mean intensity of fluorescence in the same area was also quantified with both the early and late phase images. With subjects in their second and third decades, choroidal arterioles began to fluoresce preferentially in the subfoveal area. Subsequently, feeding arterioles and choriocapillaris filled rapidly. Water-shed zones running vertically through the optic disc were often observed. In eyes of subjects over fifty, it took longer to fill the choroidal vasculature with the dye. Eventually, the margin of the water-shed zone became indistinct. Quantitative analysis disclosed that the number of choroidal arterioles and the fluorescence intensities in the macular region were reduced significantly with age (p<0.005 and p<0.001 respectively). In late phase angiograms, the mean intensity of fluorescence in the macula increased with subject age, through a time course ending 24 hours after dye injection.
One hundred and one eyes, including normal eyes as aged-matched control subjects, were used to study choroidal vascular changes in AMD. Early dye filling was observed in 23 eyes and 17 fellow eyes of AMD patients (66.5±11.6 and 67.8±11.2 years old) and in 18 eyes of normal volunteers (62.4±9.4 years old). AMD eyes presenting hemorrhagic RPE detachment or disciform scar were excluded from this study. Initially, large choroidal arterioles outside of the macula began to fluoresce and took a straight course with few branches in diseased eyes. Dye filling of the arterioles was also slow. Eventually, either water-shed or focal hypofluorescence was observed in the macula. Choroidal neovascularization developed in areas of hypofluorescence in all AMD eyes. Focal hypofluorescence in the macula was observed and this turned into negative fluorescence by 24 hours after dye injection. Computer assisted image analysis (performed as above)demonstrated a significant decrease in the number of choroidal arterioles and reduced fluorescence in the macula (p<0.005 and p<0.01 respectively). These observations of Yoneya et al may support the hypothesis that poor perfusion of the choroid in the macula plays an important role in the development of choroidal neovascularization in patients with AMD . Poor perfusion of the choroid might also accelerate the deposition of hydrophobic lipids, such as triglycerides.
Foveolar choroidal blood flow in aging and in AMD
Grunwald et al  investigated the effect of aging on foveolar choroidal blood flow (ChBFlow) and compared foveolar choroidal blood circulation of subjects with nonexudative AMD with that of control subjects. Choroidal blood flow was determined using laser Doppler flowmetry . Twenty-nine normal subject eyes (ages ranged from 15 to 76 years; mean±SD, 42±18 years) were included in the aging study. Relative choroidal blood velocity (ChBVel), choroidal blood volume (ChBVol), and ChBFlow were determined in the foveolar region by asking subjects to fixate on the probing laser beam. Measurements were also obtained in eyes of 20 subjects having 10 or more large drusen, visual acuity of 20/32 or better, and no evidence of CNV . Findings obtained in these subjects were compared with those of eyes from 10 age- and blood pressure-matched control subjects with no large drusen.
In normal eyes, significant negative correlations were observed between ChBVol and the subject's age (R=-0.52, p=0.004) and between ChBFlow and the subject's age (R=-0.54, p=0.003). No significant correlation was detected between ChBVel and the subject's age (R= 0.07, p=0.70). Significant differences were observed in ChBVol and ChBFlow between younger normal subjects aged 15 to 45 years (mean±SD, 0.48±0.20 arbitrary units [AU] and 18.9±5.8 AU, respectively) and the older normal ones aged 46 to 76 years (mean±SD, 0.34±0.11 AU and 13.3±3.3 AU, respectively; unpaired Student t-test, p=0.04 and p=0.007, respectively). In the second study, no significant differences in average age, blood pressure, or intraocular pressure were observed between subjects with AMD and control subjects. In subjects with AMD, average ChBVol was 0.24±0.08 (±SD) arbitrary units (AU): this value was 33% lower than that of control subjects (0.36±0.11 AU: two-tailed, independent Student's t-test, p=0.005). Average ChBVel, conversely, was not significantly different from normal (0.44±0.10 AU). Average ChBFlow in subjects with AMD (8.7±3.1 AU) was 37% lower than that of control subjects (13.7±3.5 AU; p=0.0005). Average blood flow pulsatility was 6% higher in subjects with AMD (0.71±0.15) than in control subjects (0.66±0.14), but this difference was not statistically significant (p=0.42). In the subjects studied, foveolar ChBFlow decreased with age . This change was probably related to the decrease in density and diameter of the choriocapillaris that occurs with increasing age. Average ChBFlow in the nonexudative stages of AMD was lower than that of age-matched controls, and the effect was caused mainly by a decrease in ChBVol [8,9]. Throughout the body, decreased circulation can lead to the formation of neovascularization. Further studies are needed to elucidate whether decreased ChBFlow plays a role in the development of choroidal neovascularization, and whether ChBFlow measurements may help identify subjects with AMD at risk for developing choroidal neovascularization.
