Current Eye Research
Volume 1 1 number 9 1992, 849-862 ~
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Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas
M.Elizabeth Fini, Beatrice Y.J.T.Yuel and Joel Sugar' MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital and Department of Dermatology, Harvard Medical School, Boston, MA and 'Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, IL, USA
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ABSTRACT
components.
C e l l s of keratoconus c o r n e a s have been r e p o r t e d t o produce h i g h e r l e v e l s of collagenolytic/gelatinolytic enzymatic a c t i v i t i e s t h a n do c e l l s of normal corneas. The c u r r e n t s t u d y i n v e s t i g a t e s t h e c o n t r i b u t i o n of 1) s p e c i f i c enzyme gene p r o d u c t s , and 2 ) t h e degree t o which these p r o t e i n s a r e present i n t h e a c t i v a t e d forms, t o t h e i n c r e a s e d enzymatic a c t i v i t i e s . We demonstrate t h a t two n e u t r a l g e l a t i n o l y t i c enzymes, a 6 6 / 5 9 kD form and a 9 2 k D form, can be d i r e c t l y e x t r a c t e d from both normal and keratoconus corneas. These enzymes a r e i d e n t i f i e d a s t h e pro- and a c t i v a t e d forms of MMP-2 and a s t h e pro-form of MMP-9, s p e c i f i c members of t h e m a t r i x m e t a l l o p r o t e i n a s e f a m i l y . Normal and keratoconus c o r n e a s show no s i g n i f i c a n t d i f f e r e n c e s i n amounts o r t y p e s of e x t r a c t a b l e n e u t r a l g e l a t i n a s e s , nor i n t h e amounts o r t y p e s t h a t t h e y s y n t h e s i z e i n c u l t u r e . Furthermore, i n both t h e normal and keratoconus c o r n e a s , g e l a t i n a s e s a r e found p r i m a r i l y i n t h e i n a c t i v e form. These s t u d i e s s u q q e s t t h e p o s s i b l e importance of changes i n proteinase inhibitor levels t o the c h a r a c t e r i s t i c biochemical f e a t u r e s of keratoconus c o r n e a s .
R e s u l t s from p r e v i o u s
i n v e s t i g a t i o n s on keratoconus have indicated that the basic biologic defect may l i e i n t h e s y n t h e s i s ( 2 ) a n d / o r
d e g r a d a t i o n ( 3 - 8 ) of p r o t e i n and c o l l a g e n molecules.
Evidence f o r abnormal m a t r i x
d e g r a d a t i o n h a s been o b t a i n e d from enzymatic s t u d i e s . C u l t u r e d t i s s u e e x p l a n t s , o r primary c e l l c u l t u r e s d e r i v e d from keratoconus c o r n e a s , have been found
t o s e c r e t e i n c r e a s e d l e v e l s of n e u t r a l enzymatic a c t i v i t i e s a g a i n s t n a t i v e c o l l a g e n t y p e s 1 and I V ,
and a g a i n s t
g e l a t i n s (denatured c o l l a g e n s ) , i n t o t h e i r c u l t u r e medium ( 3 - 7 ) .
Also,
lysosomal
a c i d hydrolase l e v e l s a r e e l e v a t e d i n keratoconus c o r n e a s ( 8 ) . Net enzymatic a c t i v i t y i s a combination of s e v e r a l f a c t o r s i n c l u d i n g t h e enzyme t y p e s p r e s e n t , t h e degree t o which i n a c t i v e proenzymes have been converted t o t h e i r a c t i v a t e d forms, and t h e p r e s e n c e of
INTRODUCTION
specific inhibitors.
Keratoconus i s a non-inflammatory
In t h i s s t u d y , we
c o r n e a l d i s o r d e r t h a t i s a s s o c i a t e d with
i n v e s t i g a t e t h e c o n t r i b u t i o n of t h e f i r s t
l o s s of t e n s i l e s t r e n g t h , t h i n n i n g , and
two f a c t o r s t o t h e i n c r e a s e d g e l a t i n o l y t i c
e c t a s i a o f t h e c e n t r a l cornea ( 1 ) . I t s
a c t i v i t y produced by keratoconus c o r n e a s .
e t i o l o g i c a l b a s i s i s not understood
W e demonstrate t h a t two n e u t r a l
The
d i s o r d e r can sometimes be a s s o c i a t e d w i t h
g e l a t i n o l y t i c enzymes, a 66/59 kD and a 92
i n h e r i t e d connective t i s s u e d i s o r d e r s s u c h
kD enzyme, can be d i r e c t l y e x t r a c t e d from
a s Ehlers-Danlos syndrome t y p e I V and V I ,
b o t h normal and keratoconus c o r n e a s .
Marfan syndrome, and o s t e o g e n e s i s
These enzymes a r e i d e n t i f i e d a s t h e pro-
imperfecta suggesting a p o s s i b l e
and a c t i v a t e d forms of MMP-2 and a s t h e
involvement i n t h e s t r u c t u r e or
pro-form of MMP-9,
maintenance of e x t r a c e l l u l a r m a t r i x
matrix metalloproteinase family.
s p e c i f i c members of t h e Normal
Received on April 30, 1992; accepted on August 21, 1992
0 Oxford University Press
849
Current Eye Research ~~
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and k e r a t o c o n u s c o r n e a s show no
c u l t u r e ( s e e b e l o w ) , t h e 8 mm c e n t r a l
~
significant. differences i n the t o t a l
c o r n e a l b u t t o n s were c u t i n t o f o u r e q u a l -
amount o r t-ypes of e x t r a c t a b l e n e u t r a l
sized pieces.
g e l a t i n a s e s , nor i n t h e amounts o r t y p e s
immediately i n t o c u l t u r e .
of g e i a t i n a s e s t h a t t h e y s y n t h e s i z e i n Furt.hermore, i n b o t h normal and
cult u r e .
Half of 7 . 5 -
The p i e c e s were t h e n p l a c e d t o 8 mm c o r n e a l b u t t o n s
w e r e obtained within 3 hours
Of
keratocoiius c o r n e a s , g e l a t i n a s e s a r e found
p e n e t r a t i n g k e r a t o p l a s t y from p a t i e n t s
p r i , m a r i l y i n t h e i n a c t i v e form.
with keratoconus.
The a g e s , d u r a t i o n of
t h e d i s e a s e , and o t h e r c l i n i c a l information f o r keratoconus p a t i e n t s a r e
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MATERIALS AND METHODS
shown i n Table 1 .
of c o r n e a l t . i s s u e S 2 2 normal haman c o r n e a s was
A t o t a l . of
o b t a i n e d from t h e I l l i n o i s Eye Bank (Chicago).
F i v e of t h e c o r n e a s were
I n a d d i t i o n , another
set of c o r n e a s was c o l l e c t e d at. p e n e t r a t i n g k e r a t o p l a s t y from s i x p a t i e n t s with o t h e r corneal d i s e a s e s .
T h e ages,
r e c e i v e d as a u t o p s y g l o b e s from donors
d i a g n o s i s , and o t h e r i n f o r m a t i o n on t h e s e
( i 3 - , 66-, 7 5 - - , 71-, and 8 0 - y e a r s - o l d )
p a t i e n t s a r e summarized i n Table 2 .
w i t h i n 2 4 h c u r s of d e a t h .
Seventeen
For s t u d i e s on t i s s u e 1 o c a l i z a t : i o n of
o t h e r s were r e c e i v e d a j c o r n e a l b u t t o n s
gelatinases,
t h a t had beer! e n u c l e a t e d from donors (mean
m e c h a n i c a l l y from t h e h a l f c o r n e a s .
a g e , 3 6 . 6 .i 2 1 . 7 y e a r s ,
range, 1 3 t o 7 6
t i s s u e l a y e r s were s e p a r a t e d Each
t i s s u e fragment was p l a c e d i n a s e p a r a t e I n a d d i t i o n , 2-mm but.t.ons
y e a r s ) aria soaked i n c o r n e a l s t o r a g e
tube a s above.
mediurr. ( 0 p t j . s o l o r Dexsol) f o r 1 4 to 1 1 5
were a l s o p r e p a r e d from some specimens f o r
hours.
d i r e c t e x t r a c t i o n zymography and
A l i cionors had no known s y s t e m i c
and 1/4
o r o c u i a r d i s e a s e s , a n t i t h e c o r n e a s were
c o r n e a l p i e c e s f o r organ c u l t u r e s t u d i e s
clear.
a s d e s c r i b e d above.
FOL s t o d i e s on t i s s u e l o c a l i z a t i o n of
g e l a t i n a s e s , an 8-mm t r e p h i n e was used t o punch oat
d
h u t t o n of c e n t r a l c o r n e a and
&traction
of u e l a t i n a s e s from t i s s u e
When a l l samples were c o l l e c t e c i f o r a
p a r t i c u l a r experiment, t h e t i s s u e : j were
t h e c e n t r a l butcon and p e r i p h e r a l r i n g
thawed a t room t e m p e r a t u r e .
were c u t ir: h a l f w i t h a s c a l p e l .
immediately e x t r a c t e d f o r a n a l y s i s b y
Epithel i a i ,
v o r t e x i n g t h e t i s s u e i n a s o l u t i c n of 2 . 0 %
:.issue
s t r o m a l , and e n d o t h e l i a l
l a y e r s were t h e n s e p a r a t e d
mechanical!y
SDS a s d e s c r i b e d
from c e n t r a l and p e r i p h e r a l
P r o t e i . n s were
( 9 ) . Each t i s s u e sample
was e x t r a c t e d w i t h an e q u a l volume of SDS
h a l v e s and each t i s s u e fragment was p l a c e d
(20 u l p e r t i s s u e s a m p l e ) . The ?C ul
in
volume of e x t r a c t from each t i s s u e sample
3
separate tube.
