December 2003
Volume 44, Issue 12
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Retinal Cell Biology  |   December 2003
RPE Cells Modulate Subretinal Neovascularization, but Do Not Cause Regression in Mice with Sustained Expression of VEGF
Author Affiliations
  • Hisashi Ida
    From the Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan; and the
  • Takao Tobe
    From the Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan; and the
  • Hiroyuki Nambu
    Department of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland.
  • Miyo Matsumura
    From the Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan; and the
  • Masanobu Uyama
    From the Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan; and the
  • Peter A. Campochiaro
    Department of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland.
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5430-5437. doi:10.1167/iovs.03-0609
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      Hisashi Ida, Takao Tobe, Hiroyuki Nambu, Miyo Matsumura, Masanobu Uyama, Peter A. Campochiaro; RPE Cells Modulate Subretinal Neovascularization, but Do Not Cause Regression in Mice with Sustained Expression of VEGF. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5430-5437. doi: 10.1167/iovs.03-0609.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Previous studies using models of choroidal neovascularization (CNV) in which the angiogenic stimulus is not sustained, have concluded that the retinal pigmented epithelium (RPE) causes regression of neovascularization (NV). However, the withdrawal of angiogenic stimuli may actually be the major modulator of NV, and RPE cells may simply be responding to withdrawal of the angiogenic stimuli or something released by NV because of the withdrawal. In this study, the long-term course of NV and the behavior of the RPE in rhodopsin/VEGF transgenic mice, in which there is a sustained angiogenic stimulus, was investigated.

methods. Hemizygous mice from the V-6 line were killed at 0.75, 1, 3, 6, and 12 months after birth, and at each time point mRNA for VEGF, VEGF-R1, and VEGF-R2 was measured by RT-PCR. Some mice were perfused with fluorescein-labeled dextran and retinal flatmounts were examined by fluorescence microscopy. Light and electron microscopy was performed on Epon-embedded eyes.

results. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 remained constant from the earliest to the latest time point. Retinal flatmounts showed numerous small areas of subretinal NV at 3 weeks and at 1 month, and there were a similar number of larger lesions. By 6 months, many of the individual NV lesions had grown together to form large networks of new vessels. At 12 months, NV networks were similar to those at 6 months, but some of the vessels were not perfused. Light microscopy showed serous retinal detachments overlying NV lesions in mice up to 3 months of age, but at 6 and 12 months, the RPE completely surrounded new vessels and formed tight junctions to reestablish the outer blood–retinal barrier, and there were no serous detachments. Electron microscopy showed that compared with more acute NV lesions, chronic lesions contained thinner endothelial cells, similar to those of the choriocapillaris in that they had scant cytoplasm and numerous fenestrations, or pinocytotic vesicles with thick basement membrane surrounded by extracellular matrix (ECM). Bruch’s membrane remained intact.

conclusions. Despite persistent high expression of VEGF and its receptors, NV stopped growing and reached a plateau in older V-6 mice. RPE cells modulated the NV by surrounding it and reestablishing the blood–retinal barrier, but did not cause regression, although some vessels in chronic lesions were not perfused. These data do not support the conclusion of several previously reported studies, that RPE cells cause regression of CNV.

