Synthesis of extracellular matrix components by human ciliary muscle cells in culture

V o l u l l l e I I I111I1111CI 4 I Y Y 7 . 333-341

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Synthesis of extracellular matrix components by human ciliary muscle cells in culture

E. Tamm. A . Baur and E. Lii[.jen-Drecoll

1)cpartmcnt 01' Anatomy. University ol' lirlangen-Nurnbcrg, Krankenhausstrasse 9, 8520-Erlangen, Germany

ABSTRACT

The production and s p a t i a l o r g a n i z a t i o n o f connective t i s s u e components i n c i l i a r y muscle c e l l c u l t u r e s was s t u d i e d w i t h immunohisto- chemical and u l t r a s t r u c t u r a l methods. A n t i b o d i e s against collagen types I V and V I , f i b r o n e c t i n and

l a m i n i n were used.

Laminln s t a i n s as p e r i c e l l u l a r network surrounding i n d i v i d u a l muscle c e l l s . Type I V collagen shows p o s i t i v e cytoplasmic s t a i n i n g and o n l y small f o c i o f e x t r a c e l l u l a r immuno-

fluorescence. S t a i n i n g f o r type V I c o l l a g e n and f i b r o n e c t i n i s seen near t h e ends o f t h e b i p o l a r c e l l s , w h i l e t h e l a t e r a l s i d e s o f t h e c e l l s remain unstained.

muscle c e l l s are surrounded by an incomplete basal lamina. I n a d d i t i o n , bundles o f 5-20 nm t h i c k e x t r a c e l l u l a r m i c r o f i b r i l s a r e seen. The bundles are o r i e n t e d p a r a l l e l t o t h e a x i s o f t h e c e l l s and are i n c l o s e c o n t a c t w i t h t h e c e l l membrane i n areas where membrane-bound dense bands are formed.

Immunoelectronmicroscopy

i n d i c a t e s t h a t t h e bundles c o n t a i n f l b r o n e c t i n and type V I c o l l a g e n f i b r i l s . While t h e

f i b r o n e c t i n f i b r i l s approach t h e c e l l membrane d i r e c t l y , type V I c o l l a g e n f i b r i l s are u s u a l l y separated from t h e c e l l membrane by f i n e

f i b r i l l o u s m a t e r i a l o f d i f f e r e n t nature. Q u a l i t y and s p a t i a l o r g a n i z a t i o n o f t h e e x t r a c e l l u l a r m a t e r i a l i n c i l i a r y muscle c e l l c u l t u r e s shows marked s i m i l a r i t i e s w i t h t h e e x t r a c e l l u l a r m a t r i x o f c i l i a r y muscle i n s i t u .

Electronmicroscopy shows t h a t c u l t u r e d c i l i a r y

INTRODUCTION

A c o n s i s t e n t f i n d i n g i n o l d human eyes

is

t h e marked increase i n h y a l i n i z e d connective t i s s u e between t h e c i l i a r y muscle bundles ( 1 - 4 ) . The increase c o r r e l a t e s w i t h t h e onset o f presbyopia and might w e l l have i n f l u e n c e on t h e f u n c t i o n o f t h e c i l i a r y muscle i n accommodation. Moreover, t h e a n t e r i o r p a r t o f t h e c i l i a r y muscle e x h i b i t s an age-related increase i n "plaque m a t e r i a l " , which i s even more pronounced i n eyes w i t h primary open angle glaucoma ( 5 ) . Recently we

e s t a b l i s h e d and c h a r a c t e r i z e d several c e l l l i n e s d e r i v e d from human c i l i a r y muscle ( 6 ) . I t i s c l e a r t h a t i n v i t r o procedures p r o v i d e a means o f s t u d y i n g t h e s y n t h e t i c c a p a b i l i t i e s o f c i l i a r y muscle c e l l s and t h e f a c t o r s which i n f l u e n c e these a c t i v i t i e s .

