LAB()RATORYSCIEI'IC!';\i;A
Cytoprotective effects of a blue light- filtering
intraocular lens on human retinal pigment
epithelium by reducing phototoxic effects
on vascular endothelial growth factor-a,
Bax, and Bcl-2 expression
Marcus Kernt, MD, Aljoscha S. Neubauer, MD, Raffael Liegl, Kirsten H. Eibl, MD, Claudia S. Alge, MD,
Carla A. Lackerbauer, MD, Michael W. Ulbig, MD, Anselm Kampik, MD
PURPOS E: To compare the possible protective effects of the ultraviolet (UV)-filtering and blue lightfiltering SN60AT intraocular lens (IOl) and the untinted UV-filtering SA60AT IOl with regard to
light-induced stress on human retinal pigment epithelium (RPE).
SETTING: Department of Ophthalmology, ludwig-Maximilians-University, Munich, Germany.
METHOOS: Primary human RPE cells were exposed to white light, and a tinted or untinted IOl was
placed in the light beam. Alter 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazo lium dye-red uction assay) and a microscopic live/dead assay. The expression
of vascular endothelial growth factor- (/. (VEGF-u.), Bax , and Bcl-2 and their mRNA was determined
by reverse -transc ription polymerase chain reaction (RT-PCR) and Western blotting.
RESUlTS: Without an IOl, white-light exposure decreased cell viability compared with the decrease
with the nonirradiated control in a time-dependent manner, Light-induced cell death was significantly reduced by both the tinted IOl and untinted IOl. The combined UV and blue-light filtering
attenuated light-induced cell damage significantly more than UV filtering alone. Results of RTPCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in
8ax and VEGF-u. that were significantly less with the tinted IOl than with the untinted IOl.
CONClUSIONS: 80th IOls reduced light-induced RPE damage. The UV- and blue light- filtering IOl
reduced damage more than the conventional IOl. This supports the hypothesis that blue lightfiltering IOls may prevent retinal damage in clinical use .
J Cataraet Retraet Surg 2009; 35,'354-362 © 2009 ASCRS and ESCRS
Age-related mac ul ar degeneration (ARMO) is the
lead ing cause of lega l blindness in people older than
60 yea rs in developed countries. Ap proximately 10%
of these patients have wet form s of ARMO due to choroidal neovasculari za tion that often lead to a rap id 1055
of vision.1.2 The other 90% of patients ha ve dry fonns
of ARMO such as geographic atrophy.1,3-S Geogra phic
atrophy is charac teri zed by atrophy Df retinal pig ment
epitheliul11 (RPE) cells a nd consecuti ve photoreceptor degeneration. 6 A t present, there is no sa tisfactory treatment fo r geog raphi c atrophy .
Even if ep id emiologic studies are inconclusive/,8
there is ev idence that cUJ11u la tive lig ht ex pos ure is si¡t
nifica ntl y associated with progression of ARMD .8 - o
354
© 2009 ASCRS and ESCRS
Published by Elsevier Inc.
Both the cornea a nd the crysta lline lens help protect
the retina aga inst the hazards of lig ht by filtering
most ultrav iol e t (UV) radiation. 11 ,12 In oIcter humans,
in w hom the retina mig ht already be compromised,
the crys ta lline lens also filters potentiall y phototoxic
proportions of light w ithin the blue ran ge by progressive ye llowing. 13 - 15 This filterin g fllllction of the ag ing
lens is particularly important as this fraction of light is
known to induce photochemical damage to the
RPE. '6•17
Cata ract s urgery is a sa fe, common, and beneficial
procedure that helps prevent blindness in millions 01
people. However, asid e from the benefits from removing the crysta lline lens and implan ting an intraocular
0886-3350/09/$- see fronl matter
doi: 10.1 01 6/j.jcrs.200B.l 0.052
L¡\BO RATORY SC I ENCE: CYTOPROTECTI VE EFFECTS OF B LUE L1GHT- F1LTER ING IOL
lens (IOL), this procedure can result in an increase of
rad iatio n reaching the retina and comprornisin§ the
ocular protective shield against Iight damage. ' ·- oTo
protect the retina from possible toxic effects of increased radiation after cataract extraction, yellowtinted, UV-filtering, and blue light-filtering IOLs
ha ve recently been developed 2l The Iight-transmission spectrulll of UV-fiItering and blue Iight-fi ltering
IOLs differs from that of conventional IOLs that filter
UV Iight onl y because these rOLs contain chJO mophores that filter light in the 400 to 500 nm range, adding this Iight¡,rotection to that already provided in the
UV range. 22 , 3 Within the visible spectrum aboye 500
nm, both types of rOLs provide even transmission
comparable to that of the clear crystalline lens n ,23
The RPE's own pigment, lipofuscin, is known to act
as a photosensitizer and to be involved in the pathogenesis of exudati ve ARMD 2 4 - 2• To test the protective
effects of UV-fiItering and blue Iight- filtering IOLs, experimental studies exa mined Iipofuscin-triggered
phototoxic reactions in the RPE. Light-induced toxic
effects were studied in RPE celllines artificially laden
with lipofusci n 29,30 The results support the theory that
UV-filtering and blue light- filtering rOLs may be more
protective aga inst photochemica l damage.29,30
This study compared the possible protective effects
of a UV-fil tering and blue Iight- filtering IO L with
those of an untinted UV-fiItering rOL with regard to
Iight-induced stress on human RPE. Early passages
of primary human RPE cells were studied. Early passages of RPE cells are known to still possess some of
the cells' own lipofu scin, which is lost when ceJls are
cultured long tenn .31 - 33 We exposed the cells to
wide-band white Iight and placed the IOLs in the Iight
beam. The viability of the cells was determined. The
expression of vascular endothelial gro wth factor-u.
Su bmilted : August 9, 2008.
Final revision submilted : October 17, 2008.
Accepted: October 21, 2008.
From the Department 01 Ophthalmology, Ludwig Maxi milian University, Munich, Germany.
No author has a financial or proprietary interest in any material or
method mentioned.
Supported by Alcon, Freiburg, Germany.
Ulrich Welge-Lüssen, MD, provided laboratory equipment. Hamamatsu Photonics, Herrsching am Ammersee, Germany, provided
the light source. Katja Obholzer provided technical assistance.
Corresponding author: Marcus Kernt, MD, Department of Ophthalmology, Ludwig-Maximilians-University, Mathilden Strasse 8,
80336 Munich , Germany. E-mail: marcus.kernt@med. lIni-m uenchen.de.
355
(VEGF-Cl,), a major StilllUlus in the development of exudalive ARMD,2-3' and Ihe expression of Ihe pro-a poptotic and anti-apoptotic proteins Bax and BcI-2 were
determined.
MATERIALS ANO METHOOS
Ethics
The methods of sec uring human ti ssue complied w ith the
Declaration of J-Ielsinki and were approved by the loca l
ethics CO llllllittee. Written infor med consent wa s ob tained
from the tiss ue donors.