In developed countries, CNV due to age-related macular degeneration is the most common cause of severe vision loss in patients 60 years of age or more. 1 CNV is also responsible for a substantial amount of visual morbidity in young patients with ocular histoplasmosis, angioid streaks, degenerative myopia, and several other diseases that affect the Bruch’s membrane–RPE interface. Laser photocoagulation and photodynamic therapy have been demonstrated to provide benefit in certain subgroups of patients with CNV, but for most patients the benefit is simply to slow visual loss, and few patients have improvement and long-term maintenance of vision. 1 2 New treatments are needed. 
The development of new treatments would be facilitated by a better understanding of the pathogenesis of CNV. Recent studies have suggested that VEGF is a necessary stimulus for CNV, 3 and therefore VEGF is an important target for intervention and VEGF antagonists are being examined in clinical trials (Guyer DR, et al. IOVS 2001;42:ARVO Abstract 2810; Schwartz SD, et al. IOVS 2001;42:ARVO Abstract 2807). Studies using gene transfer have demonstrated that pigment epithelium-derived factor (PEDF) suppresses and causes regression of ocular neovascularization, 4 5 and this too is being investigated in clinical trials. 6 Because PEDF is normally produced by the RPE, the role of the RPE in CNV is in question. Is it part of a natural defense or barrier against CNV, and does healthy RPE promote regression of CNV? 
Numerous studies have addressed the interaction between the RPE and the choriocapillaris. 7 8 9 10 11 12 Destruction of the RPE by sodium iodate or some other toxin that differentially ablates the RPE is associated with atrophy of the choriocapillaris, suggesting that RPE cells may provide a necessary survival factor for the choriocapillaris. After hereditary or toxin-induced photoreceptor degeneration, it is common for new vessels to sprout from retinal vessels, extend through the photoreceptors, and grow between the RPE cells. In some degenerations, particularly the Royal College of Surgeons (RCS) rat model, once the new vessels enter the RPE, vessels with surrounding RPE cells grow back through the entire retina and break into the vitreous cavity. 13 These observations suggest that after photoreceptor degeneration, RPE cells become proangiogenic. After mild laser application to Bruch’s membrane in rats, CNV occurs with relatively low frequency, and after laser-induced rupture of Bruch’s membrane in monkeys, rats, or mice, CNV occurs with high frequency at rupture sites. In all these situations, the CNV is surrounded by RPE cells. 14 15 16 17 If a selective RPE toxin is used to damage RPE cells simultaneously with rupture of Bruch’s membrane or soon after rupture, development of CNV is suppressed. 18 19 This observation also suggests that RPE cells promote angiogenesis. However, if a selective RPE toxin is given 2 weeks after rupture of Bruch’s membrane, excessively large areas of CNV develop, suggesting a possible antiangiogenic role of the RPE. 20  
In human diseases associated with CNV, such as age-related macular degeneration (AMD), the angiogenic stimulus is sustained, but in animal models of CNV induced by rupture of Bruch’s membrane, there is transient expression of angiogenic factors, and therefore it is difficult to determine whether the behavior of CNV is due to changing levels of angiogenic factors or to influences of RPE cells. Recently, we have generated transgenic mice in which the rhodopsin promoter drives expression of VEGF in photoreceptors. 21 22 In these mice (rho/VEGF transgenics), subretinal neovascularization develops that is surrounded by RPE cells. In this study, we sought to examine the long-term interaction of subretinal neovascularization and RPE cells in rho/VEGF transgenic mice in which there is sustained expression of VEGF. 
Materials and Methods
Rho/VEGF Transgenic Mice
Mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice that express VEGF in photoreceptors (rho/VEGF mice) have been described. 21 22 At 0.75, 1, 3, 6, and 12 months of age (postnatal days [P] 21, P1 month [M], P3M, P6M, P12M), mice were killed for various types of analyses: semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) to measure mRNA levels in the retina, measurement of subretinal neovascularization on retinal flatmounts after perfusion with fluorescein-labeled dextran, and evaluation of morphology by light and electron microscopy of plastic-embedded eyes. 
Assessment of mRNA Levels by RT-PCR
Mice were killed and eyes were rapidly removed. Anterior segments of the eyes were removed, and retinas were dissected and homogenized in extraction reagent (TRIzol; Invitrogen-Life Technologies, Inc., Gaithersburg, MD). Total RNA was isolated according to the manufacturer’s protocol. Reverse transcription was performed with 0.2 μg of total retinal RNA, reverse transcriptase (Superscript II; Invitrogen-Life Technologies), and 5.0 mg of oligo d(T) primer. Aliquots of the cDNAs were used for PCR amplification with primers for human VEGF (forward: TGC AGA TGT GAC AAG CCG AG; reverse: GAT GTG GCG AGA TGC TCT TGA AGT CTG GTA), VEGF receptor-1 (forward: AGC TCT CCG TGG ATC TGA AA; reverse: CCA AGA ACT CCA TGC CCT TA), or VEGF receptor-2 (forward: TCG GCT GCA GTG TGT AAG TC; reverse: CTT TTG TGG GAA AAG GGA CA). Titrations were performed to ensure that PCR reactions were performed in the linear range of amplification. Mouse S16 ribosomal protein primers (forward: CAC TGC AAA CGG GGA AAT GG; reverse: TGA GAT GGA CTG TCG GAT GG) were used to provide an internal control for the amount of template in the PCR reactions. 
Measurement of Retinal NV
Mice were anesthetized, the descending aorta was clamped, the right atrium was cut, and 1 mL of phosphate-buffered saline containing 50 mg/mL of fluorescein-labeled dextran (2 × 106 average molecular weight; Sigma-Aldrich, St. Louis, MO) was infused through the left ventricle. 22 The eyes were removed and fixed for 1 hour in 10% phosphate-buffered formalin. The cornea and lens were removed, and the entire retina was carefully dissected from the eyecup, radially cut from the edge of the retina to the equator in all four quadrants, and flat-mounted in aqueous medium (Aquamount; BCH, Poole, UK) with photoreceptors facing upward. 
Retinal flatmounts were examined by fluorescence microscopy at 400× magnification, which provides a narrow depth of field so that when we focused on NV on the outer edge of the retina, the remainder of the retinal vessels were out-of-focus allowing easy delineation of the NV. The outer edge of the retina, which corresponds to the subretinal space in vivo, is easily identified and therefore there is standardization of the focal plane from slide to slide. Images were digitized with a three-color CCD video camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber. Commercial software (Image-Pro Plus software; Media Cybernetics, Silver Spring, MD) was used to delineate each of the lesions and calculate the number in each retina, the area of each lesion, and the total area of neovascularization per retina. Measurements were repeated three times for each retina, and the mean was used for one experimental value. There was little variability among triplicate measurements. 
Light and Electron Microscopy
