Binding of Basic Fibroblost Growth Factor to Normal and Neovascularized Rabbit Cornea

Transcription

1 Investgatve Ophthalmology & Vsual Scence, Vol. 31, No. 2, February 1990 Copyrght Assocaton for Research n Vson and Ophthalmology Bndng of Basc Fbroblost Growth Factor to Normal and Neovascularzed Rabbt Cornea Gsele 5oubrane,*f Jance Jerdan4 lans Karpouzas, Ncole A. Fayen,* Bert Glaser,:J: Gabrel Coscos,t Yves Courtos,* and Jean Claude Jeanny* The labelng pattern of frozen sectons of rabbt cornea ncubated wth radoodnated basc fbroblast growth factor (bfgf) was nvestgated n normal corneas and prostaglandn-nduced neovascularzed corneas by autoradography followed by mage analyss. 125 I-bFGF bnds to Bowman's, Descemet's, and vascular basement membranes n a dose-dependent manner. The specfcty of the bndng of bfgf to basement membrane was demonstrated by the followng experments: 1) an excess of unlabeled growth factor dsplaced the labelng; 2) hstones dd not modfy the labelng; and 3) 2 M NaCl washng and enzymatc treatment wth hepartnase prevented bndng of labeled growth factor wthout apparent destructon of the overall structure of the basement membrane. Our results suggest that bfgf bnds to the heparan sulfate proteoglycan of basement membranes. Both normal lmbal vessels and the newly formed corneal vessels exhbted the same type of labelng but wth dfferent ntenstes, accordng to the degree of maturaton of the new vessels. bfgf bndng also s located clearly on the endothelal cells n both types of vessels. Ths second bndng ste could correspond to the hgh affnty receptors on the cell surface and suggests a drect nteracton of bfgf wth endothelal cells durng new vessel formaton. Invest Ophthalmol Vs Sc 31: , 1990 The transparent rabbt cornea has been used prevously as an expermental model for the study of angogenc stmul. 1 ' 2 Many substances have been shown to nduce or stmulate vessel prolferaton n the normally avascular cornea, among them prostaglandns (manly prostaglandn E [PGE]) and fbroblast growth factors (FGF). 3 Acdc and basc fbroblast growth factors (afgf and bfgf) are two structurally and functonally related polypeptdes of molecular weght 15,000-18,000 D. 4 They were solated orgnally from bovne bran, ptutary, and retna, 56 but are present also n other tssues, ncludng the eye. These factors have been gven varous names, dependng on the tssue of orgn (eg, eye-derved growth factor), on ther target cells (eg, fbroblast growth factor), or on ther chemcal affnty used for purfcaton (eg, heparn-bndng growth factor). 78 Sequence data analyss has demon- From the *Unte de Recherches Gerontologques, Inserm U. 118 et Cnrs U.A. 630, Developpement et Senescence cellulares, Pars, France; the funversty of Cretel, Department of Ophthalmology, Cretel, France; tthe Center of Vtreoretnal Research, Wlmer Ophthalmologcal Insttute, Johns Hopkns Unversty, Baltmore, Maryland; and the Laboratore d'ophtalmologe, Inserm U. 86, Hotel Deu, Pars, France. Reprnt requests: G. Soubrane, MD, Inserm U. 118, 29 rue Wlhem, Pars, France. Supported by a grant from Ipsen Research Foundaton. Submtted for publcaton: July 1, 1988; accepted June 29, strated a 55% amno acd sequence homology n the algned regon. 9 " 12 Both mtogens dsplay hgh affntes for heparn, a property that has allowed purfcaton of both factors wth heparn-sepharose affnty chromatography. 13 " 15 In vtro, they stmulate the prolferaton and support the dfferentaton of a varety of mesoderm-, ectoderm-, and neuroectoderm-derved cells, n partcular vascular endothelal cells wth bfgf beng fold more potent than afgf n the absence of heparn. 4 They bnd to the same cell surface receptors. 19 ' 20 In vvo, these soluble factors can nduce neovascularzaton, and therefore, angogeness. 21 ' 22 In the mouse eye, bfgf bnds specfcally to the heparan sulfate proteoglycan 23 of all basement membranes, 24 ncludng the lens capsule and nner lmtng membrane of the retna. Descemet's membrane, located beneath the corneal endothelum, s known to contan FGF n vvo. 25 Extracellular matrx s suggested to be a regulator of angogeness through ts ablty to support cell mgraton and prolferaton selectvely n response to stmulaton by these soluble mtogens. Furthermore, endothelal cells n culture synthesze bfgf. 26 " 27 We have studed and quantfed by mage analyss the bndng of exogenous bfgf on nonvascularzed cornea, ncludng mature establshed vessels of the normal lmbus (as a control) and PGE 1-nduced new vessels at dfferent steps of maturaton (well establshed corneal new vessels 323 Downloaded From: on 03/02/2018