I n a p r e v i o u s study we have demonstrated t h a t f i b r o n e c t i n , l a m i n i n , and t y p e I V c o l l a g e n form l a r g e p a r t s o f t h e e x t r a c e l l u l a r m a t r i x o f t h e human c i l i a r y muscle ( 7 ) . I n a d d i t i o n , t y p e V I c o l l a g e n i s present i n considerable amounts i n t h e "plaque m a t e r i a l " i n t h e r e g i o n o f t h e a n t e r i o r i n s e r t i o n o f t h e c i l i a r y muscle (7,8).

I n t h e present study we i n v e s t i g a t e d p r o d u c t i o n and s p a t i a l o r g a n i z a t i o n o f c o l l a g e n types I V and V I , l a m i n i n and f i b r o n e c t i n i n c i l i a r y muscle c e l l c u l t u r e s .

MATERIAL AND METHODS

C i l i a r y muscle c e l l c u l t u r e s were e s t a b l i s h e d from 10 human donors (16-91 years). The immunocytochemical and u l t r a s t r u c t u r a l c h a r a c t e r i z a t l o n o f t h e c u l t u r e s has been

described i n d e t a i l elsewhere ( 6 ) . I n b r i e f , t h e c u l t u r e d c e l l s showed t h e t y p i c a l growth p a t t e r n o f c u l t u r e d smooth muscle c e l l s , s t a i n e d f o r smooth muscle s p e c i f i c a - a c t i n and desmin, and e x h i b i t e d u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s o f c i l i a r y muscle c e l l s . A p p l i c a t i o n o f 10 mmol/l BaCls t o t h e c u l t u r e d c i l i a r y muscle c e l l s r e s u l t e d i n a membrane d e p o l a r i z a t i o n w i t h superimposed a c t i o n p o t e n t i a l s ( 9 ) .

The c e l l s were maintained a t 37'C i n a 53 C 0 2 athmosphere. C u l t u r e medium (medium 199

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supplemented wlth 10% f e t a l c a l f serum, 50 U m l - l penicillin, 50 pg m l - 1 streptomycin and 50 u9

m l - 1 ascorblc acid, a l l Gibco L t d , P a l s l e y , Scotland) was exchanged t w l c e a week. Confluent c e l l s were passaged a t a r a t i o o f about 1:4 u s l n g trypsln-EDTA. For t h e present study 3 r d t o 6th- passage c u l t u r e s , one week a f t e r confluency were I n v e s t i g a t e d .

Antlbodv s t a i n i n g

C e l l s were seeded I n t i s s u e c u l t u r e chambers mounted on PermanoxR m l c r o s l l d e s (Lab Tek*, Nunc

Inc., N a p e r v l l l e , I L ) . C u l t u r e medium was removed by r l n s i n g t h r e e tlmes w l t h phosphate-buffered s a l i n e (PBS). The c e l l s were f l x e d w l t h

ethanol/ether ( 1 : l ) a t -2O'C f o r 3 mln. The c e l l s were again r i n s e d t h r e e tlmes w l t h PBS and then Incubated f o r 90 mln a t room temperature wlth t h e prlmary antlbody. For demonstration o f t y p e I V collagen, monoclonal mouse a n t i b o d i e s from Heyl, B e r l l n , Germany (antl-human IgH, Kappa) and from Dakopatts, Hamburg, Germany (anti-human, I g G l Kappa, CIV22,) (10) were used, b o t h a t a d i l u t i o n of 1:lOO. For l a m i n l n p o l y c l o n a l r a b b l t a n t l - human a n t l b o d l e s from Heyl (11) were a p p l i e d t o t h e c u l t u r e chambers a t a d i l u t l o n o f 1:lO.

Demonstration o f t y p e V I c o l l a g e n was performed using a monoclonal mouse antlbody (anti-human, I g l , Kappa) and p o l y c l o n a l r a b b i t anti-human antlbodles, (both from Hey11 (12) a t a d i l u t i o n of 1:50. F i b r o n e c t l n was s t a i n e d wlth a

monoclonal mouse antibody against c e l l u l a r f i b r o n e c t l n (Sigma Chemical Company, S t . Louis MO, clone no. FN-3E2). T h l s product l o c a l i z e s t h e 240 KD bands o f c e l l u l a r f l b r o n e c t i n . The c e l l s were r i n s e d t h r e e tlmes w l t h PBS then Incubated f o r another 60 min w l t h f l u o r e s c e i n - l a b e l l e d r a b b l t anti-mouse IgG o r sheep a n t l - r a b b i t IgG ( b o t h Dakopatts) d i l u t e d I n PBS (1:20). The c e l l s were washed again t h r e e tlmes w i t h PBS, then t h e t l s s u e chambers were removed from t h e s l l d e s . The s l l d e s were mounted I n g l y c e r o l c o n t a i n i n g 2.5%