Eyes were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 24 hours at 4°C. The anterior segments were removed, and the posterior eyecups were divided into four parts. The segments of tissue were post fixed with 1% osmium tetroxide/cacodylate buffer (pH 7.4), dehydrated through a series of graded alcohols, and embedded in resin (Quetol 812; Nisshin EM Co., Ltd., Tokyo, Japan). One-micrometer serial sections were cut, stained with toluidine blue, and examined by microscope (model C35A; Olympus Co., Tokyo, Japan). Sections were photographed, scanned, labeled, and printed as just described. For electron microscopy, ultrathin sections were cut, counterstained with 2.0% uranyl acetate and 0.3% lead citrate, and examined with a transmission electron microscope (H-7000; Hitachi Co. Ltd., Tokyo, Japan). 
Results
Expression of mRNA for VEGF, VEGF Receptor-1, and VEGF-Receptor-2 is in the Retinas of Rho/VEGF Transgenics
The mRNA level for VEGF was elevated in the retinas of rho/VEGF transgenics compared with control mice and was very similar at each of the time points tested between 3 weeks and 1 year of age (Fig. 1) . The mRNA levels for VEGF receptor-1 and VEGF receptor-2 were also somewhat elevated, but clearly were not reduced, in the retinas of rho/VEGF transgenic animals compared with the control and were roughly the same at all time points. These data demonstrate that there was sustained elevation of VEGF expression for at least 1 year and that there was no evidence of downregulation of the receptors. 
Spontaneous Regression of Subretinal NV in Rho/VEGF Mice
Retinal flatmounts of fluorescein dextran-perfused retinas mounted with the photoreceptor side up, provide a panoramic view of the three-dimensional structure of subretinal new vessels. As noted in several studies, 22 23 there were numerous small foci of NV in the photoreceptor layer that were partially surrounded by RPE cells at P21 (Fig. 2A , arrowheads). Small feeder vessels connected the new vessels to retinal vessels that were out of focus in the background. At 1 month, small connecting vessels had formed between the foci of NV, resulting in some lesions that were larger (Fig. 2B , arrowheads surround a large lesion consisting of small foci of new vessels that are interconnected). At 3 months, large interconnected fronds of vessels with surrounding RPE (Fig. 2C , arrowheads) were interspersed between small isolated foci of NV. At 6 months, tortuous, closely packed clumps of new vessels (Fig. 2D , arrowheads) were present within dense envelopes of RPE cells. Some connections between clumps could be inferred based on relative position, but were obscured by RPE cells. At 1 year, the RPE covering of NV was somewhat less dense than that at 6 months, and interconnecting vessels were more apparent between tortuous clumps of new vessels (Fig. 2E , arrowheads). Image analysis supported the conclusion that numerous small foci of NV at P21 gradually became interconnected, resulting in a decreasing number of lesions and increasing size of each lesion between P21 and P3M (Figs. 2F 2G) . Between 3 months and 1 year, the number and size of lesions did not show any significant changes. Throughout the entire period between 3 weeks and 1 year, there was not a significant difference in the total area of NV per retina (Fig. 2H) , indicating that the changes in NV were primarily remodeling, and there clearly was no regression of NV, as has been demonstrated in other models of subretinal NV. 
RPE Cells and the Blood–Retinal Barrier
As noted in previous studies, 21 22 P21 rho/VEGF mice showed NV originating from the deep capillary bed of the retina and extending through the outer nuclear layer into the subretinal space (Fig. 3A) . At 1 month, feeder vessels passed through and partially disrupted the outer nuclear layer and connected to a network of vessels in the subretinal space (Fig. 3B) . The subretinal vessels were partially surrounded by RPE cells. At 3 months, the network of vessels in the subretinal space was more extensive and more completely surrounded by RPE cells, but there was still subretinal fluid indicating breakdown of the blood–retinal barrier (Fig. 3C)
The subretinal vessels were similar in extent at 6 (Fig. 3D) and 12 months (Fig. 3E) compared with 3 months, but they were completely surrounded by RPE cells, and there was no longer any subretinal fluid, indicating that the blood–retinal barrier had been reestablished. There was also prominent extracellular matrix associated with NV, particularly at 12 months (a higher resolution view of the extensive extracellular matrix is shown in Figs. 4D 4E ). 
Interaction of RPE Cells and Subretinal Vessels
The new vessels among the rod outer segments at P21 were made up of thick, immature-appearing endothelial cells connected by tight junctions (Fig. 4A) . Polarized RPE cells were oriented normally on Bruch’s membrane and had normal ultrastructure. At 1 month, RPE cells had begun to migrate around the new vessels and in areas of close contact with RPE cells, endothelial cells were thinner and exhibited fenestrations (Fig. 4B , inset). At 3 months, RPE cells surrounded new vessels in the subretinal space, but had not formed competent tight junctions, because there was still some overlying serous detachment (Fig. 4C) . There was an increase in extracellular matrix surrounding thin endothelial cell processes, which exhibited numerous fenestrations (Fig. 4C , inset, arrows). 
At 6 months, subretinal NV was completely surrounded and isolated by RPE cells. There was no subretinal or intraretinal fluid (Fig. 4D) . The RPE cells on top of the NV had basal infoldings adjacent to the new vessels and microvilli along their apical surface that interdigitated with outer segments. There was no identifiable subretinal fluid. The RPE cells beneath the NV had no microvilli, but rather had small basal infoldings along both surfaces. Few fenestrations and many pinocytotic vesicles were seen compared with earlier time points. Pericytes were associated with new vessels, and some had thick surrounding extracellular matrix, which was even more prominent at 12 months (Fig. 4E) . RPE cells located between NV and photoreceptors were still polarized, but neither microvilli nor basal infoldings were as prominent as at previous time points. 
Discussion
Investigators in many studies have observed the interactions between the choriocapillaris and/or CNV and RPE cells. These studies have suggested that RPE cells produce survival factors that are necessary for survival of the choriocapillaris 7 8 9 ; that the choriocapillaris influences RPE cells, particularly with respect to their polarity 9 ; that when Bruch’s membrane is ruptured, RPE cells play an important role in promoting CNV and in the absence of RPE cells, CNV does not occur 18 19 ; and that RPE cells are attracted to vessels that invade the subretinal space where they surround the vessels, reestablish the blood–retinal barrier, and promote involution of the CNV. 14 20 These conclusions are based on studies in which RPE cells were damaged by selective RPE toxins and/or Bruch’s membrane was ruptured by intense laser photocoagulation. In these model systems, angiogenic stimuli are induced by the insults, but they are transient. This differs from human diseases associated with CNV, as demonstrated by the high incidence of recurrent CNV after ablative treatments. The recurrences continue to mount over time, with many occurring years after treatment. 