2 324 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1990 Vol. 31 ["hghly vascularzed"] and growng sprouts ["lghtly vascularzed"]). In the three types of corneas, we have examned these vessels for the presence of two recently descrbed bndng stes 28 whch correspond to the hgh affnty of cell surface receptors and the low affnty of heparan sulfate proteoglycan. Chemcals and Reagents Materals and Methods The followng reagents were used: heparn-sepharose, PD 10 column (Pharmaca, St. Quentn en Yvelnes, France), Na I25 I (Amersham, Les Uls, France), optmal cuttng temperature medum (OCT; Mles Scentfc, Puteaux, France) hstones (Sgma, La Verpllere, France), hepartnase (Mles Scentfc), PGE1 (Upjohn, Kalamazoo, MI) (1 jg/pellet) n ethylene-vnyl-acetate polymer (Elvax 40; Dupont, Wlmngton, DE). Growth Factor Preparaton and Iodnaton bfgf was purfed from bovne bran. Tssues were homogenzed wth phosphate-buffered salne (PBS; Ca- and Mg-free, ph 7.4). The homogenate was centrfuged for 30 mn at 10,000 g, and then fractonated wth ammonum sulfate (20-60%). The last precptate was suspended n PBS and dalyzed for 6 hr at 4 C n 0.1 M acetc acd, and then centrfuged for 30 mn at 20,000 g, at 4 C. The supernatant was appled to a column of heparn-sepharose. The frst eluton was performed wth 0.65 M NaCl n PBS. Then the two peaks of mtogenc actvty were eluted wth 1.1 M and 2.0 M NaCl consecutvely n PBS. All of these steps were performed at 4 C. bfgf was labeled wth I25 I by the chloramne T method, as already publshed. 24 When ths odnated soluton was subjected to electrophoress on sodum dodecyl sulfate (SDS)-polyacrylamde gels, a sngleband mgraton n the poston of purfed bovne bran bfgf was observed on autoradography. Full bologc actvty, as tested by 3 H-thymdne ncorporaton n bovne epthelal lens cells (BEL) 29 was recovered after odnaton. Expermental Corneal Neovascularzaton Corneal neovascularzaton was nduced by usng a modfcaton of a technque descrbed prevously. 3 ' 30 Pellets of the slow-release ethylene-vnyl-acetate copolymer wth 1 ng PGE1, were mplanted n the corneal stroma at a dstance of 1-2 mm from the lmbus. Preparaton of Rabbt Corneal Sectons Freshly excsed corneas were trmmed and postoned n OCT on a metal block and frozen n lqud ntrogen. Samples from normal rabbt cornea were excsed at the lmbus so that mature vessels would be ncluded n these control sectons. Sectons were taken from the neovascularzed corneas at three dfferent locatons: near the lmbus, where the new vessel trunks were largest ("hghly vascularzed" samples); n the regon of growng sprouts, close to the nsert ("lghtly vascularzed" samples); and n the central avascular zone of the cornea ("nonvascularzed" samples). Sectons 5-/zm thck were cut at -20 C n a cryostat (2800 Frgcut N, Rechert Jung, Buffalo, NY) perpendcular to both the new vessel and corneal epthelal cell layer, collected on serum albumn coated sldes, and stored at -20 C. Autoradography The unfxed corneal sectons were dred for 5 mn at room temperature after OCT extracton n PBS, and ncubated for 1 h at room temperature n a water-saturated atmosphere wth 20 /x\ 125 I-labeled bfgf dluted n PBS (20 ng/ml except n dose-dependence experments). After the ncubaton, the sldes were washed sx tmes each wth PBS, and dehydrated. They then were dpped n photoemulson K2 (Ilford) dluted 1:1 wth twce-dstlled water. After exposure at 4 C, the sldes were