RESEARCH ARTICLE

A Potential Role for �- and �-Crystallins in theVascular Remodeling of the EyeCheng Zhang,1 Peter Gehlbach,1 Celine Gongora,4 Marisol Cano,1 Robert Fariss,5 Stacey Hose,1

Avindra Nath,2 William R. Green,1 Morton F. Goldberg,1 J. Samuel Zigler, Jr.,5 andDebasish Sinha1,3*

We demonstrate that expression of �- and �-crystallins is associated with intraocular vessels during normalvascular development of the eye and also in the Nuc1 rat, a mutant in which the hyaloid vascular systemfails to regress normally. Real-Time RT PCR, Western blot and metabolic labeling studies indicate anincreased expression of �- and �-crystallins in Nuc1 retina. The increased expression of crystallins waslocalized to the astrocytes surrounding the intraocular vessels. A similar pattern of crystallin expressionwas also observed in the retinal vessels during normal development. Cultured human astrocytes exposed to3-nitropropionic acid, an established model of neuronal hypoxia, increased VEGF expression, as expected,but also increased expression of crystallins. Our data suggest that crystallins may function together withVEGF during vascular remodeling. Interestingly, in human PFV (persistent fetal vasculature) disease,where the hyaloid vasculature abnormally persists after birth, we show that astrocytes express both VEGFand crystallins. Developmental Dynamics 234:36–47, 2005. © 2005 Wiley-Liss, Inc.

Key words: hyaloid and retinal vessels; �/�-crystallins; VEGF; astrocytes; vascular remodeling

Received 17 March 2005; Revised 11 May 2005; Accepted 16 May 2005

INTRODUCTION

In mammalian eyes, the pupillarymembrane and hyaloid vessels, in-cluding the hyaloid artery, tunica vas-culosa lentis, and vasa hyaloidea pro-pria, nourish the immature lens,retina, and vitreous (Fig. 1). Thesevessels are known to regress duringthe later stages of ocular develop-ment, pre-natally in humans andwithin the first few weeks of post-na-tal development in rodents, to providean optically clear path between the

lens and retina (Ito and Yoshioka,1999). The development of the retinalvasculature coincides with the start ofthe regression of the hyaloid vascula-ture (Zhu et al., 2000).

There are many critical moleculesinvolved in the physiological regula-tion of blood vessel formation as wellas regression (Saint-Geniez andD’Amore, 2004). Vascular endothelialgrowth factor (VEGF), for example,has been implicated in both the nor-mal development and maintenance of

the vasculature as well as in its dys-function (Thurston and Gale, 2004;Ferrara and Davis-Smyth, 1997).Mice with only one copy of the VEGFgene die before birth because of bloodvessel abnormalities, indicating thatnormal amounts of VEGF are abso-lutely required for normal vasculardevelopment (Carmeliet et al., 1996).

Vasculogenesis and angiogenesisare the two processes by which vascu-lar development occurs. The retina isvascularized by a two-step process,

1Department of Ophthalmology, School of Medicine, Johns Hopkins University, Baltimore, Maryland2Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland3Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland4CNRS UMR5160, Faculte de Pharmacie, Montpellier, France5National Eye Institute, National Institutes of Health, Bethesda, MarylandGrant sponsor: Helena Rubeinstein Foundation; Grant sponsor: Knights Templar Eye Foundation, Inc.; Grant sponsor: Juvenile DiabetesResearch Foundation International; Grant sponsor: Alexander and Margaret Stewart Trust; Grant sponsor: NIH; Grant number:KO8EY13420; Grant sponsor: Guerrieri Retinal Research Fund; Grant sponsor: Research to Prevent Blindness.*Correspondence to: Debasish Sinha, Department of Ophthalmology (School of Medicine), & Environmental Health Sciences(Bloomberg School of Public Health), The Johns Hopkins University, Baltimore, MD 21287. E-mail: Debasish@jhmi.edu

DOI 10.1002/dvdy.20494Published online 7 July 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 234:36–47, 2005

© 2005 Wiley-Liss, Inc.

initially by vasculogenesis, followedby angiogenesis (Flower et al., 1985;Chan-Ling et al., 1990; McLeod et al.,1987). Whether vessels form by vascu-

logenesis or angiogenesis, the primi-tive vessels are subsequently remod-eled. Vascular remodeling, includingthe growth of new vessels and regres-

sion of others, is a complex processthat involves several critical factors; itplays a major role in the early devel-opment of the vascular system (Yan-copoulos et al., 2000).

Crystallins are the most abundantsoluble proteins of the lens of the eye(Piatigorsky, 2002). Although highlyspecialized for lens, the crystallins arealso expressed in other tissues (Srini-vasan et al., 1992; Deretic et al., 1994;Head et al., 1995; Sinha et al., 1998;Magabo et al., 2000; Jones et al., 1999;Crabb et al., 2002; Xi et al., 2003).Three major families of crystallins, �,�, and �, are ubiquitously representedin all vertebrates (Hejtmancik and Pi-atigorsky, 1994). There are also taxon-specific crystallins that serve as met-

Fig. 1.

Fig. 2.