1 In this study, we investigated the interaction of RPE cells and subretinal NV when there is a sustained angiogenic stimulus, just as occurs in human diseases. Our findings support most of the conclusions described earlier, obtained with model systems in which there is transient production of angiogenic stimuli, except RPE cells do not cause regression of subretinal NV. Once the subretinal NV was established 3 weeks after birth, the total area of NV remained unchanged over the course of a year of sustained expression of VEGF in the retina. However, there was substantial remodeling of the NV, due in part to establishment of connections between previously independent foci of NV and also due to influences of RPE cells. RPE cells surrounded the subretinal NV, reestablished the blood–retinal barrier, and induced the formation of a layer of vessels that resembled a second choriocapillaris sandwiched between two layers of RPE cells. The new, innermost layer of RPE cells became polarized with microvilli that interdigitated with photoreceptor outer segments and basal infoldings that bordered the neovascularization. The outermost layer of RPE cells lost their polarity and exhibited basal infoldings bordering both the neovascularization on the apical side and the original choriocapillaris on the basal side. 
The subretinal blood vessels in rho/VEGF transgenic mice originate from the deep capillary bed of the retina and therefore technically constitute retinal NV. It is not clear whether they behave like CNV, which originates from the choroid and is present in the subretinal space. This caveat must be kept in mind in the interpretation of our findings, but it should also be noted that blood vessels are strongly influenced by their surrounding microenvironment. If human umbilical vein endothelial cells, transfected with a gfp cDNA so that they are easily identified, are transplanted into the brain, they form vessels with tight junctions and other barrier characteristics that are phenotypically identical with brain microvessels. 25 Astrocytes are responsible for inducing endothelial cells to adopt central nervous system (CNS) characteristics. 26 Thus, surrounding cells may be a more powerful influence on behavior of endothelial cells and blood vessels than tissue of origin. Based on this information, it is our opinion that blood vessels that invade the subretinal space are likely to behave similarly, whether they originate from retinal or choroidal vessels; however, we acknowledge that there is no definitive proof of this, and caution should be exercised when drawing conclusions from our data regarding CNV. 
The source of VEGF in patients with CNV due to AMD has been suggested to be the RPE, although this is not known with certainty. Because VEGF is a secreted protein, it would make little difference whether it is produced by photoreceptors or RPE cells, because in both cases the result would be increased levels of VEGF in the subretinal space, unless VEGF were exclusively secreted from the basal surface of RPE cells. There is some evidence suggesting that under normal circumstances, VEGF may be secreted only from the basal surface of RPE cells, 27 but under normal circumstances CNV does not occur. One possible reason for the occurrence of CNV is that the normal polarized secretion of VEGF from RPE cells becomes disturbed so that VEGF is secreted apically. This, in combination with disturbance of the Bruch’s membrane/RPE barrier, may result in CNV. In fact, it has been demonstrated that rupture of Bruch’s combined with increased secretion of VEGF into the subretinal space results in much more severe CNV than rupture of Bruch’s membrane alone. 28 In contrast, increased expression of VEGF from normal, polarized RPE does not result in CNV. 29 Therefore, expression of VEGF by photoreceptors, resulting in increased levels of VEGF in the subretinal space is likely to mimic the situation in human disease quite well. 
Keeping these cautions in mind, it still may be useful to consider how the behavior of subretinal vessels in rho/VEGF mice can provide insight to help understand the clinical course of subfoveal CNV in some young patients. Before the development of photodynamic therapy, most patients with subfoveal CNV due to ocular histoplasmosis, high myopia, or essentially any disease other than age-related macular degeneration, were observed without treatment or treated with steroids. A small subgroup of such patients had decreased vision and metamorphopsia due to leakage from subfoveal CNV that over the course of many months or years improved. 30 These patients often exhibit hyperpigmented rings surrounded by hypopigmented rings due to migration of RPE cells to surround new vessels and their replacement by proliferated RPE cells deficient in melanin. The development of this funduscopic appearance was often accompanied by conversion of a lesion that leaked fluorescein to one that stained with fluorescein, but did not leak. This was interpreted as spontaneous involution of CNV, but involution may not be a good term, because it implies elimination of the NV. However, as demonstrated in rho/VEGF transgenics in the present study and in primates with rupture of Bruch’s membrane in another study, 14 despite reestablishment of the blood–retinal barrier, the NV may not be eliminated, but rather enveloped by the RPE, which forms tight junctions that prevent leakage. The present study may help to explain why some patients in whom this remodeling occurs regain good central vision. The RPE cells that surround the subretinal NV not only reestablish the blood–retinal barrier, but also reestablish a polarized epithelial monolayer that separates the new vessels from photoreceptors and engulfs the photoreceptors in microvilli. Visual acuity may remain stable for quite some time, but further follow-up may also demonstrate loss of vision from recurrent NV (Campochiaro PA, unpublished observations, 1995). Thus, RPE-induced remodeling of subretinal NV may not be a permanent solution in the setting of persistent angiogenic stimuli, but it suggests that neutralization of such stimuli, even without regression of NV, may be consistent with regain of visual function. 
The course we have described is rarely, if ever, seen in patients with age-related macular degeneration (AMD). In that setting, RPE cells appear incapable of productive remodeling, but rather tend to proliferate excessively, resulting in extensive subretinal scarring (disciform scarring) that when sufficiently thick is accompanied by photoreceptor cell death and permanent loss of vision. Whether this is due to loss of RPE cell function in the elderly, perturbation of RPE function by the underlying AMD disease pathogenesis, or excessive or different proliferative stimuli in AMD compared with ocular histoplasmosis, is unknown. In any case, it is clear that prevention of scarring as well as inhibition of neovascularization is an important goal of treatment of subretinal NV. 
One component of subretinal scarring is excessive and disorganized proliferation of cells. Another component is excessive production of extracellular matrix. Rho/VEGF transgenic mice do not exhibit excessive proliferation of RPE cells through 1 year of age, but they show excessive production of extracellular matrix by 6 months. Understanding the molecular signals responsible for the excessive deposition of extracellular matrix could provide insight regarding scarring that accompanies subretinal NV and provide additional therapeutic targets. 
 