developed n Kodak D19 and fxed n Hypam Ilford fxatve. All experments were run n trplcate, and each treatment was performed on a mnmum of three sldes. The sectons were exposed for 1-2 weeks, dependng on the specfc actvty of the odnated bfgf used n each experment. Controls were run smultaneously wth expermental samples. The comparsons were done on sectons of the same experment developed after the same exposure tme. The photographs were taken under the same condtons usng PAN-F flm (Ilford) on a Zess (Oberkochen, West Germany) photomcroscope. Competton Experments Unlabeled purfed bfgf (0.5, 2, and 5 jug/ml) ether was mxed wth the odnated FGF before deposton on the sectons or was deposted n sequence for 1 hr. In the latter case, the labeled factor was deposted ether before or after the unlabeled factor. Chemcal and Enzymatc Pretreatments The sectons were prencubated for 1 hr wth hstones (0.5, 2, and 5 /g/ml) or hepartnase (390 U/mg, and 1, 2, 5, 10, and 20 ng/m\ n presence of Ca ++ ) before ncubaton wth the odnated bfgf. Downloaded From: on 03/02/2018

3 No. 2 bfgf DINDING TO NORMAL AND NEOVA5CULARJZED CORNEA / Soubrone er ol 325 A B IP Fg. 1. Cornea sectons ncubated wth the same dose 20 t\ of odnated bfgf (20 ng/ml; SA = 114,000 cpm/ng), n the same experment, on nonvascularzed secton (A) on lghtly neovascularzed wth growng neovascular sprouts (B) and on hghly neovascularzed wth large vessels trunks (C). The labelng of Bowman's membrane (sold stars) s stronger n nonvascularzed cornea (A) than n lghtly (B) and hghly (C) vascularzed sectons. The bndng on Descemet's membrane (open stars) s smlar n the three types of tssues. The labelng of new vessels (arrow heads) s more ntense n hghly vascularzed (C) versus lghtly vascularzed corneal sectons (B). (XI00). Analyss System Trr analyss system 3 ' conssted of a camera lnked to montors; data were nput drectly nto a computer system. The ensemble was controlled by a seres of Imagena (Bocom, les Uls, France) software. After adjustng the mage of the montor, mage focusng and mage treatment, the computer analyzed automatcally the labelng ntensty of dfferent structures from normal or neovascularzed corneas. Table 1. In stu bndng of odnated bfgf (20 ng/ml) to dfferent types and structures of corneas: mean gray levels Corneal structure Type of cornea Bowman's membrane Vessels Descemet 's membrane SJonvascularzed Lghtly vascularzed Hghly vascularzed 58.0 ± ±5.5n 45.8 ±4.0J ± ± ± ± 4.9=j 5l.2±8.6-J Mean gray levels determned by mage analyss. Data are presented as the mean ± SD of 10 measurements performed on negatves correspondng to Fgure I. Statstcal sgnfcance was assessed between the mean values by Fsher's test: sold lne, dfference statstcally sgnfcant at P < 0.05; dashed lne, dfference not statstcally sgnfcant at P < Downloaded From: on 03/02/2018

4 326 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1990 Vol. 31.*. s * B ;3 * * * / '*"* As> «.', 1 ''< < * ' *? " Fg. 2. Normal lmbal vessels (A) and large corneal new vessels (C, E) ncubated wth odnated bfgf at 20 ng/ml (SA = 100,000 cpm/ng). The labelng s localzed both to the endothelal cell surface and to the surroundng basement membrane n the vessels (E). The numbers allow dentfcaton of the vessels on the sectons labeled (A, C, E) or bleached wth potassum ferrcyande and counterstaned for nucle wth hematoxyln (B, D, F) (A, B, C, D) X250; (E, F) X625. Data Acquston Images were acqured on flm negatves by a charge couple devce (CCD) camera, 512 X 512 pxels and 100 gray levels (expressed from 0 to 255). The negatves from each fgure were placed on a lght box and held flat by a weghted glass slde. The analogc vdeo mages were dgtzed n the computer, where the gray Downloaded From: on 03/02/2018