Fig. 1. Schematic diagram of the intraocularvessels. During development of the mammalianeye, the nourishment of the immature lens, ret-ina, and vitreous is ensured by the hyaloid vas-cular system, including the pupillary membrane,tunica vasculosa lentis, vasa hyaloidea propria,and the hyaloid artery as shown in this sche-matic diagram. The hyaloid vessels regress by aprogrammed cell death process during oculardevelopment, pre-natally in humans and withinthe first few weeks of post-natal development inrodents. In rats, the hyaloid vessels undergoinvolution, starting at post-natal day 7, whenretinal vessels start developing. The transienthyaloid vessels in the rat normally regresswithin 3 weeks after birth and as the retinamatures, the inner part of the retina becomesvascularized by retinal vessels. The mature lensand vitreous remain avascular to provide anoptically clear path to the retina.

Fig. 2. Defective regression of embryonic vas-culature in Nuc1 mutant rat. In wild type animals(a), the hyaloid artery had completely regressedby 5 weeks of age, showing a normal opticnerve head (ONH). In 5-week-old Nuc1/Nuc1rats (b,c), the hyaloid artery and adjacent tissuewere still present on the surface of the opticnerve head projecting into the vitreous (arrow).Note in c, the thick vessel wall of the artery athigher magnification showing cellular morphol-ogy of the retained vessel. Representative H & Estained sections from 25-day-old animals (d)show normal lens (L), iris (I), ciliary body (CB),and cornea (C). e: The pupillary membrane isstill evident in the Nuc1 homozygous animals(arrows), whereas it has fully regressed in thewild type eye (d). Iris hyperplasia was alsonoted in the Nuc1/Nuc1 eyes (arrowheads). Thelens shows abnormal shape and disorganiza-tion of structure. f: The normal eye structure at120 days in wild-type eyes. In Nuc1 homozy-gote (g), the ciliary process (arrow) is draggedcentrally towards the disrupted lens, resulting intraction, which causes peripheral retinal drag-ging and folding (arrowhead). Scale bar � 50�m.

CRYSTALLINS IN VASCULAR REMODELING 37

abolic enzymes in other tissues. Thetwo �-crystallins (�A and �B) belongto the small heat shock protein familyof molecular chaperones (Sax and Pi-atigorsky, 1994; Horwitz, 1992). The�- and �-crystallins are evolutionarilyand structurally related members of a�� superfamily, which also includesmicro-organism stress proteins aswell as vertebrate proteins that ap-pear to be associated with processes ofcell differentiation and morphologicalchange (Clout et al., 1997; Ray et al.,1997). The non-lens functions of �-and �-crystallins are yet to be fullydetermined.

Astrocytes have multiple functionsthat include regulation of blood vesselstructure and function as well as in-volvement in pathologic processes(Nedergaard et al., 2003). While oligo-dendrocytes of the glial cell familyhave been shown to express crystal-lins, there has not been a report indi-

cating expression of � and �-crystal-lins by astrocytes (Dabir et al., 2004).The glial cells of the optic nerve con-tain the same cell types found in thewhite matter throughout the CentralNervous System (CNS) (Mi et al.,2001). Type-1 astrocytes develop inthe embryonic optic nerve from astro-cyte precursor cells (APC) (Raff et al.,1984; Miller et al., 1985, 1989; Mi andBarres, 1999). The majority of theAPCs differentiate into GFAP-immu-nopositive astrocytes.

Astrocytes modulate the differenti-ation of vascular endothelial cells(Lattera et al., 1990) and contribute tothe glial limitans that lines blood ves-sels (Stewart and Tuor, 1994; Rung-ger-Brandle et al., 1993). Astrocytesare also involved in the formation andpreservation of the blood-brain andblood-retinal barriers (Janzer andRaff, 1987). It has been postulatedthat astrocytes guide and modulatevascular growth (Jiang et al., 1995).They migrate ahead of the vessels(vascular “front”), and are thus in aposition to respond to local environ-mental signals (Provis, 2001). Al-though all of the vascular cells mayparticipate in the remodeling process,the astrocytes may play a prominentpart, because they are capable of sens-

Fig. 3. Crystallin expression in the retina. Histograms (a, b) of real-time RT-PCR analysis of totalRNA (1 �g) from 25-day-old retina clearly show upregulation of �-crystallins (�A1/A3, �A4, �B1,�B3) and �-crystallins (�A-�F) in Nuc1 homozygous rats (dark gray bar) compared to wild-typecontrols (gray bar). Data shown as mean � SEM. Autoradiograph (c) of 4–12% Bis-Tris Nu-PAGEgel shows that new synthesis of proteins in the 20–30-kD range is markedly increased in Nuc1retina compared to age-matched wild-type retina. Western blotting (d) using a cocktail of anti � and�-crystallin antibodies, clearly identifies �, �-crystallins as major components in the upregulatedprotein fraction. This is consistent with the upregulation in mRNA levels in Nuc1 homozygousretina.