Human Relinal Pigment EJlithe lium
Retinal pigment epitheli al cells fl"Om human donors v..'ere
obtai ned 3 to 10 hours postlllortelll fl'OIn the eye bank at Lud w ig Maxjtni lian Uni versity and prepared as p reviously descri bed ?':> In briet whole eyes were thoroughly clea nsed in
0.9% sodi ulll chlo ride (NaCl) solutio n, immersed in 5% polyv inyl pyrroli d ine iodi ne, élnd rinsed élgaí n in the NaCl so luHon. The anterior segment of each donor eye ",as removed,
and the posterior po les ",ere exa mined with a binocu lar s tereom icroscope to cOlúi rlll the élbsence of g ross reti nal disease. The neura l retina was then carefully peeled away
frolll the RPE-choroid-sc1era l;vith a fine forceps. The eye
cup \Vas rinsed with Ca 2 + -free a nd Mg2 + -free H,mks balanced sa lt Sollltion (BSS) and fiHed with 0.25 % trypsin (Invi trogen) for 30 minutes at 3TC. The trypsin was ca refllll y
asp irated and replaced l;vith DlIlbecco mod ified Eagle mediulll (DMEM, Biochrom) s uppl e mented with 20% fetal
ca lf serum (FCS, Biochrom). The m edium \Va s gently ag itated \Vi th a pipette, releasing the RPE cells into the medi1l1l1
w ithout damaging Bruch 1l1e1l1brane. The RPE ceH solution
was transferred to a 50 m L fl ask (Faleon) containing 20 mL
o f DMEM sup plemented with 20% FCS alld ma intained a t
37°Cand 5% ca rbo n dioxide. Epithelia l origin Wél s colÚinned
by imlllunohistochemica l s tainin g for cytokera tin w ith
a pa n-cytokeratin antibody (Sigma-A ldrich).36 The cells
were tes ted él nd found to be free o f conta minati ng Inacl"Ophages (anti-CD1L Sig llla-Aldrich) él nd end ot heli al cells
(a nt i-von Willebrand fac tor, Sig llla-Aldrich) (da ta not
shown). After growing to conflu ence (100%), primary RPE
cells were sllbcllltured and 1l1aintained in DMEM supp l e~
Illented with 10 % FCS at 37°C and 5% carbon dioxide. Primary RPE cells of passages 2 a nd 3 were lI sed in the
exper iments. AH investiga ted RPE cell cultures still presented intracellul ar pigm ented g ra nules in phase-contrast
microscopy.1t ha s been showll th at beside melanin and other
pigments, these granules con tain a certai n amou nt of ceJlow n li pofllscin. This characteristic is lost wh en RPE cells
are cu ltured long terll1 ?1 -33
InlraoclIlar Lenses
Two s ingle-piece acrylic fo ld able IOLs were tested: a yellow-ti nted UV- and btue light- fi lte ring IOL (SN60AT; tinted
JOL) and a UV- filtering JOL (SA60AT; untinted JOL). Both
IO Ls (Alcon Laboratories) were + 20.0 di opters.
IIll1mination 01 Ce lis
A spot-light source (LC-8, Hamamatsu Photonics) fro1l1
él mercllry-xenon lamp llsing a n opt ic-fiber as the light gll id e
(spectra l ran ge 400 nm to 700 nm) was lI sed fOI" illllmination.
J CATARACT REf RACT SURG . VOL 35, fEBRUARY 2009
356
LABORATORY SC IENCE : CYTOPROTECTIVE EFF'ECTS 01" BLUE L1GHT- FILTERING [OL
The ceH-cult u re medium \Vas replaced ,vith phosphate-buffered sa line (POS) sol ution jus t before iIIumination. The p lastic cover o f the ill umi na ted ceH-culture \Vell was removed,
éllld the cells w e re illu1llinated f1'ol11 abo ve. They we re ill ulllj naled for 15 to 60 minutes (350 mW /c nl ) in the presence or
abse nce of the ¡OLs. The IOLs we re applied to the li ght-emitting o ut put of lhe optic fib er (diameter 3.5 mm), where lhe)'
remailledattachecl \vitho ut .lI1aie! . The illumin a ti oll powe r
anc! spec tra l ra nge were measurecl with él spec trom eter
(CI 0083MD, Ha mal1latsll Photonics). Directly after iII umi natí OIl, PBS was replaced by serum-free ceH -cultu re l1lediull1
and cells were kept in d arkness for ano ther 24 hours. Then,
the tetrazo lium d ye-red uctio n assay (M1T; 3-[4,5-dimethy lth ia zol-2-y IJ-2,5-diphenyl tetra zolium bromide), live/
dead assay, ph ase-contras t microsco py, RNA isolation, 0 1'
protein ex tra ction \Vas perfonned. Illuminati on times of '15,
30, 45, and 60 minutes were chosen for all exper im ents to
eva lu ate poss ible protec ti ve effects of th e untinted IO L and
the tinted IO L.
MTT Assay
The MTI assay \<vas used to d etermine lhe cell survival
rateoThe MIT test, which is well established for lhe assessment of cell vi abi lity, \Vas perfo rmed as described by Mosmann,37 with some modifications. The medium \Vas
removed; Ihe cells were washed wit h PBS, and 1000 Il1L of
MTT solution (1.5 mL MTT stoc k, 2.0 mg/ mL in PB5, plus
28.5 mL DM EM) IVas ad d ed to eaeh IVell. The RPE eells
',vere incuba ted at 37°C for 1 ho ur. The forll1a zan crys la ls
that fonned were dissolved by the addition of 1000 IllL of di methylsulfoxid e per well. Absorption was measured by
a scanning multiw eJl spectrophotometer at 550 11m (Mol ec ular Probes). Th e res ults we re ex pressed as lhe mean percentage of proli feratio n in the control. The experilllents v\'ere
performed in triplica te and repeated 3 limes. The co ntrol
cells were Ihe RPE cells of the sa llle passage, incubated
wilh ba lanced sa line, kept in darkness, and without exposure lo a ny J"ad ia lion.
Live/Dead Cel! Viability Assay
Confluent RPE cell s \vere prepa red and treated as described. Cell v ia bi lity \Vas quant ified based on a 2-color f1u orescence assay in which the nuclei o f nonviable cell s
appeared red du e lo slaining by the ll1embrane-im penneable
dye pro pidillm iodide (Sigma-A ldrich) while the nucl ei o f all
cells were sta ined w ith the membrane-penneable d ye
Hoec hsl 33342 (Intergen). Con flu ent cultures of RPE cell s
growin g on cover slips in 24-well ti ssll e culture plates were
exposed lo \v hite light in Ihe absence 01' presence of the
IO Ls, as d escribed for lhe MIT assay. To eva lll ate cell viabilit)', Ihe cells were \vashed in PBS and incubated wit h 2.0 mg/
mL p ro pidium iodide and 1.0 mg/ mL Hoechst 33342 for 20
minutes al 37°C. Subsequent ly, Ihe cells were ana lyzed wit h
an ep ifl uo rescence microscope (Ar is toplan, Leitz). The labeled nllcl ei were then cou nted in f1uorescence photo microg raphs. Dead ceHs \vere expressed as a percenta ge of the
total nucl ei in the field. The data are based on counts in 3 experiments performed in duplicate w ells, with 3 to 5 d ocumented representa ti ve field s per well. The RPE cell s o f the
same passage incubated with DMEM served as controls.