5 No. 2 bfgf BINDING TO NORMAL AND NEOVA5CULARIZED CORNEA / Soubrane er al 327 B Fg. 3. Dfferent doses of odnated bfgf (SA = 112,000 cpm/ng) deposted on hghly neovascularzed cornea sectons. The factor was deposted at 4 ng/ml (A, B), 8 ng/ml (C, D), and 20 ng/ml <E, F). The labelng of each of the basement membranes {Bowman's, vessels, Descemet's) s sgnfcantly dependent on the concentraton of l23 I-bFGF used. (A, C, E XI00) Phase contrast; (B, D,F) brght feld (x 100). D levels were calculated. Ten measurements were taken from each corneal structure (epthelum, Bowman's membrane, stroma, vessels, and Descemet's membrane) and also from the background (out of the sectons). Data were presented as the mean ± standard devaton. The Fsher test was carred out between dfferent structures and types of cornea. Results In Stu Bndng of bfgf to Rabbt Corneal Sectons Autoradography after ncubaton wth odnated bfgf at the same concentraton (20 ng/ml) showed a specfc localzaton of slver grans on corneal basement membranes (e, Bowman's and Descemet's membranes n nonvascularzed cornea, and Bowman's, Descemet's and vessel membranes n vascularzed cornea) (Fg. 1). The entre depth of all basement membranes was labeled as a contnuous band. Quantfed analyss of Bowman's membrane showed a statstcally sgnfcant ncrease of the labelng (Table 1) n nonvascularzed cornea (Fg. 1A) compared to the lghtly (Fg. IB) and hghly (Fg. 1C) vascularzed samples. The ntensty of the labelng of Descemet's membrane was quanttatvely comparable n the three types of sectons (Table 1). In well Downloaded From: on 03/02/2018

6 328 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / February 1990 Vol. 31 vascularzed cornea (Fg. 1C), both large and small vessels have a more pronounced labelng than n lghtly vascularzed samples (Fg. IB, Table 1). Measurements of the background were found not to be statstcally dfferent. (Fg. 1 A, 26.9; B, 24.6; C, 25.4). The gray levels of the corneal epthelum and stroma were hgher than those of the background, but the mean values were close (to compare wth the hgh values of Bowman's, Descemet's and vessel's membranes). No statstcal dfference n the three types of tssues was observed for corneal epthelum labelng (Fg. 1 A, 34.4; B, 27.8; C, 31.0) as for stromal labelng (Fg. 1A, 27.1; B, 28.1; C, 28.8). The affnty of bfgf for lmbal vessels of normal corneas was compared to nduced vessels (Fg. 2). Labelng ntensty was smlar n both lmbal (Fg. 2A) and establshed new vessels (Fg. 2C, E). A ste of bndng other than basement membrane could be dentfed towards the vessel lumen, whch by hstologc stanng was demonstrated to be on the endothelal cells (Fg. 2E, F). Incubaton wth three concentratons of bfgf (4, 8, and 20 ng/ml) demonstrated that the ntensty of the labelng was dose dependent n each of the three types of corneal sectons studed. In Fgure 3, whch shows hghly vascularzed samples, the sgnal ntensty ncreased n parallel wth an ncrease n radolabeled bfgf concentraton, 4 ng/ml (Fg. 3A, B), 8 ng/ml (Fg. 3C, D), and 20 ng/ml (Fg. 3E, F) at the dfferent bndng stes; at the lowest concentraton (4 ng/ml), labelng was hardly vsble (Fg. 3B). Ths ncrease was statstcally sgnfcant for Bowman's and Descemet's membranes as well as for the vessels (Table 2). The labelng of the corneal stroma (Fg. A, B, 74.0; C, D, 82.2; E, F, 85.4) and of the epthelum (Fg. 3A, B, 67.2; C, D, 91.6; E, F, 99.6) ncreased sgnfcantly when 8 and 20 ng/ml were compared to 4 ng/ml, but there was no statstcal dfference between the two former concentratons (8 and 20 ng/ml). The gray levels of background were ndependent of the concentraton of labeled factor used (Fg. 3A, B, 65.2; C, D, 60.6; E, F, 56.6). Bndng Specfcty Studes To nvestgate the bndng specfcty of labeled bfgf to corneal tssue, the bndng of 125 I-bFGF to sectons n the presence of ncreasng concentratons of natve bfgf was examned. These studes showed that labelng decreased wth ncreasng concentratons of competng unlabeled factor (0.5, 2, and 5 /g/ml) (Fg. 4) n a statstcally sgnfcant way (Table 3). Ths dose dependence was more evdent when sectons were ncubated wth 125 I- bfgf pror to unlabeled bfgf (Fg. 4) rather than Table 2. Dose dependence of odnated bfgf bndng to dfferent