Figure 1.
 
Persistent increased expression of mRNA for VEGF, VEGF receptor-1, and VEGF-R2 for 1 year in rho/VEGF transgenic mice. Mice were killed at several different time points after birth (n = 6 at each) including P21, P1M, P3M, P6M, and P12M. Retinas were dissected, and retinal RNA was isolated. RT-PCR demonstrated that retinal mRNA levels for VEGF were similar at each of the time points in rho/VEGF mice. The same was true for retinal mRNA levels for VEGF-R1 and VEGF-R2. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 were lower in mice that did not carry the rho/Vegf transgene. The mRNA level for S16 ribosomal protein was similar in each lane, demonstrating equal loading.
Figure 1.
 
Persistent increased expression of mRNA for VEGF, VEGF receptor-1, and VEGF-R2 for 1 year in rho/VEGF transgenic mice. Mice were killed at several different time points after birth (n = 6 at each) including P21, P1M, P3M, P6M, and P12M. Retinas were dissected, and retinal RNA was isolated. RT-PCR demonstrated that retinal mRNA levels for VEGF were similar at each of the time points in rho/VEGF mice. The same was true for retinal mRNA levels for VEGF-R1 and VEGF-R2. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 were lower in mice that did not carry the rho/Vegf transgene. The mRNA level for S16 ribosomal protein was similar in each lane, demonstrating equal loading.
Figure 2.
 
Retinal flatmounts from fluorescein-dextran perfused rho/VEGF transgenic mice show prominent neovascularization (NV) through 1 year of age. (A) At P21, there are numerous small foci of NV partially surrounded by RPE cells (arrowheads) at the outer border of the retina. The retinal vessels are out of focus in the background. (B) At 1 month, the tufts of NV are larger (arrowheads) compared with those at P21, and many are connected. (C) At 3 months, most of the tufts are connected to form a complex network of NV along the outer border of the retina (arrowheads). The NV is surrounded by RPE cells. (D) At 6 months, clumps of tortuous new vessels (arrowheads) were partially obscured by a dense coating of RPE cells. (E) At 12 months, clumps of tortuous NV loops (arrowheads) with dense surrounding RPE cells were connected by less tortuous NV with less prominent RPE cell coating. (F) Image analysis (n = 6 at each time point) showed that the number of neovascular lesions per retina decreased between P21 and P3M and remained stable thereafter. This decrease in the number of lesions corresponds to the coalescence of smaller lesions into larger ones over time. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month, by ANOVA for populations with unequal variances. (G) The average area of NV increased between P21 and 3 months and then stabilized. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month by ANOVA for populations with unequal variances. (H) There was no significant difference in the total area of NV per retina between P21 and 1 year.
Figure 2.
 
Retinal flatmounts from fluorescein-dextran perfused rho/VEGF transgenic mice show prominent neovascularization (NV) through 1 year of age. (A) At P21, there are numerous small foci of NV partially surrounded by RPE cells (arrowheads) at the outer border of the retina. The retinal vessels are out of focus in the background. (B) At 1 month, the tufts of NV are larger (arrowheads) compared with those at P21, and many are connected. (C) At 3 months, most of the tufts are connected to form a complex network of NV along the outer border of the retina (arrowheads). The NV is surrounded by RPE cells. (D) At 6 months, clumps of tortuous new vessels (arrowheads) were partially obscured by a dense coating of RPE cells. (E) At 12 months, clumps of tortuous NV loops (arrowheads) with dense surrounding RPE cells were connected by less tortuous NV with less prominent RPE cell coating. (F) Image analysis (n = 6 at each time point) showed that the number of neovascular lesions per retina decreased between P21 and P3M and remained stable thereafter. This decrease in the number of lesions corresponds to the coalescence of smaller lesions into larger ones over time. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month, by ANOVA for populations with unequal variances. (G) The average area of NV increased between P21 and 3 months and then stabilized. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month by ANOVA for populations with unequal variances. (H) There was no significant difference in the total area of NV per retina between P21 and 1 year.
Figure 3.
 
Toluidine blue–stained 1-μm-thick retinal cross sections demonstrate persistent subretinal neovascularization (NV) through 1 year of age in rho/VEGF transgenic mice. (A) At P21, there was NV (arrows) extending from dilated vessels in the deep capillary bed ( Image not available ) to photoreceptor outer segments (arrowhead). (B) At 1 month, NV extended through and disrupted portions of the outer nuclear layer into the subretinal space. Feeder vessels (arrows) connected to networks of subretinal vessels (arrowheads) partially surrounded by RPE cells. (C) At 3 months, there were large networks of NV in the subretinal space almost completely surrounded by RPE cells. There were pockets of subretinal fluid overlying the NV (arrowheads). The outer nuclear layer was irregular, and occasional feeder vessels (arrow) extended from the outer nuclear layer into the subretinal NV. (D) At 6 months, the network of NV in the subretinal space was completely surrounded by RPE cells. Photoreceptors were in close contact with the carpet of RPE overlying the NV (arrowheads) and there was a conspicuous absence of subretinal fluid. Occasional feeder vessels (arrow) were still extending through a thinned outer nuclear layer into the network of subretinal NV. (E) At 12 months, prominent extracellular matrix ( Image not available ) was present within the subretinal NV complex, which was completely surrounded by a thick layer of RPE cells. Encapsulating RPE cells were also prominent around feeder vessels (arrows).
Figure 3.
 