RNA Isolation and Real·Time Polymerase Chain
Reaction
The RPE cells from 5 d ono rs were used to assess mR NA
expression of Bax, Bcl-2, a nd VEG F-u.; Bax, BcI-2, and
VEGF-u. messenger RNA expressio n was d etec led in every
sa mpl e. AII d etec ted Il1R NA levels of Bax, BcI-2, and
VEG F-u; were nOl'mali zed to those of 185 rRNA, and the
va lll es were expressed as the rela live ra ti o of Bcl-2/18S,
Bax/185, or VEGF.,,/185.
Total RNA \Va s isola ted from 10 cm Petri dishes by theguanidiu m thioc)'anate-phenol-ch loro fo rm extraction method
(Stra tagene). The structural integrity o f Ihe RNA samples
was confir med by electropho resis in 1 % Tris-acetate-ethy lenediaminetetraace tic acid (TAE) aga rose gels. The yield and
pul'ity were d etermined photometrica ll y (Bio Pho tometer, Eppendol'f). Real-time polymerase chain reaction (PCR) enables
quantitative d etection of s mal! amount s of mRNA. After the
usual isolation of mRNA, the mRNA \Vas tran sferred to
cONA via reverse transcri p tase. Th is cDN A ,vas fhen used
fo r the s pecific PC R. Q uantifica tion o f BcI-2, Ba x, and
VEGF-u, mR NA ,vas perfonned w ith specific p rimers (rabie
1) wi th the LightCycler system (Roche Oiag nostics). Primers
and probes were fo und w ith ProbeFinder 2.04 (Roche Oia gnostics). Al! primers and probes were d esig ned lo cross intron/ exon boun da ries to avoid am plifi cati on of ge noll1 ic
ONA. AH PC R produ cts were seqllenced to ensure product
va lidity. Each 14 'lL reac tion voJume co ntained 1 x FastStart
ONA Mas ter Hybrid ization Probes Mi x (Roche Oiagnostics),
4.0 m M Illagnesilllll d ichloride, 0.5 In M of each primer,
0.2 m M TaqMan probe, and 2.0 ,tL cONA.
The amplification s ignals . ."ere d etec ted in rea l time,
which enabled accura le quantifica tio n o f Ihe <l. ll1ounts of
the initia l RNA template beca use the sys tem can selec t signals easil)' durin g the expo nen tial a mp lificat ion p ha se of
PCR. The cONA of cell s exposed to wh ite li ght in the absence
0 1' presence of the IO Ls wa s ampli fi ed with spec ific prim ers
fol' 40 cyeles. Two o ligo nucleo tid es with d ifferen t labeled fluorop hores were hybridi zed lo Ihe am plified fra g ment during
the annea ling phase. When Ihe 2 probes came in close proximity, f1 uo rescence reso nance energy tra nsfer developed between the 2 fluo ropho res. The em itted flu orescence was then
measured by the LightCycler ins trull1ent. Hybridi zation
probes were dis pla ced during lhe ex tens ion ste p. Oepending
on the initia l co nce ntrati on of target ge nes, the s ignal intensity increased in different cycles; these cycles were used as
the cross ing poin!. The standard curve ,vas made wilh 3
Table 1. Primers used for RT-PCR.
Télrget/Length Positioll AT (oC) %GC
Sequence
Bcl-2
19
20
3465-3483
3505-3524
60
60
SO ccaggtMlcaélaélccccélca
58
526-543
592-609
59
59
61
56
caélgélccaggglggttgg
cactcccgccacaaagat
1540-1557
1592-1609
60
60
56
6]
tgcccgctgctgtcaat
tctccgctctgagcMlgg
aga gglcacggggctaa t
Bax
18
18
VEGF ·~
18
18
AT = anncal ing tempcrature; CC = G-C nuc1eolidc pairs; VEGF-a =
vascular endolhclial growlh factor-a
J CATARACT REFRACT SURG . VOL 35, FEBRUAR Y 2009
LABORATORY SCIENCE: CYTOPROTECTIVE EFFECTS 0 1" BLUE
To veri fy that Ihe li ght-induced increase in Bax and decrease in Bc1-2 in mR NA tra nscriptio n Iransla les inlo incl'eased protei n sy nthes is, whole cellular protein ext racts of
non ill umi nated cont rol cells and cells that were illumina ted
in Ihe presence 01' absence oE the tested IOLs (untinted and
tinted) were ana lyzed by Western blotting.
Primary RP E cells grow n on 35.0 111m ti ss ue culture dishes
were washed Iwice "vilh ice-cald PBS, collec ted, and Iysed in
rad ia immunoprecipitation assay ceH Iysis buffer. After centrifugation for 30 n'linutes at 19000 x g in a microfuge
(S8l0R, Eppendorr) in the cold, the superna tant was tra nsferred to fresh tu bes a nd stored at - 70°C for future use.
The protein content \Vas Illeasured by Ihe bicinchoni nic
ac id pro tein assay (Pierce). Denalured proteins (le2 mg)
''''ere separated by electrophoresis und er red ucing conditions tl sing a 5% sodiul11 dodecy l sul fate-polyacry lamid e
staek ing gel and a 12% sod ium dodecyl sulfate- polyacry lam ide separating ge l, transferred with semidry blotting
onto a polyv inyl difluorid e membrane (Roche), a nd probed
wi th a mouse anti-BcI-2-a nti body 0 1' a mouse anti-Bax-antibody as desc ribed prev iously.38 Chemil umi nescence was detec ted with the imager (LAS-IOOO, RayTest) and genera ted
Iighl units. Exposure times ra nged between 1 minute and
10 minutes. Qua ntifica tion was performed on a co mpute!'
(A lOA software, Ra yTest). AH experiments were perfoJ'med
al least in triplicate in RP E ceJls cultures from S do nors. An
even protei n load in each lane was confirm ed by staining
the pol yv inyl difluorid e membranes with Coomassie brilliant blue after Ihe blotting procedu re.
45mln
B
The study evalua ted RPE cells from 5 human donors
aged 42 yea rs, 52 yea rs, 58 yea rs, 64 yea rs, and 72
yea rs.