structures of hghly neovascularzed corneas: mean gray levels Doses of l25 I-bFGF (ng/ml) Bowman's membrane 82.8 ± ± ± 12.2 Corneal structures Vessels 74.0 ± ± ± 14.6 Descemet 's membrane 88.2 ± ± ± 10.6 Mean gray levels determned by mage analyss. Data are presented as the mean ± SD of 10 measurements of negatves correspondng to Fgure 3. Statstcal sgnfcance was assessed between the mean values by Fsher's test: for each structure, the dfference between gray levels for any two doses tested was statstcally sgnfcant at P < wth unlabeled pror to 125 I or wth both together (data not shown). We dd not observe complete suppresson of the labelng, even wth a 250-fold excess of unlabeled growth factor (Fg. 4E, F). A smlar result has been descrbed prevously for whole preparatons of mouse eye. 24 When compettve nhbton n bfgf bndng to the dfferent corneal structures was compared, the extent of dsplacement of bfgf bndng was not equal n all basement membranes. Bndng to corneal vessels (45%) was more easly competed away wth unlabeled bfgf than was the FGF bndng to ether Descemet's (25%) or to Bowman's membranes (21%) (Fg. 4B, D, F; Table 3). When statstcally sgnfcant results were consdered, competton wth unlabeled bfgf was more effcent on nonvascularzed than on vascularzed corneas (Fg 4; Table 3). Nature of Bndng Because bfgf s a basc proten, the bndng of bfgf to vascularzed cornea was examned n the presence of other basc protens (hstones) (Fg. 5 A). The sectons were prencubated wth hstone protens at 0.5, 2 and 5 /ug/ml (Fg. 5A) for 1 hr. Even hgh concentratons of hstone protens had no effect on subsequent bndng of the labeled bfgf to the corneal tssue (Table 4). No statstcal dfference was found n gray levels n any corneal structure, when compared to the control (Fg. 5D). Because bfgf bnds to a heparn-sepharose column wth strong affnty and can be eluted only wth NaCl at hgh onc strength (2 M), 7 the release of bfgf from corneal basement membranes under smlar condtons was examned. Incubaton of the sectons wth odnated bfgf was followed by a washng n the presence of 2 M NaCl. The basement membrane labelng was sgnfcantly decreased but not totally abolshed (Fg. 5B compared wth Fg. 5D). Des- Downloaded From: on 03/02/2018

7 # * * ' No. 2 bfgf DINDING TO NORMAL AND NEOVASCULAPJZED CORNEA / 5oubrone er ol 329 Fg. 4. Competton wth ncreasng amounts of unlabeled bfgf (0.5 Mg/ml (A, B), 2 Mg/ml (C, D), and 5 Mg/ml (E, F) performed on nonvascularzed (A, C, E) and hghly neovascularzed (B, D, F) sectons prevously ncubated wth the same concentraton of odnated growth factor (20 ng/ml; SA = H4,000cpm/ng[A,C,E] and 112,000 cpm/ng [B, D, FJ. The reference secton (not treated wth unlabeled factors) for (A, C, E) s Fgure 1A, and for (B, D, F) s Fgure 3F. The labelng decreases wth ncreasng concentraton of natve bfgf. Labelng s more competed away on vessels than on Bowman's and Descemet's membranes and on non vascularzed than on vascularzed samples (XI00). B cemet's (44%) and Bowman's (38%) membranes appear about equally affected (Table 4). Vessel stanng (54%) s more affected than the above-lsted structures. Basement membranes contan several extracellular matrx components Heparan sulfate proteoglycan s consdered to be the most probable bndng ste of bfgf because of ts analogy wth heparn at the polysaccardc chans. Therefore, the sectons were dgested wth hepartnase at varous concentratons (1, 2, 5, 10, and 20 Mg/ml). The results demonstrated that hepartnase treatment can prevent bfgf bndng to basement membranes (Fg. 5C) n a dosedependent manner. Descemet's membrane (29%) Downloaded From: on 03/02/2018