Choriocapillaris degeneration in the human choroid
McLeod and Lutty have developed a method to study the human choroidal vasculature using alkaline phosphatase (APase) activity. They demonstrated that loss of APase activity in choriocapillaris indicates a loss of viable endothelial cells and, therefore, choriocapillaris degeneration (CCD) and compromise . Lutty and associates have recently used this technique to analyse choriocapillaris dropout in occlusive disorders like diabetes mellitus and sickle cell disease. In diabetic subjects, the area of CCD was more than four fold greater than in the nondiabetic group (p<0.001). The area of CCD in the submacular choroid was 4.9 fold greater in diabetic subjects than in nondiabetic subjects (p<0.001), while the mean age of the nondiabetic subjects was 14 years greater than the subjects with diabetes. The CCD in diabetic subjects was more prominent in the posterior pole than in the peripheral choroid. CCD in diabetics appeared in two different patterns: diffuse CCD (partial loss of APase activity in a poorly defined area) and focal CCD (complete loss of APase activity in a relatively well defined area). The thickness of basal laminar deposits (BLD; a pathologic change in Bruch's membrane) was correlated with the severity of CCD in the diabetic choroid; focal CCD areas had thicker BLD than diffuse CCD areas . Choroidal neovascularization was associated with diffuse CCD and half of the CNV formations lacked APase and a viable endothelium suggesting they were infarcted.
We have also investigated the association of polymorphonuclear leukocytes (PMNs) with CCD using a double staining technique: APase and nonspecific esterase (NSE) for analysis of the choroidal vasculature and PMNs respectively. The total number of PMNs was increased within the choriocapillaris in diabetic eyes (170.9±12.9 PMNs/mm2 of choroid) compared to nondiabetic eyes (84.2±16.9 PMNs/mm2; p<0.001). In the diabetic choroid, increased numbers of PMNs were present in areas of the choriocapillaris with CCD compared with nonpathologic choriocapillaris (205.1±46.9 PMNs/mm2 in pathologic vs 152.3±23.4 PMNs/mm2 in nonpathologic areas; p<0.001). PMNs were often queued up within the lumens of capillaries, demonstrating loss in APase activity. Finally, there was a strong correlation between the area of CCD and number of PMNs . In subjects with sickle cell disease, there were also areas of choriocapillaris degeneration which were greater in size than age-comparable normal subjects. The choriocapillaris dropout, however, was not associated with increased numbers of PMNs. The occluded vascular segments instead contained packed red blood cells.
In conclusion, significant CCD occurs in subjects with both diabetes and sickle cell disease, two vaso-occlusive disorders. PMNs may participate in the choroidal vaso-occlusive process in subjects with diabetes due to elevated ICAM-1 and P-selectin in diabetic choroidal vasculatures . RBCs appear to initiate the occlusions in sickle cell subjects. Although subjects with AMD have not yet been studied using these techniques, two pathological features of AMD are associated with choriocapillaris dropout in diabetes; basal laminar deposits and choroidal neovascularization.
The role of RPE in choroidal neovascularization (CNV)
Uyama et al hypothesize that RPE cells promote the progression of CNV in the early stage of its development, however, in the late or involution stage of CNV, proliferating RPE enclose CNV and cause its regression . To clarify the role of RPE in the progression or involution of CNV, Uyama et al evaluated CNV lesions histopathologically in experimentally induced CNV. CNV was induced in monkey and rat eyes by intense laser photocoagulation of the retina in the posterior pole . Using light and electron microscopy, these laser photocoagulation areas were observed histopathologically in order to demonstrate the relationship of RPE to CNV. The role of RPE was confirmed in vivo using the following substances. To damage the RPE selectively, ornithine was administered intravitreally or sodium iodate was administered intravenously at different stages in the development of CNV . Also, interferon (IFN-ß) was administered systemically to the animals with CNV, as it promotes the proliferation of RPE. The expression of some growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-ß), and their receptors were revealed using in situ hybridization and immunohistochemistry in sections from the animal models. In addition, VEGF was localized immunohistochemically in surgically removed fibrovascular membranes beneath the macula in human eyes having age-related macular degeneration.