Fig. 4. Persistence of transient vessels in Nuc1 and expression of crystallins. a: The retainedhyaloid artery in Nuc1 at post-natal day 25 shows multiple branches, known as the vasa hyloideapropria, that are configured like the struts of an umbrella or guide ropes of a parachute (arrow).Such a structure has also been reported in human PFV disease. Note, red fluorescence indicatingpresence of �-crystallin in the retained hyaloid tissue. In rats, the intraocular vessels normallyregress within 3 weeks after birth. b: The persistent pupillary membrane (PM) in Nuc1 showsexpression of crystallins (red) in the retained vessels (v). The section was stained with isolectin B4(green) and Hoechst (blue). Isolectin B4 has previously been shown to localize rat blood vessels.Hoechst was used for staining nuclei. Note that there was crystallin immunoreactivity also in thelens epithelium (LE) and lens capsule (LC, short arrow) with some scattered staining in thehyperplastic iris (I). Lectin staining was also evident in the anterior part of the iris (large arrows). Inthe degenerate lens shown in this section, the area beneath the epithelium was devoid of cells (seeFig, 1e). c: Crystallin staining is discretely localized peripheral to the vessels of the retained hyaloidtissue (arrows) relative to isolectin B4 that stains the vessel wall (d). The merged image (e)demonstrates that the crystallin staining and the lectin staining are discrete with virtually no overlap.In d and e, a portion of image c is shown at a slightly higher magnification. For orientation, thelumen of each major vessel visible in these images is marked with an asterisk. Scale bar� 50 �m.

Fig. 5. Crystallin expression in the normal and Nuc1 homozygous rats during development.Confocal microscopy of 9-day-old normal (a) and Nuc1 homozygous (b) hyaloid artery showsdistinct crystallin expression surrounding the vessels (red, arrows). The normal rat (a) was FITC-Dextran perfused (green, asterisk) and was counterstained with DAPI (blue, arrowhead). The Nuc1homozygous hyaloid artery was stained with Isolectin-B4 (b). Note, in both wild-type and Nuc1homozygous hyaloid artery, crystallin is expressed surrounding the vessel wall. A similar pattern ofcrystallin expression was also observed in the retinal vasculature during development of bothwild-type and Nuc1 homozygous rats. By 20 days, in normal rats (c) and Nuc1 homozygous (d),crystallin expression was mostly localized surrounding the vessels of the inner limiting membraneof the retina (red, arrows). The rats were perfused with FITC-Dextran (green, asterisk) and coun-terstained with DAPI (blue, arrowhead).

38 ZHANG ET AL.

ing changes within their immediatemilieu.

In a microarray analysis comparinggene expression in the retina of Nuc1

mutant rats (Sinha et al., 2005) withwildtype littermates, we found in-creased expression of a number of �-and �-crystallin genes in Nuc1. These

crystallins were subsequently foundto be localized near vessels both inNuc1, in which the hyaloid vascula-ture is abnormally retained, as well asin the wildtype retina. Specifically,the astrocytes at the vascular frontexpress not only VEGF, as expected,but also �- and �-crystallins. We fur-ther demonstrate that in human PFV(Persistent Fetal Vasculature) disease(Goldberg, 1997), in which the hyaloidvascular system also fails to regressnormally, astrocytes similarly express�- and �-crystallins, as well as VEGF.In an in-vitro model of chemical hyp-oxia, we show that astrocytes express-ing VEGF also express �- and �-crys-tallin. Our data suggest, for the firsttime, that �- and �-crystallins may beinvolved in mediating vascular stabi-lization, remodeling, or survival in thedeveloping mammalian eye.

RESULTS

Persistent Hyaloid VascularSystem in Nuc1 HomozygousRats

In the wild-type rats, the hyaloid ar-tery, a constituent of the transientvasculature that nourishes the imma-ture lens, retina, and vitreous, re-gressed by day 35 (Fig. 2a). In con-trast, in Nuc1 homozygous rats, thehyaloid artery persisted at the surfaceFig. 4.

Fig. 5.

CRYSTALLINS IN VASCULAR REMODELING 39

of the optic nerve head projecting intothe vitreous until adulthood (Fig.2b,c). The pupillary membrane (PM),a temporary capillary network on theanterior surface of the lens, alsoknown as the anterior tunica vascu-losa lentis, normally regresses duringthe second week after birth. As shownin Figure 2d, in the normal eye, thePM was absent by post-natal day 25.However, it persisted in Nuc1 at thesame post-natal stage (Fig. 2e, ar-rows). Nuc1 rats also displayed irishyperplasia (Fig. 2e, arrowhead) anddisrupted lens structure (Fig. 2e). Byfour months of age, the iris and ciliarybody were dragged towards the centerof the posterior chamber (Fig. 2g, ar-row), inducing dragging and folding ofthe peripheral retina (Fig. 2g, arrow-head).

�- and �-CrystallinExpression in the Retinaand Hyaloid Vessels

To confirm microarray data (notshown) that indicated a more thantwofold increase in the expression of�- and �-crystallins in Nuc1 homozy-gous rat retina, we performed Real-time RT-PCR on 25-day-old retinas ofNuc1 homozygous (inclusive of the hy-aloid artery) and wild-type SpragueDawley rats. As shown in Figure 3aand b, mRNA levels of �A1/A3, �A4,�B1, �B3, �A, �B, �C, �D, and �EFcrystallins were significantly in-creased in Nuc1 retina compared tothe wild-type retina. Incorporation of35S-amino acids by organ cultured 20-day retina also indicated a marked in-crease in crystallin synthesis in Nuc1homozygotes compared to wild-type(Fig. 3c). Crystallin protein expressionwas confirmed by Western blotting(Fig. 3d).