1,4-dlazobicyclo-octane (Herck, Darmstadt, Germany). Control experlments were performed u s l n g e l t h e r PBS, mouse o r r a b b l t p r e - l m u n e serum Instead o f t h e prlmary antlbody. Stalned

c u l t u r e s were vlewed wlth a L e l t z AriStOplan photomlcroscope ( E r n s t L e l t z GmbH, Wetzlar, Germany) equipped w l t h eplfluorescence o p t i c s and a p p r o p r l a t e f l l t e r s . A Kodak 1-max 400 b l a c k and w h l t e f l l m (Kodak L i m i t e d , Hempstead, England) was used f o r photography.

E~ectronmlcroscoDy

The c e l l s were grown I n uncoated, p l a s t i c p e t r l dlshes o r I n t l s s u e c u l t u r e f l a s k s and f l x e d w l t h

I t o ' s s o l u t i o n (13) f o r 4 hr. C e l l s were p o s t f i x e d w i t h 1% osmlum t e t r o x l d e , dehydrated w l t h graded a l c o h o l s and embedded I n Epon.

Tangential and p e r p e n d i c u l a r s e c t i o n s o f t h e C e l l s were c u t on an ultramlcrotome. The s e c t i o n s

F l g u r e 1: Confluent c l l l a r y muscle c e l l c u l t u r e s c h a r a c t e r i s t i c a l l y c o n s i s t o f l o n g l t u d l n a l and s l l g h t l y curved bands o f p a r a l l e l b l p o l a r c e l l s (donor age 16 years, 3 r d passage, phase-contrast micrograph, x 250, bar: 40 pm).

2

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were contrasted w i t h lead c i t r a t e and u r a n y l incubated f o r 8 h r s i n a 1:20 d i l u t i o n o f goat acetate and viewed u s i n g a Zeiss (EM 902) anti-mouse ( 1 0 nm, Sigma) o r goat a n t i - r a b b i t

electronmicroscope. immunogold ( 5 nm, Sigma), 1% BSA and protease

For immunoelectronmicroscopy, c e l l s were f i x e d i n h i b i t o r s . Unbound antibody was removed by t h r e e i n phosphate-buffered

paraformaldehyde-lysine-

30 min changes i n PBS. The c e l l s were then f i x e d p e r i o d a t e (PLP) ( 1 4 ) s o l u t l o n a t room temperature w i t h 2% g l u t a r a l d e h y d e f o r 20 min, washed i n PBS f o r 3 min. A f t e r washing t h r e e times 10 min with and embedded i n Epon as descrlbed above.

PBS, t h e c e l l s were incubated w l t h t h e primary

a n t i b o d i e s ( f i b r o n e c t i n , t y p e V I c o l l a g e n ) f o r 8 RESULTS

h r s a t 4'C. The antibody s o l u t i o n contained 1% Immunohistochemlstrv

bovine serum albumln (BSA) and v a r i o u s protease Confluent c i l i a r y muscle c e l l c u l t u r e s

i n h i b i t o r s ( 1 5 ) . A f t e r t h r e e 30 min changes i n c h a r a c t e r i s t i c a l l y c o n s i s t o f l o n g i t u d i n a l and PBS t o remove unbound antibody, t h e c e l l s were s l i g h t l y curved bands o f p a r a l l e l b i p o l a r c e l l s

Figure 2: Immunofluorescence o f a c o n f l u e n t c i l i a r y muscle c e l l c u l t u r e (same donor as i n Fig. 1, 3rd passage). A: S t a i n i n g f o r l a m i n l n

reveals a p e r l c e l l u l a r network which o u t l i n e s t h e

f u s i f o r m shape o f t h e c e l l s . B: Type I V c o l l a g e n shows p o s i t l v e cytoplasmic s t a i n i n g (arrowheads) and o n l y small f o c i o f p o s i t i v e e x t r a c e l l u l a r

immunofluorescence (arrows). (x 300, bar: 33

w).