Viability 01 Cells
With up to 45 minu tes of illumination, no gross abno rmalities were d etected by phase-co ntras t microscopy in primary RPE cells in the presence of the
tested tOLs or w hen cells were ex posed to unfiltered
ligh!. After 60 minutes of light exposure, only
cells tha t were illuminated with unfiltered white light
showed distinct morphologic changes and signs of cell
dea th. The number of cells, counted in phase-contrast
microscopy, correla ted well w ith the results of the
MTI tes t w here quantification was pe rformed
(Figure 1).
MTT Assay
W he n cells were illuminated \v ith unfiltered w hite
light (350 mW /cm>¡' a time-dependent reduction of
RPE cell via bility was detected that was rapid and
Tinted ¡Ol
E
45ml n
45m in
F
D
60min
RESUlTS
Untinted IOl
e
60ml n
357
AH data were ana lyzed llsing SPSS fOl" Windows (version
13.0, SPSS, Ine). For aU stat isti cal tes ts, a P va lue less than
0.05 \Vas considered signifi ca nt. Results of Ihe MTT assay
are presented as Illean ± SO units of absorba nce. Ten individua l sa mples per group were measured in triplica te. The
Ma nn-Wh itney test was lIsed. Results of the RT-PCR are presented as mea n ± SO ratios of the investigated mRNA and
18S rRNA. Again, Mann-W hitney tes ting was app lied; all experiments were perfonned in Iriplica te and repea ted 3 times.
Western blot analysis \v as performed analogo usly; expe riments were performed a t leasl in triplica te in RPE ceH cultures frolll 3 donors.
Protein Extraction and Western Blotting
A
IOL
Statistical Evaluation
different probes of lInt rea ted RPE ee lls. To normalize for differences in the amOlll1 t of total RNA added to each reactiol1,
18S rRNA \Vas silllultaneo llsly processed in th e sa me sa mple
as an internal control. The level of BcI-2, Bax, and VEGF(j. mRNA \va s determined as the rela li ve rati o, \vh ich was
ca lculated by d ivid ing Ihe level of Bc1-2, Bax, and VEGFrJ. IllRNA by the level o( Ihe 185 rRNA housekeeping gene
in Ihe sallle sa mples. The ratios are expressed in a deci mal
for ma toAII experiments \Vere performed a l least in tr iplicate
and repeated 3 times.
Without IOl
LlGHT- F I LTEI~I NG
60 min
J CATARACT REFRACT SURG . VOL 35, FEBRUARY 2009
Figure 1. Phase-conl mst microscop)' of pr imar)' hum ,m RPE cel1 s.
There wcre no morphologica l
changes in Ihe p rescnce of Ihe 2
IOLs (C and E) or with unfiltcred illum inOltion (A) aher 45 m inutes of
illu m ination. When iIl um inated for
60 minutes, cells cxposed to unfillered wh ite li ghl showcd marked
signs of destruction éll1d cell dea lll
(8) w hile cells protected by eHller
IOL showed no significélnt morphologica l change (D él nd F) (IOL = intraocular lens) (scOl le x l 0).
358
LABORATORY SClENCE: CYTOPROTECTlVE EFFECTS OF BLUE LlGHT-FlLTERlNG lOL
significant beginning from 45 minutes of illumination.
When an untinted rOL was placed in the light beam
during illumination, this decrease in viability was sig­
nificant less pronounced compared with cells that
were illuminated with unfiltered white light; however,
this stillled to a significant reduction in RPE viability
decrease after 60 minutes of illumination. In the pres­
ence of the tinted IOL, the reduction in viability de­
crease was even more distinct. After 60 minutes of
illumination, RPE cell viability was significantly less
reduced compared with that of cells that were illumi­
nated with unfiltered white light and cells that were
illuminated in the presence of the untinted rOL
(Figure 2) .
live/Dead Assay
When the cells were illuminated 15 or 30 minutes,
no significant increase in nonviable cells was detected
com pared with the control in the presence of the tested
IOLs or when cells were exposed to unfiltered light
(data not shown). After 45 minutes of unfiltered illu­
mination, the RPE cells showed a significant increase
in nonviable cells compared with the nonirradiated
control. Cells that were exposed to light that was fil­
tered by the tinted or untinted rOL showed no de­
crease in the number of viable cells after 45 minutes
of illumination compared with the nonirradiated con­
trol. After 60 minutes, cells that were illuminated in the
presence of the tinted IOL showed no significant re­
duction in viable cells compared with the nonirradi­
ated control. In contrast, cells that were exposed to
unfiltered white light showed a significant decrease
100
.!!)
80
Oi
u
al
:oro
60
.;;
'O
~
e
40
QJ
:::
QJ
c.
20
Control
• Without IOl
15 min
•
30 min
Untinted IOl
45 min
60 min
• Tinted IOl
Figure 2. Viability of primary human RPE cells after iIlumination
with plain white light in the presence of an untinted rOL or a tinted
rOL as measured by a colorimetric test (MTI). The time of illumina­
tion decrease in cel! viability that was dependent on time of illumi­
nation significantly reduced when light was filtered by the 10Ls
and it was significantly more reduced with the tinted rOL than
with the untin ted rOL after 45 or 60 minutes of illumination (rOL
= intraocular lens) (error bars = SD).
in viable RPE cells compared with the nonirra diated
control. Cells that were illuminated in the presen ce
of the untinted rOL showed significantly fewer dead
cells than those with the unfiltered illumination but
also showed a significant decrease in viable cells com­
pared with the nonirradiated control (Figure 3).
Expression of Bax, Bcl-2, and VEGF-IX rnRNA
Illumination with unfiltered white light led to
a time-dependent decrease in Bcl-2 mRNA expression.
In contrast, expression of Bax and VEGF-a mRNA was
increased in primary human RPE cells (Figures 4 to 6).
rn comparison to unfiltered illumination, filtering
light with either tested IOL significantly reduced the
light-induced in crease in Bax and VEGF-a mRNA after
15, 30, 45, or 60 minutes of illumination. In addition,
this reduction of light-induced increase in Bax mRNA
was significantly higher when light was filtered by the
tinted IOL than when filtered by the untinted IOL
after 15, 30, 45, or 60 minutes of illumination. Ihe
VEGF-a mRNA overexpression was significantly
reduced after 45 or 60 minutes of illumination
when light was filtered by the tinted IOL compared
with overexpression with the untinted rOL (Figures 4
and 6) .
After 15, 30, 45, and 60 minutes of unfiltered illumi­
nation of RPE cells, a significant reduction in Bcl-2
mRNA expression was detected. Ihis phototoxic de­
crease in Bcl-2 mRNA express ion was significantly re­
duced by both tested IOLs. In addition, after 45 and 60
minutes of illumination, the decrease was significantly
less when light was filtered by the tinted IOL than
when filtered by the untinted IOL (Figure 5).
Protein-Expression of Bax and Bcl-2
After 60 minutes of unfiltered illumination, Bcl-2 ex­
pression decreased 3.3x in primary human RPE cells
compared with the nonirradiated control. Ihis decrease
was reduced to 1.4 x in the presence of the untinted IOL
and to 1.25 x in the presence of the tinted IOL (Figure 7).
