Increased Expression of KDR/Flk-1 (VEGFR-2) in Murine Model of Ischemia-Induced Retinal Neovascularization

Increased Expression of KDR/Flk-1 (VEGFR-2)in Murine Model of Ischemia-InducedRetinal Neovascularization

Kiyoshi Suzuma, Hitoshi Takagi, Atsushi Otani, Izumi Suzuma,and Yoshihito HondaDepartment of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine,Kyoto 606, Japan

Received February 17, 1998

Although the vascular endothelial growth factor (VEGF)/VEGF receptor system plays a critical role in the patho-genesis of ischemic retinal neovascular diseases such asdiabetic retinopathy, regulation of VEGF receptor ex-pression in ischemic retina has not been fully investi-gated in vivo. Accordingly, we studied the regulation ofFlt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) expressionin a mouse model of ischemia-induced retinal neovascu-larization. Immunohistochemistry for Flt-1 and KDR/Flk-1 revealed that, in hypoxic retina, the immunoreac-tivity of KDR/Flk-1 was increased in both intensity andextent of involvement in the vessels near the avasculararea, particularly at the neovascular tufts, but that thepattern of Flt-1 expression in hypoxic retina was almostthe same as that of control animals. The number of KDR/Flk-1-positive vessels was significantly increased in hy-poxic retina (P < 0.01). In addition, expression of bothFlt-1 and KDR/Flk-1 was observed in nonvascular cells ofthe neural retina. Northern blot analysis demonstratedthat the mRNA levels of KDR/Flk-1 were greater in theneovascular retina of hypoxic animals than in controlanimals. We suggest that the increased expression ofKDR/Flk-1 in vascular cells might potentiate the VEGF-mediated angiogenesis that accompanies many ischemicretinal diseases. © 1998 Academic Press

INTRODUCTION

Neovascular diseases of the retina are one of themajor causes of blindness, and recent evidence hasmost strongly implicated vascular endothelial growthfactor (VEGF) in the pathogenesis of several ischemicretinal neovascular diseases, including diabetic reti-nopathy (Adamis et al., 1994; Aiello et al., 1994, 1995;Amin et al., 1997). VEGF is a potent angiogenic andvasopermeability factor (Leung et al., 1989; Berse et al.,1992) whose expression is increased by hypoxia (Sh-weiki et al., 1992), which is one of the primary stimulifor ocular neovascularization. The effects of VEGF aremediated through highly endothelial cell-specific,high-affinity phosphotyrosine kinase receptors: Flt-1(VEGFR1) and KDR/Flk-1 (VEGFR2) (de-Vries et al.,1992; Ferrara et al., 1992; Millauer et al., 1993; Quinn etal., 1993). In vitro studies have shown that KDR/Flk-1is expressed in microvascular endothelial cells andthat Flt-1 is expressed in both endothelial cells andpericyte (Nomura et al., 1995; Takagi et al., 1996).VEGF binding sites and the KDR/Flk-1 protein levelhave been shown to be increased by hypoxia in cul-tured retinal microvascular endothelial cells (Thiemeet al., 1995; Takagi et al., 1996) and human umbilical

Microvascular Research 56, 183–191 (1998)Article No. MR982111

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vein endothelial cells (HUVEC) (Waltenberger et al.,1996). A paracrine upregulation of KDR/Flk-1 wasalso suggested in cultured HUVEC subjected to hyp-oxia (Brogi et al., 1996), and both Flt-1 and KDR/Flk-1mRNAs were upregulated in an in vivo model ofchronic lung hypoxia (Tuder et al., 1995). To ourknowledge, however, the regulation of VEGF receptorexpression has not yet been investigated in retinalneovascularization induced by hypoxia in vivo.

Accordingly, we evaluated the expression of Flt-1and KDR/Flk-1 in a mouse model of ischemia-in-duced retinal neovascularization and observed the up-regulation of KDR/Flk-1 expression in the vascularcells.