Toluidine blue–stained 1-μm-thick retinal cross sections demonstrate persistent subretinal neovascularization (NV) through 1 year of age in rho/VEGF transgenic mice. (A) At P21, there was NV (arrows) extending from dilated vessels in the deep capillary bed ( Image not available ) to photoreceptor outer segments (arrowhead). (B) At 1 month, NV extended through and disrupted portions of the outer nuclear layer into the subretinal space. Feeder vessels (arrows) connected to networks of subretinal vessels (arrowheads) partially surrounded by RPE cells. (C) At 3 months, there were large networks of NV in the subretinal space almost completely surrounded by RPE cells. There were pockets of subretinal fluid overlying the NV (arrowheads). The outer nuclear layer was irregular, and occasional feeder vessels (arrow) extended from the outer nuclear layer into the subretinal NV. (D) At 6 months, the network of NV in the subretinal space was completely surrounded by RPE cells. Photoreceptors were in close contact with the carpet of RPE overlying the NV (arrowheads) and there was a conspicuous absence of subretinal fluid. Occasional feeder vessels (arrow) were still extending through a thinned outer nuclear layer into the network of subretinal NV. (E) At 12 months, prominent extracellular matrix ( Image not available ) was present within the subretinal NV complex, which was completely surrounded by a thick layer of RPE cells. Encapsulating RPE cells were also prominent around feeder vessels (arrows).
Figure 4.
 
Ultrastructural changes in subretinal neovascularization (NV) over time. (A) At P21, new vessels were seen among the rod outer segments (OS) between the outer nuclear layer (ONL) and the RPE. The RPE cells were oriented normally on Bruch’s membrane (Br) and had normal ultrastructure. The new vessels had lumens ( Image not available ) lined with thick, immature endothelial cells (En), connected by tight junctions (arrows). (B) At 1 month, RPE cells had begun to migrate around the new vessels and in some areas contacted glial processes (white arrow). In areas of close contact with RPE cells, endothelial cells were thinner and exhibited fenestrations (inset, arrowheads). ( Image not available ) The lumen of the new vessel; small arrows: a tight junction between endothelial cells. (C) At 3 months, a new vessel appears to be surrounded by RPE cells, but there was still an overlying serous detachment ( Image not available ). There was an increase in extracellular matrix surrounding thin endothelial cell processes, which exhibited numerous fenestrations (inset, arrows). An endothelial cell nucleus protruded into a flattened lumen. (D) At 6 months, subretinal NV was completely surrounded by RPE cells. The RPE cells on top of the NV had basal infoldings adjacent to the new vessels and microvilli along their apical surface that interdigitated with outer segments. There was no identifiable subretinal fluid. The RPE cells beneath the NV had no microvilli, but rather had small basal infoldings along both surfaces. Fewer fenestrations and many more pinocytotic vesicles (inset, arrows) were visible than at earlier time points. Pericytes (P) were associated with new vessels, and some had thick surrounding extracellular matrix ( Image not available ). (E) At 12 months, extracellular matrix ( Image not available ) surrounding new vessels was more prominent than at previous time points. Endothelial cell processes lining lumens of vessels were very thin making it difficult to discern fenestrations or pinocytotic vesicles (inset). No pericytes were seen. RPE cells on top of the NV were polarized and had microvilli that interdigitated with rod outer segments, but neither microvilli nor basal infoldings were as prominent as at previous time points. RPE cells beneath the NV were flattened and nonpolarized.
Figure 4.
 
Ultrastructural changes in subretinal neovascularization (NV) over time. (A) At P21, new vessels were seen among the rod outer segments (OS) between the outer nuclear layer (ONL) and the RPE. The RPE cells were oriented normally on Bruch’s membrane (Br) and had normal ultrastructure. The new vessels had lumens ( Image not available ) lined with thick, immature endothelial cells (En), connected by tight junctions (arrows). (B) At 1 month, RPE cells had begun to migrate around the new vessels and in some areas contacted glial processes (white arrow). In areas of close contact with RPE cells, endothelial cells were thinner and exhibited fenestrations (inset, arrowheads). ( Image not available ) The lumen of the new vessel; small arrows: a tight junction between endothelial cells. (C) At 3 months, a new vessel appears to be surrounded by RPE cells, but there was still an overlying serous detachment ( Image not available ). There was an increase in extracellular matrix surrounding thin endothelial cell processes, which exhibited numerous fenestrations (inset, arrows). An endothelial cell nucleus protruded into a flattened lumen. (D) At 6 months, subretinal NV was completely surrounded by RPE cells. The RPE cells on top of the NV had basal infoldings adjacent to the new vessels and microvilli along their apical surface that interdigitated with outer segments. There was no identifiable subretinal fluid. The RPE cells beneath the NV had no microvilli, but rather had small basal infoldings along both surfaces. Fewer fenestrations and many more pinocytotic vesicles (inset, arrows) were visible than at earlier time points. Pericytes (P) were associated with new vessels, and some had thick surrounding extracellular matrix ( Image not available ). (E) At 12 months, extracellular matrix ( Image not available ) surrounding new vessels was more prominent than at previous time points. Endothelial cell processes lining lumens of vessels were very thin making it difficult to discern fenestrations or pinocytotic vesicles (inset). No pericytes were seen. RPE cells on top of the NV were polarized and had microvilli that interdigitated with rod outer segments, but neither microvilli nor basal infoldings were as prominent as at previous time points. RPE cells beneath the NV were flattened and nonpolarized.
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Figure 1.
 