A l l samples were
t e l y f a s t . - f r o z e n by dropping i n
was immediately d i l u t e d 2 : 5 w i t h Laemmli
Liquid n i t r o g e n ; t - i s s u e s were s t o r e d a t
g e l sample b u f f e r and 2 5 ul (out of t h e
-?DOC f o r \ J F t o s e v e r a l months.
t o t a l of 5 0 u l ) of each sample was loaded
For
d i r e c t q u a n t i t a t i v e s t u d i e s ( s e e below), a 2-mm
t r e p h h e was used t o punch o u t two or
c h r e e c e n t r a l b u t t o n s of t i s s u e from t h e 8
i n a s i n g l e g e l lane (without heating o r r e d u c t i o n ) on s t a n d a r d zymograns
s e c t i o n ) f o r a n a l y s i s of g e l a t i n o l ~ y t i c
m bT2t.ton; t i s s u e l a y e r s were n o t
enzymes p r e s e n t .
separared.
Zvmocrrar>hv
Each b u t t o n was p l a c e d ,
...n t a c t ,
i n a s e p a r a t e t u b e and f r o z e n a s
The method of zymography
above.
F o r q u a n t i t a t i v e a n a l y s i s by organ
analyze t h e content of s p e c i f i c
8 50
( s e e next
(10) was used t o
Current Eye Research TABLE 1 Ages a n d O t h e r C l i n i c a l C h a r a c t e r i s t i c s of P a t i e n t s w i t h K e r a t o c o n u s
1 2 3 4 5 6
Race
Age a t S u
F
white black white white white black white white white white white white white black black white
27 40 59 42 43 37 31 37 48 25 34 50 31 48 30 72
F F F
M M M F M M M M
7
8 9 10 11 Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
Sex
12 13 15
M F F
16
M
14
r
g
D u r a t i o n of e r s
n
g
+ + + + + + + + + + +
10
)A, ;.a! ! r
.-
Virtually the
o n l y g e l a t i n a s e species p r e s e n t i n t h e
The t i . s s u e l o c a l i z a t i o n of
t h e 6 6 kD g e l a t i n a s e w a s s i m i l a r tci t h a t of t h e normal corneas with very l i t t l e i f a n y enzyme f o u n d i n t h e e p i t h e l i u m .
1 c o r n e a s o b t a i n e d af t e r Penet-rat.lny
keratoDlastv The s t u d i e s r e p o r t e d a b o v e d e m o n s t - r a t e d a
. i n d c h e 9 2 kD y e l a t i n a s e i?
I
Ariiist~riiin
( F i g . 2A, L a n e
c l e a r d i f f e r e n c e i n t h e types of
s p e c i f i c f o r human MMP-9
g e l a t i n a s e s p r e s e n t i n normal corneas
e c i p i t a t e d t h e 92 kD
o b t a i n e d f r o m t h e Eye Bank a n d t h e
. 2B) arid a n t i s e r u m r n r ~ ~ r o e n z y nform e o f human
i::sily
p r e c i p i t a t e d t h e 6 6 kD
keratoconus corneas obtained a f t e r penetrating keratoplasty.
I t seemed
p o s s i b l e t h a t t h i s d i f f e r e n c e c o u l d be an a r t i f a c t o f a l t e r n a t i v e h a n d l i n g ; t h e Eye Bank c o r n e a s u n d e r w e n t a someti.mes
~
e x t e n s i v e s t o r a g e p e r i o d i n l i q u i d (up t o 5 d a y s ) while t h e keratoconus c o r n e a s were
Table 3 : G e l a t i n a s e s P r e s e n t i n Other Diseased Corneas Obtained a t t h e Time of Penetrating Keratoplasty
p r o c e s s e d w i t h i n t h r e e hours a f t e r surgery.
elatmases
To t e s t t h i s h y p o t h e s i s , t h e
gelatinolytic species present i n other diseased corneas, obtained a f t e r
1
MMP-2 proenzyme
2
MMP-2 proenzyme
3
MMP-2 proenzyme
p e n e t r a t i n g k e r a t o p l a s t y , were examined by d i r e c t e x t r a c t i o n and zymography.
Since
4 0 kD g e l a t i n a s e
i n t h i s experiment, t h e t i s s u e l o c a l i z a t i o n of enzymes was not of
4
MMP-2 proenzyme MMP-2 a c t i v a t e d
5
MMP-2 proenzyme MMP-2 a c t i v a t e d
6
MMP-2 proenzyme MMP-2 a c t i v a t e d
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r e l e v a n c e , e x t r a c t i o n s were performed on complete c o r n e a l b u t t o n s .
The r e s u l t s a r e
summarized i n Table 3 . Unlike Eye Bank c o r n e a s , no MMP-9 was found i n any of t h e 6 corneas o b t a i n e d a f t e r penetrating keratoplasty.
However,
i n 3 of t h e c o r n e a s , l a r g e amounts of t h e a c t i v a t e d form of MMP-2 were p r e s e n t , r e p r e s e n t i n g g r e a t e r t h a n 1 0 % of t h e t o t a l
MMP-2 enzyme.
These 3 corneas a l s o had a
C l i n i c a l i n f o r m a t i o n on each p a t i e n t i s as numbered i n Table 2 . Enzyme was e x t r a c t e d from c o r n e a l t i s s u e samples w i t h SDS and t h e enzyme forms p r e s e n t were analyzed by zymography. The p r e s e n c e of MMP-2 a c t i v a t e d enzyme w a s not l i s t e d h e r e u n l e s s i t r e p r e s e n t e d g r e a t e r t h a n 1 0 % of t h e t o t a l MMP-2 i n t h e sample.
s u b s t a n t i a l degree of s c a r r i n g i n common. I n a d d i t i o n , t h e cornea t h a t had been
removed because of i n t e r s t i t i a l k e r a t i t i s
keratoconus corneas a r e t h i n n e r t h a n
c o n t a i n e d a 4 0 kD g e l a t i n a s e ; t h e i d e n t i t y
normal, it i s not c l e a r whether t h i s
of t h i s enzyme i s unknown.
r e p r e s e n t s a s e l e c t i v e l o s s of p a r t i c u l a r p r o t e i n s , o r a generalized p r o t e i n l o s s .
normal and keratoconus c o r n e a s
I f t h e former c a s e i s c o r r e c t , t h e n
The l e v e l s of MMP-2 proenzyme which was
important d i f f e r e n c e s between normal and
e x t r a c t a b l e from normal and keratoconus
keratoconus c o r n e a s might be missed by
c o r n e a s were q u a n t i f i e d from zymograms
comparing enzyme l e v e l s t o t o t a l p r o t e i n
s i m i l a r t o t h o s e shown i n f i g u r e 3 .
alone.
E x t r a c t i o n s were performed on p i e c e s of
normal c o r n e a s were analyzed and t h e
c o r n e a l b u t t o n s with equal s u r f a c e a r e a ,
r e s u l t s a r e r e p o r t e d i n Table 4 .
s o t h a t v a l u e s from a l l samples could be compared based on c o r n e a l a r e a .
Half of
Four keratoconus corneas and I
A s was observed i n our p r e v i o u s
experiment, t h e o n l y g e l a t i n a s e t y p e
t h e e x t r a c t from each c o r n e a l p i e c e was
p r e s e n t i n keratoconus e x t r a c t s was MMP-2,
analyzed by zymography, and h a l f was a l s o
almost a l l of which was i n t h e pro-form.
used f o r t o t a l p r o t e i n d e t e r m i n a t i o n s o
The same r e s u l t was o b t a i n e d w i t h 6 of t h e
t h a t t h e amount of g e l a t i n a s e i n samples
7 normal c o r n e a s ; i n t h e s e v e n t h , a s m a l l
could be compared w i t h r e s p e c t t o t o t a l
amount of 92 kD g e l a t i n a s e could be
protein as well.
observed.
We b e l i e v e t h a t both
Q u a n t i t a t i o n of MMP-2 proenzyme
comparisions a r e important t o c o n s i d e r .
w i t h r e s p e c t t o s t a n d a r d s demonstrated
O u r " t o t a l p r o t e i n " r e p r e s e n t s SDS s o l u b l e
t h a t t h e r e was no s t a t i s t i c a l l y
proteins
-
insoluble.
polymerized m a t r i x would remain While i t i s c l e a r t h a t
s i g n i f i c a n t d i f f e r e n c e i n t h e amount of
MMP-2 proenzyme e x t r a c t a b l e from 2 mm
855
Current Eye Research
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E
2
1
3
4
5
7
6
S
N
E
8
S
N
9
R
i n t e n s i t i e s coi.:ld be Size standards are apparent molecular i n kiloDalLz.)n:3 ( k D ) ,
G e l a t i n a s e s e : < t r a c t e d from r:issues s e p a r a t e d from k e r a t o c o n u s corneas. E : E p i t h e l i a l T i s s u e , S : Stromal Zymography Lissiie, N : EndotheLial t i s s u e . was p e r f o r m e d on a l l s a m p l e s a t t h e same
t i m e s u c h t h a t band v i s u a l l y compared. rabbit gelatinases; s i z e s are i n d i c a t e d
G u y t.ons
even when t h e enzyme l e v e l s w e r r ccrre::ted
Fig. 3 :
keratoconus o r normal c o r n e a s
(Table 4 ) .
for total protein.
In a d d i t i o n , t h e r e was no
s i g r : i f i c a n t d i f f e r e n c e i n t h e amount o f e x t r a c t a b l e erizyme when compared t o t o t a l
rxt ractabie p r o t e i n .
DISCUSSION C o l l a g e n s form h i g h l y p o l y m e r i z e o ,
P r e v i a z s st,!idi e s d e r n o n s t r a t i n g
m a c r o m o l e c u l a r s t r u c t u r e s which can be
d i f f e r e n c e s in t.he l~evels of y e l a t i n o l y t i c
removed from t i s s u e s o n l y throhi3h
a c t i v i t y between normal and k e r a t o c o n u s
s p e c i a l i z e d b i o l o g i c a l m e c h a n i s m s . The
,:orn+zs u s c d c u l t u r i n g a s a method t o
best u n d e r s t o o d i s t h e mechanism for
]?.ate crude enzyme f o r a n a l y s i s
(3-
To l e a r n w h e t h e r c u l t u r i n g c o u l d
? ) ,
r-eves.!! s i:. f f e r e n c e b e t ween normal o r k e r a t o c o n u s c 3 r n e a s n o t o b s e r v e d by d i r e c t r2xt z
on, :.he amount of proenzyme
Frc;ciuc,eci in e x p l a n t c u l t u r e by e q u a l a r e a piece:: of :Ior.ma!
and k e r a t o c o n u s c o r n e a s
h*as i i i i o q u a c t i t a t e d .