MATERIALS AND METHODS

Animal Model

The experiments described herein adhered to theARVO Standards for the Use of Animals in Ophthal-mic and Vision Research. To produce ischemia-in-duced retinal neovascularization, litters of 7-day-old(postnatal day 7, P7) C57BL/6J mice with nursingmothers were exposed to 75 6 2% oxygen for 5 daysand then returned to room air at age P12, as described(Smith et al., 1994; Pierce et al., 1995). Mice of the sameage kept in room air were used as controls. Flat-mounted, fluorescein-conjugated dextran-perfusedretinas were examined to assess the retinal vasculature(Smith et al., 1994).

Northern Blot Analysis

For Northern blot analysis, the total RNA was iso-lated from retinas of mice at four different time points(10 retinas from five mice at each time point, P12immediately after return to room air, P14, P19, andP26) using guanidium thiocyanate (Chomczynski etal., 1987). The Northern blot analysis was performedon 15 mg total RNA after 1% agarose–2 M formalde-hyde gel electrophoresis and subsequent capillarytransfer to Biodyne nylon membranes (Pall BioSup-port, East Hills, NY) and ultraviolet cross-linking us-

ing a FUNA-UV-LINKER (FS-1500, Funakoshi, Tokyo,Japan). Radioactive probes were generated using Am-ersham Megaprime labeling kits and 32P-dATP (Am-ersham, Arlington Heights, IL). Blots were prehybrid-ized, hybridized, and washed in 0.5 3 SSC, 5% SDS at65°C with four changes over 1 h in a rotating hybrid-ization oven (TAITEC, Koshigaya, Japan). All signalswere analyzed using a densitometer (BAS-2000II, FujiPhoto Film, Tokyo, Japan), and lane loading differ-ences were normalized using a 36B4 cDNA probe(Masiakowski et al., 1982; Liang et al., 1992). The hu-man Flt-1 and KDR/Flk-1 cDNA were generous giftsfrom Dr. Masabumi Shibuya and Dr. Lloyd P. Aiello,respectively. The human VE-cadherin cDNA was ob-tained from American Type Culture Collection (Rock-ville, MD).

Immunohistochemistry

Mice at different time points (P17, P18, P19, P20, andP21, n 5 5 for each time point) were deeply anesthe-tized with pentobarbital sodium (100 mg/kg) and sac-rificed by cardiac perfusion of 4% paraformaldehydein phosphate-buffered saline (PBS). Eyes were enucle-ated and fixed in 4% paraformaldehyde at 4°C over-night, and embedded in paraffin. Serial 5-mm sectionsof the whole eyes were placed on microscope slides,and the slides were stored at 4°C. Several slides fromeach eye were also stained with hematoxylin–eosin.

For immunohistochemical analysis, all incubationsteps were performed in a moist chamber, and rinseswere performed by immersing the slides in a PBSbath. Paraffin was then removed from the sections bytreatment with xylene, and the sections were rehy-drated through a graded series of alcohol and rinsedwith PBS. To block endogenous peroxidase, 3% hydro-gen peroxide was applied to each section for 10 min-utes, after which the sections were incubated for 20min with blocking serum (Vector, Burlingame, CA).The specimens were then incubated overnight at 4°Cwith the primary antibody (rabbit polyclonal anti-Flt-1, rabbit polyclonal anti-Flk-1; Santa Cruz Biotech-nology, Santa Cruz, CA, or rabbit polyclonal anti-vonWillebrand factor; Dako, Glostrup, Denmark) andwashed for 10 min with PBS. A standard indirectimmunoperoxidase procedure using the ABC Elite kit

184 Suzuma et al.

Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

(Vector) was performed with 3,39-diamino-benzidinetetrahydrochloride (DAB, Dako) as the substrate. Fi-nally, the slides were rinsed with tap water, dehy-drated through a graded series of alcohol, clarifiedwith xylene, and coverslipped with xylene-based per-manent mounting medium for viewing.

For the negative control, the primary antibody pre-incubated with the immunizing peptide (Santa CruzBiotechnology) was used. Other staining procedureswere the same as described above.