The retained hyaloid artery andvasa hyaloidea propria in Nuc1 as-sumed a configuration typical of thestruts of an umbrella or the guideropes of a parachute (Fig. 4a). Distinctstaining patterns with the crystallinantibodies were obtained for the re-tained hyaloid artery (Fig. 4a,c, ar-row) and pupillary membrane (Fig.4b) in Nuc1. When the �-crystallinstaining pattern (similar data wereobtained with �-crystallin) in the re-tained hyaloid artery (Fig. 4c, arrow)was compared to the section stained

with isolectin-B4 (Fig. 4d), it clearlyindicated that crystallin was localizedaround the blood vessels [see mergedpicture with �-crystallins and Isolec-tin-B4 (Fig. 4e). Isolectin B4 has beenshown to label blood vessels in the rat(Ashwell et al., 1989).

The transient hyaloid artery atpost-natal day 9 in normal (Fig. 5a)and Nuc1 homozygous rats (Fig. 5b)showed distinct crystallin expression(Fig. 5,a,b, red, arrows) surroundingthe blood vessels (Fig. 5a,b, green, as-terisk). In rats, the hyaloid arterystarts involuting around post-natalday 7 and regresses completely by 3weeks of post-natal development(Cairns, 1959). While the hyaloid ves-sels undergo involution, the retinalvessels continue growing and reachthe adult form by the time the hyaloidvasculature has completely regressed.The vascularization of the retina isrestricted to the inner part of the ret-ina. At post-natal day 20, crystallinexpression is also localized surround-ing the retinal vessels in both normal(Fig. 5c) and Nuc1 homozygous rats(Fig. 5d).

VEGF and �- and �-Crystallin Expression inGFAP-ImmunopositiveAstrocytes of the RetainedVasculature in Nuc1Homozygote Rats

We further analyzed the cellular iden-tity of crystallin-immunopositive cellsin the retained hyaloid vasculature ofthe 5-week-old Nuc1 homozygote. Pre-vious studies have shown that astro-cytes migrate ahead of the vessels andare present on the external surface ofthe blood vessels (Stone et al., 1995).Therefore, we used antibodies toGFAP to localize the astrocytes in theretained hyaloid artery and retinalvessels of Nuc1 homozygotes. VEGFhas also been shown previously to beexpressed by astrocytes (Wechsler-Reya and Barres, 1997) and has animportant function in vascular remod-eling. Our data demonstrate VEGFimmunopositive reactivity in the re-tained hyaloid tissue of Nuc1 (Fig. 6a),co-localizing with GFAP (Fig. 6b,c).Co-localization of GFAP and �-crystal-lin reactivity (Fig. 6d,f) clearly demon-strated that astrocytes in the retained

hyaloid of Nuc1 also expressed crys-tallins.

In the Nuc1 homozygous retina,confocal microscopy with crystallinspecific antibodies also showed ex-pression at the internal limiting mem-brane (Fig. 7b) and in ganglion cells(Fig. 7b, arrowheads). GFAP immu-nopositive staining was also presentin the internal limiting membrane ofthe Nuc1 retina but not in the gan-glion cells (Fig. 7a). Co-localization ofcrystallins and GFAP (Fig. 7c) showedthat GFAP� cells associated with ves-sels at the inner limiting membranealso expressed crystallins. VEGF ex-pression was localized primarily atthe internal limiting membrane (Fig.7e). A similar pattern of staining wasobserved with antibodies to GFAP(Fig. 7d) and co-localization indicatedthat GFAP� astrocytes do expressVEGF (Fig. 7f, arrows).

VEGF and �- and �-Crystallin Expression inHuman PFV

Failure of regression of the hyaloidvasculature in the human eye leads topersistent fetal vasculature (PFV) dis-ease. The ocular phenotypes of Nuc1and human PFV are similar. To deter-mine if �- and �-crystallins are alsoexpressed in the GFAP� astrocytes ofthe retained hyaloid tissue of PFV pa-tients, we used a similar approach asindicated above for Nuc1. We immu-nolocalized VEGF, GFAP, and crystal-lins from human PFV patients, on se-rial paraffin sections, all of whichshowed a positive staining patterncomparable to our Nuc1 rat data. Fig-ure 8 shows a representative PFV tis-sue section (one of five patient eyesexamined) from a female who died sixdays after birth with a diagnosis oftrisomy 13 (Patau’s syndrome) withmicrophthalmia and multiple congen-ital malformations, including PFV.There was a persistent pupillarymembrane, and the lens showed cata-ractous changes with posterior distor-tion (not shown). Firmly attached tothe capsule was a mesenchymal fibro-vascular tissue, including the well-preserved hyaloid system, as shown inpart in Figure 8a and at higher mag-nification in Figure 8b. Within thattissue, many well-differentiated ro-

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Fig. 6. VEGF and Crystallin expression in the astrocytes. a: Immunofluorescent labeling (red) of sections from 5-week-old Nuc1 homozygotes withVEGF antibodies shows positive staining surrounding the vessels of the retained hyaloid artery. b: Positive staining is shown with antibody to GFAP(green). c: The merged image shows co-localization of VEGF and GFAP (arrows). d: �-crystallin antibodies also show positive staining surrounding thevessels of the retained hyaloid artery of 5-week-old Nuc1 homozygotes (red). e: The hyaloid vasculature was immunopositive for GFAP (green). f: Thetwo images (d,e) are merged to reveal co-localization of the �-crystallin and GFAP immunoreactivity in the cells surrounding the retained vasculature.The nuclei are stained with Hoechst. Scale bar � 50 �m.