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Figure

3:

Imunofluorescence of a confluent unstained.

C :

Staining for fibronectin shows a ciliary muscle cell culture (same culture as in

Fig. 2 ) . A:

Staining for type V I collagen

i s

VI collagen, but appears to be more pronounced confined to the ends of the bipolar cells,

( x 300,

bar:

33

m).

whereas the lateral sides of the cells remain

similar spatial organization as staining for type

(Fig.

1).

Staining of confluent cultures for laminin (Fig.

2 A )

shows a well established pericellular network which outlines the fuslform shape of the muscle cells. Immunofluorescent staining to demonstrate type IV collagen (Fig.

26)

shows only small foci of extracellular fluorescence. The cells exhibit, however, an intense posltlve cytoplasmic staining. Type VI collagen

I s

confined to the ends of the bipolar cells, whereas the lateral sides of the cells remain unstained (Fig.

3 A ) .

Staining for

fibronectin shows a similar spatial organization as staining for type VI collagen, but appears to

be

more pronounced

(Fig. 3 8 ) .

Electronmicroscow

A t

confluency, the ciliary muscle cells are surrounded by an incomplete basement membrane

(Fig.

4 A ) .

In addition, the cultures show a matrix of extracellular fine fibrils which appear to be aggregated into loose bundles. The fibrils measure

5-20

nm in cross sections and show no apparent evidence of periodicity. Most

o f

this

.

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Figure 4: Electronmicrographs o f c u l t u r e d c i l i a r y muscle c e l l s (Same donor as i n F l g . 1 , 3 r d passage). A: A t confluency t h e c e l l s a r e

surrounded by an Incomplete basal lamina (arrow).

M. Myofllaments ( x 60000, bar: 1 6 , 6 nm). 8. Near t h e ends o f t h e c e l l s e x t r a c e l l u l a r m i c r o f i b r i l s

a r e observed (arrows). The f i b r l l s a r e i n c l o s e c o n t a c t w i t h t h e c e l l membrane i n areas where dense bands a r e formed (arrowheads). The o r i e n t a t i o n o f t h e f i b r i l s i s p a r a l l e l t o t h e myofllaments

(M)

w i t h i n t h e c e l l s ( x 8800, bar:

1 . 1 3 p n ) .

m a t e r l a l I s present near t h e elongated ends o f t h e b i p o l a r muscle c e l l s (Fig. 4 8 ) . Here, t h e o r i e n t a t l o n o f t h e e x t r a c e l l u l a r f i b r l l s i s p a r a l l e l t o t h e a x i s o f t h e i n d i v i d u a l muscle c e l l s and t h e r e f o r e a l s o p a r a l l e l t o t h e

myofllaments w i t h i n t h e c e l l s . The e x t r a c e l l u l a r f i b r i l s are I n c l o s e contact w i t h t h e membrane o f t h e muscle c e l l s . I n these areas o f c o n t a c t , t h e c e l l membrane forms dense bands with adhering myofilaments.

With

immunoelectronmicroscopy,

i n t e n s e

l a b e l l i n g f o r f i b r o n e c t i n i s seen throughout t h e

whole e x t r a c e l l u l a r f i b r i l l a r m a t e r i a l (Fig. 5 ) . The f i b r o n e c t i n p o s l t l v e f i b r i l s come I n c l o s e c o n t a c t w i t h t h e c e l l membrane o f t h e muscle c e l l s . I n a d d l t i o n t o f i b r o n e c t l n , f i b r i l s which are p o s i t i v e l y l a b e l l e d f o r t y p e V I c o l l a g e n are present i n t h e bundles o f e x t r a c e l l u l a r f i b r l l s near t h e ends o f t h e muscle c e l l s . I n c o n t r a s t t o t h e f i b r o n e c t i n f l b r i l s , however, t h e t y p e V I c o l l a g e n f i b r i l s o n l y o c c a s s l o n a l l y approach t h e muscle c e l l membrane d i r e c t l y , b u t are u s u a l l y

separated from t h e c e l l s by u n l a b e l l e d f i n e f i b r i l l a r m a t e r i a l o f d i f f e r e n t n a t u r e (Fig. 6 ) .