In contrast, Bax expression was increased l.4 x com­
pared with the nonirradiated control, in which cells
were exposed to unfiltered irradiation for 60 minutes.
In the presence of the untinted IOL, this light-induced
overexpression of Bax protein decreased to 1.2 x .
When light was filtered with the tinted rOL, no increase
in Bax expression was detected (Figure 7).
DlSCUSSION
Several epidemiological studies4.8.9.39,40 seem to indi­
cate that aphakic and pseudophakic eyes have an in­
creased risk for development and progression of
ARMD. Photochemical damage and light-induced ap­
optosis due to cumulative light exposure have been
J CATARACT REFRACT SURG - VOL 35, FEBRUARY 2009
I
10C
~ 90
Qi
u 80
~
-e
.8
70
o
ID
60
~
ID
a. 50
CIJ
ID
40
U
.l!! 30
.o
ro
.:;
20
e
O
e 10
O
implü
RPEc
the Rl
totie e
andsl
Thi:
a UV
IOLrl
huma
filtere
tion d
explal
cell dé
of the
sion ti
range
phore
actly
prono
matel]
Ina
minati
LABORATORY SClENCE: CYTOPROTECTIVE EFFECTS OF BLUE LlGHT- FILTERIN G lOL
359
Hoechst 33342
With o ut ¡O L
Untint ed IOL
e
A
E
60min
4Smon
B
Tintad IOL
Control
L
G
N
60min
D
K
H
F
M
O
propidium iodide
100
~ 90
Q;
u 80
:§
.8
*
*
70
*
Co
45min
60min
Without ¡Ol
45min
60min
Untinted ¡Ol
im plicated as risk factors fo r atrophic degeneration of
RPE cells. 41 Light-induced apoptotic cell d eath within
the RPE is even more serious as RPE cells are postmitotic cells tha t do no t di vide under normal conditions
a nd should persist for the enti re Iife of a n individual42
This in vitro stu dy lo und that filtering light w ith
a UV- and blue Iight-filtering ¡O L or a UV-fil tering
¡OL resulted in red uced RPE cell da mage in primary
h uman RPE cells compared with the da mage from unfiltered illumination. The light we used for illurnination d id not inelude the UV spectruIl1. Therelore, olle
ex pla nation of w hy both ¡OLs led to a reduction in
cel! damage could be the light-attenuating properties
of the acrylic lOL material, w hich reduces transmission to approximately 90% within the 500 to 700 nm
range 01 light. 22 In addition, the UV-liltering e1u-omophore that both tested ¡OLs contain d oes not cut off exactly a t 400 nm wavelength but provides a less
prollounced, decreasing absorption up to approxima tely 430 nm ?2.23
In our experimental setup, after 60 minutes of illumination, the reduction in light-induced cell death
45min
Fig ure 3. A fter 45 min utes of il1um ination, Ihe number of v iable RPE
cel1s was significantly red uced
when cel ls were exposed to unfil tered lighl (A and B); there was no
signi ficant increase in nonviable
cells w hen cell s were il1 umi na led
in Ihe presence of bolh ¡OLs (E, F,
J, and K) . After 60 minutes of il1um ination, cells tha! were il lumi na ted
in Ihe presence of Ihe untinted ¡O L
showed significantly fewe r dead
cells Ihan those wil h Ihe unfillered
il luminatio n (e, o, G, and 1-1) bul
also showed a significant decrease
in viable cells compared w ith Ihe
no nirradia ted control (N and O).
lIl umi natiotl in the presence of Ihe
tinted ¡OL (L and M) d id no! increase Ihe number of nonviable
cells comp¡wed w it h the nonirmdiated control (N and O). Compa ri son
lOcontrols is show n above the ba rs,
w il h sig nifica nt differences rnarked
by an aster isk (*) (Co = control;
[OL = intraocular lens) .
60min
Tinted ¡Ol
was significantl y less in the presence 01 the tinted
¡OL than with the untinted IOL. Apoptosis is a cel!autonomolls mechanism that is genetica lly controlled
by the dying cel!, resulting in the acti vation of tumor
suppressor proteins (TSP)43 a nd a poptosis-initiating
caspases'4 A rece nt stud y 45 fOllnd that the UVand blue light- liltering properti es 01 the SN60AT
IO L red llced RPE cel! apo ptosis in vitro. In ad d ition,
our resu lts show tha t the d ecrease in RPE cell d ea th
is accom panied by a significant red uction in phototoxic deCl·ease 01 the a ntiapo ptotic pro tein BeI-2.
BeI-2 is general! y considered a c ustodian of mitochondrial functional integrity, as it stabilizes the
mitochondrial membrane aga inst the release of
cytocrome c. TI is able to interrupt apo ptosis through
its inhibi tory effect on caspase ac ti va tion. 46,47, It has
been show n that Bcl-2 med iates cell surviva l and
decreases apoptosis in human RPE cells48 and other
ocular and nonocular cell lines. 49 - S1 Sparrow and
Ca i52 demonstrated that Bcl-2 also can reduce bluelight-induced apo ptotic cell d eath in human RPE
cells in vitro .
J CATARACT REFRACT SURG - VOL 35, FE8AUAAY 2009
360
LABORATORY SCIENCE: CYTOPRQTECT IVE EFFECTS OF BLUE LlGHT- FI LTER1NG IOL
BAX mRNA
,
,
,
,
c;-;'
c;-;'
c;-;'
c;-;'
""
""
""
""
BCI·2 mRNA
2
,
,
,
c;--'
c;--'
';7
';7
"
"
""
""
•
0.5
o
Co
15min
• Without IOl
30min
45min
60min
O Tinted lel
• Untinted IOl
Figure 4. Q Uéln titative RT-PCR <l na lysis o f Bax mRNA exp ress ion of
primélry RPE cells a fl er iIIum illél l ion \V ilh plain white ligh l (Co) in
Ihe prescnce of <In untinled IOL and linled IO L. The mRNA levels
are expresscd as Ihe Tatioof BcI -2 mR NA/185 rRNA (Co
= con lro l;
[Ol = in traocula r Icns;.h'lx is = re lal ive ratioof Bax m RNA norma l·
ized lo 18s rR NA, cxpressed in d eci méll forma l; y-axis = tim e o f
ill umination),
In our ex perimental set-u p the Iight-induced d ecrease of a ntia poptotic BcI-2 was significantl y redu ced
by both tested IOLs. Furthermore, afler 45 or 60 minutes of illumina tion, the BcI-2 le veIs were sig nifica ntly
higher w hen Iight was filtered by the ti nted IOL tha n
by the untinted TOL.