Fig. 7. VEGF and Crystallin expression in the retinal astrocytes. a: Confocal microscopy shows GFAP positive staining at the internal limitingmembrane of 5 week old Nuc1 homozygote retina. b: �-crystallin also stained positive at the internal limiting membrane and ganglion cells(arrowheads). c: The merged image shows co-localization in the internal limiting membrane (arrows). In 5-week-old Nuc1 homozygote, the internallimiting membrane of the retina is positive for both GFAP (d) and VEGF (e) staining. f: The merged image shows co-localization (arrows). The nucleiare stained with Hoechst. Scale bar � 50 �m.

CRYSTALLINS IN VASCULAR REMODELING 41

settes were present (Fig. 8a, short ar-rows).

Immunohistochemical study showedthat cells surrounding the hyaloid tis-

sue were positive with VEGF (Fig. 8cand d), GFAP (Fig. 8e), and with crys-tallin antibodies (Fig. 8f), clearly indi-cating an expression pattern of VEGF

and �, �-crystallins by GFAP� astro-cytes similar to that seen in our Nuc1model.

Induction of VEGF and �-and �-Crystallins inCultured Human AstrocytesExposed to 3-NitropropionicAcid

It has been shown that astrocytes aresensitive to hypoxia, which inducesthem to release VEGF (Chow et al.,

Fig. 8. VEGF and Crystallin expression in human PFV disease. PASstaining of human PFV tissue showed centrally dragged retina withrosette formation (a, short arrows) and a retained hyaloid artery project-ing into the vitreous chamber (a and b, long arrow). b: The highermagnification view of the hyaloid artery shown in a. Histologically, thestructure is similar to Nuc1 (Fig. 2) although no rosette formation hasbeen observed in Nuc1. c: Immunofluorescent labeling (red) with VEGFshows staining surrounding the vessel wall of the retained hyaloid artery.d: The section is counterstained with Hoechst to label nuclei. Stainingpatterns similar to that of VEGF were obtained using antibodies to GFAP(e) and �-crystallin (f). Note that the staining pattern of GFAP-positiveastrocytes, VEGF, and �-crystallin in PFV are similar to that observed inNuc1 (Figs. 4, 6, and 7). Scale bar � 50 �m.

Fig. 9. VEGF and crystallin expression in cultured astrocytes exposedto 3-nitropropionic acid (3-NP). Human astrocyte cell line (SVG) wastreated with 10 �M 3-NP, and cells were harvested for immunostainingwith antibodies to VEGF and crystallins after 6, 12, and 24 hr. Photomi-crographs (counterstained with DAPI) show that normal astrocytes donot express �-crystallins or VEGF (top). When cells are exposed to 3-NPfor 6 hr, distinct cytoplasmic staining (red) for both �-crystallin and VEGFhas been observed. Fluorescence with both of the antibodies is in-creased after 12 hr of exposure to 3-NP in culture. By 24 hr, most of thecells have died. Nuclei counterstained with DAPI.

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2001; Sandercoe et al., 2003). To de-termine if astrocytes also express �-and �-crystallins in response to hyp-oxia, we used 3-nitropropionic acid, anirreversible inhibitor of mitochondrialsuccinate dehydrogenase activity(Cavaliere et al., 2001). It can be con-sidered a model substance to studyhypoxic neuronal damage (Riepe etal., 1996). As shown in Figure 9, whenfetal human astrocytes in culturewere exposed to 3-NP, there was in-duction not only of VEGF within 6 hr,as expected, but also �- and �-crystal-lins. Strong cytoplasmic staining wasdemonstrated with both VEGF and�-crystallin antibodies (�-crystallinalso showed a similar staining pat-tern); nuclei were counterstained withDAPI. Control cells showed little or noVEGF or �, �-crystallin staining.

DISCUSSION

Programmed hyaloid vascular regres-sion is an essential element of the de-velopment of the eye; apoptosis playsan important role in this process. Vas-cular regression normally occurswithin 3 weeks after birth in rats (Lat-ker and Kuwabara, 1981). In Nuc1,the programmed regression of the vas-cular network is inhibited. We haveshown that the Nuc1 mutation alsoprevents the normal programmed lossof nuclei from lens fiber cells and af-fects the regulation of cell numbersand maturation of retinal neurons(Sinha et al., 2005). At present, themechanistic relationship among thesevarious phenotypes is unclear. How-ever, it appears that Nuc1 may be aneye-specific regulator of apoptosis,since the mutation has shown no ob-vious effects outside of the eye. Evenhomozygous mutants appear grosslynormal, except for their eyes, and bothmales and females are able to repro-duce. Nuc1 may govern the matura-tion of several ocular tissues. The onlyother animal model that shows simi-lar features is the Apaf-1 (CED-4 ho-molog) null mouse (Cecconi et al.,1998). That model similarly shows al-teration in lens development and ab-normal cell number regulation in theretina; it inhibits hyaloid regression,but also exhibits delayed inter-digitalmesenchymal cell death, which is notseen in Nuc1.