Persistent increased expression of mRNA for VEGF, VEGF receptor-1, and VEGF-R2 for 1 year in rho/VEGF transgenic mice. Mice were killed at several different time points after birth (n = 6 at each) including P21, P1M, P3M, P6M, and P12M. Retinas were dissected, and retinal RNA was isolated. RT-PCR demonstrated that retinal mRNA levels for VEGF were similar at each of the time points in rho/VEGF mice. The same was true for retinal mRNA levels for VEGF-R1 and VEGF-R2. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 were lower in mice that did not carry the rho/Vegf transgene. The mRNA level for S16 ribosomal protein was similar in each lane, demonstrating equal loading.
Figure 1.
 
Persistent increased expression of mRNA for VEGF, VEGF receptor-1, and VEGF-R2 for 1 year in rho/VEGF transgenic mice. Mice were killed at several different time points after birth (n = 6 at each) including P21, P1M, P3M, P6M, and P12M. Retinas were dissected, and retinal RNA was isolated. RT-PCR demonstrated that retinal mRNA levels for VEGF were similar at each of the time points in rho/VEGF mice. The same was true for retinal mRNA levels for VEGF-R1 and VEGF-R2. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 were lower in mice that did not carry the rho/Vegf transgene. The mRNA level for S16 ribosomal protein was similar in each lane, demonstrating equal loading.
Figure 2.
 
Retinal flatmounts from fluorescein-dextran perfused rho/VEGF transgenic mice show prominent neovascularization (NV) through 1 year of age. (A) At P21, there are numerous small foci of NV partially surrounded by RPE cells (arrowheads) at the outer border of the retina. The retinal vessels are out of focus in the background. (B) At 1 month, the tufts of NV are larger (arrowheads) compared with those at P21, and many are connected. (C) At 3 months, most of the tufts are connected to form a complex network of NV along the outer border of the retina (arrowheads). The NV is surrounded by RPE cells. (D) At 6 months, clumps of tortuous new vessels (arrowheads) were partially obscured by a dense coating of RPE cells. (E) At 12 months, clumps of tortuous NV loops (arrowheads) with dense surrounding RPE cells were connected by less tortuous NV with less prominent RPE cell coating. (F) Image analysis (n = 6 at each time point) showed that the number of neovascular lesions per retina decreased between P21 and P3M and remained stable thereafter. This decrease in the number of lesions corresponds to the coalescence of smaller lesions into larger ones over time. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month, by ANOVA for populations with unequal variances. (G) The average area of NV increased between P21 and 3 months and then stabilized. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month by ANOVA for populations with unequal variances. (H) There was no significant difference in the total area of NV per retina between P21 and 1 year.
Figure 2.
 
Retinal flatmounts from fluorescein-dextran perfused rho/VEGF transgenic mice show prominent neovascularization (NV) through 1 year of age. (A) At P21, there are numerous small foci of NV partially surrounded by RPE cells (arrowheads) at the outer border of the retina. The retinal vessels are out of focus in the background. (B) At 1 month, the tufts of NV are larger (arrowheads) compared with those at P21, and many are connected. (C) At 3 months, most of the tufts are connected to form a complex network of NV along the outer border of the retina (arrowheads). The NV is surrounded by RPE cells. (D) At 6 months, clumps of tortuous new vessels (arrowheads) were partially obscured by a dense coating of RPE cells. (E) At 12 months, clumps of tortuous NV loops (arrowheads) with dense surrounding RPE cells were connected by less tortuous NV with less prominent RPE cell coating. (F) Image analysis (n = 6 at each time point) showed that the number of neovascular lesions per retina decreased between P21 and P3M and remained stable thereafter. This decrease in the number of lesions corresponds to the coalescence of smaller lesions into larger ones over time. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month, by ANOVA for populations with unequal variances. (G) The average area of NV increased between P21 and 3 months and then stabilized. *P < 0.005 for difference from P21; †P < 0.05 for difference from 1 month by ANOVA for populations with unequal variances. (H) There was no significant difference in the total area of NV per retina between P21 and 1 year.
Figure 3.
 
Toluidine blue–stained 1-μm-thick retinal cross sections demonstrate persistent subretinal neovascularization (NV) through 1 year of age in rho/VEGF transgenic mice. (A) At P21, there was NV (arrows) extending from dilated vessels in the deep capillary bed ( Image not available ) to photoreceptor outer segments (arrowhead). (B) At 1 month, NV extended through and disrupted portions of the outer nuclear layer into the subretinal space. Feeder vessels (arrows) connected to networks of subretinal vessels (arrowheads) partially surrounded by RPE cells. (C) At 3 months, there were large networks of NV in the subretinal space almost completely surrounded by RPE cells. There were pockets of subretinal fluid overlying the NV (arrowheads). The outer nuclear layer was irregular, and occasional feeder vessels (arrow) extended from the outer nuclear layer into the subretinal NV. (D) At 6 months, the network of NV in the subretinal space was completely surrounded by RPE cells. Photoreceptors were in close contact with the carpet of RPE overlying the NV (arrowheads) and there was a conspicuous absence of subretinal fluid. Occasional feeder vessels (arrow) were still extending through a thinned outer nuclear layer into the network of subretinal NV. (E) At 12 months, prominent extracellular matrix ( Image not available ) was present within the subretinal NV complex, which was completely surrounded by a thick layer of RPE cells. Encapsulating RPE cells were also prominent around feeder vessels (arrows).
Figure 3.
 