Half o f t h e
r e m o v a l o f s t r u c t u r e s c o r i t a i n i n q t:ype i collagen.
Cells i n t i s s u e s undergoing
r e s o r p t i o n o r r e m o d e l l i n g o f cc.lliigen s t r u c t u r e s a c t i v e l y mediate t y p e 1 collaqenolysis
(16). T h i s activity h a s
been a t t r i b u t e d t 3 secret-ion
C j i
i n t e r s t i t i a l c o l l , z i g e n a s e s , t h e only enzymes which c a n c a t a l y z e d e g r a d s t i o n o f
2ond.i t icr,eil mediurr; p r o d u c e d from e a c h
t h e native type I collagen t r i p l e h e l i x a t
cult.:.~rt. w a s a n a l y z e d by zymography, a n d
t h e n e u t r a l pH of t h e e x t r a c e l l u l 3 r s p a c e .
s f.or total p r o t e i n
haif
:leterrnti-jac io::.
n ? ' i l k ic.
Type I c o l l a g e n o l y t i c a c t i v i t y h a s been
The r e s u l t s are reported
a f t e r i n j u r y ( 1 7 ),
?!
A q n i n rtc? s i g n i f i c a n t . d i f f e r e n c e e x i s t e d
r ni- anount: of
KMP-2 proenzyme
.i !)\/ k e r a t o c o n u s o r normal
856
d e m o n s t r a t e d i n c o r n e a s undergoj.ng r e p a i r
corneas,
i n d i c a t i n g o p e r a t i o n of
t h i s b a s i c c o 11agen o 1y t i c m e c ha ~i ?. :jm i n cornea, a s i n o t h e r t i s s u e s . I n t e r s t i t i a l c o l l a g e n a s e can deqrade
Current Eye Research Table 4: Levels of MMP-2 Proenzyme i n Corneas Obtained a t t h e Time of Penetrating Keratoplasty ~~
~
~-
KERATOCONUS sample MMP - 2 1 10 2 19 3 75 4 45
mean
37.25
protein 172.2 145.0 -
158.6
ratio 0.06 0.13
KRRATOCONUS sample 'vity act1 1 90.5 2 41.5 3 43.5
876 713 1020
ratio 0.103 0.054 0.043
889. 7
0.067
3 4 5
ivitv 42.5 51.0 27.5 26.5 98.0
Drotein 889.5 658.5 801.0 708.5 798.5
r a t 1~ 0.048 0.077 0.035 0.033 C. 123
mean
49.0
771.2
0.062
-
mean
0.094
NORMAL
NORMAL
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
Table 5: Levels of MMP-2 Proenzyme S e c r e t e d by Organ-Cultured Corneas
sample
MMP - 2
1
2 3 4 5 6 7
78 27 15 32 55 28 22
protein 221.5 160.0 293 .0 242.0 224.4 174,5 236.5
ratio 0.35 0.17 0.05 0.13 0.24 0.16 0.09
mean
36.7
221.7
0.17
C l i n i n c a l i n f o r m a t i o n on keratoconus p a t i e n t s a r e l i s t e d i n Table 1 . Enzyme was e x t r a c t e d from cornea d i s k s of e q u a l a r e a w i t h SDS and enzyme forms were v i s u a l i z e d by zymography. The o p t i c a l d e n s i t y volume of t h e zymogram c l e a r i n g produced by MMP-2 proenzyme was q u a n t i t a t e d for each sample and t h e r e l a t i v e amount of enzyme producing t h i s a c t i v i t y was determined by comparison t o a s t a n d a r d curve of 2 - f o l d enzyme d i l u t i o n s (1-1009; of s t a n d a r d s a m p l e ) . The t o t a l p r o t e i n i n each sample was a l s o determined a s a r e l a t i v e v a l u e with respect t o a standard curve. A r e l a t i v e comparison between enzyme and p r o t e i n l e v e l s ( r a t i o ) f o r each sample was c a l c u l a t e d by d i v i d i n g t h e r e l a t i v e enzyme l e v e l v a l u e by t h e t o t a l p r o t e i n v a l u e . I n keratoconus samples 3 and 4, t e c h n i c a l d i f f i c u l t i e s prevented t h e determination of t o t a l p r o t e i n .
sample 1 2
59.0 ,
I
Dr o t e i n
C l i n i c a l i n f o r m a t i o n on keratoconus p a t i e n t s i s l i s t e d i n Table 1 . Enzyme i n samples of medium c o n d i t i o n e d by c o r n e a l e x p l a n t s was v i s u a l i z e d b y zymography. The o p t i c a l d e n s i t y volume of t h e zymogram c l e a r i n g produced by MMP-2 proenzyme was q u a n t i t a t e d f o r each sample and t h e r e l a t i v e amount of enzyme producing t h i s a c t i v i t y was determined by comparison t o a s t a n d a r d curve of 2-fold enzyme d i l u t i o n s (1-100% of s t a n d a r d s a m p l e ) . The t o t a l p r o t e i n i n each sample was a l s o determined a s a r e l a t i v e v a l u e w i t h respect t o a standard curve. A r e l a t i v e comparison between enzyme and p r o t e i n l e v e l s ( r a t i o ) f o r each sample was c a l c u l a t e d by d i v i d i n g t h e r e l a t i v e enzyme l e v e l v a l u e by t h e t o t a l p r o t e i n v a l u e .
t h e h e t e r o t y p i c i n t e r a c t i o n between type 1 and V may be a t l e a s t p a r t i a l l y
r e s p o n s i b l e f o r c r e a t i n g t h e narrow d i a m e t e r f i b r i l s (18), t h e unique s t r u c t u r e of which i s thought t o be
o n l y n a t i v e c o l l a g e n t y p e s I , 11, and 111.
important f o r c o r n e a l t r a n s p a r e n c y . I t
While t y p e I i s t h e major c o l l a g e n
seems c e r t a i n t h a t t h e h e t e r o t y p i c f i b r i l
component of t h e c o r n e a l stroma, a number
s t r u c t u r e w i l l be found i n mammalian
of minor c o l l a g e n t y p e s a r e a l s o found
cornea a s i n a v i a n s p e c i e s ; t y p e V
which c o n f e r on t h e cornea many of i t s
c o l l a g e n makes up about 11%of t h e t o t a l
unique p r o p e r t i e s .
Recent s t u d i e s i n
c o l l a g e n i n r a b b i t stroma ( 1 9 ) .
Type V I
chick cornea show t h a t t h e f i b r i l s of t h e
c o l l a g e n i s a l s o found i n t h e c o r n e a l
s t r o m a l l a m e l l a e , which have p r e v i o u s l y
stroma.
It exists i n tendril-like
been thought t o be composed e n t i r e l y of
s t r u c t u r e s which may s e r v e t o s t a b i l i z e
t y p e I c o l l a g e n , a r e copolymers of t y p e I
t h e corneal lamellae
and V c o l l a g e n s .
t h e r e have been a few r e p o r t s on t h e
Experiments suggest t h a t
(20). I n a d d i t i o n ,
857
Current Eye Research p r ~ s e ~ n c iof - t y p e 111 co1.lagen a s a minor ,-ampc:!e:~t ..:i- c o r n e a l s t r o m a . ,..I
O t h e r zcirneal basement membranes
c o n t a i n o t h e r minor t y p e s of c o l l a g e n s . T yp i c 3 i b a serne n t niemb r a n e s
.zol.lageii, c o p o l y m e r i z e d w i t h l a m i n i n a n d rieparan s i ~ i f a i ep r o t e o y l y c a n
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
i n h i b i t e d by b i n d i n g t o members o f t h e T i s s u e I n h i b i t o r of M e t a l l o p r o t e i n a s e (TIMP) f a m i l y .
u n d e r 1y i n g
a l tlssues contain type I V
(21) .
t n e r e i s some c o n t r o v e r s y a s t o
iiowever,
a l l members o f t h e MMP f a m i l y can h e
To d a t e ,
s e v e n d i f f e r e n t MMPs w i t h
s t r u c t u r a l s i m i l a r i t y t o c o l l a q e n a s e have b e e n c h a r a c t e r i z e d by c l o n i n g arid sequencing.
The c l a s s i c a l ,
type 1
c o l l a g e n a s e p r o d u c e d by f i b r o b l a s t i c c e l l s
whether t . y p I V c o l l a g e n i s found i n t h e
h a s b e e n d e s i g n a t e d MMP-1,
fjasement membrane of c c i r n e a l e p i t h e l i u m
c o l l a g e n a s e p r o d u c e d by n e u t r o p h i l s i s now
;2:j
r?n ? h e a t h e r h a n d , t h e r e seems t o
.
i ~ er\-o quest L G I I
concerning t h e presence of in t h e s p e c i a l i z e d
C C J L6;q;ii-I ~
membrane b a c k i n g t h e e n d o t h e l i a l
Desceir,er ' s membrane.
it.'?i,
LITI I . 1
. i l l
iilso c o n t a i n s c o l l a g e n type
ir-
F u r t h e r m o r e , t h e r e i s good
{L'ji.
fi:r t h e p r e s e n c e o f t y p e VII 5
o i Licjeii ir: I :it?
cornea.1 basement membrane.
l < , ~ i . ~ i.: je
localized t o the
emcnl: membrane to t h e u n d e r l y i n g :.:r!3rTa
25)
(;)/;,
.n:.sn.
A
11,
iiii:i
besides types I,
11, a n d
t e e n s u q q e s c e d by i d e n t i f i c a t i o n
In ;i 1. mat: r i x - deg r a d i n g en zyme s
3f
r-e~.ar.eil t c ;
i r i t e r s t i t i a l c o l l a g e n a s e (26).