Confocal Microscopy

All specimens were examined with a Zeiss scanninglaser confocal microscope (LSM 410 inverted LaserScan Microscope, Zeiss, Oberkochen, Germany) in thetransmitted-light mode. Digitized images were cap-tured by computer and stored on an optical disk forsubsequent display. Photographic images were takenfrom the computer with a digital printer (Pictrogra-phy, Fuji Photo Film, Tokyo, Japan).

Quantitation of KDR/Flk-1-Positive Vessels

Over 50 serial 5-mm paraffin-embedded axial sec-tions were obtained, starting at the optic nerve head.Ten intact sections of equal length, each 25mm apart,were evaluated for a span of 250 mm. After immuno-staining, the numbers of KDR/Flk-1-positive vesselswere counted in each section according to a fullymasked protocol. The mean of all 10 counted sectionsyielded the average number of KDR/Flk-1-positivevessels per 5-mm section per eye.

To quantify the incidence of KDR/Flk-1-positivevessels, serial sections next to the sections stained forKDR/Flk-1 were stained for von Willebrand factor(vWF). The numbers of vWF-positive vessels werecounted in each section, and the percentage was cal-culated by dividing the number of KDR/Flk-1-posi-tive vessels by the number of vWF-positive vessels.

Statistical Analysis

Data are expressed as the mean 6 standard devia-tion (SD). Statistical analysis was performed by using

the Mann–Whitney test. A P value less than 0.05 wasregarded as significant.

RESULTS

Localization of Flt-1 and KDR/Flk-1Immunoreactivity in Hypoxic Retina

Similar to previous studies (Smith et al., 1994; Pierceet al., 1995), histologic observation of hematoxylin–eosin-stained sections showed neovascular tufts, par-ticularly in the mid-periphery, extending above theinternal limiting membrane into the vitreous after 5days of hypoxia. These neovascular tufts were mostprominent on P17–P19, and after P23 the neovascular-ization regressed, and the vascular pattern normalizedby P26. The vascular endothelial cells were identifiedas the result of immunoreactivity to vWF in bothhypoxic (Fig. 1H) and control (Fig. 1I) retinas.

KDR/Flk-1 immunoreactivities were observed in thevascular cells, ganglion cell layer, and internal and outernuclear layers in retinas from both hypoxic P19 mice(Fig. 1C) and normal control P19 mice (Fig. 1B). NoKDR/Flk-1 immunoreactivity was seen in the negativecontrol (Fig. 1A). Analysis of the pattern of KDR/Flk-1protein expression demonstrated that not all of the ves-sels in hypoxic and control retinas expressed KDR/Flk-1; rather, in hypoxic retina, the immunoreactivity ofKDR/Flk-1 was increased in both intensity and in num-ber of vessels involved near the avascular area, particu-larly at the neovascular tufts (Fig. 1D).

Flt-1 immunoreactivity was observed primarily inthe nerve fiber layer and the inner plexiform layer andwas not remarkable in vascular cells either in hypoxicretina (Fig. 1G) or normal retina (Fig. 1F). Flt-1 proteinexpression was also unremarkable at neovascular tuftsin hypoxic retina, and the staining pattern was similarin the hypoxic and control specimens (Fig. 1G, 1F).There was no Flt-1 immunoreactivity in the negativecontrol (Fig. 1E).

Increased Expression of KDR/Flk-1 Protein inIschemia-Induced Retinal Neovascularization

We studied also the regulation of VEGF receptorexpression in retinal vascular cells. Since Flt-1 immu-

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noreactivity was not remarkable in vascular cells, wefocused on KDR/Flk-1, and since the retinal neovas-cularization was most prominent on P17–P19, we usedP19 mice for this evaluation. To assess KDR/Flk-1expression in retinal vessels, we quantified the num-ber of KDR/Flk-1-positive vessels in both hypoxic andnormal retinas. As shown in Fig. 2A, the mean numberof KDR/Flk-1-positive vessels was 59 6 14 in hypoxicretina and 30 6 9 in control retina; the number ofKDR/Flk-1-positive vessels was significantly in-creased in hypoxic retinas (P , 0.01, n 5 5). We alsoquantified the percentage of KDR/Flk-1-positive ves-sels by dividing the number of KDR/Flk-1-positivevessels by the number of vWF-positive vessels. Asshown in Fig. 2B, the percentage of KDR/Flk-1-posi-tive vessels was 65.9 6 11.1% in hypoxic retinas and34.3 6 6.4% in control retinas; the percentage of KDR/Flk-1-positive vessels significantly increased in hy-poxic retinas (P , 0.01, n 5 5). No obvious increase inKDR/Flk-1 was observed in the ganglion cell layer orinternal and outer nuclear cell layers in hypoxic retinaas compared with normal retina.