Several mouse models with persis-

tent hyaloid vessels have been re-ported (Saint-Geniez and D’Amore,2004). In these models, the hyaloidvessels might persist to compensatefor the absence or lack of retinal ves-sels. In contrast, the Nuc1 mutantrats exhibited the initial network ofretinal vessels but with a possible ab-normality in the remodeling process(Gehlbach et al., unpublished data).Further, Nuc1 exhibits inhibition ofthe normal regression of the entire fe-tal intraocular vasculature and notjust part of it, as reported in thosemouse models.

Another factor believed to be impor-tant in the regression of the hyaloidvascular system is the macrophagepopulation. Persistent vasculaturewas reported in transgenic micewhere macrophages were disrupted bydirected diphtheria toxin expression,using macrophage-specific promoterelements (Lang and Bishop, 1993).That study suggested that macro-phages are required for normal hya-loid vessel regression. Our studies doindicate that macrophages may playan essential role in the pathophysiol-ogy of Nuc1 (Hose et al., 2005), al-though we have no evidence to suggestdirect involvement in the persistenceof the fetal vasculature.

Crystallins, initially regarded aslens-specific structural proteins, noware thought to be multifunctional pro-teins with physiological roles in non-lens tissues as well. A recent studyhas shown that �A and �B-crystallinsfunction as distinct anti-apoptotic reg-ulators (Mao et al., 2004). Severalother laboratories have also reportedthe possible involvement of �-crystal-lins in cellular apoptosis (Mehlen etal., 1996; Golenhofen et al., 1999;Hoover et al., 2000; Andley et al.,2000; Mo et al., 2001; Kamradt et al.,2001; Bruey et al., 2000). While ourmicroarray data (not shown) showedupregulation of �B-crystallin, a ubiq-uitously expressed small heat shockprotein known to be induced by stress,it also showed a marked upregulationof �, �-crystallins. Real-time RT PCRand Western blots confirmed this re-sult. Interestingly, our studies showedthat while many members of the �-and �-crystallin family are upregu-lated in Nuc1, they are also differen-tially regulated. Moreover, metaboliclabeling studies with 35S amino acids

indicated that newly synthesized crys-tallin proteins were increased in or-gan-cultured Nuc1 retina relative towildtype retina. Double-labeling ex-periments with isolectin B4 and �-and �-crystallin antibodies of the re-tained hyaloid artery in Nuc1 clearlyshowed that �-and �-crystallin ex-pression is concentrated surroundingthe vasculature. During normal devel-opment, � and �-crystallin expressionwas also associated with the hyaloidartery as shown in the 9-day wildtypeeye. Moreover, the retinal vasculaturein both normal and Nuc1 rats showedcrystallin expression surrounding theblood vessels. It is possible that �- and�-crystallin have a role in vascular de-velopment and/or survival.

Several investigators have analyzedVEGF expression during normal andpathological vascular development.Our studies demonstrate that the re-tained hyaloid artery in Nuc1 expressVEGF as well as �-and �-crystallin.We localized both crystallin andVEGF expression in the retained hya-loid artery in Nuc1 to the GFAP-posi-tive astrocytes. Astrocytes are nowregarded as multifunctional house-keeping cells that interact with thevasculature to form a gliovascular net-work. While, expression of both VEGFand crystallins was seen in the GFAP-positive astrocytes of the inner limit-ing membrane of the Nuc1 retina,crystallin expression was evident inother parts of the retina including theganglion cells. Crystallins have beenshown by others to be expressed in theretina (Deretic et al., 1994; Sinha etal., 1998; Jones et al., 1999; Xi et al.,2003); however, the function of crys-tallins in the neural retina remains tobe determined. We have recentlyshown that in Nuc1 the retina isthicker than normal and shows re-duced programmed cell death duringdevelopment (Sinha et al., 2005). Oth-ers have shown that the formation ofthe retinal vasculature is associatedwith the thickening of the retina (Dre-her et al., 1992). Crystallin productionmay be upregulated in the Nuc1 ret-ina as a result of increased vascular-ization, to meet the higher metabolicdemand of the abnormally thickenedtissue.

Apoptosis plays a major role in theremodeling of organs. We have re-cently shown that in Nuc1, the normal

CRYSTALLINS IN VASCULAR REMODELING 43

apoptotic-like process in the lens andretina appeared to be inhibited (Sinhaet al., 2005). It is possible that �- and�-crystallins synthesized by the astro-cytes foster vessel survival or stabili-zation and thus inhibit regression ofthe hyaloid system. The vasculature iscapable of sensing changes within itsmilieu and remodels itself, when nec-essary, through local production ofmediators that influence structure aswell as function. Vascular remodelingis an active process of structural alter-ations that may subsequently contrib-ute to the pathophysiology of vasculardiseases (Gibbons and Dzau, 1994).

