Cloning and mRNA Expression of Vascular EndothelialGrowth Factor in Ischemic Retinas of Macaca fascicularis
David T. Shima,* Anne Gougos,-\ Joan W. Miller,% Michael Tolentino,%Gerald Robinson,$ Anthony P. Adamis,^X and Patricia A. DAmore*-f\\
Purpose. To identify and isolate cDNAs for the alternatively spliced vascular endothelial growthfactor (VEGF) mRNAs present in retina and to compare the relative levels of the splicevariants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas.
Methods. Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the mainbranch retinal veins. Reverse transcription-polymerase chain reaction was used to amplifythe VEGF coding region of RNAfrom ischemic and control retinas, and amplification productswere analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing.Analysis of VEGF mRNA expression was accomplished by in situ hybridization.
Results. Control and ischemic retinas produce mRNAs that correspond to the 121 and 165amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively splicedVEGF transcripts between control and ischemic retinas. Cloning and sequencing revealedthat simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteinsidentical to their respective human homologues. In situ hybridization of nonischemic retinasrevealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer.VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion,when there was a small but reproducible increase in signal. The expression peaked at approxi-mately 13 days, coincident with maximal iris neovascularization, and was significantly reducedby 28 days, when the iris vessels largely regressed.
Conclusions. The elevation of simVEGFi2i and VEGF165 in ischemic retinas is consistent witha role for diffusible, retina-derived angiogenic factors in the development of ocular neovascu-larization. Invest Ophthalmol Vis Sci. 1996; 37:1334-1340.
Vascular endothelial growth factor (VEGF) is an en-dothelial-specific mitogen that has potent angiogenicand permeability-enhancing properties in vivo. Theexpression of VEGF and its receptors has been corre-lated with embryonic, physiological, and pathologicangiogenesis.' We have hypothesized that VEGF playsa role in ischemia-associated ocular neovasculariza-
From the * Program in Biological and Biomedical Sciences, Harvard Medical School;the f laboratory for Surgical Research, Children's Hospital; the % Retina Service,Department of Ophthalmology, Harvard Medical School/Massachusetts Eye and EarInfirmary; the ̂ Department of Pathology, Harvard Medical School, Boston; and\\Hybridon, Inc., Worcester, Massachusetts.Sxif>p<nted l/y National Institutes of Health grants EY05985 and CA45548 (PD),the American Diabetes Association (JWM), and the Massachusetts Lion's ResearchFund (JWM, APA).Submitted for publication May 9, 1995; revised February 7, 1996; acceptedFebruary 9, 1996.Proprietary interest category: N.Reprint requests: Patricia A. D'Amore, Uiboratory for Surgical Research, Children'sHospital, Enders 1060, 300 Longiuood Avenue, Boston, MA 02115.
tion, and we have demonstrated2 that increased VEGFprotein levels in the aqueous are correlated spatiallyand temporally with experimental iris neovasculariza-tion in adult primates. In the primate neovasculariza-tion model, the retina is rendered ischemic by laserocclusion of the main branch retinal veins. Within 5to 7 days, new iris blood vessels are reproducibly ob-served and can be quantified by their angiographicappearance. Retinas that receive laser injury withoutproducing ischemia do not develop neovasculariza-tion.
It has been postulated that the induction of angio-genic factors is a direct response to the decreasedoxygen levels in the ischemic retina.3 In support ofthis hypothesis, VEGF mRNA has been shown to beelevated in the presumed hypoxic regions of tumors,4
and retinal cells have been shown to increase theirlevels of VEGF mRNA, protein, and bioactivity in re-
sponse to hypoxia.' Moreover, elevated levels of VEGFprotein have been demonstrated in the vitreous fromeyes with proliferative diabetic retinopathy, a neovas-cular disease clinically associated with decreased reti-nal vascular perfusion/1"8
VEGF meets many of the criteria necessary for con-sideration as an ischemia-induced retinal angiogenic fac-tor. First, VEGF is a well-documented angiogenic factorand is present in the retina.9 Second, the expression ofVEGF in the retina is elevated in response to ischemia.2
Third, the level of VEGF protein is correlated with thetiming and degree of neovascularization.2 Yet, in spiteof these observations, little is known of how VEGF isregulated in the retina. An investigation of the biosynthe-sis and localization of VEGF mRNA, in the normal andischemic adult primate eye, is critical to understandinghow changes in local retinal metabolism trigger neovas-cular pathologies.
