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Further observations on the characteristics of primary cultures of human lens epithelium
Thursday, 5:30-7:00 P.M., Sep 24, 1992 Palazzo Dei Congressi
X ICER Abstracts 649
19 INDUCTION PYRUVATE Dept.
OF CATARACTS
M.. arma. of Ophtbknology,
Univ.
BY SELENITE:
of Maryland,
PREVENTION
Baltimore,
Maryland
BY
USA
Previous studies from this laboratory have demonstrated that keto acids such as pyruvic acid can prevent the lens against oxidative stress in Y&Q. In addition, pynwate has been shown to prevent competitively the aldose reductase catalyzed synthesis of sugar alcohols and consequent prevention of such cataracts. The present investigations were directed towards finding the effect of pyruvate against selenite induced cataracts in rat pups. That oxidative stress is at least partly involved in this form of cataracts was evident from the observed preventive effect of ascorbate. Cataracts were induced in rat pups by a single lP dose of sodium selenite, 0.5 micromoles/pup. Nuclear cataracts developed within a week. Such cataract development was prevented if the animals received pyruvate (0.5 mmoles/day) starting three days prior to selenite administration and continued thereafter. The cataract development was delayed at least by two weeks, when the experiments were terminated. The cataract was monitored by Scheimpflug photography. Biochemically, the levels of ATP (1985 nmole g-’ lens) and GSK (4.9 pmole g-’ lens) were higher in the pyruvate plus selenite group as compared to the group given selenite alone (ATP = 966 nmole g.’ lens, GSH = 2.7 pmole g.’ lens). Pyruvate also stimulated the activity of the HMP shunt in a. Further studies on the mechanisms involved in the prevention of sclenite cataracts by pyruvate are in progress.
Human lens epithelial cells obtained from senile cataractous patients during cataract surgery were cultured with lens capsules. The outgrowth of cultured cells was observed by phase contrast microscopy. Mitotis in outgrowing cells was investigated via anti-bromodeoxyuridine(BrdU) immunohistochemistry. BrdU, an analogue of thymidiic, can be incorporated specifically into synthesizing DNA (i.e. in the S-phase of cell cycle). Lens epithelial cells began to outgrow on the culture plate from capsular edge at third day after incubation. They actively outgrew for hvo weeks, but stopped after four weeks. The cells near the capsular edge showed elongated shape in an early culture period, but formed small polygonal shape afterwards. At the middle outgrowing zone the cells showed polygonal shape, and they were surrounded by large and irregularly shaped cells. BrdU-positive cells could be seen in outgrowing cells between third day and second week, and disappeared within four weeks. These results showed that human lens epithclial cells from senile cataractous patients outgrew definitively, but they had low proliferative potency.
650
20 CALP?OXILrN A,\J'lACONISTS AND 'KANSPARENCY
INDUCE
CHANGES
IN LENS CATIOtj
652
22 TISSUE CULTURE OF HUMAN LENS EPITHELIAL CELLS FROM SENILE CATARACTOUS PATIENTS Nobuhiro lbaraki M.D., Kunitoshi Ohara M.D. and Hiroyuki Shbnizu M.D. Department of Ophthalmology, Jichi Medical School, Tochigi, Japan
FERME1BII.ITY
653
23 FURTHER OBSERVATIONS ON THE CHARACTERISTICS OF PRIMARY CULTURES OF HUMAN LENS EPITHELIUM. V.N. Rcddy, T. Arita, T. Tsuji, Y. Murata and L-R. Lin. Eye Research Institute, Oakland University, Rochester, MI 48309-4401, U.S.A.