Eie ic !.hose l
i
present.
of n o r m a l c o r n e a s a n d
r r i e a c t i v a t e d f o r m of MMP-2
was
Possibly t h i s i s because t h e keratoconus corneas is thi.r! a l i i r q e amount o f nonit
is a l s o p o s s i b l e t h a t
t i v a t e d MMP-2
860
i n keratoconus
ACKNOWLEDGEMENTS W e g r a t e f u l l y acknowledge t h e a s s i s t a n c e
of W i l l i a m R i n e h a r t a n d P e t e r Malen i n p r e p a r a t i o n o f t h e f i g u r e s , and tile t e c h n i c a l a s s i s t a n c e o f I s a b e l Chanq a n d Jean Kawa.
W e a l s o t h a n k D r s . K a r e n Hasty
Current Eye Research (U. of Tenn.) and Margaret Hibbs (VA
Medical Center, Newington, Conn.) for their generous gifts of antisera. Supported by grants from the National Institutes of Health to MEF (EY08408) and to BYJTY (EY03890). Additional funds were provided through an agreement with the Shiseido Company of Japan.
a,
CORRESPONDING AUTHOR Elizabeth Fini, Ph.D.; Massachusetts Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
10. Heussen, C. and Dowdle, E.B. (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. U , 196202. 11. Fini, M.E. and Girard, M.T. (1990) Expression of collagenolytic/gelatinolytic metalloproteinases by normal cornea. Invest. Ophthalmol. Vis. Sci. 1779-1788. 12. Fini, M.E. and Girard, M.T. (1990) The pattern of metalloproteinase expression by corneal fibroblasts is altered with passage in cell culture. J. Cell Sci. 97, 373-383. 13. Girard, M.E., Matsubara. M. and Fini, M.E. (1991) Transforming growth factor-beta and 11-1 modulate expression of metalloproteinases by corneal stromal cells. Invest. Ophthalmol. Vis. Sci. 2 , 2441-2454. 14. Hibbs, M.S., Hoidal, J.R. and Kang, A.H. (1987) Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J. Clin. Invest. 1644-1650. 15. Smith, P .K., Krohn, R. I ., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N .M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. 76-85. Biochem. 16. Gross, J. (1982) An essay on biological degradation of collagen. In "Cell Biology of the Extracellular Matrix, (Ed. Hay, E.D.) pp. 217-258. Plenum Press, New York. 17. Brown, S.I. and Weller, C.A. (1970) Cell origin of collagenase in normal and wounded corneas. Arch. Ophthalmol. 74-77. 18. Birk, D.E., Fitch, J.M., Babiarz, J.P., Doane, K.J. and Linsenmayer, T.F. (1990) Collagen fibrillogenesis in vitro: interaction of type I and V collagen regulates fibril diameter. J. Cell Sci. B,649-655. 19. Cintron, C., Hong, B-S and Kublin, C.L. (1981) Quantitative analysis of collagen from normal developing corneas and corneal scars. Curr. Eye Res. 1, 1-7. 20. Bruns, R.R., Press, W., Engva, E., Timpl, R. and Gross, J. (1986) Type VI collagen in extracellular, 100 nm periodic filament and fibrils: Identification by immunoelectron 393microscopy. J. Cell Biol. 404. 21. Timpl, R. (1989) Structure and biological activity of basement
General Hospital, CBRC, Building 149 - 3rd Floor, 13th Street, Charlestown, MA 02129. REFERENCES 1. Bron, A.J. (1988) Keratoconus. Cornea. 2, 163-169. 2. Yue, B.Y.J.T., Sugar, J. and Benveniste, K . (1984) Heterogeneity in keratoconus: possible biochemical mechanism. Proc. SOC. Exp. Biol. Med. 175, 336-341. 3. Rehany, U., Lahav, M., and Shoshan, S. (1982) Collagenolytic activity in keratoconbs. Ann. Ophthalmol. 14, 751754. 4. Kao, W.W.Y., Vergnes, J-P., Ebert, J., Sundar-Raj, C.V. and Brown, S.I. (1982) Increased collagenase and qelatinase activities in keratoconus . Biochem. Biophys. Res. Commun. 107, 929-936. 5. Ihalainen, A., Salo, T., Forsius, H . and Peltonen, L. (1986) Increase in type I and type IV collagenolytic activity in primary cultures of keratoconus cornea. Eur. J . Clin. 78-84. Invest. 6. Critchfield, J.W., Calandra, A.J., Nesburn, A.B. and Kenney, M.C. (1988) Keratoconus I. Biochemical studies of normal and keratoconus corneas. Exp. Eye Res. G,953-963. 7. Kenney, M.C., Chwa, M., Escobar, M. and Brown, D . (1989) Altered gelatinolytic activity by keratoconus corneal cells. Biochem. Biophys. Res. 353-357. Comm. 8. Sawaguchi, S, Yue, B.Y.J.T., Sugar, J. and Gilboy, J.E. (1989). Lysosomal enzyme activities in keratoconus. Arch. Ophthalmol. m, 1507-1510. 9. Matsubara, M. , Girard, M.T., Kublin, C.L., Cintron, C. and Fini, M.E. (1991) Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea. Dev. Biol. 425-439.
s,
m,
m,
m,
m,
'I
u,
m,
86 1
Current Eye Research ~~
nembrane p r o t e i n s . E u r . J. Biochem. 48'1-501. 22. K o l e q a , J . , M a n a b e , M . a n d S u n , T-T. ( 1 9 8 9 ) Basement membrane h e t e r o g e n e i t y and varia?.ion i n c o r n e a l epithelial differentiation. D i f f e r e n t i a t i o n , 9-2, 54-63. 2 3 . S a w a d a , H., K o n o m i , H . , a n d Hirosawa, K , ( 1 9 9 8 ) C h a r a c t e r i z a t i o n of t h e c o i i a g e r ! i n t h e h e x a g o n a l l a t t i c e of Descen?et's m e m b r a n e : I t s r e l a t i o n t o t - y p e ' J I r 1 c o l l a g e n . J . C e l l B i o l . Us,
m,
219--127 24. P i c r g e s o ~ ~X.E. , (1988) New c o l l a g e n s , n e x c:oncl.:pts. A n n . R e v . C e l l B i o l . 4, c~\ --cJ7;, 25. i p s o n , 1 .K., S p u r r - - M i c h a u d , s . J . , a n d &isdale, A.S. ( 1 9 8 8 ) Hemidesmosomes a n d a n c h o r i n g f i b r i l c o l l a g e n appear s y n cii r cn ou s I y d u r i n g d e v e 1opme n t a n d womd h e , . i i . i n q . D e v e l . B i o l . 126, 2 5 3 -
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
i-
26. W o e s s n e r , ,J.E'.
(1991) Matrix inecai lopco::.einases a n d t h e i r lnhibitors i n connective t i s s u e r e m o d e l l irig. F A S E B J . 5, 2 1 4 5 - 2 1 5 4 . 2 s . B r i n c k e r h o f f , C.E. a n d F i n i , M . E . (1989) Molecular c l o n i n g o f c o i l a g e n a s e a n d a c t i v a t o r / strome l y s i n , members o f a c o o r d i n a t e l y r e g u l a t e d gene f a r n i l y of m e t a l l o p r o t e i n a s e s i m p o r t a n t : i.n t h e m o d u l a t i o n o f corinecLive t i s s u e metabolism. I n "Zol.iagen: Volume I V , M o l e c u l a r Bioloqy" (Eds. Olsen, B. and Nimmi, M . E . ) p p 6 5 - 8 6 . CRC P r e s s , Boca P l u c i n s k a , I . M . , Mayer, R. H. and Brinckerhoff , C.E. (1987) A g e n e f o r r a b b i t s y n o v i a l c e l l z o l i a q e n a s e : member of a f a m i l y of m e t a l l . o p r o t e i n a s e s w h i c h d e g r a d e s t-h e C O I - ~ I E ' C Ki v e t i s s u e m a t r i x . Ejochemistry, 6156-6164. 29. Erowri, D . , Chwa M . , E s c o r b a r , M . a n d K e n n e y , M.C. (1991) C h a r a c t e r i z a t i o n of t h e r r a j o r m a t r i x d e g r a d i n g m e t a l l o p r o t e i n a s e of human c o r n e a l stroina: evidence f o r an enzyine/inhihitor complex. E x p . Eye I
s,
z,
Re$. 2,5-16. Matsskmr,, M., Z i e s k e , J . a n d F i n i , M.E. ( i 9 9 1 ) Mechanism o f b a s e m e n t membrane d i s s o l u t i o n p r e c e d i n g c o r n e a l u1.cci a r l o n . I n v e s t . O p h t h a l m o l . V i s . S C ~ .2,9 2 - 1 0 7 . F.ini, M . E . , Girard, M.T. a n d Matsubara, M . (1992) i70 l 1a q e n c 1y t. i c / g e 1a t i n o 1y t i c e n z yme s i n c o r n e a l wound h e a l l n g . A c t a O p h t h a l m o l . ( s u p p l ) , 24, 2 6 - 3 3 . Al.e:.:ander, I: . J . , S a m p l e s , J . R . , Van B u s k l r k , E.M. a n d A c o t t , T . S ( 1 9 9 0 ) Expression of matrix metalloproteinase a n d I n L i t i i r o r b y human r e t i n a l p i g m e n t . Ophthalmol. V i s .
862
33. A l e x a n d e r , P . J . , B r a d l e y , J . M . B .
,
Gabourel, J.D. and A c o t t , T . S . (1991) Expression of matrix metalloproteinase a n d i n h i b i t o r b y human t r a b e c u l a r meshwork. I n v e s t . O p h t h a l m o l . V i s . S c i . 2,1 7 2 - 1 8 0 . 3 4 . S a w a g u c h i , S . , T w i n i n g , S . S . , Yu e , B . Y.J . T . , W i l s o n , P . M . , S u g a r , J . a n d Chan, S.-K. (1990) A l p h a - 1 p r o t e i n a s e i n h i b i t o r levels i n keratoconus. Exp. E y e R e s . 54, 5 4 9 - 5 5 4 . 3 5 . Yue, B . Y . J . T . , L o u s i n , G . , S a w a g u c h i , S . , Twining, S.S. and Sugar, J . (1992) levels i n Alpha-2-macroglobulin keratoconus. I n v e s t . Ophthalmol. V i c . S c i . U u p p l), 9 7 6 .