In the early or growing stages of experimentally induced CNV, the CNV developed in association with hyperplastic RPE. In the late or involutionary stage of CNV, the RPE proliferated in a monolayer to enclose CNV and appeared to cause its regression. In eyes which ornithine or sodium iodate was administered, the RPE was damaged and did not proliferate. When the drugs were administered in the early stage, CNV developed poorly. When they were administered in the late stage, the RPE did not proliferate and envelop the CNV, but rather the CNV persisted for a long period of time and grew to a large size. When IFN-ß was administered, the RPE proliferated to enclose the CNV, causing it to regress in a short period of time. The expression of VEGF, bFGF, TGF-ß, and their receptors, was up-regulated in the early stage and down-regulated in the later stages in CNV areas. In the surgically removed fibrovascular membranes, VEGF was demonstrated immunohistochemically at sites of RPE proliferation.
These results suggest that RPE behave in a different manner at different stages in the progression and involution of CNV. In the early stages of CNV development, RPE promotes its progression. VEGF and bFGF may be produced by the RPE in the lesions in an autocrine or paracrine manner, promoting CNV development. However, in the late stage of CNV, RPE proliferate and envelop CNV, causing it to regress.
Surgical Model of RPE debridement: Inhibition of choriocapillaris regeneration by Genistein
The choriocapillaris has been shown to atrophy after retinal pigment epithelial cell loss in clinical and experimental studies [17,18]. Retinal pigment epithelium regenerates in the rabbit after iodate-induced retinopathy, resulting in choriocapillaris regeneration . Majji and associates have developed a surgical model of choriocapillaris atrophy by surgically debriding the RPE in rabbits. In their model, the RPE regenerates in a centripetal manner. Retinal pigment epithelium covers the wound area by day 7. The choriocapillaris revascularizes, along with regeneration of the RPE. Choriocapillaris regeneration was nearly complete by 4-8 weeks. With this model, the area of RPE cell loss can be controlled and the effects of pharmacological agents on choriocapillaris can be measured in comparative studies.
Genistein, a naturally occurring isoflavone isolated from soybean , has been shown to inhibit proliferation of vascular endothelial cells and tumor cell lines . Genistein exerts its varied tissue effects at different concentrations. These effects include inhibition of angiogenesis, tyrosine kinase phosphorylation, DNA synthesis and cell cycle arrest. The effects of Genistein on choriocapillaris regeneration were examined in this surgical model and compared with the effects of cycloheximide and dimethyl sulfoxide on choriocapillaris regeneration. Genistein inhibited choriocapillaris regeneration without affecting RPE wound healing. The inhibitory effects of Genistein were significant compared to DMSO, the vehicle for Genistein. Tyrosine kinase inhibitors such as Genistein may be useful as a pharmacological approach in the treatment of choroidal neovascularization. CNV occurs in a variety of diseases like AMD, histoplasmosis, and pathologic myopia leading to central loss of vision.
Choriocapillaris blood flow in the fovea decreases with age and further decreases in subjects with AMD. Filling defects in choriocapillaris can be observed with ICG angiography, which demonstrates choroidal defects in AMD, often associated with CNV and lipid deposits. There is histological evidence that dropout of choriocapillaris is associated with deposits in Bruch's membrane and CNV in diseases such as diabetes. Experimental models demonstrate that RPE loss can result in choriocapillaris dropout; as RPE cells repopulate Bruch's membrane, the choriocapillaris reforms. Finally, RPE cells may produce substances that stimulate both the formation and regression of CNV in animal models. The latter two studies intimate a key role for RPE in maintaining and controlling choriocapillaris and choroidal neovascularization.
Dr. Lutty's studies were supported by a fellowship from Fight for Sight, Research Division of Prevent Blindness America (Fellowship for Jingtai Cao), the American Heart Association, NIH grant EY01765 (Wilmer Institute), and the Titus and Brownstein Foundations. Dr. Lutty is an American Heart Association Established Investigator and a recipient of a Research to Prevent Blindness Lew R. Wasserman Merit Award. Dr Grunwald's studies were supported by the Vivian Simkins Lasko Retinal Vascular Research Fund, the Pennsylvania Lions Sight Conservation and Eye Reseach Foundation, Research to Prevent Blindness, the Nina Mackall Trust, and a Grant from the National Institutes of Health, NEI R21 EY10964.
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