ives et al., 1986, mechanical effects on endothlial cell morphology_in vitro assessment

8/14/2019 Ives Et Al., 1986, Mechanical Effects on Endothlial Cell Morphology_In Vitro Assessment

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IN VITRO CELLULAR& DEVELOPMENTALBIOLOGYVolume 22, Num ber 9, September 19869 1986 Tissue Culture Association, nc.

M E C H A N I C A L E F F E C T S O N E N D O T H E L I A L C E L LM O R P H O L O G Y : I N V I T R O A S S E S S M E N T

C. L. IVES , S. G . ESK IN, ANDL.V . M c I N T I R E 1Department o[ Surgery, Baylor College o] Medicine, Houston, Texas 77030 (C; L. L , S. G. E.) andBiomed ical Enginee ring Labo ratory, Rice University, Houston, Texas 77251 (L. I1".M c I . )

(Received 25 Jhly 1985; accepted 13 March 1986)

SUMMARYE n d o t h e l i a l c e li s a r e s u b j e c t ed t o f lu i d m e c h a n i c a l f o r c e s w h i c h a c c o m p a n y b l o o d f l o w . T h e s e c e ll s

becom e e longa t ed and o r i en t t he i r l ong axes pa ra l l e l t o t he d i r ec t i on o f shea r s t r e s s w hen t he cu l t u r edc e l l s a r e s u b j e c t e d t o f l o w i n a n i n v i t r o c i r c u l a t o r y s y s t e m . W h e n t h e s u b s t r a t e i s c o m p l i a n t a n dcyc l i ca l l y de fo r f f ied , t o s im u la t e e f f ec t s o f p r e s su re i n t he vascu l a tu r e , t he ce l l s e l onga t e an o r i en tp e r p e n d i c u l a r t o t h e a x i s o f d e f o r m a t i o n . C e l l s h a p e c h a n g e s a r e r e f l e c t e d i n t h e a l i g n m e n t o fm ic ro tubu l e ne t~ *orks. Th e sys t em s desc r i bed p rov ide t oo l s f o r a s ses s ing t he i nd iv idua l r o l e s o f shea rs t r e s s , p r e s su re , and m echan i ca l s t r a in on vascu l a r ce l l s t r uc tu r e and func t i on .K e y w o r d s: endo the l i a l c e l l s ; com pl i ance ; shea r s t r e s s ; p r e s su re ; a l i gnm en t ; m ic ro tubu l e s .

I NTRODUCTIONB lood f l ow p roduces a t angen t i a l f o r ce { shea r s t r e s s ) on

the l um ena l su r f ace o f the ves se l w a l l . Th e pu l sa t i len a t u r e o f t h e f l o w i n a n a r t e r y , t h r o u g h i t s p r e s s u r ew a v e f o r m a c t i n g o n t h e c o m p l i a n c e o f t h e w a l l, p r o d u c e sa pe r iod i c va r i a t i on i n ves se l r ad ius , r e su l t i ng i n cyc l i cs t r e t ch ing o f t he ves se l w a l l c e l l s . The endo the l i a l c e l l sl i n ing t he ves se l w a l l a r e sub j ec t ed t o bo th f l u id shea rs t re s s a n d t h e p r e s s u r e - i n d u c e d s t r a i n c o m p o n e n t s o f th ef l ow .

M e c h a n i c a l f o r c e s h a v e b e e n p r o p o s e d a s c a u s a t i v ef a c t o r s i n c a r d i o v a s c u l a r d i s e a s e a n d h a v e b e e n i m p l i c a t -e d i n m o d u l a t i n g e n d o t h e l i a l c e ll m o r p h o l o g y a n df u n c t i o n ( 1 , 3 - 9 , 1 1 , 1 7 , 2 2 ) . H o w e v e r , i n v i v o r e s e a r c h t oe s t ab l i sh t he i r s i gn i f i cance i s l im i t ed by t he i nab i l i t y t oa c c u r a t e l y m e a s u r e h e m o d y n a m i c f a c t o r s i n t h e v i c i n i t yo f t he ves se l w a i l . I n add i t i on , t he ab i l i t y t o m on i to r andc o n t r o l a l l o f t h e a p p r o p r i a t e p h y s i c a l a n d b i o c h e m i c a lva r i ab l e s i s no t pos s ib l e a t p r e sen t i n i n v ivo m ode l s .