Volume 1 1 number 9 1992, 849-862 ~
~~
~
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~
Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas
M.Elizabeth Fini, Beatrice Y.J.T.Yuel and Joel Sugar' MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital and Department of Dermatology, Harvard Medical School, Boston, MA and 'Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, IL, USA
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ABSTRACT
components.
C e l l s of keratoconus c o r n e a s have been r e p o r t e d t o produce h i g h e r l e v e l s of collagenolytic/gelatinolytic enzymatic a c t i v i t i e s t h a n do c e l l s of normal corneas. The c u r r e n t s t u d y i n v e s t i g a t e s t h e c o n t r i b u t i o n of 1) s p e c i f i c enzyme gene p r o d u c t s , and 2 ) t h e degree t o which these p r o t e i n s a r e present i n t h e a c t i v a t e d forms, t o t h e i n c r e a s e d enzymatic a c t i v i t i e s . We demonstrate t h a t two n e u t r a l g e l a t i n o l y t i c enzymes, a 6 6 / 5 9 kD form and a 9 2 k D form, can be d i r e c t l y e x t r a c t e d from both normal and keratoconus corneas. These enzymes a r e i d e n t i f i e d a s t h e pro- and a c t i v a t e d forms of MMP-2 and a s t h e pro-form of MMP-9, s p e c i f i c members of t h e m a t r i x m e t a l l o p r o t e i n a s e f a m i l y . Normal and keratoconus c o r n e a s show no s i g n i f i c a n t d i f f e r e n c e s i n amounts o r t y p e s of e x t r a c t a b l e n e u t r a l g e l a t i n a s e s , nor i n t h e amounts o r t y p e s t h a t t h e y s y n t h e s i z e i n c u l t u r e . Furthermore, i n both t h e normal and keratoconus c o r n e a s , g e l a t i n a s e s a r e found p r i m a r i l y i n t h e i n a c t i v e form. These s t u d i e s s u q q e s t t h e p o s s i b l e importance of changes i n proteinase inhibitor levels t o the c h a r a c t e r i s t i c biochemical f e a t u r e s of keratoconus c o r n e a s .
R e s u l t s from p r e v i o u s
i n v e s t i g a t i o n s on keratoconus have indicated that the basic biologic defect may l i e i n t h e s y n t h e s i s ( 2 ) a n d / o r
d e g r a d a t i o n ( 3 - 8 ) of p r o t e i n and c o l l a g e n molecules.
Evidence f o r abnormal m a t r i x
d e g r a d a t i o n h a s been o b t a i n e d from enzymatic s t u d i e s . C u l t u r e d t i s s u e e x p l a n t s , o r primary c e l l c u l t u r e s d e r i v e d from keratoconus c o r n e a s , have been found
t o s e c r e t e i n c r e a s e d l e v e l s of n e u t r a l enzymatic a c t i v i t i e s a g a i n s t n a t i v e c o l l a g e n t y p e s 1 and I V ,
and a g a i n s t
g e l a t i n s (denatured c o l l a g e n s ) , i n t o t h e i r c u l t u r e medium ( 3 - 7 ) .
Also,
lysosomal
a c i d hydrolase l e v e l s a r e e l e v a t e d i n keratoconus c o r n e a s ( 8 ) . Net enzymatic a c t i v i t y i s a combination of s e v e r a l f a c t o r s i n c l u d i n g t h e enzyme t y p e s p r e s e n t , t h e degree t o which i n a c t i v e proenzymes have been converted t o t h e i r a c t i v a t e d forms, and t h e p r e s e n c e of
INTRODUCTION
specific inhibitors.
Keratoconus i s a non-inflammatory
In t h i s s t u d y , we
c o r n e a l d i s o r d e r t h a t i s a s s o c i a t e d with
i n v e s t i g a t e t h e c o n t r i b u t i o n of t h e f i r s t
l o s s of t e n s i l e s t r e n g t h , t h i n n i n g , and
two f a c t o r s t o t h e i n c r e a s e d g e l a t i n o l y t i c
e c t a s i a o f t h e c e n t r a l cornea ( 1 ) . I t s
a c t i v i t y produced by keratoconus c o r n e a s .
e t i o l o g i c a l b a s i s i s not understood
W e demonstrate t h a t two n e u t r a l
The
d i s o r d e r can sometimes be a s s o c i a t e d w i t h
g e l a t i n o l y t i c enzymes, a 66/59 kD and a 92
i n h e r i t e d connective t i s s u e d i s o r d e r s s u c h
kD enzyme, can be d i r e c t l y e x t r a c t e d from
a s Ehlers-Danlos syndrome t y p e I V and V I ,
b o t h normal and keratoconus c o r n e a s .
Marfan syndrome, and o s t e o g e n e s i s
These enzymes a r e i d e n t i f i e d a s t h e pro-
imperfecta suggesting a p o s s i b l e
and a c t i v a t e d forms of MMP-2 and a s t h e
involvement i n t h e s t r u c t u r e or
pro-form of MMP-9,
maintenance of e x t r a c e l l u l a r m a t r i x
matrix metalloproteinase family.
s p e c i f i c members of t h e Normal
Received on April 30, 1992; accepted on August 21, 1992
0 Oxford University Press
849
Current Eye Research ~~
~~~
~~
and k e r a t o c o n u s c o r n e a s show no
c u l t u r e ( s e e b e l o w ) , t h e 8 mm c e n t r a l
~
significant. differences i n the t o t a l
c o r n e a l b u t t o n s were c u t i n t o f o u r e q u a l -
amount o r t-ypes of e x t r a c t a b l e n e u t r a l
sized pieces.
g e l a t i n a s e s , nor i n t h e amounts o r t y p e s
immediately i n t o c u l t u r e .
of g e i a t i n a s e s t h a t t h e y s y n t h e s i z e i n Furt.hermore, i n b o t h normal and
cult u r e .
Half of 7 . 5 -
The p i e c e s were t h e n p l a c e d t o 8 mm c o r n e a l b u t t o n s
w e r e obtained within 3 hours
Of
keratocoiius c o r n e a s , g e l a t i n a s e s a r e found
p e n e t r a t i n g k e r a t o p l a s t y from p a t i e n t s
p r i , m a r i l y i n t h e i n a c t i v e form.
with keratoconus.
The a g e s , d u r a t i o n of
t h e d i s e a s e , and o t h e r c l i n i c a l information f o r keratoconus p a t i e n t s a r e
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
MATERIALS AND METHODS
shown i n Table 1 .
of c o r n e a l t . i s s u e S 2 2 normal haman c o r n e a s was
A t o t a l . of
o b t a i n e d from t h e I l l i n o i s Eye Bank (Chicago).
F i v e of t h e c o r n e a s were
I n a d d i t i o n , another
set of c o r n e a s was c o l l e c t e d at. p e n e t r a t i n g k e r a t o p l a s t y from s i x p a t i e n t s with o t h e r corneal d i s e a s e s .
T h e ages,
r e c e i v e d as a u t o p s y g l o b e s from donors
d i a g n o s i s , and o t h e r i n f o r m a t i o n on t h e s e
( i 3 - , 66-, 7 5 - - , 71-, and 8 0 - y e a r s - o l d )
p a t i e n t s a r e summarized i n Table 2 .
w i t h i n 2 4 h c u r s of d e a t h .
Seventeen
For s t u d i e s on t i s s u e 1 o c a l i z a t : i o n of
o t h e r s were r e c e i v e d a j c o r n e a l b u t t o n s
gelatinases,
t h a t had beer! e n u c l e a t e d from donors (mean
m e c h a n i c a l l y from t h e h a l f c o r n e a s .
a g e , 3 6 . 6 .i 2 1 . 7 y e a r s ,
range, 1 3 t o 7 6
t i s s u e l a y e r s were s e p a r a t e d Each
t i s s u e fragment was p l a c e d i n a s e p a r a t e I n a d d i t i o n , 2-mm but.t.ons
y e a r s ) aria soaked i n c o r n e a l s t o r a g e
tube a s above.
mediurr. ( 0 p t j . s o l o r Dexsol) f o r 1 4 to 1 1 5
were a l s o p r e p a r e d from some specimens f o r
hours.
d i r e c t e x t r a c t i o n zymography and
A l i cionors had no known s y s t e m i c
and 1/4
o r o c u i a r d i s e a s e s , a n t i t h e c o r n e a s were
c o r n e a l p i e c e s f o r organ c u l t u r e s t u d i e s
clear.
a s d e s c r i b e d above.
FOL s t o d i e s on t i s s u e l o c a l i z a t i o n of
g e l a t i n a s e s , an 8-mm t r e p h i n e was used t o punch oat
d
h u t t o n of c e n t r a l c o r n e a and
&traction
of u e l a t i n a s e s from t i s s u e
When a l l samples were c o l l e c t e c i f o r a
p a r t i c u l a r experiment, t h e t i s s u e : j were
t h e c e n t r a l butcon and p e r i p h e r a l r i n g
thawed a t room t e m p e r a t u r e .
were c u t ir: h a l f w i t h a s c a l p e l .
immediately e x t r a c t e d f o r a n a l y s i s b y
Epithel i a i ,
v o r t e x i n g t h e t i s s u e i n a s o l u t i c n of 2 . 0 %
:.issue
s t r o m a l , and e n d o t h e l i a l
l a y e r s were t h e n s e p a r a t e d
mechanical!y
SDS a s d e s c r i b e d
from c e n t r a l and p e r i p h e r a l
P r o t e i . n s were
( 9 ) . Each t i s s u e sample
was e x t r a c t e d w i t h an e q u a l volume of SDS
h a l v e s and each t i s s u e fragment was p l a c e d
(20 u l p e r t i s s u e s a m p l e ) . The ?C ul
in
volume of e x t r a c t from each t i s s u e sample
3
separate tube.