Toluidine blue–stained 1-μm-thick retinal cross sections demonstrate persistent subretinal neovascularization (NV) through 1 year of age in rho/VEGF transgenic mice. (A) At P21, there was NV (arrows) extending from dilated vessels in the deep capillary bed ( Image not available ) to photoreceptor outer segments (arrowhead). (B) At 1 month, NV extended through and disrupted portions of the outer nuclear layer into the subretinal space. Feeder vessels (arrows) connected to networks of subretinal vessels (arrowheads) partially surrounded by RPE cells. (C) At 3 months, there were large networks of NV in the subretinal space almost completely surrounded by RPE cells. There were pockets of subretinal fluid overlying the NV (arrowheads). The outer nuclear layer was irregular, and occasional feeder vessels (arrow) extended from the outer nuclear layer into the subretinal NV. (D) At 6 months, the network of NV in the subretinal space was completely surrounded by RPE cells. Photoreceptors were in close contact with the carpet of RPE overlying the NV (arrowheads) and there was a conspicuous absence of subretinal fluid. Occasional feeder vessels (arrow) were still extending through a thinned outer nuclear layer into the network of subretinal NV. (E) At 12 months, prominent extracellular matrix ( Image not available ) was present within the subretinal NV complex, which was completely surrounded by a thick layer of RPE cells. Encapsulating RPE cells were also prominent around feeder vessels (arrows).
Figure 4.
 
Ultrastructural changes in subretinal neovascularization (NV) over time. (A) At P21, new vessels were seen among the rod outer segments (OS) between the outer nuclear layer (ONL) and the RPE. The RPE cells were oriented normally on Bruch’s membrane (Br) and had normal ultrastructure. The new vessels had lumens ( Image not available ) lined with thick, immature endothelial cells (En), connected by tight junctions (arrows). (B) At 1 month, RPE cells had begun to migrate around the new vessels and in some areas contacted glial processes (white arrow). In areas of close contact with RPE cells, endothelial cells were thinner and exhibited fenestrations (inset, arrowheads). ( Image not available ) The lumen of the new vessel; small arrows: a tight junction between endothelial cells. (C) At 3 months, a new vessel appears to be surrounded by RPE cells, but there was still an overlying serous detachment ( Image not available ). There was an increase in extracellular matrix surrounding thin endothelial cell processes, which exhibited numerous fenestrations (inset, arrows). An endothelial cell nucleus protruded into a flattened lumen. (D) At 6 months, subretinal NV was completely surrounded by RPE cells. The RPE cells on top of the NV had basal infoldings adjacent to the new vessels and microvilli along their apical surface that interdigitated with outer segments. There was no identifiable subretinal fluid. The RPE cells beneath the NV had no microvilli, but rather had small basal infoldings along both surfaces. Fewer fenestrations and many more pinocytotic vesicles (inset, arrows) were visible than at earlier time points. Pericytes (P) were associated with new vessels, and some had thick surrounding extracellular matrix ( Image not available ). (E) At 12 months, extracellular matrix ( Image not available ) surrounding new vessels was more prominent than at previous time points. Endothelial cell processes lining lumens of vessels were very thin making it difficult to discern fenestrations or pinocytotic vesicles (inset). No pericytes were seen. RPE cells on top of the NV were polarized and had microvilli that interdigitated with rod outer segments, but neither microvilli nor basal infoldings were as prominent as at previous time points. RPE cells beneath the NV were flattened and nonpolarized.
Figure 4.
 
Ultrastructural changes in subretinal neovascularization (NV) over time. (A) At P21, new vessels were seen among the rod outer segments (OS) between the outer nuclear layer (ONL) and the RPE. The RPE cells were oriented normally on Bruch’s membrane (Br) and had normal ultrastructure. The new vessels had lumens ( Image not available ) lined with thick, immature endothelial cells (En), connected by tight junctions (arrows). (B) At 1 month, RPE cells had begun to migrate around the new vessels and in some areas contacted glial processes (white arrow). In areas of close contact with RPE cells, endothelial cells were thinner and exhibited fenestrations (inset, arrowheads). ( Image not available ) The lumen of the new vessel; small arrows: a tight junction between endothelial cells. (C) At 3 months, a new vessel appears to be surrounded by RPE cells, but there was still an overlying serous detachment ( Image not available ). There was an increase in extracellular matrix surrounding thin endothelial cell processes, which exhibited numerous fenestrations (inset, arrows). An endothelial cell nucleus protruded into a flattened lumen. (D) At 6 months, subretinal NV was completely surrounded by RPE cells. The RPE cells on top of the NV had basal infoldings adjacent to the new vessels and microvilli along their apical surface that interdigitated with outer segments. There was no identifiable subretinal fluid. The RPE cells beneath the NV had no microvilli, but rather had small basal infoldings along both surfaces. Fewer fenestrations and many more pinocytotic vesicles (inset, arrows) were visible than at earlier time points. Pericytes (P) were associated with new vessels, and some had thick surrounding extracellular matrix ( Image not available ). (E) At 12 months, extracellular matrix ( Image not available ) surrounding new vessels was more prominent than at previous time points. Endothelial cell processes lining lumens of vessels were very thin making it difficult to discern fenestrations or pinocytotic vesicles (inset). No pericytes were seen. RPE cells on top of the NV were polarized and had microvilli that interdigitated with rod outer segments, but neither microvilli nor basal infoldings were as prominent as at previous time points. RPE cells beneath the NV were flattened and nonpolarized.
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