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Figure

5:

Imunoelectronmicroscopy of cultured extracellular fibrillar materlal (arrowheads).

ciliary muscle cells (same donor as in Fig. 1, The flbronectin positive fibrils come in close 3rd passage). Intense imnunogold labelling for contact with the cell membrane (arrows) (x

48000,

fibronectin

i s

seen throughout the whole bar: 210 nm).

There were no obvious qualitative changes in culture

i s

incomplete. Formation of an incomplete extracellular matrix production between cultures basal lamina, however,

i s

regarded as typical for derived from donors of dlfferent ages. cultured viszeral and vascular smooth muscle

cells (20-24).

DISCUSSION In addition, Immunostaining Indlcates that

Ciliary muscle cells in culture produce

considerable amounts of extracellular fibrillar extracellular flbrlllar materlal synthesized by material. The cultured cells are surrounded

b y

cultured cillary muscle cells. The fibronectin-

lamlnln and type

XV

collagen, both macromolecules labelled fibrils approach the ciliary muscle cell essential for basement membranes (16,171. In membrane directly. Indeed, it has been shown that contrast to ciliary muscle cells in situ (18,19), flbronectln mediated cell adhesion occurrs via the basement membrane of ciliary muscle cells in cell surface proteins (25,261. In tissue culture

fibronectln

i s

a major component of the

C

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Figure 6: Innnunoelectronmicroscopy o f c u l t u r e d t y p e V I c o l l a g e n f i b r i l s o n l y o c c a s s i o n a l l y c l l i a r y muscle c e l l s (same donor as i n F i g . 1, aproach t h e c e l l membrane d i r e c t l y (arrowheads), 3 r d passage). Immunogold l a b e l l i n g f o r t y p e V I b u t a r e u s u a l l y separated from t h e c e l l ( C ) by collagen I s a l s o seen i n t h e e x t r a c e l l u l a r u n l a b e l l e d f i n e f l b r l l l a r m a t e r i a l o f d i f f e r e n t f l b r l l s near t h e ends o f t h e muscle c e l l s . The n a t u r e (arrow) ( x 56000, bar: 180 nm).

c o n d i t i o n s , analogous b u t s m a l l e r transmembrane form apparent c l o s e transmembrane a s s o c i a t i o n s a s s o c i a t l o n s which were termed " f i b r o n e x u s " have w l t h t h e cytoplasmic myofllaments and may

be

been described between f i b r o n e c t l n - c o n t a i n i n g regarded as m i n i a t u r e muscle c e l l - t e n d o n f i b r i l s and t h e i n t r a c e l l u l a r s t r e s s f i b e r s o f j u n c t i o n s which p r o v i d e an anchoring f u n c t i o n . hamster and human f i b r o b l a s t s ( 2 7 ) . I n s l t u , These s t r u c t u r e s c o n t a i n n o t o n l y f i b r o n e c t l n - these flbronexus have been demonstrated a t t h e l a b e l l e d f i b r i l s , b u t a l s o t y p e V I collagen. I n surface o f m y o f l b r o b l a s t s i n g r a n u l a t i o n t i s s u e c o n t r a s t t o t h e f i b r o n e c t i n f i b r i l s , t y p e V I

( 2 8 ) . c o l l a g e n f i b r i l s a r e u s u a l l y separated from t h e

muscle c e l l membrane by f i n e f l b r l l l a r m a t e r i a l The e x t r a c e l l u l a r f l b r l l s synthesized by

c u l t u r e d c i l i a r y muscle c e l l s are c h a r a c t e r i s t l - o f d i f f e r e n t nature. E l e c t r o n m i c r o s c o p i c a l l y , we c a l l y deposited i n t h e r e g i o n o f t h e elongated

ends o f t h e b i p o l a r muscle c e l l s . The f i b r i l s i n d i v i d u a l c i l i a r y muscle c e l l s i n s t t u (19) and r e c e n t l y described s i m i l a r s t r u c t u r e s between t h e

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suggested t h a t these connections between muscle c e l l s and surrounding stroma may p l a y an

important r o l e i n m a i n t a i n i n g mechanical t e n s i o n and supporting t h e shape changes o f c i l i a r y muscle d u r i n g accormodation.