In contrast, light-exposure led to an increased expression of the apoptotic Bax protein. Bax is a key protein w ithin a majar pathway of the cel! death progra mo
It is up-reg ulated by the TSP p53 a nd is highly associated w ith a poptotic ceH dea th .53 - 55 An im balance
w ithin the ra tio of Bax and BcI-2 towa rd a n excess of
,
,
,
c;--'
c;--'
';7
';7
"
"
""
""
•
VE GF-a mRNA
4
o
Co
15min
• Without IOl
, <111
•••_
1
Co
--
BcI -2
0,8
0,7
Tinted
Untinted
27kD- ' _ _
Co
• Without IOl
15min
30min
• Untinted IOl
45min
60min
Tinted IOl
Figure 6. QU<lntitéltive RT-PCR an<l lysis of VEGF-rJ. mRNA expressio n of priméll")' RPE cel ls after il1 11m infl lion wilh plain wh ile lig hl
(Co) in Ihe presence o f an unl inled 10 L a nd a linled ¡O L (Co = control; 10 L = inlrilOC U]¡U lens; VEG F·(I. = vasc ula r endolhelia l g ro", l11
fa ctoNJ., x-élxis = rela ti ve féltiO of Bax mRNA nonna lized lo 185
rR NA, ex prcssed in decimél l fo n néll; Y·élxis = lime of il1 ulll infl tio n).
60min
(] Tinted IOl
Bax leads to apopto tic cell death in many cellula r systems. 46,47,49-S·¡,S3-S5 In our testing, the Iight-induced
overexpression of a poptotic Bax was significantly
reduced by both IOLs. Howeve r once aga in, the
light-induced overexpression o f proapoptotic Bax
was significantly Illore reduced by the tinted than by
the untinted IOL.
The main ca use fOl" rapid loss of central vision in
ARMD patients is choroidal neovasc ulari zation . .vithin
the macular region. Vasc ular endothelial grow th factor(j. is known to bea maja r sti.mulus for neovascu.lariz ation
in exuda ti ve ARM0 2 ,34 Cumula ti ve light ex posure is
27 kD -
o
45min
Figure S. Q uantita ti ve RT-PCR a nfl l)'sis of &1-2 mR NA ex p ression
of prinmr), RPE cells a her iIlum ina lion w ilh plélin ",h ile 1ig h! (Co)
in Ihe presencc o f él n unlinled !Ol a nd él linlOO IOL (Ca = contro l;
x-a xis = relat ive ra lio of Bax mRNA norméllizcd lo 18s rRNA, expressed in d ec ima l for mal ; y-a xis = time of iIIu mina lion).
3
2
30min
• Untinted 10l
1
1
1.2
0.3
No IOl
-
BAX
lA
Figure 7. Representa ti ve Western bl ots showing Ihe expression of
13c1-2 <l nd l3<l x in no n-irradiated RPE cells (Co) a nd arte!" 60·m ill llte
exposlll"c to white light, unfiltered (no 10 1..) in lhe presence of a tin ted
[O L and an untinted !OL. Ten microgra ms of prole in were loadOO
per !¡me (IO L = intraoc ula r lens).
J CATARACT REFRACT SURG . VOL 35, FEBRUARY 2009
LABORATORY SCIENCE: CYTOPROTECTIVE EFI'ECTS OF BLUE LIGHT- FILTER ING IOL
thought to induce proangiogenic factors, such as VEGF(j., in RPE cells and promotes the development of exudati ve ARMD 2 ,56 Studies ha ve shown that unfiltered light
expos ure ca n lead to an increased production of VEGFa. in RPE cells. 3O The reason for this could be tha t continuous light exposure induces sublethal cellula r stress,
which results in an up-regulation of proangiogenetic
VEGF-(j, secretion 30 In agreemen t with these find ings,
our res ults show that lUlfiltered light exposure directl y
stimulates RP E cells to a time-of-irrad iation dependent
overexpression ofVEGF-a. mRNA.111is might bea possible explanation of why cataract surgery is implicated in
the increase in the incidence of exudative ARMD. 57•58
In our study, this inc rease in VEGF-(j, !URNA expression
was significantl y reduced in the presence of either IOL;
however, the effect was significantly sh'onger in the
presence of the tinted IOL.
In sllmma ry, our in vitro findin gs s upport the theory
tha t UV- a nd blue light- filtering rOLs might be usefu l
in patients wit h increased ris k for p rogression of
ARMD. Ultraviolet- and blue light-filtering IOLs probably have properties tha t can help to prevent apoptotic
cell d ea th in the human RPE du e to extensive light exposure. Therefore, they might be able to redu ce the ris k
for develop ment and progression of atrophic ARMD.
Further c1 inica ] investi ga tions w i]] have to slIbstantiate
these findin gs in vivo. Nevertheless¡ res ults indicate
that UV- and blue light- filterin g rOLs wo ul d be beneficial to patients w ith a high risk for ARMD and to those
tha t have ARMD that may progress.
REFERENCES
1. Ferris FL 111, Fine SL, Hyman lo Age-related macular degeneration and blindness due lo neovascular maculopalhy. Arch OphIhalmol1 984; 102: 1640-1642
2. Witmer AN , Vrensen GFJM, Van Noorden CJf, Schlingemann Aa.
Vascu lar endothelial growth laclors and angiogenesis in eye
disease. Prog Aetin Eye Aes 2003; 22: 1-29
3. Th e Eye Oiseases Prevalence Aesearch Group. Causes and
prevalence 01 visual impairment among adults in th e United
Slales. Arch Ophlhalmol2004; 122:477- 485
4. Klein A, Klein BEK, Unton Kl. Prevalence 01age- related maculopalhy; Ihe Beaver Oam Eye Sludy. Ophlh almology 1992;
99:933-943
5. The Eye Oiseases Prevalence Aesearch Group. Preva lence 01
age-related macular degeneration in the United Sta tes. Arch
Ophlhalmol 2004; 122:564- 572
6. Knudtson MO, Klein A, Kleln BEK, Lee KE , Meuer SM,
Tomany SC. Location 01lesions associaled wilh age-related maculopalhy over a 1O-year perlod: the Beaver Oam Eye Sludy. lnvesl
Ophthalmol Vis Sci 2004; 45:2135-2 142. Available al: hllp:/1
www.iovs.orgfcgi/reprinV4517/2 135. Accessed November 4, 2008
7. Oarzins P, Mitchell P, Heller AF. Sun exposure and age- related
macular degene ration; an Australian case-control study. OphIhalmology 1997; 104:770- 776
8. Cruicks hanks KJ, Klein R, Klein BEK. Sunlight and age- related
macul ar degeneration; the Beaver Oam Eye Study. Arch OphIhalmol1 993; 111:514- 518
361
9. Cru ickshanks KJ , Klein A, Klein BEK, Nondahl OM. Sunlight and
the 5-year incidence of early age-relaled maculopalhy; the Beaver Oam Eye Sludy. Arch Ophlhalmol 2001 ; 11 9:246-250
10. Tomany SC, Cruickshanks KJ , Klein A, Klein BEK,
Knudtson MO. Sunlight and the 10-yea r incidence o, age-related
maculopalhy; the Beaver Dam Eye Sludy. Arch Ophlhalmol
2004; 122:750-757
11 . Norren OV, Vos JJ. Spectral transmission of the human ocular
media. Visíon Res 1974; 14:1 237-1244
12. van den Berg T JTP, Spekreijse H. Near infrared light absorption
in the human eye medi a. Vision Aes 1997; 37:249- 253
13. Bron AJ , Vrense n GFJM , Koretz J, Maraini G, Harding JJ. The
ageing lens. Ophthalmologica 2000; 214:86-104
14. Mellerio J. Yellowing 01 the hum an lens: nuclear and cortical contri butions. Vision Res 1987; 27: 1581- 1587
15. Wea le AA. Age and the transmittance 01 the human crystalline
lens. J Physiol1988; 95:577- 587
16. Ham WT Jr, Mueller HA, Ruffolo JJ Jr, Millen JE, Cleary SF,
Guerry AK, Guerry O 111 .. Basic mechanisms underlying Ihe production 01photochemical lesions in the mammalia n relina. Curr