Vascular endothelial growth factor is a family ofprotein isoforms that exhibits different biochemicalproperties and tissue-specific gene expression. At leastthree isoforms are known, each derived from alterna-tive splicing of a single VEGF gene.10 The differentisoforms are active in experimental models of angio-genesis and permeability in vitro and in vivo. VEGFi^i(numbers refer to the amino acids present in the ma-ture protein) is a secreted, diffusible protein found inthe conditioned media of tissue culture cells. VEGF165
has moderate heparin affinity and is detected both inconditioned media and attached to the cell surfaceand extracellular matrix. VEGF|8c, contains an addi-tional heparin-binding domain that tethers the pro-tein heparan sulfate on the cell surface and in thebasement membrane. Bioactive VEGF189 can be liber-ated from these sites by partial plasmin proteolysis orby disruption of its interaction with heparan sulfateproteoglycans. The functional significance of the dif-ferent VEGF isoforms is not understood, though it hasbeen hypothesized that the 165 and 189 amino acidisoforms bound to the extracellular matrix may act asa biologic reservoir for angiogenic or permeability-enhancing activity.''
We have characterized the structure and expres-sion of VEGF mRNAs in ischemic retinas from eyesin which iris neovascularization has developed. Thealternatively spliced forms of VEGF in control andischemic retina were identified by reverse transcrip-tion -polymerase chain reaction (RT-PCR), Southernblot analysis and molecular cloning of simian VEGF(simVEGF) cDNAs. These cDNAs were used as molec-ular probes for in situ hybridization analysis of VEGFmRNA expression in normal and ischemic monkeyretinas.
METHODSAll animals were cared for in accordance with theARVO Statement for the Use of Animals in Ophthal-
mic and Vision Research and with guidelines estab-lished for animal care at the Massachusetts Eye andEar Infirmary. Cynomolgous monkeys (Macaco, fascicu-laris) were anesthetized for all procedures with intra-muscular injection of a mixture of 20 mg/kg keta-mine, 1 mg/kg diazepam or 0.25 mg/kg acepromaz-ine maleate, and 0.125 mg/kg atropine sulfate.Supplemental anesthesia of 5 to 6 mg/kg of ketaminehydrochloride was administered as needed. Propara-caine hydrochloride (0.5% topical anesthetic drops)was administered before placement of lid speculaeand for pneumotonometry. Pupils were dilated asneeded with 2.5% phenylephrine and 0.8% tropicam-ide drops. Animals were placed in a comfortable re-straint device to allow head positioning for photogra-phy and angiography. Deeply anesthetized animalswere killed with an intravenous injection of Somn-lethal (JA Webster, Sterling, MA), a phentobarbital-based euthanasia solution approved by the AmericanVeterinary Medical Association.
Retinal ischemia and the resultant iris neovascu-larization were induced by laser occlusion of thebranch retinal veins, as previously described.2 For non-ischemic controls (referred to as sham-lasered eyes),laser photocoagulation was directed adjacent to theretinal vessels to produce retinal injury without retinalischemia. Iris neovascularization was followed by serialfluorescein angiography and was graded using stan-dardized angiograms and photographs. In one series,the animals were anesthetized and the eyes obtainedon days 4, 13, and 28 after laser occlusion. In a secondseries, untreated eyes were obtained, as were eyes 24hours after occlusion.