Our recent studies showed that human lens epithetial (HLE) cells in express alpha and beta crystallins and can be made to undergo differentiation in a predictable manner. The differentiated cells (lentoids) not only express gamma crystallin and MP2 characteristic of terminal differentiation, but continue to express a Ppha and beta crysrallins analogous to lens fibers tn situ (Reddy. et al, Exp. Eye Res. 53~367, 1991). HLE cells in tissue culture were also found to synthesize basement membrane as a continuous sheet. Transmission electron microscopy (TEM) showed that in monolayer cultures of 450 days, the thickness of the basement membrane was 2 to 2.5 microns which is
culture
similar to the thickness of capsule seen in human lens in the first decade of life. The identity of the membrane sheet, formed between the monolayer and the culture dish, with lens capsule was established by immunohistochemistry. These studies demonstrated the presence of both laminin and Type IV collagen, the characteristic basement membrane proteins. Immunohistochemical localization of Na+-K+ATPase with TEM has shown a change in the polarized distribution of the enzyme. In situ. contrary to earlier reports, Na+-K+ATPasc is detected only in the basal plasma membrane whereas the enzyme is localized in the apical (facing the media) plasma membrane in cultured cells. Explants of capsule-epithelium showed translocation of the enzyme from basal to apical side upon culture for only three hours. In cells grown on permeable biopore membrane the enzyme was observable on both plasma membrane surfaces in contact with the medium suggesting that the distribution of the transport enzyme in cultured cells is dictated by the need to extrude Na ions across the cell membrane facing the media.
651
21
EPITHELIAL (HLE) CELLS IN TISSUE CULTURE: MODIFICATION EVALUATED SY FLUSS CYTOFLUORIMETRIC STUDIES RASI,V..SINIBALDI-VALLESONA P RASI G..EALACCOr p------m-__IGABRIELI,C. Institute of Ophthalmology, Chear of Physiopathologic
TIiZ EFFECT OF ZGF GIN THE GROWTH 3?OlWti’IAL CELLS FROM HUMAN l.ZNS Riyoyuki Majima,~sashi Kohnakq Department of Ophthalmology, Sch@ol of Health University. Toyoake,Japan
optics, Policlinico.1
The
HUMAN
LENS
DIFFERENCES
Cataract
AND
University 00161
“La Rome,
Sapienza” Italy.
Rome,
Viale
de1
is the most common disease of lens and one of, the most frequent cause of blindness. Mechanisms of lens opacification is still large-ly unknown. Even if several etiologies (diabetes, UV/X-irradiation, steroidas )I under1 ie cataract geneses, common mechanisms should be hypotized. The aim of this work is to studymechanisms of lens opacification employing HLE cells culture. A two phases study was started. first phase consisted in HLE primary cultures setting up; lenses were obtained cataract patients from stratified for age, type of cataract systemic disease; lenses from healthy and donors served as controls. HLE were plated in 25 cm2 cultures flasks with DMEM supplemented with 10% FCS incubated in 95% humidity / 5% CO2 at 37-C; growth rate and proliferative capacity were determinated by phasecontrast inverted microscope. Second phase consisted in Cytofluorlmetric studies (“Becton Dickinson FACScan-plus analyzer” was employed). Cells size, shape, granularity and density variations were measured twice weekly. Wi tht the same technique and monoclonal antibodies DNA cell cycle and surface antigenes will be studied.
growth
promoting
effect
(EGF) was studied on cultures human normal and &taractous tial of lens epitbelial cells assav. f The KTT assav wan a tontlal of cells on the culture tity of MTT fonnazan formed The xmcentratios mitochondria. urn (Eagle's MEH supplenentcd 1% MEN) were classified into With the result of analysis centration of EGF rancied from i.qduceC z-1 increase oi growth co:lr. conparing with an EGF
S.192
of
epidernal
OF EPIZHXLIAL Nedicine. grow::l
Fuji% factor
of epithelial cells fmm Lenses. The growth potenwas measured h7 "rhe HTY nethod vnrifvi:w qxovtll pnby nr?osu;ln; ;_he gna&hv ex.yn&tio reaction in of WGF i? culture nediwit:-! 1% fetal bovine .r,ermm: 7 gro*qw (3-10' m~/nl). of UTT ,ssay, when the con1 na/ml to lO~na/ml. EGF pot&tial of epithelial free group.