T h e a i m o f th i s w o r k i s t o e s ta b l i s h i n v i t ro m o d e l s i nw h i c h t h e e f f e c t s o f f l u i d m e c h a n i c a l f o r c e s o ne n d o t h e l i a l c e l l s c a n b e i n d e p e n d e n t l y s t u d i e d t ou n d e r s t a n d t h e m e c h a n i s m b y w h i c h s h e a r s t r e s s a n ds t r a in m o d u l a t e m o r p h o l o g y a n d m e t a b o l i s m o f t h e sece l ls . I n s t ud i e s desc r i bed he re in , endo the l i a l c e l l s w e re

To whom correspondence should be addressed at Department ofChemical Engineering, Rice University, P. O. Box 1892, Houston, T X77251.

g r o w n o n a c o m p l i a n t s u b s t r a t e a n d t h e n s u b j e c t e d t ow e l l - c h a r a c t e r i z e d l e v e l s o f e i th e r s h e a r s t r e s s o rm e c h n i c a l s t r a i n . T h e g o a l w a s t o d e t e r m i n e t h e s e p a r a t ee f f ec t s o f shea r s t r e s s and pu l sa t i l e s t r a in on endo the l i a lc e ll m o r p h o l o g y .

M A TER IA LS A N D M ETH O D SC el l cu l t u re . H u m a n u m b i l i c a l v e i n e n d o t h e l i a l c e l l s

( H U V E C ) w e r e h a r v e s t e d f r o m u m b i l i c a l c o r d s o b t a i n e df r o m L a b o r a n d D e l i v e r y , M e t h o d i s t H o s p i t a l . T h e c o r d sw e r e k e p t a t r o o m t e m p e r a t u r e a n d u t i l i z e d w i t h i n 3 t o 4 ho f d e l i v e r y . C u l t u r e p r o c e d u r e s w e r e a d a p t e d f r o m t h o s eo f G i m b r o n e { 1 0 ) . T o r e m o v e t h e e n d o t h e l i a l c e ll s , t h ev e i n s w e r e c a n n u l a t e d , r i n s e d w i t h 5 0 m l o f p h o s p h a t ebu f f e r ed s a l i ne (PB S) , and t hen f i l l ed w i th 0 . 03? /0c o ll a g en a s e i n M e d i u m 1 99 ( G I B C O , G r a n d I s l a n d , N Y )a n d i n c u b a t e d f o r 2 0 m i n . A f t e r in c u b a t i o n t h e e n z y m es o l u t i o n w a s f l u s h e d t h r o u g h t h e c o r d w i t h 4 0 m l o f P B S ,t h e e f f l u e n t w a s c o l l e c t e d a n d c e n t r i f u g e d a t I 0 0 0 r p m f o rI0 m in . A f t e r cen t r i f uga t i on , t he ce l l pe l l e t w asr e s u s p e n d e d i n M e d i u m 1 9 9 , s u p p l e m e n t e d w i t h 2 0 %f e ta l b o v i n e se r u m ( F B S , H y C l o n e , L o g a n U T ) , I 0 0 U / m lp e n i ci ll i n a n d I 0 0 ~ g / m l s t r e p t o m y c i n . B e t w e e n 7 . 4 X104 and 1 . 5 X 104 ce l l s / cm 2 w ere s eeded o n to t hes u b s t r a t e f o r e x p e r i m e n t s .

B o v i n e a o r t i c e n d o t h e l i a l c e l l s ( B A E C ) w e r e o b t a i n e dus ing t echn iques p r ev ious ly desc r i bed (6 ) . The ce l l s w e rec l o n e d a n d u s e d i n t h e 4 t h t h r o u g h t h e 7 t h p a s s a g e . F o ra n e x p e r i m e n t , c e l l s f r o m a T - 2 5 f l a s k w e r e d e t a c h e d ,u s i n g 0 . 2 5 % t r y p s i n in 1 : 50 0 0 E D T A , c e n t r i f u g e d a t I 0 0 0r p m f o r I 0 m i n , a n d t h e p e l l e t r e s u s p e n d e d i n c o m p l e t e

5 0 0


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MECHANICAL EFFECTS ON CELLS 501

FIG. 1. Schematic drawing of the stretch chambe r. I n chamber 1, the compliant memb rane experiences the samefluid motion as in experimental chamber 2, but it is not stretched. Fo r details, se e text.