Retention of fetal vasculature in hu-mans is characteristic of PFV disease(Goldberg, 1997). We provide evidencethat GFAP-immunopositive astro-cytes in the retained vessels of PFVpatients express VEGF and also �-,�-crystallins. Thus, increased expres-sion of VEGF by GFAP� astrocytes inthe retained hyaloid tissue, a findingnot previously reported, suggestsVEGF-mediated remodeling of thevessels in PFV. Moreover, expressionof the crystallins by the same astro-cytes suggests that crystallins mayalso participate in the vascular re-modeling process.

It has been postulated that “physio-logical hypoxia” is required for forma-tion and survival of the transient em-bryonic vasculature of the eye (Chowet al., 2001). Physiological levels ofhypoxia are also the stimulus for nor-mal development of the retinal vascu-lature (Chan-Ling and Stone, 1993). Itis possible that a defect in hyaloid vas-cular regression in both Nuc1 and hu-man PFV may lead to increased VEGFand �, �-crystallin production due to ahypoxic environment. To determinewhether hypoxia could induce crystal-lin synthesis in astrocytes, we cul-tured human astrocytes and exposedthe cells to 3-nitropropionic acid (3-NP), a substance shown to induce neu-ronal hypoxia (Chauhan et al., 2003).Interestingly, the astrocytes ex-pressed high levels of VEGF and �-,�-crystallins within 6 hr of exposure to3-NP. Indeed, it has also been shownthat astrocytes respond to the chang-ing physiological levels of hypoxiaduring development and secreteVEGF. Although VEGF is known to beregulated by hypoxia, there is as yetno other evidence that hypoxia regu-

lates �- and �-crystallins. However,the presence of a hypoxia-response el-ement (HRE) in the promoter of �s(unpublished observation), a �-crys-tallin family member that is stress in-ducible in the retina (Sinha et al.,1998), raises the possibility that �-and �-crystallins could also be regu-lated by hypoxia.

In conclusion, it is tempting to spec-ulate that �, �-crystallins may func-tion in mediating blood vessel remod-eling and/or survival in the developingeye. While cellular functions of �-crys-tallins in non-lens tissues have beenestablished, the function of �/� crys-tallins remains unclear. The possibil-ity that they may have a role in vas-cular remodeling is important,because such remodeling is funda-mental to normal ocular developmentand to the pathogenesis of numerousdiseases. Any influence that crystal-lins may have on such processes couldhave potential clinical importance.

EXPERIMENTALPROCEDURES

Specimen Preparation

Experiments were performed usingpostnatal Nuc1 and wild-type SpragueDawley rats, as described earlier(Sinha et al., 2005) in accordance withthe Guide for the Care and Use of Lab-oratory Animals (National AcademyPress). Experiments involving normaland PFV human tissue conformed tothe guidelines set forth in the Decla-ration of Helsinki for the use of hu-man tissue in research. Post-mortemsamples were fixed in 10% formalin,and 5-�m sections were cut and eitherstained with PAS or processed for im-munofluorescence.

Real Time RT- PCR

Real time RT- PCR was used to deter-mine the expression of �- and �-crys-tallins in wild type and Nuc1 homozy-gote retinas. Total RNA from sampleswas reverse transcribed using Super-Script II Reverse Transcriptase (In-vitrogen, La Jolla, CA). For Real-timePCR analysis, LightCycler FastStartDNA Master SYBR Green kit (RocheDiagnostics) and the Light Cyclerfrom Roche Diagnostics were used.Primer sets for �- and �-crystallin

family members were taken from pub-lished sequences and are availableupon request. Since �- and �-crystal-lin family members share close homol-ogy, we selected non-homologous re-gions using the ComAlign software.Primer pairs based on the respectiverat sequences of each crystallin genefrom the Ensembl database were thendesigned using the primer 3 software.Hypoxanthine PhosphoRibosyl Trans-ferase (HPRT) was used as an internalcontrol. SYBR green was incorporatedinto the reaction mixture to facilitatemeasurement of product. The integ-rity of PCR product was verified bymelting curve analysis. Real-timePCR values were determined by refer-ence to a standard curve that was gen-erated by Real-time PCR amplifica-tion of serially diluted cDNAs using �-and �-crystallin and HPRT primers.Values obtained for levels of �- and�-crystallins were normalized to thelevels of HPRT mRNA.

Metabolic Labeling

To determine if new synthesis ofcrystallins in Nuc1 homozygotes wasdifferent from that in the wild type,20-day-old retinas in organ culturewere incubated with 200 �ci of 35S-labeled amino-acids (Easy Tag Pro-tein labeling mix, Perkin Elmer LifeSciences, Oak Brook, IL) for 3 hr.Retinal samples were then rinsedand homogenized in 20 mM Tris (pH7.1) containing protein inhibitors(Roche) in a loose-fitting plastic tis-sue grinder to disrupt cells, but notto disrupt the nuclei. The sampleswere centrifuged at maximum speedin an Eppendorf microfuge for 15min to remove debris and cell nuclei.To each supernatant was added afew microliters of DNase (Roche) todigest any remaining DNA. After 30min of incubation at room tempera-ture, SDS sample buffer with reduc-ing agent (Novex) was added. Thesamples were placed in a boiling wa-ter bath for 2 min and then loaded ona Nu-PAGE (4 –12%) Bis-Tris gradi-ent gel (Invitrogen). Gels werestained with Coomassie brilliantblue, dried, and autoradiographedusing Kodak Biomax film.