Eye Res 1984; 3: 165- 174
17. Ham WT Jr, Ruffolo JJ Jr, Mueller HA, Clarke AM , Moon ME.
Hislologic analysis o, photochemicallesions produced in rhesus
retina by short-wavelenglh lighl. Inves! Ophthalmol Vis Sci
1978; 17:1029-1035. Ava ilable al: hllp://www. iovs.org/egi/
reprinV17/ 10/ 1029. Accessed November 4, 2008
18. Main sler MA, Sparrow JR . How much blue light shou ld an 10 l
transmit? [perspective] Br J Ophthalmol 2003; 87: 1523-1529
19. l awrence HM, Reynolds TA . Erythropsial phototoxicity associated with nonultraviolet-liltering intraocula r lenses. J Cata ract
Refrael Surg 1989; 15:569- 572
20. Komatsu M, Kanagami S, Shim izu K. Ultraviolet-absorbing intraocular lens versus non-UV-absorbing intraocu lar lens: comparison of angiographic cysloid macular edema . J Cataract Relract
Surg 1989; 15:654- 657
21. Braunstein AE, Sparrow JA. A blue-blocking intraocular lens
should be used in cataract surgery [controversies]. Arch OphIhalmol 2005; 123:547-549
22. Ernesl PH. Light-transmission-speclrum comparison of foldable
intraocular lenses. J Cataract Aefrac! Surg 2004 ; 30: 1755-1758
23. Brockmann C, Sehulz M, Laube T. T ransmillance cha racteristics of ultraviolet and blue-light-l iltering intraocular lenses. J Cataract Refraet Surg 2008; 34: 11 61- 11 66
24. Algvere PV, Se regard S. Age-related maculopathy: palhogenetic fealures and new Irealment modalilies. Acta Ophlhalmol
Seand 2002; 80: 136- 143
25. Aoth F, Bindewald A, Holz FG. Key pathophysiologic palhways
in age- related macular disease. Graeles Arch Cli n Exp Ophthalmol 2004; 242:7 10-716
26. Wing Gl , Blanchard GC, Wei ler JJ. Th e topography and age relationship of lipofusein concentralion in Ihe retinal pigment epithelium. Invest Ophlhalm ol Vis Sci 1978; 17:60 1- 607.
Available al: hllp://www. iovs.o rglcgi/reprinV17/7/60 1. Accessed
November 4, 2008
27. Weiter JJ, Oelori FC, Wing GL, Fitch KA . Aelinal pigmenl epilheliallipofuscin and melanin and choroidal melanin in human eyes.
Invest Oph!halmol Vis Sci 1986; 27: 145- 152. Available al: hllp://
www.iovs.orglcgilreprinV27/2/145. Accessed November 4,
2008
28. Holz FG, Bellmann C, Margaritidis M, Schüll F, 0110 TP,
Vblcker HE. Patterns 01 increased in vivo lundus aulolluorescence in the junctional zone 01 geog raphic atrophy of Ihe retinal pigmen! epith elium associated with age-related macular
degeneration. Graefes Arch Clin Exp Ophthalmol 1999;
237: 145- 152
J CATARACT REfRACT SURG . VOL 35, fEBRUARV 2009
362
LABORATORY SC1ENCE: CYTO PROTECTIVI1: EF FECT S OF BLUE LlGHT- FILTERING [QL
29. Spa rrow JR, Miller AS, Zhou J. Blue light -absorbing intraoc ular
lens and rel inal pigment epith elium protection in vitro. J Cataract
Refrael Surg 2004; 30:873-878
30. Yanagi Y, Inoue Y, Iriyam a A, Jang W-D. Eflecls 01yellow intraocular tenses on lighl-induced upregulal ion 01vascular endothelial growth factor. J Cataract Refrae! Surg 2006 ; 32:1540-1544
31. Barishak YR. In vitro behaviou r 01 the pigmented cells 01Ih e rel¡na and uvea 01 Ihe adult human eye . Acta O phthalmol (Copenh)
1960; 38:339-346
32. Flood MT, Gouras P, Kjeldbye H. Growth characteristics and ultra-
47.
48.
struclure 01 human retina! pigment epil helium in vitro. Inves! OphIhalmol
Vis
Sei
1980;
19: 130S-1320.
Available
al:
hllp:/!
www.iovs.orgfcgilreprinV19/1111309. Accessed November 4, 2008
33. Nilsson SEG, Sundelin SP. Wihlmark U, Brunk
ur.
49.
Aging 01 cul-
tured retinal pigmenl epilhelial cells: oxidative reaclions, lipofuscin
formation and blue lighl damage. Doc Ophlhalmol2003; 106: 13-16
34. Waisbourd M, Loewenstein A, Goldstein M , Leibovitch 1. Targeting
vascular endothelial growlh factor; a promising Slrategy for lreating
age-related macular degeneration. Drugs Aging 2007; 24:643-662
35. Kernt M, Neubauer AS , Ulbig MW, Kampik A, Welge-Lül3en U.ln
vitro safety 01inlravitreal m oxifloxacin lor endophlhalmitis Irealmen!. J Catarae! Relrae! Surg 2008; 34:48G-488
36. Leschey KH , Hackett SF, Singer JH, Campochiaro PA. G rowth
factor responsiveness 01human retinal pigment epithelial cells. lnvest OphthalmoJ Vis Sci 1990; 3 1:839-846. Available at: http://
www.iavs.arglcgilreprinV3 1/5/839. Accessed November 4,2008
37. Masmann T . Rapid colorimetric assay for cellular growth and
sU/viva1: app1ication to proliferation and cytotoxicily assays . J
Im munol Me1hods 1983; 65:55-63
38. Welge- Lür3en U, May CA, Eichhorn M , Bloemendal H , Lü ljenDrecoll E. aB-cryslallin in Ihe trabecular m eshwark is inducible
by transforming growth fac tor-p. Invest Ophthalmol Vis Sci
1999; 40:2235--2241. Available al: hllp://www.iavs.orglcgi/
reprinV40/ 10/2235. Accessed November 4, 2008
39. Hawkins BS, Bird A, Klein R, West SK. Epidemialagy of age- re lated macular degeneration. Mol Vis 1999; 5:26. Available al:
hllp://www.malvis.orglmo lvis/v5/a26/v5a26·hawkins.pdf.Accessed Navember 4 2008
40 . Klein A, Klein BE K, Jense n SC, Meuer SM. The five-year incidence and progression of age-related maculopalhy; Ihe Beaver
Oam Eye S1udy. Ophlhalmology 1997; 104:7-2 1
4 1. Algvere PV, Marshall J, Seregard S. Age-re1ated macu lopalhy
and Ihe impact 01 blu e light ha zard. Acta Ophlhalmol Scand
2006; 84:4- 15
42. Katz ML. Potential role 01 retinal pigm ent epilheliaJ lipoluscin accumulation in age-related macular degeneration. Arch Gerantol
Gerialr 2002; 34:359-370
43 . Levin e AJ. p53, the cellular gatekeeper lor growth and division.