FI(~. 2. Bovine aortic endothelial cells cultured on Mitr athan e for 3 d -- th en sub jected to 66 h of a, movement of themedium over the cells ~control) and b, 10% cyclic stretching of the Mitrath ane at 1H z. Axis of stretch is horizontal.XI20.


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502 IVES ET AL.

FIG. 3. Human umbil ical vein endothel ial cel ls (primary) cul tured on Mitrathane for 3 d then subjected to 48 h of a,mov eme nt Of the me dium over the cells and b , 10% cycl ic st retching of the Mitr atha ne at 1 Hz, Axis of st retch ishorizontal . X 120.

FIG. 4. Histogra m of cel ls in Fig. 3 b shows dist ribut ion of angles made by the cel ls ' max imum dimensions wi th theaxis of st retch, defined as 0 to 180 ~ The cel ls al igned most freque nt ly 90~ away from the di rect ion of st retch. The cel lswere photographed wi th t he me mbrane re l axed .


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M E CHANICAL E F F E CT S ON CE L LS 503

medi um {Dul becco ' s modi f i ca t i on o f E ag l e ' s medi umwi t h 10% F BS and an t i b i o t i c s ) . Be t ween 3 .0 X 104 and 5 .9X 104 ce lls/ cm 2 were seeded on t o t he subs t r a t e .Substrate. F or t he shea r s t r e s s s t ud i es , po l ye t he rure -t h a n e u r e a { M i t r at h a n e R , s u p p l i ed b y M i t r a l M e d i c a l ,Denver , CO) was so l ven t cas t on t o a 3 .81 by 7 .64-cm g l as ss l i de (F i she r S c i en t i f i c , P i t t sburg , P A) . F or t he ax i a ls t r a i n s t ud ies , t he M i t r a t h ane R wa s s i mi l a r l y cas t , bu tthe f i lm was removed f rom the glass and used as a 200- /~mt h i c k m e m b r a n e . M i t r a t h a n e R i n t h i s f o r m w a st r anspa ren t , smoot h , compl i an t , and hydrophi l i c .Stretch experiments. T he appara t us fo r s t r e t ch i ng t hecel l s resem bled th at of L eung et a l . {13). I t consis t s of ap o l y e a r b o n a t e c h a m b e r d i v i d e d i n t o t w o c o m p a r t m e n t s{F i g. 1 ). I n t he exper i men t a l com par t m ent ( c hamber 2 ) ,t he subs t r a t e was mount ed wi t h c l amps a t e i t he r end o ft he cham ber so t ha t i t was pos i t i oned c l ose to (w i t h i n 1mm) and pa ra l l e l t o t he f l oor o f the chamb er . I n t hec o n t ro l c o m p a r t m e n t ( c h a m b e r 1 ), t h e m e m b r a n e w a sf i xed t o t he bo t t om of t he chamber be t ween t he c l amps .A f t e r m o u n t i n g t h e m e m b r a n e s i n th e c o m p a r t m e n t s , t h echamber was au t oc l aved . Ce l l suspens i ons {approx i mat e -ly 1 X 10~ ce l ls i n 2 ml ) were seeded on t o t he m emb ranei n each compar t ment and a l l owed t o a t t ach fo r 30 mi n .T h e n , 1 3 m l of c o m p l e te m e d i u m w a s a d d e d t o e a c hc o m p a r t m e n t a n d t h e c h a m b e r p l a c e d i n t h e i n c u b a t o r .Af ter 3 to 4 d in s ta t ion ary cul ture , when the cel l s hadreached conf l uence , t he chamber was r emoved f rom t he

i ncuba t or and mount ed on a f r ame a t t ached t o t he s t age o fa n i n v e r t e d p h a s e m i c r o s c o p e ( N i k o n D i a p h o t ) . T h em o v a b l e c l a m p s i n b o t h c o m p a r t m e n t s w e r e t h e nconnec t ed , w i t h a cord v i a a pu l l ey , t o a mot or -d r i vencamshaf t . Var i a t i on o f t he cam eccen t r i c i t y con t ro l l edt he s t r e t ch ampl i t ude . M ovement o f t he c l amps i n bo t hchamb er s was aga i ns t a sp r i ng whi ch ensured t ha t t hec l amp fo l l owed t he pos i t i on o f t he cam t h roughout t hecyc l e . I n t he expe r i ment a l chamb er t he mov ement o f t hec l amp produced a cyc l i c s t r e t ch i ng and r e l axa t i on o f t hem e m b r a n e t o g e t h e r w i t h v i s c o u s d r a g - i n d u c e d m o v e m e n to f t h e m e d i u m b a t h i n g t h e m e m b r a n e . I n t h e c o n t r o lc o m p a r t m e n t t h e m o v e m e n t o f t h e c l a m p a f f e c t e d o n l yt he medi um. A l t hough t he membrane was no t s t r e t ched i nt he con t ro l chamber , f l u i d mot i on due t o c l amp mot i onwas nea r l y i den t i ca l i n bo t h cham ber s .