X ICER Abstracts 649
19 INDUCTION PYRUVATE Dept.
OF CATARACTS
M.. arma. of Ophtbknology,
Univ.
BY SELENITE:
of Maryland,
PREVENTION
Baltimore,
Maryland
BY
USA
Previous studies from this laboratory have demonstrated that keto acids such as pyruvic acid can prevent the lens against oxidative stress in Y&Q. In addition, pynwate has been shown to prevent competitively the aldose reductase catalyzed synthesis of sugar alcohols and consequent prevention of such cataracts. The present investigations were directed towards finding the effect of pyruvate against selenite induced cataracts in rat pups. That oxidative stress is at least partly involved in this form of cataracts was evident from the observed preventive effect of ascorbate. Cataracts were induced in rat pups by a single lP dose of sodium selenite, 0.5 micromoles/pup. Nuclear cataracts developed within a week. Such cataract development was prevented if the animals received pyruvate (0.5 mmoles/day) starting three days prior to selenite administration and continued thereafter. The cataract development was delayed at least by two weeks, when the experiments were terminated. The cataract was monitored by Scheimpflug photography. Biochemically, the levels of ATP (1985 nmole g-’ lens) and GSK (4.9 pmole g-’ lens) were higher in the pyruvate plus selenite group as compared to the group given selenite alone (ATP = 966 nmole g.’ lens, GSH = 2.7 pmole g.’ lens). Pyruvate also stimulated the activity of the HMP shunt in a. Further studies on the mechanisms involved in the prevention of sclenite cataracts by pyruvate are in progress.
Human lens epithelial cells obtained from senile cataractous patients during cataract surgery were cultured with lens capsules. The outgrowth of cultured cells was observed by phase contrast microscopy. Mitotis in outgrowing cells was investigated via anti-bromodeoxyuridine(BrdU) immunohistochemistry. BrdU, an analogue of thymidiic, can be incorporated specifically into synthesizing DNA (i.e. in the S-phase of cell cycle). Lens epithelial cells began to outgrow on the culture plate from capsular edge at third day after incubation. They actively outgrew for hvo weeks, but stopped after four weeks. The cells near the capsular edge showed elongated shape in an early culture period, but formed small polygonal shape afterwards. At the middle outgrowing zone the cells showed polygonal shape, and they were surrounded by large and irregularly shaped cells. BrdU-positive cells could be seen in outgrowing cells between third day and second week, and disappeared within four weeks. These results showed that human lens epithclial cells from senile cataractous patients outgrew definitively, but they had low proliferative potency.
650
20 CALP?OXILrN A,\J'lACONISTS AND 'KANSPARENCY
INDUCE
CHANGES
IN LENS CATIOtj
652
22 TISSUE CULTURE OF HUMAN LENS EPITHELIAL CELLS FROM SENILE CATARACTOUS PATIENTS Nobuhiro lbaraki M.D., Kunitoshi Ohara M.D. and Hiroyuki Shbnizu M.D. Department of Ophthalmology, Jichi Medical School, Tochigi, Japan
FERME1BII.ITY
653
23 FURTHER OBSERVATIONS ON THE CHARACTERISTICS OF PRIMARY CULTURES OF HUMAN LENS EPITHELIUM. V.N. Rcddy, T. Arita, T. Tsuji, Y. Murata and L-R. Lin. Eye Research Institute, Oakland University, Rochester, MI 48309-4401, U.S.A.