The q u a l i t y and s p a t i a l o r g a n i z a t i o n o f t h e e x t r a c e l l u l a r m a t e r i a l which i s produced by c u l t u r e d c i l i a r y muscle c e l l s show marked s i m i l a r i t i e s w i t h t h e e x t r a c e l l u l a r m a t r i x o f c i l i a r y muscle i n s l t u . T h i s i n d i c a t e s t h a t t h e c e l l s have maintained some o f t h e i r d l f f e r e n t l a - t e d f u n c t i o n s . I n s i t u , type V I c o l l a g e n i s n o t o n l y found between t h e i n d i v i d u a l c i l i a r y muscle c e l l s , b u t i s a l s o a major component o f t h e a n t e r i o r tendons o f t h e c i l i a r y muscle, which i n s e r t t o t h e t r a b e c u l a r meshwork ( 7 ) . The type V I collagen f i b r i l s form a sheath which surrounds these a n t e r i o r e l a s t i c tendons ( 7 , 8 ) . With age, t h e sheath t h i c k e n s s i g n i f i c a n t l y , thereby forming p a r t o f t h e "plaque m a t e r i a l " ( 5 ) . Q u a n t i t a t i v e s t u d i e s have shown t h a t both i n t h e t r a b e c u l a r meshwork ( 2 9 ) and i n t h e a n t e r i o r p a r t o f t h e c i l i a r y muscle ( 5 1 , t h e amount o f t h i s type V I c o l l a g e n c o n t a i n i n g "plaque m a t e r i a l " i s s i g n i f i c a n t l y increased i n eyes with primary open angle glaucoma.

I m u n o h i s t o c h e m i c a l l y , we d i d n o t observe obvious q u a l i t a t i v e changes between c u l t u r e s d e r i v e d from donors o f d i f f e r e n t ages, b u t q u a n t i t a t i v e measurements have s t i l l t o be done.

We t r u s t t h a t t h i s c e l l c u l t u r e model provides an important t o o l f o r such q u a n t i t a t i v e s t u d i e s on c o n t r o l mechanisms modulating t h e p r o d u c t i o n o f e x t r a c e l l u l a r m a t r i x by c i l i a r y muscle c e l l s . These s t u d i e s might be h e l p f u l i n c l a r i f y i n g t h e reasons f o r t h e increase i n e x t r a c e l l u l a r

m a t e r i a l i n t h e c i l i a r y muscle i n o l d and glaucomatous eyes.

ACKNOWLEDGEMENTS

We would l i k e t o thank J u t t a Gehr f o r her e x p e r t assistance w i t h t i s s u e c u l t u r e s . We would a l s o

l i k e t o thank Simone K l e l n f o r h e r e x c e l l e n t h e l p i n electronmicroscopy and Marc0 G6Rwein f o r h i s i n v a l u a b l e p r e p a r a t i o n o f t h e photographs. The

study was supported by g r a n t Dre 124/6-1 from t h e Deutsche Forschungsgemeinschaft.

CORRESPONDING AUTHOR

D r . E r n s t Tamm, Anatomisches I n s t i t u t der

U n i v e r s i t l t Erlangen-NOrnberg, Krankenhausstr. 9 , 8520 Erlangen, Germany.

REFERENCES 1 .

2 .

3.

4.

5.

6.

7 .

a.

9.

10.

11.

Fuchs, E. (1928) uber den Z i l i a r m u s k e l . A l b r e c h t v. Graefes Arch. Ophthalmol.

120,

S t i e v e , R. ( 1 9 4 9 ) uber den Bau des menschlichen C i l i a r m u s k e l s , seine

physiologischen Verlnderungen wlhrend des Lebens und seine Bedeutung f u r d i e

Akkomnodation. 2 . mikroskop. anatom. Forsch.