D u r i n g a n e x p e r i m e n t t h e a p p a r a t u s w a s m a i n t a i n e d a t37 ~ C us i ng an a i r cur t a i n i ncuba t o r , and med i um p H wascon t ro l l ed by gas s i ng t he un i t w i t h 95% a i r : 5% CO2.

F or t he cyc l ic s t r e t ch exper i ment s , t he mot or speed andcam eccen t r i c i t y were ad j us t ed t o sub j ec t t he exper i men-t a l membr ane t o cyc li c de form at i on o f 10% of it suns t r e t ched l eng t h a t 1 Hz . T he movem ent o f t he c l amp i nt he con t ro l com par t m ent was se t t o g ive t he same me di umm o v e m e n t a s i n t h e e x p e r i m e n t a l c o m p a r t m e n t . A t t h es t a r t and compl e t i on o f an exper i ment pho t ographs o f t hece l ls on t he con t ro l and exper i men t a l mem branes weret aken i n bo t h t he uns t r e t ched and s t r e t ched pos i t i ons .

FIG. 5. Bovine aortic endothelial cells (P6) cultured for 3 d under stationary conditions on Mitrathane {a) thensubjected to a step stretch (b). No te the elongation of the cells parallel to the ho rizontal axis of stretch.


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5 0 4 I V E S E T A L .

A f t e r t e r m i n a t i o n o f a n e x p e r i m e n t t h e c e l l m o n o l a y e rw a s p h o t o g r a p h e d a n d t h e n u s e d f o r e i t h e r m o r p h o m e t r i co r cy toske l e t a l s t ud i e s .

F o r t h e s t e p s t r e t c h s t u d i e s , t h e c h a m b e r w a sa s s e m b l e d a s d e s c r i b e d a b o v e . H o w e v e r , a l l m e m b r a n em o v e m e n t s w e r e p r o d u c e d b y m a n u a l r o t a t i o n o f t h e c a m .T h e b a r e m e m b r a n e w a s s t e p s t r e t c h e d t o d e t e r m i n e t h ec o m p o n e n t s o f st r a i n b o t h p e r p e n d i c u l a r a n d p a r a l le l t ot h e p r i n c i p a l a x i s o f s t r et c h . B A E C w e r e c u l tu r e d i n t h ed e v i c e a s f o r t h e c y c l i c s t r e tc h e x p e r i m e n t s . W h e n t h ec e l l s r e a c h e d c o n f l u e n c e , t h e y w e r e p h o t o g r a p h e d , s t e ps t r e t ch e d , a n d p h o t o g r a p h e d i n t h e s t re t c h e d s t a te .

I n t h e r e v e r s e e x p e r i m e n t t h e m e m b r a n e w a s s t r e t c h e dand subsequen t l y s eeded w i th ce l l s i n t he s t r e t chedp o s i t i o n , w h i c h w a s m a i n t a i n e d t h r o u g h o u t t h e s t a t i o n a r ycu l t u r e pe r i od w h i l e t he ce l l s g r ew to con f luence ; t hent h e c e l l s w e r e p h o t o g r a p h e d , t h e m e m b r a n e w a s r e l a x e da n d t h e c e l l s r e p h o t o g r a p h e d .Shear stress experiments. B A E C c u l t u r e s w e r e s u b j e c t -ed t o shea r s t r e s s i n pa r a l l e l p l a t e f l ow cham ber (11 ) w i thw e l l - c h a r a c t e r i z e d f l u i d d y n a m i c p r o p e r t i e s . T h e u p p e rs u r f a c e o f t h e c h a m b e r w a s m a d e o f p o l y c a r b o n a t e . T h eM i t r a t h a n e - c o a t e d g l a s s s l i d e ( p l u s c e l l m o n o l a y e r ) s e r v e da s t h e l o w e r s u r f a c e a n d t h e s i d e w a l l s w e r e f o r m e d b y as i l a s t i c g a s k e t t h a t s e p a r a t e d t h e u p p e r a n d l o w e r p a r a l l e lp l a t e s t o g iv e a c h a m b e r 2 0 0 / ~ m d e e p , 7 . 6 2 c m l o n g , a n d3 . 8 1 c m w i d e .