44 ZHANG ET AL.

SDS-PAGE and Western BlotAnalysis

The eyes were enucleated from 20-day-old wild type and Nuc1 homozy-gous rats after euthanization. The ret-ina from each eye was dissected andrinsed in PBS and homogenized inSDS sample preparation buffer. Afterthe supernatant fractions were heatedin a boiling waterbath for 2 min, ap-proximately 100 �g protein from eachpreparation was loaded on 4–12% Bis-Tris Nu-PAGE gels (Invitrogen). Thegels were stained with Coomassiebrilliant blue. For Western blotting,proteins were transferred to Nitrocel-lulose membranes (Bio-Rad Laborato-ries, Richmond, CA), blocked with10% milk diluent, and incubated withthe primary antibody overnight at4°C. HRP-conjugated secondary anti-bodies and 4-CN substrate (Kirkeg-aard and Perry Laboratories) wereused for visualization. A cocktail con-taining �- and �-crystallin antibodies,each at 1:800 dilution was used. Theantisera were raised in rabbits usingcalf �- or �-crystallin protein as anti-gen.

Immunofluorescence

The primary antibodies used in thisstudy included rabbit polyclonal anti-bodies to � and �-crystallin (1:500),VEGF (Santa Cruz, sc-152; 1:100),GFAP (glial fibrillary acidic protein)(Dako; 1:1,000) for single labeling andthe mouse monoclonal GFAP (SantaCruz; 1:200) for double labeling. Fro-zen sections were incubated with pri-mary antibodies overnight at 4°C,washed with PBS, and incubated withdonkey anti-rabbit secondary antibod-ies conjugated to either Cy-2 or Cy-3(Jackson ImmunoRes, West Grove,PA, 1:200) for 1 hr at room tempera-ture. Crystallin antibodies were alsoimmunoabsorbed with respectivecrystallin proteins and used as an ad-ditional control in the present study.For double labeling with biotinylatedisolectin B4 (Sigma, St. Louis, MO)and crystallin, streptavidin-Cy2 andCy3 conjugated secondary antibodies(Jackson ImmunoRes, 1:200) wereused. For double labeling with otherprimary antibodies (two primary anti-bodies from two different species), Cy2or Cy3 conjugated secondary antibod-

ies were used. The sections were fi-nally counterstained with Hoechstand mounted with DAKO fluorescentmounting medium. For visualizationof blood vessels, rats were anesthe-tized and perfused with PBS contain-ing 50 mg/ml of fluorescein-labeleddextran (average molecular weight500,000; Sigma, St. Louis, MO) as pre-viously described (Tobe et al., 1998).The eyes were removed, immersed inOCT compound without fixation, andsectioned. The 7-�m sections werethen immunolabeled with either �- or�-crystallin antibodies (1:1,000 dilu-tion). Fluorescent digital images weretaken with a Zeiss microscope (Axios-kop II). Confocal microscopy was doneon Zeiss LSM 510.

Culture of HumanAstrocytes and3-Nitropropionic AcidTreatment

The SVG cell line used in this studywas derived from human fetal astro-cytes transformed with SV40 large Tantigen (Major et al., 1985; Tornatoreet al., 1996). Cultures of the humanfetal astrocyte cell line (SVG) weremaintained in Dulbecco’s modified Ea-gle’s medium with 2 mM L-glutamine,10% fetal bovine serum, and strepto-mycin-penicillin-fungizone solutions.Cells grown in 60-mm dishes weretreated with 10 �M 3-nitropropionicacid (3NP) for 6, 12, and 24 hr at 37°Cand then fixed with paraformaldehyde(4%) for 15 min and blocked with 3%BSA at 4°C overnight. The fixed cellswere processed following standardtechniques for immunofluorescenceusing VEGF and crystallin antibodiesas indicated earlier.

ACKNOWLEDGMENTSThis work was supported in part byHelena Rubeinstein Foundation (toD.S.), a pediatric ophthalmology re-search grant from Knights TemplarEye Foundation, Inc. (to D.S.), Juve-nile Diabetes Research FoundationInternational (to P.G.), Alexander andMargaret Stewart Trust (to P.G.),NIH KO8EY13420 (to P.G.), GuerrieriRetinal Research Fund (to M.F.G.),and Research to Prevent Blindness(an unrestricted grant to Wilmer EyeInstitute). We thank Drs. Peggy

Zelenka, Paul Russell, Panagiotis Tso-nis, and Gerard Lutty for critical read-ing and discussion regarding thismanuscript. We are grateful to Dr.Ashok Chauhan for his help and sup-port with the cell culture studies andMs. Rhonda Grebe of the Wilmer Con-focal facility.

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