- 5 5 , 3-88.

van der Zypen, E. ( 1 9 7 0 ) Licht-und

elektronenmikroskopische

Untersuchungen uber d i e Altersverlnderungen am M. c i 1 i a r i s i m menschlichen Auge. A l b r e c h t v. Graefes Arch.

K l l n . Exp. Ophthalmol.

179,

332-357.

T a m , S . , Tamm, E. and Rohen, J.W. ( 1 9 9 2 ) Age-related changes o f t h e human c i l i a r y muscle. A q u a n t i t a t i v e morphometric study.

Mech. Ageing Develop. i n press.

LOtjen-Drecoll, E., Shimizu, T . , Rohrbach,

M.

and Rohen, J.W. ( 1 9 8 6 ) Q u a n t i t a t i v e a n a l y s i s o f "plaque m a t e r i a l " between c i l i a r y muscle t i p s i n normal and

glaucomatous eyes. Exp. Eye Res. 42, 457- 465.

Tam, E., F l t i g e l , C., Baur, A . and L u t j e n - D r e c o l l , E. ( 1 9 9 1 ) C e l l c u l t u r e s o f human c i l i a r y muscle: Growth, u l t r a s t r u c t u r a l and immunocytochemical c h a r a c t e r i s t i c s . Exp. Eye Res. 53, 375-387.

R i t t i g ,

M.,

L t i t j e n - D r e c o l l , E., Rauterberg, J . , Jander, R. and Mollenhauer, J. ( 1 9 9 0 ) Type V I c o l l a g e n i n t h e human i r i s and c i l i a r y body. C e l l Tissue Res.

259,

305-312.

LOtjen-Drecoll, E., R i t t i g ,

M.,

Rauterberg, J., Jander, R. and Mollenhauer, J. ( 1 9 8 9 ) Immunomicroscopical study o f t y p e V I c o l l a g e n i n t h e t r a b e c u l a r meshwork o f normal and glaucomatous eyes. Exp. Eye Res.

S t a h l , F., Lepple-Wienhues, A., Kupplnger,

M.,

Tamm, E. and Wiederholt, M. ( 1 9 9 2 ) E l e c t r o g e n i c sodium-bicarbonate c o t r a n s p o r t i n human c i l i a r y muscle c e l l s . Am. J.

P h y s i o l . i n press.

Odermatt, B.F., Lang, A.B., ROttner, J.W., W i n t e r h a l t e r , K.H. and Trileb, B. ( 1 9 8 4 ) Monoclonal a n t i b o d i e s t o human type I V c o l l a g e n : u s e f u l reagents t o demonstrate t h e h e t e r o t r i m e r i c n a t u r e o f t h e molecule. Proc.

N a t l . Acad. Sci. E l l , 7343-7347.

Becher, J., Schuppan, D., Hahn, E.G., A l b e r t , G. and R e i c h a r t , P. ( 1 9 8 6 ) The

immunohistochemical d i s t r i b u t i o n o f

c o l l a g e n s t y p e I V , V, V I and o f l a m i n i n i n 733-741.

- 4 8 , 139-147.

Research Eye

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

the human o r a l mucosa. Arch. Oral. 6101.

1,

179-186.

Schuppan, D., Rtihlmann, T. and Hahn, E.Q.

(1985) Radioimunoassay f o r human type V I collagen and i t s a p p l i c a t i o n t o t i s s u e and body f l u i d s . Annal. Biochem.

149,

238-247.

I t o , S. and Karnovsky, H.J. (1968)

Formaldehyde-glutaraldehyde

f i x a t i v e s

containing t r i n i t r o compounds. J. C e l l 6101. 28.

-

39, 168a-169a.

McLean, I . W . and Nakane, P.K. (1974)

Periodate-lysine-paraformaldehyde f i x a t i v e : a new f i x a t i v e f o r innnuno-electron

microscopy. J. Histochem. Cytochem.

22,

1077-1083.

Lutjen-Drecoll, E., Schenholm, M., Tam. E. 29.

and Tengblad, A. (1990) V i s u a l i z a t i o n o f hyaluronic a c i d i n the a n t e r i o r segment o f r a b b i t and monkey eyes. Exp. Eye Res.