KDR/Flk-1 and Flt-1 mRNA Levels in HypoxicRetina

To investigate mRNA expression of VEGF receptorsin retinal neovascularization, Northern blot analysiswas performed using total RNA. Figure 3A shows anincrease in KDR/Flk-1 mRNA levels from P14 to P19.RNA from age-matched control animals raised inroom air demonstrated a comparatively constant andlow level of KDR/Flk-1 mRNA. Figure 3B shows thefold increase in KDR/Flk-1 mRNA at each time pointcompared with age-matched controls after normaliza-

tion to the 36B4 signal in each lane. Densitometerquantitation of the KDR/Flk-1 mRNA signal on theNorthern blot shown in Figs. 3A and 3B showed anincrease in the KDR/Flk-1 mRNA level from P12through P26, with a maximal 1.7-fold increase on P19compared with that seen in normal age-matched con-trols. In contrast, the Flt-1 mRNA increased 1.2-fold atday 14; however, the increase was not as marked asthat of KDR/Flk-1. Because the hypoxic retina devel-ops more angiogenic vessels, it is possible that theobserved increased mRNA levels of VEGF receptorsmight result from an increased number of neovascularendothelial cells. To determine if the increase in KDR/Flk-1 is related to an increased number of vascularendothelial cells, we determined mRNA levels of VE-cadherin, a specific marker for vascular endothelialcells using the same mRNA blots. The increase inVE-cadherin was less than that of KDR/Flk-1 (Figs. 3Aand 3B).

DISCUSSION

In ischemic retinal diseases such as diabetic retinop-athy, the retinal nonperfusion is followed by patho-logic angiogenesis that leads to visual loss. We havenow demonstrated an increased expression of KDR/Flk-1 (VEGFR-2) in such ischemia-induced retinal an-giogenesis in vivo and found also that Flt-1 and KDR/Flk-1 were expressed in nonvascular cells in the neuralretina, suggesting a potential role of the VEGF/VEGFreceptor system in neural retina.

In this mouse model, retinal neovascularization isthought to be mediated predominantly through hy-

FIG. 1. Localization of Flt-1 and KDR/Flk-1 protein expression in murine retina. No labeling for KDR/Flk-1 was seen in negative control (A).KDR/Flk-1 immunoreactivity was observed in the vascular cells and in ganglion cell layer (GCL) and internal (INL) and outer nuclear layers(ONL) in both hypoxic retina (C) and control retina (B). In hypoxic retina, the immunoreactivity of KDR/Flk-1 was increased in both intensityand number of the vessels involved near the avascular area, particularly at the neovascular tufts (C and D, arrow). No labeling for Flt-1 wasseen in negative control (E). Flt-1 immunoreactivity was observed in the nerve fiber layer and the inner plexiform layers, but was notremarkable in vascular cells in both hypoxic retina (G) and control retina (F). Flt-1 expression was also not remarkable at the neovascular tufts(G, arrowhead) in hypoxic retina, and the pattern of Flt-1 expression in hypoxic retina was almost same as that of control mice. The vascularendothelial cells were detected as immunoreactivity to vWF in both hypoxic (H) and control (I) retina. In hypoxic retina, immunoreactivity tovWF showed neovascular tufts, particularly in the mid-periphery, extending above the internal limiting membrane into the vitreous (H,arrowheads). A, B, C, E, F, and G are at the same magnification. H and I are at the same magnification. Bar, 50 mm in A and H, 10 mm in D.