Reverse Transcription-Polymerase ChainReaction and cDNA analysis
Animals were anesthetized, eyes were enucleated andbisected at the equator, and retinas were dissectedwith forceps. Retinal tissue from day 13 ischemic reti-nas and nonischemic controls was flash frozen in liq-uid nitrogen until RNA extraction. CsCl-purified totalRNA (100 ng) was reverse transcribed using MMLVreverse transcriptase (Superscript II; Gibco BRL,Grand Island, NY). Approximately 1 % of RT productwas used for PCR amplification with primers based onthe human VEGF translational start and stop codons(5'-ACCATGAACTTTCTGCTGTC-3' and 5'-TCACC-GCCTCGGCTTGTCAC-3'). Amplification was per-formed using standard GeneAmp PCR buffer with 200fjM dNTP, 50 pmol primer, and 2.5 U Amplitaq (Per-kin Elmer, Norwalk, CT). The parameters for amplifi-cation were: 3-minute initial denaturation at 94°C; 30cycles of 94°C, 45 seconds each; 1-minute denatur-ation at 55°C; 1-minute denaturation at 72°C; and 3-minute final extension at 72°C. Products separatedin agarose gels and visualized by ethidium bromide
staining either were excised from gels, electroeluted,and cloned into the pCRII plasmid using a T/A clon-ing kit (Invitrogen, San Diego, CA), or they were trans-ferred to nylon for Southern blot analysis with a 520-bp Ncol/Bglll fragment of the human VEGF cDNA.Nucleotide sequence information was determined formore than 10 independently amplified cDNAs usingdideoxy-terminated sequencing. Nucleotide and pro-tein sequence comparisons were performed usingMacDNAsis (Hitachi Software, San Bruno, CA) se-quence analysis software.
In Situ Hybridization
Retinas, removed as described above, were immersedin 4% paraformaldehyde in phosphate-buffered saline(PBS), pH 7.4, at 4°C for 4 hours and then transferredto buffer and embedded in paraffin. Untreated and24-hour ischemic retinas were from the right and lefteye, respectively, of two animals. Days 4, 14, and 28ischemic retinas were from two eyes of the same ani-mal. One sham-lasered retina was examined. Six-mi-crometer sections were deparaffinized, rehydrated,and fixed in 4% paraformaldehyde-PBS for 1 hour.After washing in PBS, the sections were treated with20 //g/ml of proteinase K (Boehringer Mannheim,Indianapolis, IN) for 15 minutes at room temperature,washed in PBS, fixed in 4% paraformaldehyde-PBSfor 45 minutes, and acetylated in 0.1 M triethanol-amine, pH 8, containing 0.15 M NaCl and 0.13% ace-tic anhydride. After dehydration, sections were prehy-bridized in 50% formamide, 2 X SSC, 1 X Denhardt's,10% dextran sulfate, and 0.5% salmon sperm DNAfor 2 hours at 37°C. Digoxigenin-labeled VEGF anti-sense and sense riboprobes were transcribed from lin-earized plasmids containing full-length simianVEGF121 cDNA, using a Boehringer Mannheim Geniuskit. Prehybridization fluid was removed by a brief rinsein 2 X SSC and 40 //I of probe diluted to 1 /xg/ml inprehybridization solution was applied to each section,after which the tissue coverslipped to prevent evapora-tion. Tissue RNA and riboprobe were denatured insitu by incubation of slides at 95°C for 20 minutes ina humidified chamber, and hybridization was thenallowed to proceed for 16 hours at 42°C. Slides wereagitated gently in 2 X SSC to remove coverslips,washed twice in 2 X SSC for 15 minutes each, andincubated for 30 minutes at 37°C in 1 mg/ml RNase A(Boehringer Mannheim) diluted in 2 X SSC. Sectionswere washed twice in 0.1 X SSC for 20 minutes eachat 42°C, then once for 10 minutes at room tempera-ture, and rinsed in Genius Buffer 1 (100 mM Tris-HC1, pH 8, 150 mM NaCl). Immunohistochemical de-tection of specific hybridization was carried out byincubation for 2 hours with an anti-digoxigenin alka-line phosphatase conjugate (1:500; Boehringer Mann-heim) and visualization of the complex using the
Boehringer Mannheim nucleic acid detection kit. Allexperiments were repeated at least twice (two inde-pendent in situ hybridization runs) with duplicateslides for both sense and anti-sense hybridizations.Sections were visualized using Nomarski optics on aZeiss Axiophot (Carl Zeiss, Oberkochen, Germany).