8 330 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1990 Vol. 31 Table 3. Competton experment of natve bfgf wth odnated bfgf bndng (20 ng/ml) on dfferent structures and types of corneas: mean gray levels Type of cornea Nonvascularzed Hghly vascularzed Concentraton of natve bfgf(tg/ml) Bowman's membrane ± ± ± 12.4 J ± ± ±21.3 J Mean gray levels determned by mage analyss. Data are presented as the mean ± SD of 10 measurements of negatves correspondng to fgure 4. Statstcal sgnfcance was assessed between the mean values by Fsher's test: all pars of data wthn one type of cornea and wthn one structural type 33% 21% Corneal structures Vessels ± ± ± 16.8 J 45% Descemet 's membrane ± ± ± 13.0 ' ± ±33.7, 55% 25% yelded a dfference statstcally sgnfcant at P < 0.05, wth the excepton of Descement's membrane n the hghly vascularzed corneas at 0.5 and 2.0 /g/ml bfgf, whch yelded a dfference not statstcally sgnfcant at P < and vessel membrane (22%) agan were more affected than was the Bowmans' membrane (3%) (Table 4). Under the same condtons, experments performed on mouse eyes have shown that the other man consttuents of basement membranes (collagen type IV and lamnn) were stll present after hepartnase treatment. 24 Dscusson Drect comparsons were possble because each experment was performed wth ts own controls, e, the same amount of odnated growth factor was added on treated and untreated sectons, whch were ncubated under the same condtons, dpped n the same nuclear emulson, and developed after the same exposure. The results confrm a dose dependency and a specfc bndng of exogenous labeled bfgf to Bowman's, Descemet's and vessel membranes. Ths bndng s affected by washng wth 2 M NaCl, by dgeston wth hepartnase, and by competton wth natve bfgf. However, the labelng s not affected by - V B Fg. 5. Prencubaton wth hstores or hepartnase treatment, and NaCl postncubaton washng of hghly neovascularzed cornea sectons ncubated wth odnated bfgf (20 ng/ml; SA = 112,000 cpm/ng). Prencubaton wth hstones, 5 Mg/ m ' (A) dd not affect the labelng when compared to the reference secton (D). Postncubaton washng wth 2 M NaCl (B) and specfc enzymatc dgeston wth hepartnase, 20 Mg/ml (C) sgnfcantly decreased the sgnal. Descemet's and vessel basement membranes were more affected than was Bowman's membrane (X100). Downloaded From: on 03/02/2018

9 No. 2 bfgf BINDING TO NORMAL AND NEOVASCULARJZED CORNEA / Soubrone er ol 331 Table 4. Effect of hstone or hepartnase pretreatment and NaCl postncubaton washng on odnated bfgf bndng on dfferent structures of neovascularzed corneas: mean gray levels Comeal structure Bowman's Type of treatment membrane Vessels Descemet 's membrane Control ± "I Hstone prencubaton ± 17.4 J 38% ± 14.6-n 3% 159.8±31.7 J 54% ± l 22% ±12.0 J 44% - 29% NaCl washng ± 9.5 J 77.6 ± 6.3 J ± Hepartnase pretreatment ± ± ± 34.3 Mean gray levels determned by mage analyss. Data are presented as the mean ± SD of 10 measurements performed on negatves correspondng to Fgure 5. Statstcal sgnfcance was assessed, between the mean values by Fsher's test: sold lne, dfference statstcally sgnfcant at P < 0.05; dashed lne, dfference not statstcally sgnfcant at P < hstones. The sgnal was not suppressed completely even wth a 250-fold excess of unlabeled bfgf. The resdual bndng was presumably due to a stll nsuffcent dose of competng factor. The effectveness of pretreatment of the sectons wth hepartnase demonstrates that bfgf bnds to heparan sulfate proteoglycan 23 present n the corneal basement membranes of the rabbt. The bndng could be the storage ste of the factor or prevent ts proteolytc degradaton. The ntensty of the labelng dffered accordng to the basement membrane consdered. Vessel and Descemet's membranes were the most senstve to enzyme pretreatment and compettve nhbton n all experments. Ths mght reflect dfferences n basement membrane composton, n structural organzaton of the membrane, or n varatons n the mode of bndng. It s noteworthy that endogenous bfgf has been extracted at low concentraton from Descemet's membrane of bovne cornea. 