Our recent studies showed that human lens epithetial (HLE) cells in express alpha and beta crystallins and can be made to undergo differentiation in a predictable manner. The differentiated cells (lentoids) not only express gamma crystallin and MP2 characteristic of terminal differentiation, but continue to express a Ppha and beta crysrallins analogous to lens fibers tn situ (Reddy. et al, Exp. Eye Res. 53~367, 1991). HLE cells in tissue culture were also found to synthesize basement membrane as a continuous sheet. Transmission electron microscopy (TEM) showed that in monolayer cultures of 450 days, the thickness of the basement membrane was 2 to 2.5 microns which is
culture
similar to the thickness of capsule seen in human lens in the first decade of life. The identity of the membrane sheet, formed between the monolayer and the culture dish, with lens capsule was established by immunohistochemistry. These studies demonstrated the presence of both laminin and Type IV collagen, the characteristic basement membrane proteins. Immunohistochemical localization of Na+-K+ATPase with TEM has shown a change in the polarized distribution of the enzyme. In situ. contrary to earlier reports, Na+-K+ATPasc is detected only in the basal plasma membrane whereas the enzyme is localized in the apical (facing the media) plasma membrane in cultured cells. Explants of capsule-epithelium showed translocation of the enzyme from basal to apical side upon culture for only three hours. In cells grown on permeable biopore membrane the enzyme was observable on both plasma membrane surfaces in contact with the medium suggesting that the distribution of the transport enzyme in cultured cells is dictated by the need to extrude Na ions across the cell membrane facing the media.
651
21
EPITHELIAL (HLE) CELLS IN TISSUE CULTURE: MODIFICATION EVALUATED SY FLUSS CYTOFLUORIMETRIC STUDIES RASI,V..SINIBALDI-VALLESONA P RASI G..EALACCOr p------m-__IGABRIELI,C. Institute of Ophthalmology, Chear of Physiopathologic
TIiZ EFFECT OF ZGF GIN THE GROWTH 3?OlWti’IAL CELLS FROM HUMAN l.ZNS Riyoyuki Majima,~sashi Kohnakq Department of Ophthalmology, Sch@ol of Health University. Toyoake,Japan
optics, Policlinico.1
The
HUMAN
LENS
DIFFERENCES
Cataract
AND
University 00161
“La Rome,
Sapienza” Italy.
Rome,
Viale
de1
is the most common disease of lens and one of, the most frequent cause of blindness. Mechanisms of lens opacification is still large-ly unknown. Even if several etiologies (diabetes, UV/X-irradiation, steroidas )I under1 ie cataract geneses, common mechanisms should be hypotized. The aim of this work is to studymechanisms of lens opacification employing HLE cells culture. A two phases study was started. first phase consisted in HLE primary cultures setting up; lenses were obtained cataract patients from stratified for age, type of cataract systemic disease; lenses from healthy and donors served as controls. HLE were plated in 25 cm2 cultures flasks with DMEM supplemented with 10% FCS incubated in 95% humidity / 5% CO2 at 37-C; growth rate and proliferative capacity were determinated by phasecontrast inverted microscope. Second phase consisted in Cytofluorlmetric studies (“Becton Dickinson FACScan-plus analyzer” was employed). Cells size, shape, granularity and density variations were measured twice weekly. Wi tht the same technique and monoclonal antibodies DNA cell cycle and surface antigenes will be studied.
growth
promoting
effect
(EGF) was studied on cultures human normal and &taractous tial of lens epitbelial cells assav. f The KTT assav wan a tontlal of cells on the culture tity of MTT fonnazan formed The xmcentratios mitochondria. urn (Eagle's MEH supplenentcd 1% MEN) were classified into With the result of analysis centration of EGF rancied from i.qduceC z-1 increase oi growth co:lr. conparing with an EGF
S.192
of
epidernal
OF EPIZHXLIAL Nedicine. grow::l
Fuji% factor
of epithelial cells fmm Lenses. The growth potenwas measured h7 "rhe HTY nethod vnrifvi:w qxovtll pnby nr?osu;ln; ;_he gna&hv ex.yn&tio reaction in of WGF i? culture nediwit:-! 1% fetal bovine .r,ermm: 7 gro*qw (3-10' m~/nl). of UTT ,ssay, when the con1 na/ml to lO~na/ml. EGF pot&tial of epithelial free group.