RESULTS
Identification and Molecular Cloning of Retina-Derived simVEGF
Previously, we determined by Northern blot analysisthat VEGF mRNA levels are elevated dramatically inretinas rendered ischemic.2 To examine the produc-tion of VEGF mRNA splice variants in ischemic andnonischemic (sham-lasered) retinas, the relative abun-dance of the various VEGF transcripts was determinedby RT-PCR analysis of total RNA. In control and isch-emic retinas, two molecular species of approximately580 and 450 bp were identified. These correspond insize with VEGF cDNAs encoding the 121- and 165-amino acid forms of the protein (Fig. la). Southernblot analysis of PCR products (cycles 20 and 25), usinga human cDNA probe, corroborated the results of theRT-PCR (Fig. 2B). Whereas the product correspond-ing to VEGF121 appears to predominate in nonisch-emic retina, the products corresponding to VEGF12|and VEGFI65 appear equivalent in hypoxic retinal tis-sue (Fig. lb). In addition, with this more sensitivemethod of detection, a third product correspondingin size to the VEGFi89 transcript was detected (Fig.lb). Identical results have been obtained with samplesfrom different experimental and control retinas. Foreach experimental animal, angiography demonstratedthat the retina was ischemic, and it documented irisneovascularization (data not shown).
simVEGF amplification products from indepen-dent PCR reactions were cloned, and their nucleotidesequences were determined (Fig. 2A). The predictedVEGF proteins encoded by these cDNAs are similarin organization to the previously described VEGFs,with the simVEGF165 protein consisting of a 44-aminoacid insertion into the C-terminal portion of theVEGF,21 protein backbone (Fig. 2B). At the nucleotidelevel, simVEGFs were nearly identical (99%) to therespective human homologues (Fig. 2C; shown is thesequence for VEGF165). Three conservative nucleotidechanges in the simVEGF sequence (asterisks) encodea protein product with a predicted sequence identicalto that of human VEGF.
In Situ Hybridization Analysis of VascularEndothelial Growth Factor mRNA Expression
In situ hybridization was used to identify the cells syn-thesizing VEGF mRNA in ischemic retinas obtained
VEGF in Normal and Ischemic Primate Retinas
a.
C I N
VEGF 165- "
VEGF 121-
I R
VEGF 189/165
. VEGF
nGURE i. Vascular endothelial growth factor (VEGF) iso-forms in control and ischemic monkey retinas, (a) Reversetranscription-polymerase chain reaction analysis of totalRNA from nonischemic (N) and ischemic (I) monkey reti-nas. (C) Polymerase chain reaction (PCR) control con-taining no cDNA. Using agarose gel electrophoresis, twomajor amplification products are observed in both nonisch-emic and ischemic conditions, corresponding in size to theVEGF 121 and VEGFigs alternatively spliced mRNAs. (b)Southern blot analysis of amplification products (left panel,PCR cycle 20; right paneK PCR cycle 25) from control andischemic retinas using a human VEGF cDNA probe. Prod-ucts corresponding to mRNAs encoding the 121-, 165-, and189-amino acid VEGF isoforms are present in ischemic ret-ina. With a longer au to radiograph ic exposures, a productcorresponding to VEGF1Hy is also detectable in the controlretina samples but at drastically lower levels than VEGFm
and VEGF|65 products (data not shown).
1, 4, 13, and 28 days after laser-induced branch retinalvein occlusion and to compare these with the expres-sion pattern in sham-lasered controls. In this modelof neovascularization, the peak of new vessel growthin the iris occurs 10 to 14 days after laser vein occlu-sion, and vessel regression is observed within 25 to 35days of occlusion. We looked to see how rapidly aftervein occlusion in the retina elevated levels of mRNAmight be visualized. As early as 24 hours after occlu-sion, there was a small but reproducible increase inVEGF mRNA levels compared to those of normal, un-treated controls, particularly in the ganglion cell layer(compare Figs. 3A, 3B). Levels of VEGF mRNA, in theischemic retina 13 days after occlusion, were signifi-cantly higher than in either the day 4 or the day 28ischemic retina (Figs. 3C to 3E). Normal, untreatedretinas investigated with an antisense probe showed
1337
very low levels of signal (Fig. 3A). The highest levelof nonspecific hybridization using the sense probe wasobserved in the day 13 ischemic retina (Fig. 3F). Alow level of VEGF mRNA was detected in day 13 sham-lasered retinas (Fig. 3G). Using Northern blot analysis,we have shown that laser-induced retinal injury in theabsence of ischemia does not result in the inductionof VEGF mRNA. Consistent widi these findings, therewas only weak VEGF mRNA signals in day 13 sham-lasered retinas in ganglion cells and scattered cells ofthe inner nuclear layer (data not shown).