poxic VEGF induction, since the induction of VEGF isclosely associated with the development of neovascu-larization (Pierce et al., 1995) and since inhibition ofVEGF results in suppression of retinal neovasculariza-tion (Aiello et al., 1995; Robinson et al., 1996). In thismodel, we observed increases of KDR/Flk-1 expres-sion in both retinal and intravitreal vascular cells. Thisincrease was more evident in vascular cells into thevitreous, and the most marked expression was ob-served at the neovascular tufts adjacent to areas ofretinal nonperfusion. Although we did not determinethe proliferative markers such as incorporation ofBrDU, these cells growing into the vitreous were prob-ably angiogenic cells as many studies utilized num-bers of these cells to quantify retinal neovasculariza-tion (Aiello et al., 1995; Robinson et al., 1996). Inaddition, not all vascular cells were positive for KDR/Flk-1, suggesting that angiogenic vascular cells mightexpress more KDR/Flk-1 than do static vascular cells.Since VEGF induction is concentrated just posterior tothe neovascular tufts (Pierce et al., 1996), the proximityof the site of production of VEGF and the expressionof KDR/Flk-1 strongly suggests a paracrine role forVEGF in the development of local angiogenesis.

The expression of KDR/Flk-1 was upregulated invascular cells; remarkable upregulation was not ob-served in nonvascular cells in the neural retina. Thissuggests that vascular cells have some mechanismcapable of increasing VEGF receptors in response tohypoxia, which might facilitate the VEGF-dependentretinal angiogenesis. To further look into this, we in-vestigated the gene expression of KDR/Flk-1. Themessenger RNA levels of KDR/Flk-1 in the retinawere increased in eyes with neovascularization com-pared with the control eyes, suggesting a local up-regulation of KDR/Flk-1. This increase in mRNA,however, was unremarkable, probably because stableexpression of KDR/Flk-1 in nonvascular cells ameri-olates the changes in vascular cells. In hypoxic retinas,the number of vascular endothelial cells increasingbecause of new vessel formation and might contributeto the increase in KDR/Flk-1 mRNA. This is probablynot the case because the increase in VE-cadherinmRNA, a specific marker for vascular endothelialcells, was less than that of KDR/Flk-1 mRNA. Thesedata suggest that the increase in KDR/Flk-1 mRNA

FIG. 2. Effects of hypoxia on KDR/Flk-1 expression in retinalvessels. (A) To assess the effects of hypoxia on KDR/Flk-1 ex-pression in retinal vessels, we quantified the number of KDR/Flk-1-positive vessels in both hypoxic and normal retinas. Themean number of KDR/Flk-1 positive vessels was 59 6 14 inhypoxic retina and 30 6 9 in control retina. The number ofKDR/Flk-1-positive vessels was significantly increased in hy-poxic retina (P , 0.01, n 5 5). (B) We also quantified the rate ofKDR/Flk-1-positive vessels by dividing the number of KDR/Flk-1-positive vessels by the number of vWF-positive vessels. Therate of KDR/Flk-1-positive vessels was 65.9 6 11.1% in hypoxicretina and 34.3 6 6.4% in control retina. The rate of KDR/Flk-1-positive vessels was significantly increased in hypoxic retinas(P , 0.01, n 5 5).

188 Suzuma et al.


may be primarily the result of upregulation of thegene by endothelial cells. An in situ hybridizationstudy is planned to elucidate the local regulation ofgenes in ischemic retinas.

In contrast to KDR/Flk-1, Flt-1 staining was unre-markable in both retinal vessels and neovascular tufts.In addition, we investigated the gene expression ofFlt-1 by Northern blot analysis, and found that thelevels of Flt-1 mRNA increased slightly; however, theincreases were minimal and smaller than that ofKDR/Flk-1. These data suggest that Flt-1 expression

might not be markedly altered by ischemia-inducedretinal angiogenesis. KDR/Flk-1 and Flt-1 have beendemonstrated to be different in function. KDR-ex-pressing cells show changes in morphology, chemo-taxis, and mitogenicity on VEGF stimulation, whereasFlt-1-expressing cells lack such response (Walten-berger et al., 1994). Gene knockout experiments alsosuggest differences of these receptor functions in thedevelopment of the vascular system (Fong et al., 1995;Shalaby et al., 1995). The upregulation of KDR/Flk-1might play a more predominant role in facilitating thedevelopment of neovascularization through ligationof VEGF to this receptor than does Flt-1.