A l l samples were
t e l y f a s t . - f r o z e n by dropping i n
was immediately d i l u t e d 2 : 5 w i t h Laemmli
Liquid n i t r o g e n ; t - i s s u e s were s t o r e d a t
g e l sample b u f f e r and 2 5 ul (out of t h e
-?DOC f o r \ J F t o s e v e r a l months.
t o t a l of 5 0 u l ) of each sample was loaded
For
d i r e c t q u a n t i t a t i v e s t u d i e s ( s e e below), a 2-mm
t r e p h h e was used t o punch o u t two or
c h r e e c e n t r a l b u t t o n s of t i s s u e from t h e 8
i n a s i n g l e g e l lane (without heating o r r e d u c t i o n ) on s t a n d a r d zymograns
s e c t i o n ) f o r a n a l y s i s of g e l a t i n o l ~ y t i c
m bT2t.ton; t i s s u e l a y e r s were n o t
enzymes p r e s e n t .
separared.
Zvmocrrar>hv
Each b u t t o n was p l a c e d ,
...n t a c t ,
i n a s e p a r a t e t u b e and f r o z e n a s
The method of zymography
above.
F o r q u a n t i t a t i v e a n a l y s i s by organ
analyze t h e content of s p e c i f i c
8 50
( s e e next
(10) was used t o
Current Eye Research TABLE 1 Ages a n d O t h e r C l i n i c a l C h a r a c t e r i s t i c s of P a t i e n t s w i t h K e r a t o c o n u s
1 2 3 4 5 6
Race
Age a t S u
F
white black white white white black white white white white white white white black black white
27 40 59 42 43 37 31 37 48 25 34 50 31 48 30 72
F F F
M M M F M M M M
7
8 9 10 11 Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
Sex
12 13 15
M F F
16
M
14
r
g
D u r a t i o n of e r s
n
g
+ + + + + + + + + + +
10
)A, ;.a! ! r
.-
Virtually the
o n l y g e l a t i n a s e species p r e s e n t i n t h e
The t i . s s u e l o c a l i z a t i o n of
t h e 6 6 kD g e l a t i n a s e w a s s i m i l a r tci t h a t of t h e normal corneas with very l i t t l e i f a n y enzyme f o u n d i n t h e e p i t h e l i u m .
1 c o r n e a s o b t a i n e d af t e r Penet-rat.lny
keratoDlastv The s t u d i e s r e p o r t e d a b o v e d e m o n s t - r a t e d a
. i n d c h e 9 2 kD y e l a t i n a s e i?
I
Ariiist~riiin
( F i g . 2A, L a n e
c l e a r d i f f e r e n c e i n t h e types of
s p e c i f i c f o r human MMP-9
g e l a t i n a s e s p r e s e n t i n normal corneas
e c i p i t a t e d t h e 92 kD
o b t a i n e d f r o m t h e Eye Bank a n d t h e
. 2B) arid a n t i s e r u m r n r ~ ~ r o e n z y nform e o f human
i::sily
p r e c i p i t a t e d t h e 6 6 kD
keratoconus corneas obtained a f t e r penetrating keratoplasty.
I t seemed
p o s s i b l e t h a t t h i s d i f f e r e n c e c o u l d be an a r t i f a c t o f a l t e r n a t i v e h a n d l i n g ; t h e Eye Bank c o r n e a s u n d e r w e n t a someti.mes
~
e x t e n s i v e s t o r a g e p e r i o d i n l i q u i d (up t o 5 d a y s ) while t h e keratoconus c o r n e a s were
Table 3 : G e l a t i n a s e s P r e s e n t i n Other Diseased Corneas Obtained a t t h e Time of Penetrating Keratoplasty
p r o c e s s e d w i t h i n t h r e e hours a f t e r surgery.
elatmases
To t e s t t h i s h y p o t h e s i s , t h e
gelatinolytic species present i n other diseased corneas, obtained a f t e r
1
MMP-2 proenzyme
2
MMP-2 proenzyme
3
MMP-2 proenzyme
p e n e t r a t i n g k e r a t o p l a s t y , were examined by d i r e c t e x t r a c t i o n and zymography.
Since
4 0 kD g e l a t i n a s e
i n t h i s experiment, t h e t i s s u e l o c a l i z a t i o n of enzymes was not of
4
MMP-2 proenzyme MMP-2 a c t i v a t e d
5
MMP-2 proenzyme MMP-2 a c t i v a t e d
6
MMP-2 proenzyme MMP-2 a c t i v a t e d
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r e l e v a n c e , e x t r a c t i o n s were performed on complete c o r n e a l b u t t o n s .
The r e s u l t s a r e
summarized i n Table 3 . Unlike Eye Bank c o r n e a s , no MMP-9 was found i n any of t h e 6 corneas o b t a i n e d a f t e r penetrating keratoplasty.
However,
i n 3 of t h e c o r n e a s , l a r g e amounts of t h e a c t i v a t e d form of MMP-2 were p r e s e n t , r e p r e s e n t i n g g r e a t e r t h a n 1 0 % of t h e t o t a l
MMP-2 enzyme.
These 3 corneas a l s o had a
C l i n i c a l i n f o r m a t i o n on each p a t i e n t i s as numbered i n Table 2 . Enzyme was e x t r a c t e d from c o r n e a l t i s s u e samples w i t h SDS and t h e enzyme forms p r e s e n t were analyzed by zymography. The p r e s e n c e of MMP-2 a c t i v a t e d enzyme w a s not l i s t e d h e r e u n l e s s i t r e p r e s e n t e d g r e a t e r t h a n 1 0 % of t h e t o t a l MMP-2 i n t h e sample.
s u b s t a n t i a l degree of s c a r r i n g i n common. I n a d d i t i o n , t h e cornea t h a t had been
removed because of i n t e r s t i t i a l k e r a t i t i s
keratoconus corneas a r e t h i n n e r t h a n
c o n t a i n e d a 4 0 kD g e l a t i n a s e ; t h e i d e n t i t y
normal, it i s not c l e a r whether t h i s
of t h i s enzyme i s unknown.
r e p r e s e n t s a s e l e c t i v e l o s s of p a r t i c u l a r p r o t e i n s , o r a generalized p r o t e i n l o s s .
normal and keratoconus c o r n e a s
I f t h e former c a s e i s c o r r e c t , t h e n
The l e v e l s of MMP-2 proenzyme which was
important d i f f e r e n c e s between normal and
e x t r a c t a b l e from normal and keratoconus
keratoconus c o r n e a s might be missed by
c o r n e a s were q u a n t i f i e d from zymograms
comparing enzyme l e v e l s t o t o t a l p r o t e i n
s i m i l a r t o t h o s e shown i n f i g u r e 3 .
alone.
E x t r a c t i o n s were performed on p i e c e s of
normal c o r n e a s were analyzed and t h e
c o r n e a l b u t t o n s with equal s u r f a c e a r e a ,
r e s u l t s a r e r e p o r t e d i n Table 4 .
s o t h a t v a l u e s from a l l samples could be compared based on c o r n e a l a r e a .
Half of
Four keratoconus corneas and I
A s was observed i n our p r e v i o u s
experiment, t h e o n l y g e l a t i n a s e t y p e
t h e e x t r a c t from each c o r n e a l p i e c e was
p r e s e n t i n keratoconus e x t r a c t s was MMP-2,
analyzed by zymography, and h a l f was a l s o
almost a l l of which was i n t h e pro-form.
used f o r t o t a l p r o t e i n d e t e r m i n a t i o n s o
The same r e s u l t was o b t a i n e d w i t h 6 of t h e
t h a t t h e amount of g e l a t i n a s e i n samples
7 normal c o r n e a s ; i n t h e s e v e n t h , a s m a l l
could be compared w i t h r e s p e c t t o t o t a l
amount of 92 kD g e l a t i n a s e could be
protein as well.
observed.
We b e l i e v e t h a t both
Q u a n t i t a t i o n of MMP-2 proenzyme
comparisions a r e important t o c o n s i d e r .
w i t h r e s p e c t t o s t a n d a r d s demonstrated
O u r " t o t a l p r o t e i n " r e p r e s e n t s SDS s o l u b l e
t h a t t h e r e was no s t a t i s t i c a l l y
proteins
-
insoluble.
polymerized m a t r i x would remain While i t i s c l e a r t h a t
s i g n i f i c a n t d i f f e r e n c e i n t h e amount of
MMP-2 proenzyme e x t r a c t a b l e from 2 mm
855
Current Eye Research
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E
2
1
3
4
5
7
6
S
N
E
8
S
N
9
R
i n t e n s i t i e s coi.:ld be Size standards are apparent molecular i n kiloDalLz.)n:3 ( k D ) ,
G e l a t i n a s e s e : < t r a c t e d from r:issues s e p a r a t e d from k e r a t o c o n u s corneas. E : E p i t h e l i a l T i s s u e , S : Stromal Zymography Lissiie, N : EndotheLial t i s s u e . was p e r f o r m e d on a l l s a m p l e s a t t h e same
t i m e s u c h t h a t band v i s u a l l y compared. rabbit gelatinases; s i z e s are i n d i c a t e d
G u y t.ons
even when t h e enzyme l e v e l s w e r r ccrre::ted
Fig. 3 :
keratoconus o r normal c o r n e a s
(Table 4 ) .
for total protein.
In a d d i t i o n , t h e r e was no
s i g r : i f i c a n t d i f f e r e n c e i n t h e amount o f e x t r a c t a b l e erizyme when compared t o t o t a l
rxt ractabie p r o t e i n .
DISCUSSION C o l l a g e n s form h i g h l y p o l y m e r i z e o ,
P r e v i a z s st,!idi e s d e r n o n s t r a t i n g
m a c r o m o l e c u l a r s t r u c t u r e s which can be
d i f f e r e n c e s in t.he l~evels of y e l a t i n o l y t i c
removed from t i s s u e s o n l y throhi3h
a c t i v i t y between normal and k e r a t o c o n u s
s p e c i a l i z e d b i o l o g i c a l m e c h a n i s m s . The
,:orn+zs u s c d c u l t u r i n g a s a method t o
best u n d e r s t o o d i s t h e mechanism for
]?.ate crude enzyme f o r a n a l y s i s
(3-
To l e a r n w h e t h e r c u l t u r i n g c o u l d
? ) ,
r-eves.!! s i:. f f e r e n c e b e t ween normal o r k e r a t o c o n u s c 3 r n e a s n o t o b s e r v e d by d i r e c t r2xt z
on, :.he amount of proenzyme
Frc;ciuc,eci in e x p l a n t c u l t u r e by e q u a l a r e a piece:: of :Ior.ma!
and k e r a t o c o n u s c o r n e a s
h*as i i i i o q u a c t i t a t e d .