At each time point, VEGF mRNA was localized inganglion cell and inner nuclear layers. The most in-tense staining was associated with cells of the ganglioncell layer (based on their size, morphology, and distri-bution, we believe these to be ganglion cells). In theinner nuclear layer, there was heterogeneity in theintensity of the hybridization signal. Cells most proxi-mal to the internal plexiform layer were as stronglypositive as ganglion cells, but elsewhere in the innernuclear layer some cells appeared completely nega-tive. This heterogeneity was most apparent in the day13 retina (Fig. 3D). By day 28 after laser occlusion(Fig. 3E), a time when the iris vessels have largelyregressed, the VEGF mRNA level had diminished sig-nificantly.
DISCUSSION
Results of RT-PCR and molecular cloning of retinaVEGFs indicate that in response to ischemia, primateretinas produce primarily the diffusible isoforms,VEGF12t and VEGF|fi5, characterized by dieir low affin-ity for heparin. These data support the hypothesis thatthe primate retina responds to ischemia with a rapidincrease in the production of diffusible angiogenicfactors. Further, these findings are consistent with thebelief that a retina-derived angiogenic factor(s) is re-sponsible for neovascularization at distant sites, suchas the iris. Results of in situ hybridization revealed thatVEGF mRNA is elevated as early as 24 hours after theinduction of retinal ischemia, an observation consis-tent with the detection of a significant elevation inaqueous VEGF protein at 48 hours.2 Further, in-creased VEGF mRNA in the retina parallels the sever-ity of iris neovascularization and levels of VEGF in theaqueous, which reach their maximum between 10 and14 days after laser occlusion. After this peak of neovas-cularization, VEGF mRNA and aqueous VEGF proteinlevels decline and are followed by regression of thenew iris vessels. These temporal correlations provideadditional evidence that the ischemic retina itself pro-duces the VEGF protein found in die vitreous andaqueous of these animals.2
Relative to the human homologue, simVEGFcDNA contains three conservative nucleotide changes,
FIGURE 2. simVEGFm and simVEGFi65 nucleotide and predicted protein sequences. (A)Nucleotide sequence alignment for the simVEGF cDNAs. (B) Alignment of simVEGF cDNAspredicted protein products. (C) Alignment of the cDNA sequences for simian and humanVEGF|65. Asterisks denote three conservative nucleotide changes in the simVEGF cDNA thatencode a protein product identical to human VEGF. The same nucleotide substitutions arepresent in the SimVEGF|2i cDNA (data not shown). Shaded areas denote regions of nucleo-tide— amino acid sequence identity. It should be noted that the 5 N- and C-terminal aminoacids are derived from primers containing human VEGF sequence and do not necessarilyrepresent the actual simian sequence. VEGF = vascular endothelial growth factor.
resulting in an identical protein product. This degreeof identity has allowed us to pursue studies in thissimian model of ocular neovascularization usingmonoclonal antibodies raised against human VEGF,which had been demonstrated to be species specific(i.e., did not cross-re act with murine or avian VEGF).12
As a result, recently we have been able to show thatintraocular injection of anti-VEGF monoclonal anti-bodies prevent new iris vessel growth, demonstratingthat VEGF is a necessary factor for ischemia-inducedocular neovascularization.13
In situ analysis of VEGF mRNA expression in the
retina confirms the alterations in VEGF mRNA levelsobserved by Northern blot analysis and provides fur-ther information regarding the localization of theVEGF response to ischemia. The hybridization patternof VEGF mRNA in the ganglion cell layer stronglysuggests that ganglion cells are a major source of thegrowth factor. Ganglion cell production of VEGF inretinal ischemia could explain the clinical observationthat proliferative diabetic retinopathy does not occurin the setting of optic atrophy or ganglion cell loss.14
It also has been noted that successful panretinal pho-tocoagulation, leading to regression of optic nerve,
VEGF in Normal and Ischemic Primate Retinas 1339
a G b
ONL
m
FIGURE 3. In situ localization of vascular endothelial growthfactor (VEGF) mRNA in normal and ischemic retinas. Cel-lular localization of VEGF mRNA expression by hybridiza-tion with an antisense VEGF riboprobe in (A) normal un-treated retina, (B) 24-hour ischemic retina, (C) 4-day isch-emic retina, (D) 13-day ischemic retina, (E) 28-day ischemicretina. Sense riboprobe hybridized in (F) 13-day ischemicretina and (G) 13-day day retina. Magnification, X330.