51,

55-63.

Martinez-Hernandez, A. and Amenta, P.S.

(1983) The basement membrane i n pathology.

Lab. Invest. 48, 656-677.

Furthmayr, H. (1988) Basement membranes. I n

"The Molecular and C e l l u l a r Biology o f Wound Repair", (Eds. Clark, R.A.F and Henson P.M.). Pp. 525-558. Plenum Publishing Corporation, New York.

Ishikawa, T. (1962) Fine s t r u c t u r e o f t h e human c i l i a r y muscle. Invest. Ophthalmol.

1,

587-608.

Tam, E., Lutjen-Drecoll, E. and Rohen, J.W.

(1990) Age-related changes o f t h e human c i l i a r y muscle i n comparison w i t h changes induced by treatment w i t h prostaglandin Fza.

Mech. Ageing Develop.

51,

101-120.

Chamley-Campbell, J., Campbell, G.R. and Ross. R. ( 1 9 7 9 ) . The smooth muscle i n c e l l c u l t u r e . Physiol. Rev.

59,

1-67.

Rifas,

L.,

Fant, J., Makman, M.H. and S e i f t e r , S. (1979) The c h a r a c t e r i z a t i o n o f human u t e r i n e smooth muscle i n c u l t u r e . C e l l . Tissue Res.

196,

385-395.

Franke, W . W . , Schmid, E., Vandekerckhove, J.

and Weber, K. (1980) A permanently p r o l i f e r a t i n g r a t vascular smooth muscle c e l l w i t h maintained expression o f smooth muscle c h a r a c t e r i s t i c s , i n c l u d i n g a c t i n o f the vascular smooth muscle type. J. C e l l B i o l . 87, 594-600.

Thyberg, J., Nilsson, J., Palmberg, L. and Sjolund, M. (1985) Adult human a r t e r i a l smooth muscle c e l l s i n primary c u l t u r e : Modulation from c o n t r a c t i l e t o s y n t h e t i c phenotype. C e l l . Tissue Res.

239,

69-74.

Palmberg, L . and Thyberg, J. (1986) Uterine smooth muscle c e l l s i n primary c u l t u r e : A l t e r a t i o n s i n f i n e s t r u c t u r e , c y t o s k e l e t a l organization and growth c h a r a c t e r i s t i c s . C e l l . Tissue Res.

246,

253-262.

Brown, J. and Juliano, R.L. (1986)

Expression and f u n c t i o n o f a p u t a t i v e c e l l surface receptor f o r f i b r o n e c t i n i n hamster and human c e l l l i n e s . J. C e l l B i o l .

103,

Horwitz, A . , Duggan, R., Greggs, R., Decker, C. and Buck, C. (1985) The c e l l substrate

27.

1595-1603.

attachment (CSAT) antigen has p r o p e r t i e s of a receptor f o r l a m i n i n and f i b r o n e c t i n . J.

C e l l B i o l .

101,

2134-2144.

Singer, 1.1. ( 1 9 7 9 ) The fibronexus: a transmembrane a s s o c i a t i o n o f f i b r o n e c t i n - c o n t a i n i n g f i b e r s and bundles o f 5 nm microfilaments i n hamster and human f l b r o b l a s t s . C e l l ,

Is,

675-685.

Singer, I.I., Kawka, D.W., Kazazis, D.M. and Clark, R.A.F. (1984) I n v i v o c o - d i s t r i b u t i o n o f f i b r o n e c t i n and a c t i n f i b e r s i n

g r a n u l a t i o n t i s s u e : Imnunofluorescence and e l e c t r o n microscope studies o f t h e

flbronexus a t t h e myofibroblast surface. J.

C e l l Biol. 98, 2091-2106.

Ltitjen-Drecoll, E . , Shimizu, T., Rohrbach, M. and Rohen, J.W. ( 1 9 8 6 ) Q u a n t i t a t i v e a n a l y s i s o f "plaque m a t e r i a l " I n t h e inner- and o u t e r w a l l o f Schlenvn's canal i n nonnal- and glaucomatous eyes. Exp. Eye Res. 42, 443-455.