25 ' 34 In the three types of tssue consdered (normal avascular, lghtly vascularzed, or hghly vascularzed corneal sectons), the bndng of exogenous labeled bfgf at the same concentraton on Descemet's membrane was comparable (Table 1). However, Bowman's labelng s lght but sgnfcantly hgher n nonvascularzed compared to vascularzed samples. Competton experments were also more effcent n non vascularzed than n vascularzed sectons. On corneal vessels (normal lmbal vessels or nduced new vessels at dfferent stages of maturaton), two stes of labelng, the vessel basement membranes and the endothelal cells, were observed clearly. Immunohstochemcal studes have revealed that newly formed vessels are surrounded by an extracellular matrx contanng all of the basement membrane major components, ncludng heparan sulfate proteoglycans. 35 The co-localzaton of FGF bndng to vessels wth the presence of heparan sulfate proteoglycans n ths regon could not be demonstrated n the current study because of the studed speces (rabbt) and because of the source of the polyclonal proteoglycan antbodes (rabbt). However, the effectveness of the heparatnase pretreatment n preventng subsequent FGF bndng to the newly formed corneal vessels suggests that at least part of the bndng observed s due to the heparan sulfate proteoglycans. A promnent cellular labelng to the endothelal cell surface was demonstrated n the current experment, n both new and establshed vessels. In competton experments wth natve bfgf, by 2 M NaCl washng and hepartnase pretreatment, the labelng on the vessels s the frst to dsappear. We dd observe a dfference n the bndng of labeled FGF to mature vessels and to mmature vessel sprouts. The ablty of the vessels to bnd 125 I-bFGF ncreases as a functon of maturaton. The bndng pattern observed on the newly formed vessel sprouts could be related to the thnness of ther basement membrane, or to the smaller number of surface cell receptors. The latter bndng to vascular cells has not been descrbed prevously. A recent work 36 has demonstrated that vascular endothelal cells synthetze bfgf n vvo as well as n vtro and release bfgf nto the extracellular matrx n vtro. Furthermore, vascular endothelal cells are one of the man bfgf target cells. In addton, an ncreased labelng of the stroma n the vcnty of vascularzed areas was observed. Ths ncrease may be due to nflammatory cells present n Downloaded From: on 03/02/2018

10 332 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1990 Vol. 31 the new vessel's area, as shown by hstologc stanng. New vessels may release ether proteolytc enzymes or heparanod substances that ncrease the amount of bndng stes avalable. The current experment demonstrates two dfferent stes of bfgf bndng that can be compared wth the low- and hgh-affnty bndng stes descrbed by Moscatell. 28 The bndng to basement membranes corresponds probably to the low-affnty bndng ste, whereas bndng to vascular endothelal cells may correspond to the hgh-affnty bndng ste. Key words: cornea, neovascularzaton, odnated basc fbroblast growth factor, basement membranes, endothelal cells Acknowledgments The authors thank Dr. Raulas for hs supply of bologcally actve bovne bran bfgf and Pr Poulquen for access to the mage analyser. They are grateful to Beatrce Croquet and Vrgne Charton for ther secretaral assstance, and to Laurent Jonet, Anne Mare Maglone and Herve Coet for ther excellent techncal assstance. References 1. Gmbrone MA, Cotran RS, Leapman SB, and Folkman J: Tumor growth and neovascularzaton: An expermental model usng the rabbt cornea. J Natl Cancer Inst 52:413, Thompson P, Arrut C, Maurce D, Plouet J, Barrtault D, and Courtos Y: Angogenc actvty of a cell growth-regulatng factor derved from the retna. In Internatonal Workshop on Problems of Normal and Genetcally Abnormal Retna, Clayton R, edtor. New York, Academc Press, 1981, pp Ben Ezra D: Neovasculogenc ablty of prostaglandn growth factors and synthetc chemoattractants. Am J Ophthalmol 86:455, Gospodarowcz D, Neufeld G, and Schwegerer L: Fbroblast growth factor. Mol Cell Endocrnol 46:187, Arrut C and Courtos Y: Morphologcal changes and growth stmulaton of bovne epthelal lens cells by a retnal extract n vtro. 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