retinal, and iris neovascularization, is accompanied byoptic atrophy.'1 Panretinal photocoagulation may leadto regression of neovascularization by causing thedeath of ganglion cells, the source of the angiogenicstimulus, and not by adjusting retinal metabolism ortransretinal oxygen gradients, as has been suggested.u>
The cells synthesizing VEGF mRNA in the inner nu-clear layer have yet to be identified definitively; candi-dates include Muller cells, amacrine cells, and hori-zontal or bipolar cells.
Not surprisingly, the level of VEGF gene expres-sion was highest in the ganglion cell and inner nuclearlayers. Because the retinal circulation affected by laservein occlusion supplies the inner retina, retinal isch-emia is most severe in the inner portion of the retina,whereas the outer retina, nourished by choroidal cir-culation, does not experience as severe ischemia,17
Retinal ischemia in the inner retina secondary to laservein occlusion has been documented in a relatedmodel by Pournaras and coworkers.18 They measuredO2 concentrations of 16.5 mm Hg in tissue of theinner retina after the disruption of retinal circulation(the normal concentration is 26 mm Hg).
Interestingly, VEGF synthesis in the ganglion celllayer appeared to be confined to the ganglion cells.This is in contrast to VEGF expression in murine reti-nal vascularization. During developmental angiogene-sis in rat and cat retinas, the growth factor was shownto be produced by astrocytes and Muller cells in re-sponse to physiologic hypoxia.19 Similarly, in an exper-imental model of retinopathy of prematurity using themouse, VEGF mRNA was localized to Muller cells.20
The difference in sites of expression may be the resultof species differences. However, it is more likely thatthe degree of hypoxia is a critical variable. Chang-Ling and Stone21 have shown that astrocytes are pref-erentially lost in the experimental models of retinopa-thy of prematurity. We suspect that the severe isch-emia created in the monkey model of laser-inducedischemia leads to the loss of astrocytes and that gan-glion cells upregulate VEGF in a compensatory fash-ion. The staining in the vicinity of the outer limitingmembrane, particularly at day 13 in ischemic retinas,appears to be associated with cell processes possiblybelonging to bipolar cells, photoreceptor cells, orboth. Although it is possible that they belong to Mullercells, we did not see the distinct signal outlining theMuller cell processes that Stone19 and Pierce2" ob-served. Upregulation of VEGF mRNA in this area maybe caused by the fact that this portion of the retinareceives part of its oxygen supply from retinal vessels18
and, hence, also would experience ischemia.VEGF appears to be expressed at a low level in
the nonischemic retina. Results from PCR analysis ofVEGF from control retinas is supported by analysis ofVEGF mRNA by Northern blot and in situ hybridiza-
1340 Investigative Ophthalmology 8c Visual Science, June 1996, Vol. 37, No, 7
tion, as well as by immunodetection of VEGF protein,all of which demonstrate low but detectable VEGF.2
The increase in VEGF mRNA in ischemic retinas isnot associated with changes in alternative splicing. Ap-proximately equal amounts of VEGF12i and VEGF,65
are detectable by PCR in control retinas, and the rela-tive contribution of each splice variant to the totalamount of VEGF mRNA remained constant after theinduction of ischemia. Low levels of VEGF|89, the cell-associated isoform, are present in ischemic and non-ischemic retinas. Although our in situ analysis of nor-mal retina does not indicate significant levels of VEGFmRNA, immunohistochemical analysis9 and immuno-assay8 indicate a low level of constitutive protein pro-duction. In addition to the retina, numerous otheradult tissues are known to have low levels of VEGFexpression, suggesting that VEGF may act as a mainte-nance/survival factor for mature blood vessel beds.22
Key Words
angiogenesis, ganglion cells, hypoxia, in situ hybridization,neovascularization
Acknowledgments
The authors thank Wendy Evanko for her assistance in pre-paring the manuscript. They also thank Dr. Elio Raviolafor his useful insights identifying cell types in the in situhybridization studies.
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