Cell 1997; 88:323-33 1. Avai1able al: hllp://www.elsevier.com/
Iramework_productslChemistry/p53Ihecellular.pcll. Accessed
Novembe r 4 ,2008
44. McKinnon SJ, Leh man DM, Kerrigan-Baumrind LA , Merges CA,
Pease ME, Kerrigan DF, Aansom NL, Tahzib NG ,
Aei lsamer HA, Levkovitch-Verbin H, Quigley HA, Zack DJ . Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension. Invest Ophtha lmol Vis Sci 2002; 43: 10771087. Available al: hllp://www.iovs.o rg/cgi/reprinV43/4/1077.
Accessed November 4, 2008
45. Rezai KA , Gasyna E, Seagle B-LL, Norris JR J r, Rezaei KA.
AcrySof Natural lilter decreases blue light-induced apoptosis
in human retinal pigmen! epithelium G raefes Arch Clin Exp.
Ophlhalmol 2008; 246:671-676
46. Kluck AM , Esposli MO, Pe rkinsG, Renken C, Kuwana T , BossyWelzel E, Goldberg MG, Allen T , Barber MJ , G reen DA ,
Newmeyer DO . Th e pro-apoptatic proteins, Bid and Bax, cause
50.
5 1.
52.
53.
54.
55.
56.
57 .
58 .
a limited permeabilizalion 01 th e milochondrial outer membrane
Ihat is enhanced by cytosol. J Cell Biol 1999; 147 :809- 822.
Available
at:
http://jcb. rupress.org/cgi/reprinV147/4/809.
Accessed November 4, 2008
EspoSli MO, Hatzinisiriou 1, McLennan H, Ralph S. Bcl-2 and mitochondrial axygen radica ls; new approaches with reactive oxygen species-sensitive probes. J Biol C hem 1999; 274:29831 29837. Available al: hllp:/lwww.jbc.arg/cgi/reprinV274/42J
29831. Accessed November 4, 2008
Godley BF, Jin GF, Guo VS, Hursl JS. Bcl-2 overexpression increases survival in human retinal pigment epi thelial cells exposed lo H, O,. Exp Eye Res 2002; 74:663-669
Chen J, Flannery JG, laVail M M , Sleinberg RH, Xu J , Simon MI.
BcI-2 overexpression reduces apoptotic photoreceptor cel! death
in three differenl retinal degenerations. Proc Natl Acad Sd USA
1996; 93:7042-7047. Available a1: hllp:l/www.pnas.org/eonlenV
93/ 14n042.full .pdf + hlml. Accessed November 4,2008
Oeng G, Su JH, Ivins KJ , Van Houten B, Col man CW. Bcl-2 facilitates recovery from ONA damage after oxidative stress. Exp
Neuro11 999; 159:309- 3 18
Ryan JJ , Prochownik E, Gottlieb CA, ApeJ IJ, Merino A,
Nuñez G , Clarke MF. C-myc and bcl-2 modulate p53 lunction
by altering p53 subcellular traflicking during Ihe cel! cycle.
Proe Natl Aead Sei USA 1994; 9 1:5878- 5882. Available a1:
hllp://www. pnas.org/eonlenV9 1/ 13/5878.full.pdf + hlmI.
Aecessed Novembe r 4, 2008
Sparrow JR, Cai B. Blue light -induced apoptosis 01 A2E -cantaining RPE: involvement of caspase-3 and protection by Bcl-2. In vest Ophthalmol Vis Sci 2001; 42: 1356- 1362. Avai lable at
http://www .iovs. org/cg i/con te nVabstracV42/6/ 1356. Accessed
November 4, 2008
Chang M -V, Sun W , Ochiai W, Nakashima K, Kim S-V, Park CH , Kang JS, Shim J-W, Jo A-Y, Kang C-S, Lee Y-S, Kim J-S,
l ee S- H. Bcl-XL/Bax proteins direct the late 01 embryonic co rtical
precursor cells. Mol Cell 8io12007 ; 27:4293-4305. Available al:
htlp:/lmcb.asm.org/cgi/reprinV27/12J4293. Accessed November4,2008
Liang V, J0rgensen A, Krestel CG, Wi encke AK, lui GM, la
Cour MH , R6pke CH , Nissen MH .. Bct-2 , Bax, and c-Fos expression correlates lo RPE cell apoptosis induced by UV-light
and daunorubicin. Curr Eye Res 2000; 20:25- 34
Lindsten T, Zong W -X, Thompson CB . Delining the role 01 Ihe
BcI-2 lami ly 01 proteins in the nervous system. Neuroscientist
2005; 11: 10-15
Zhou J, Cai B, Jang VP , Pachydaki S, Schmidt AM, Sparrow JR.
Mechanisms lor the induction 01 HNE- MOA- and AGE-adducts,
AAGE and VEGF in retinal pigmen t epi thelial ce lls. Exp Eye Res
2005; 80:567-580
Klein R, Klein BEK, Wang O, Moss SE. ls age-related maculopathy
associaled with cataracts? Arch Ophthalmol1 994; 11 2: 19 1- 196
WangJJ, Klein R, Smi1h W, Klein BEK , TomanyS, Milehell P. Calaract surgery and the 5-year incidence 01 Jate-stage age-related
macuropathy; pooled findings from the Beaver Dam and Blue
Mounlains Eye Sludies. Ophlhalmology 2003; 110: 1960- 1967
J CATARACT REFRACT SURG - VOL 35, FE8RUARY 2009
First autho r:
MarclIs Kernt MD
Depnrl ll/ellt olOpIJ lllnllllology,
Ludwig Mnxillliliall Ulliversity,
MI/llich, Gerll/aJly