Half o f t h e
r e m o v a l o f s t r u c t u r e s c o r i t a i n i n q t:ype i collagen.
Cells i n t i s s u e s undergoing
r e s o r p t i o n o r r e m o d e l l i n g o f cc.lliigen s t r u c t u r e s a c t i v e l y mediate t y p e 1 collaqenolysis
(16). T h i s activity h a s
been a t t r i b u t e d t 3 secret-ion
C j i
i n t e r s t i t i a l c o l l , z i g e n a s e s , t h e only enzymes which c a n c a t a l y z e d e g r a d s t i o n o f
2ond.i t icr,eil mediurr; p r o d u c e d from e a c h
t h e native type I collagen t r i p l e h e l i x a t
cult.:.~rt. w a s a n a l y z e d by zymography, a n d
t h e n e u t r a l pH of t h e e x t r a c e l l u l 3 r s p a c e .
s f.or total p r o t e i n
haif
:leterrnti-jac io::.
n ? ' i l k ic.
Type I c o l l a g e n o l y t i c a c t i v i t y h a s been
The r e s u l t s are reported
a f t e r i n j u r y ( 1 7 ),
?!
A q n i n rtc? s i g n i f i c a n t . d i f f e r e n c e e x i s t e d
r ni- anount: of
KMP-2 proenzyme
.i !)\/ k e r a t o c o n u s o r normal
856
d e m o n s t r a t e d i n c o r n e a s undergoj.ng r e p a i r
corneas,
i n d i c a t i n g o p e r a t i o n of
t h i s b a s i c c o 11agen o 1y t i c m e c ha ~i ?. :jm i n cornea, a s i n o t h e r t i s s u e s . I n t e r s t i t i a l c o l l a g e n a s e can deqrade
Current Eye Research Table 4: Levels of MMP-2 Proenzyme i n Corneas Obtained a t t h e Time of Penetrating Keratoplasty ~~
~
~-
KERATOCONUS sample MMP - 2 1 10 2 19 3 75 4 45
mean
37.25
protein 172.2 145.0 -
158.6
ratio 0.06 0.13
KRRATOCONUS sample 'vity act1 1 90.5 2 41.5 3 43.5
876 713 1020
ratio 0.103 0.054 0.043
889. 7
0.067
3 4 5
ivitv 42.5 51.0 27.5 26.5 98.0
Drotein 889.5 658.5 801.0 708.5 798.5
r a t 1~ 0.048 0.077 0.035 0.033 C. 123
mean
49.0
771.2
0.062
-
mean
0.094
NORMAL
NORMAL
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Table 5: Levels of MMP-2 Proenzyme S e c r e t e d by Organ-Cultured Corneas
sample
MMP - 2
1
2 3 4 5 6 7
78 27 15 32 55 28 22
protein 221.5 160.0 293 .0 242.0 224.4 174,5 236.5
ratio 0.35 0.17 0.05 0.13 0.24 0.16 0.09
mean
36.7
221.7
0.17
C l i n i n c a l i n f o r m a t i o n on keratoconus p a t i e n t s a r e l i s t e d i n Table 1 . Enzyme was e x t r a c t e d from cornea d i s k s of e q u a l a r e a w i t h SDS and enzyme forms were v i s u a l i z e d by zymography. The o p t i c a l d e n s i t y volume of t h e zymogram c l e a r i n g produced by MMP-2 proenzyme was q u a n t i t a t e d for each sample and t h e r e l a t i v e amount of enzyme producing t h i s a c t i v i t y was determined by comparison t o a s t a n d a r d curve of 2 - f o l d enzyme d i l u t i o n s (1-1009; of s t a n d a r d s a m p l e ) . The t o t a l p r o t e i n i n each sample was a l s o determined a s a r e l a t i v e v a l u e with respect t o a standard curve. A r e l a t i v e comparison between enzyme and p r o t e i n l e v e l s ( r a t i o ) f o r each sample was c a l c u l a t e d by d i v i d i n g t h e r e l a t i v e enzyme l e v e l v a l u e by t h e t o t a l p r o t e i n v a l u e . I n keratoconus samples 3 and 4, t e c h n i c a l d i f f i c u l t i e s prevented t h e determination of t o t a l p r o t e i n .
sample 1 2
59.0 ,
I
Dr o t e i n
C l i n i c a l i n f o r m a t i o n on keratoconus p a t i e n t s i s l i s t e d i n Table 1 . Enzyme i n samples of medium c o n d i t i o n e d by c o r n e a l e x p l a n t s was v i s u a l i z e d b y zymography. The o p t i c a l d e n s i t y volume of t h e zymogram c l e a r i n g produced by MMP-2 proenzyme was q u a n t i t a t e d f o r each sample and t h e r e l a t i v e amount of enzyme producing t h i s a c t i v i t y was determined by comparison t o a s t a n d a r d curve of 2-fold enzyme d i l u t i o n s (1-100% of s t a n d a r d s a m p l e ) . The t o t a l p r o t e i n i n each sample was a l s o determined a s a r e l a t i v e v a l u e w i t h respect t o a standard curve. A r e l a t i v e comparison between enzyme and p r o t e i n l e v e l s ( r a t i o ) f o r each sample was c a l c u l a t e d by d i v i d i n g t h e r e l a t i v e enzyme l e v e l v a l u e by t h e t o t a l p r o t e i n v a l u e .
t h e h e t e r o t y p i c i n t e r a c t i o n between type 1 and V may be a t l e a s t p a r t i a l l y
r e s p o n s i b l e f o r c r e a t i n g t h e narrow d i a m e t e r f i b r i l s (18), t h e unique s t r u c t u r e of which i s thought t o be
o n l y n a t i v e c o l l a g e n t y p e s I , 11, and 111.
important f o r c o r n e a l t r a n s p a r e n c y . I t
While t y p e I i s t h e major c o l l a g e n
seems c e r t a i n t h a t t h e h e t e r o t y p i c f i b r i l
component of t h e c o r n e a l stroma, a number
s t r u c t u r e w i l l be found i n mammalian
of minor c o l l a g e n t y p e s a r e a l s o found
cornea a s i n a v i a n s p e c i e s ; t y p e V
which c o n f e r on t h e cornea many of i t s
c o l l a g e n makes up about 11%of t h e t o t a l
unique p r o p e r t i e s .
Recent s t u d i e s i n
c o l l a g e n i n r a b b i t stroma ( 1 9 ) .
Type V I
chick cornea show t h a t t h e f i b r i l s of t h e
c o l l a g e n i s a l s o found i n t h e c o r n e a l
s t r o m a l l a m e l l a e , which have p r e v i o u s l y
stroma.
It exists i n tendril-like
been thought t o be composed e n t i r e l y of
s t r u c t u r e s which may s e r v e t o s t a b i l i z e
t y p e I c o l l a g e n , a r e copolymers of t y p e I
t h e corneal lamellae
and V c o l l a g e n s .
t h e r e have been a few r e p o r t s on t h e
Experiments suggest t h a t
(20). I n a d d i t i o n ,
857
Current Eye Research p r ~ s e ~ n c iof - t y p e 111 co1.lagen a s a minor ,-ampc:!e:~t ..:i- c o r n e a l s t r o m a . ,..I
O t h e r zcirneal basement membranes
c o n t a i n o t h e r minor t y p e s of c o l l a g e n s . T yp i c 3 i b a serne n t niemb r a n e s
.zol.lageii, c o p o l y m e r i z e d w i t h l a m i n i n a n d rieparan s i ~ i f a i ep r o t e o y l y c a n
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
i n h i b i t e d by b i n d i n g t o members o f t h e T i s s u e I n h i b i t o r of M e t a l l o p r o t e i n a s e (TIMP) f a m i l y .
u n d e r 1y i n g
a l tlssues contain type I V
(21) .
t n e r e i s some c o n t r o v e r s y a s t o
iiowever,
a l l members o f t h e MMP f a m i l y can h e
To d a t e ,
s e v e n d i f f e r e n t MMPs w i t h
s t r u c t u r a l s i m i l a r i t y t o c o l l a q e n a s e have b e e n c h a r a c t e r i z e d by c l o n i n g arid sequencing.
The c l a s s i c a l ,
type 1
c o l l a g e n a s e p r o d u c e d by f i b r o b l a s t i c c e l l s
whether t . y p I V c o l l a g e n i s found i n t h e
h a s b e e n d e s i g n a t e d MMP-1,
fjasement membrane of c c i r n e a l e p i t h e l i u m
c o l l a g e n a s e p r o d u c e d by n e u t r o p h i l s i s now
;2:j
r?n ? h e a t h e r h a n d , t h e r e seems t o
.
i ~ er\-o quest L G I I
concerning t h e presence of in t h e s p e c i a l i z e d
C C J L6;q;ii-I ~
membrane b a c k i n g t h e e n d o t h e l i a l
Desceir,er ' s membrane.
it.'?i,
LITI I . 1
. i l l
iilso c o n t a i n s c o l l a g e n type
ir-
F u r t h e r m o r e , t h e r e i s good
{L'ji.
fi:r t h e p r e s e n c e o f t y p e VII 5
o i Licjeii ir: I :it?
cornea.1 basement membrane.
l < , ~ i . ~ i.: je
localized t o the
emcnl: membrane to t h e u n d e r l y i n g :.:r!3rTa
25)
(;)/;,
.n:.sn.
A
11,
iiii:i
besides types I,
11, a n d
t e e n s u q q e s c e d by i d e n t i f i c a t i o n
In ;i 1. mat: r i x - deg r a d i n g en zyme s
3f
r-e~.ar.eil t c ;
i r i t e r s t i t i a l c o l l a g e n a s e (26).