The mechanism of upregulation of KDR/Flk-1 inretinal vascular cells is still not clear. In cultured bo-vine retinal microcapillary endothelial cells, hypoxiadecreased the mRNA level of KDR/Flk-1, whereas the[125I]VEGF binding activity was increased (Thieme etal., 1995; Takagi et al., 1996). Similarly, HUVEC andporcine aortic endothelial cells exposed to hypoxiashowed increased KDR protein and VEGF bindingsites and a decreased level of KDR mRNA (Walten-berger et al., 1996). These data suggest that the upregu-lation of KDR/Flk-1 by hypoxia is mediated through aposttranscriptional mechanism such as reduced inter-nalization of the receptors. An interesting observationwas reported by Borgi et al. (1996); in their study,hypoxia-conditioned media increased the number ofVEGF binding sites, suggesting that ischemic tissuemight produce and release soluble factors capable oflocally upregulating VEGF receptor expression onvascular cells and thereby promote local neovascular-ization. Our finding that KDR/Flk-1 upregulation ismost evident close to the area of nonperfusion mightsupport this concept.

Although the VEGF/VEGF receptor system has notyet been investigated well in neural systems, Yang etal. (1996) reported that Flk-1 is expressed on the sur-face of neural progenitors in mouse retina. In thisstudy, KDR/Flk-1 transcripts were detected withinthe inner nuclear layer; most likely Muller glial cellsand the lacZ gene inserted into the Flk-1 gene are alsoexpressed in the inner nuclear layer, ganglion celllayer, and the outer nuclear layer of adult retina. In thepresent study, we observed KDR/Flk-1 immunoreac-tivity in the ganglion cell layer and internal and outer

FIG. 3. KDR/Flk-1 and Flt-1 mRNA expressions during hypoxiaand the development of neovascularization. Results of the Northernblot analysis of total RNA (15 mg) isolated from animals aftervarious durations of hypoxia and from age-matched normal con-trols. Northern blots and control 36B4 (A) and quantification (B). Forthese calculations, the amount of mRNA at each time point was firstnormalized to its own 36B4 signal. The fold increase over thenormalized value for the corresponding age-matched normal con-trol was then calculated. A maximal 1.7-fold increase of KDR/Flk-1mRNA was observed on P19 compared with that seen in normalage-matched controls. The levels of Flt-1 and VE-cadherin mRNAincreased 1.2-fold on P14 and 1.2-fold on P19. Similar data wereobtained from another Northern blot analysis (data not shown).



nuclear layers in retinas from P19 mice, and Flt-1immunoreactivity was primarily in the nerve fiberlayer and the inner plexiform layer. Although our dataneed to be confirmed, the correlation of these studiesprobably suggests that VEGF receptors might play arole in neurogenesis as well as in vasculogenesis. Fur-ther studies should be performed to confirm the ex-pression of VEGF receptors in the neural retina andparticularly to determine the types of neural cells thatexpress VEGF receptors.

Taken together, the present findings suggest that, inischemic retina, retinal microvascular cells, specifi-cally cells adjacent to the area of nonperfusion, up-regulate KDR/Flk-1 expression and this might facili-tate local neovascularization mediated by anautocrine/paracrine action of VEGF.

ACKNOWLEDGMENTS

We thank Ms. Hisako Okuda for her help with the histologictechniques. This study was supported by a Grant-in-Aid for Scien-tific Research from the Ministry of Education, Science, and Cultureof the Japanese Government and by the Japan Association for Inhi-bition of Blindness.

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Increased Expression of KDR/Flk-1 (VEGFR-2) in Murine Model of Ischemia-Induced Retinal Neovascularization