Eie ic !.hose l
i
present.
of n o r m a l c o r n e a s a n d
r r i e a c t i v a t e d f o r m of MMP-2
was
Possibly t h i s i s because t h e keratoconus corneas is thi.r! a l i i r q e amount o f nonit
is a l s o p o s s i b l e t h a t
t i v a t e d MMP-2
860
i n keratoconus
ACKNOWLEDGEMENTS W e g r a t e f u l l y acknowledge t h e a s s i s t a n c e
of W i l l i a m R i n e h a r t a n d P e t e r Malen i n p r e p a r a t i o n o f t h e f i g u r e s , and tile t e c h n i c a l a s s i s t a n c e o f I s a b e l Chanq a n d Jean Kawa.
W e a l s o t h a n k D r s . K a r e n Hasty
Current Eye Research (U. of Tenn.) and Margaret Hibbs (VA
Medical Center, Newington, Conn.) for their generous gifts of antisera. Supported by grants from the National Institutes of Health to MEF (EY08408) and to BYJTY (EY03890). Additional funds were provided through an agreement with the Shiseido Company of Japan.
a,
CORRESPONDING AUTHOR Elizabeth Fini, Ph.D.; Massachusetts Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
10. Heussen, C. and Dowdle, E.B. (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. U , 196202. 11. Fini, M.E. and Girard, M.T. (1990) Expression of collagenolytic/gelatinolytic metalloproteinases by normal cornea. Invest. Ophthalmol. Vis. Sci. 1779-1788. 12. Fini, M.E. and Girard, M.T. (1990) The pattern of metalloproteinase expression by corneal fibroblasts is altered with passage in cell culture. J. Cell Sci. 97, 373-383. 13. Girard, M.E., Matsubara. M. and Fini, M.E. (1991) Transforming growth factor-beta and 11-1 modulate expression of metalloproteinases by corneal stromal cells. Invest. Ophthalmol. Vis. Sci. 2 , 2441-2454. 14. Hibbs, M.S., Hoidal, J.R. and Kang, A.H. (1987) Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J. Clin. Invest. 1644-1650. 15. Smith, P .K., Krohn, R. I ., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N .M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. 76-85. Biochem. 16. Gross, J. (1982) An essay on biological degradation of collagen. In "Cell Biology of the Extracellular Matrix, (Ed. Hay, E.D.) pp. 217-258. Plenum Press, New York. 17. Brown, S.I. and Weller, C.A. (1970) Cell origin of collagenase in normal and wounded corneas. Arch. Ophthalmol. 74-77. 18. Birk, D.E., Fitch, J.M., Babiarz, J.P., Doane, K.J. and Linsenmayer, T.F. (1990) Collagen fibrillogenesis in vitro: interaction of type I and V collagen regulates fibril diameter. J. Cell Sci. B,649-655. 19. Cintron, C., Hong, B-S and Kublin, C.L. (1981) Quantitative analysis of collagen from normal developing corneas and corneal scars. Curr. Eye Res. 1, 1-7. 20. Bruns, R.R., Press, W., Engva, E., Timpl, R. and Gross, J. (1986) Type VI collagen in extracellular, 100 nm periodic filament and fibrils: Identification by immunoelectron 393microscopy. J. Cell Biol. 404. 21. Timpl, R. (1989) Structure and biological activity of basement
General Hospital, CBRC, Building 149 - 3rd Floor, 13th Street, Charlestown, MA 02129. REFERENCES 1. Bron, A.J. (1988) Keratoconus. Cornea. 2, 163-169. 2. Yue, B.Y.J.T., Sugar, J. and Benveniste, K . (1984) Heterogeneity in keratoconus: possible biochemical mechanism. Proc. SOC. Exp. Biol. Med. 175, 336-341. 3. Rehany, U., Lahav, M., and Shoshan, S. (1982) Collagenolytic activity in keratoconbs. Ann. Ophthalmol. 14, 751754. 4. Kao, W.W.Y., Vergnes, J-P., Ebert, J., Sundar-Raj, C.V. and Brown, S.I. (1982) Increased collagenase and qelatinase activities in keratoconus . Biochem. Biophys. Res. Commun. 107, 929-936. 5. Ihalainen, A., Salo, T., Forsius, H . and Peltonen, L. (1986) Increase in type I and type IV collagenolytic activity in primary cultures of keratoconus cornea. Eur. J . Clin. 78-84. Invest. 6. Critchfield, J.W., Calandra, A.J., Nesburn, A.B. and Kenney, M.C. (1988) Keratoconus I. Biochemical studies of normal and keratoconus corneas. Exp. Eye Res. G,953-963. 7. Kenney, M.C., Chwa, M., Escobar, M. and Brown, D . (1989) Altered gelatinolytic activity by keratoconus corneal cells. Biochem. Biophys. Res. 353-357. Comm. 8. Sawaguchi, S, Yue, B.Y.J.T., Sugar, J. and Gilboy, J.E. (1989). Lysosomal enzyme activities in keratoconus. Arch. Ophthalmol. m, 1507-1510. 9. Matsubara, M. , Girard, M.T., Kublin, C.L., Cintron, C. and Fini, M.E. (1991) Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea. Dev. Biol. 425-439.
s,
m,
m,
m,
m,
'I
u,
m,
86 1
Current Eye Research ~~
nembrane p r o t e i n s . E u r . J. Biochem. 48'1-501. 22. K o l e q a , J . , M a n a b e , M . a n d S u n , T-T. ( 1 9 8 9 ) Basement membrane h e t e r o g e n e i t y and varia?.ion i n c o r n e a l epithelial differentiation. D i f f e r e n t i a t i o n , 9-2, 54-63. 2 3 . S a w a d a , H., K o n o m i , H . , a n d Hirosawa, K , ( 1 9 9 8 ) C h a r a c t e r i z a t i o n of t h e c o i i a g e r ! i n t h e h e x a g o n a l l a t t i c e of Descen?et's m e m b r a n e : I t s r e l a t i o n t o t - y p e ' J I r 1 c o l l a g e n . J . C e l l B i o l . Us,
m,
219--127 24. P i c r g e s o ~ ~X.E. , (1988) New c o l l a g e n s , n e x c:oncl.:pts. A n n . R e v . C e l l B i o l . 4, c~\ --cJ7;, 25. i p s o n , 1 .K., S p u r r - - M i c h a u d , s . J . , a n d &isdale, A.S. ( 1 9 8 8 ) Hemidesmosomes a n d a n c h o r i n g f i b r i l c o l l a g e n appear s y n cii r cn ou s I y d u r i n g d e v e 1opme n t a n d womd h e , . i i . i n q . D e v e l . B i o l . 126, 2 5 3 -
Curr Eye Res Downloaded from informahealthcare.com by Chulalongkorn University on 01/02/15 For personal use only.
i-
26. W o e s s n e r , ,J.E'.
(1991) Matrix inecai lopco::.einases a n d t h e i r lnhibitors i n connective t i s s u e r e m o d e l l irig. F A S E B J . 5, 2 1 4 5 - 2 1 5 4 . 2 s . B r i n c k e r h o f f , C.E. a n d F i n i , M . E . (1989) Molecular c l o n i n g o f c o i l a g e n a s e a n d a c t i v a t o r / strome l y s i n , members o f a c o o r d i n a t e l y r e g u l a t e d gene f a r n i l y of m e t a l l o p r o t e i n a s e s i m p o r t a n t : i.n t h e m o d u l a t i o n o f corinecLive t i s s u e metabolism. I n "Zol.iagen: Volume I V , M o l e c u l a r Bioloqy" (Eds. Olsen, B. and Nimmi, M . E . ) p p 6 5 - 8 6 . CRC P r e s s , Boca P l u c i n s k a , I . M . , Mayer, R. H. and Brinckerhoff , C.E. (1987) A g e n e f o r r a b b i t s y n o v i a l c e l l z o l i a q e n a s e : member of a f a m i l y of m e t a l l . o p r o t e i n a s e s w h i c h d e g r a d e s t-h e C O I - ~ I E ' C Ki v e t i s s u e m a t r i x . Ejochemistry, 6156-6164. 29. Erowri, D . , Chwa M . , E s c o r b a r , M . a n d K e n n e y , M.C. (1991) C h a r a c t e r i z a t i o n of t h e r r a j o r m a t r i x d e g r a d i n g m e t a l l o p r o t e i n a s e of human c o r n e a l stroina: evidence f o r an enzyine/inhihitor complex. E x p . Eye I
s,
z,
Re$. 2,5-16. Matsskmr,, M., Z i e s k e , J . a n d F i n i , M.E. ( i 9 9 1 ) Mechanism o f b a s e m e n t membrane d i s s o l u t i o n p r e c e d i n g c o r n e a l u1.cci a r l o n . I n v e s t . O p h t h a l m o l . V i s . S C ~ .2,9 2 - 1 0 7 . F.ini, M . E . , Girard, M.T. a n d Matsubara, M . (1992) i70 l 1a q e n c 1y t. i c / g e 1a t i n o 1y t i c e n z yme s i n c o r n e a l wound h e a l l n g . A c t a O p h t h a l m o l . ( s u p p l ) , 24, 2 6 - 3 3 . Al.e:.:ander, I: . J . , S a m p l e s , J . R . , Van B u s k l r k , E.M. a n d A c o t t , T . S ( 1 9 9 0 ) Expression of matrix metalloproteinase a n d I n L i t i i r o r b y human r e t i n a l p i g m e n t . Ophthalmol. V i s .
862
33. A l e x a n d e r , P . J . , B r a d l e y , J . M . B .
,
Gabourel, J.D. and A c o t t , T . S . (1991) Expression of matrix metalloproteinase a n d i n h i b i t o r b y human t r a b e c u l a r meshwork. I n v e s t . O p h t h a l m o l . V i s . S c i . 2,1 7 2 - 1 8 0 . 3 4 . S a w a g u c h i , S . , T w i n i n g , S . S . , Yu e , B . Y.J . T . , W i l s o n , P . M . , S u g a r , J . a n d Chan, S.-K. (1990) A l p h a - 1 p r o t e i n a s e i n h i b i t o r levels i n keratoconus. Exp. E y e R e s . 54, 5 4 9 - 5 5 4 . 3 5 . Yue, B . Y . J . T . , L o u s i n , G . , S a w a g u c h i , S . , Twining, S.S. and Sugar, J . (1992) levels i n Alpha-2-macroglobulin keratoconus. I n v e s t . Ophthalmol. V i c . S c i . U u p p l), 9 7 6 .