F o r a n e x p e r i m e n t t h e c h a m b e r w a s p o s i t i o n e d o n t h es t a g e o f a n i n v e r t e d p h a s e m i c r o s c o p e ( N i k o n D i a p h o t )a n d s u b j e c t e d t o f l o w i n a c o n s t a n t - h e a d f l o w l o o p . T h i ss y s t e m h a s b e e n d e s c r i b e d p r e v i o u s l y ( 7 ,1 1 ) .

T h e s h e a r s t r e ss o n t h e c e l l m o n o l a y e r i n t h e f l o wc h a m b e r w a s c a l c u l a t e d a s s u m i n g f u l ly d e v e l o p e dl a m i n a r f l o w , k n o w i n g t h e d i m e n s i o n s o f t h e c h a m b e r ,v o l u m e t r i c f l o w r a te , a n d m e d i u m v i s c o si t y ( 1 4) . T h e f l o wr a t e w a s s e t a t a b o u t 4 0 m l / m i n , w h i c h g e n e r a t e d a w a l ls h e a r s t r e s s i n t h e c h a m b e r o f a p p r o x i m a t e l y 5 0d y n e s / e m 2 . L i g h t m i c r o g r a p h s o f t h e c e ll s w e r e t a k e n a tt h e b e g i n n i n g o f f lo w a n d a t v a r i o u s t i m e i n t e r v a l st h e r e a f te r . T i m e l a p s e v i d e o m i c r o s c o p y w a s a ls o d o n e i ns e l e c t e d e x p e r i m e n t s t o c o n t i n u o u s l y f o l l o w c h a n g e s i ne n d o t h e l i al c e l l m o r p h o l o g y .Cytoskeletal studies. T o d e t e r m i n e t h e e f f e c t o f f l u i dm e c h a n i c a l f o r c e s o n t h e m i c r o t u b u l e c o m p o n e n t s o f t h ec y t o s k e l e t o n , c o n t r o l a n d e x p e r i m e n t a l c e l l m o n o l a y e r s ,g r o w n o n e i t h e r g l a s s o r p o l y u r e t h a n e , w e r e p r o c e s s e d t ov i s u a l i z e t h e i r m i e r o t u b u l e n e t w o r k s u s i n g a n t i b o d yb i n d i n g . T h e c e ll m o n o l a y e r s w e r e f ix e d i n fo r m a l i n a n dt h e n l y s e d u s i n g T r i t o n X - 1 0 0 . T h e c y t o s k e l e t a l e l e m e n t sr e m a i n i n g a f t e r t h e d e t e r g e n t t r e a t m e n t w e r e t h e ni n c u b a t e d w i t h a b u f f e r s o l u t i o n o f a n t i b o d y t o t u b u l i n( g i ft o f D r . B . R . B r i n k l e y , B a y l o r C o l l e g e o f M e d i c i n e ,H o u s t o n , T X ) . A f t e r i n c u b a t i o n w i t h t h e s p e c i f i c a n t i b o d y , as e c o n d a n t i - I g G a n t i b o d y w i t h a f l u o r e s c e i n i s o t h i o - e y a n a t e( F I T C ) t a g w a s u s e d to l ab e l t h e b o u n d a n t ib o d y . T h e F I T Cl a b e l w a s v i s u a l iz e d u s i n g a N i k o n e p i f l u o r e s c e n t m i c r o s c o p ew i t h a n e x c i t a ti o n f i l te r o f 3 9 0 to 4 7 0 n m a n d a n e m i s s i o n f il t ero f 4 9 0 t o 5 3 0 n m . T h e g l a s s s u b s t r a t e p o s e d n o p r o b l e m s f o re x a m i n i n g t h e c el ls u n d e r U V l i g h t , b u t t h e p o l y u r e t h a n e h a da l o w l e v e l o f a u t o f i u o r e s c e n e e a t t h e F I T C e x c i t a t i o n

w a v e l e n g t h s . T o i m p r o v e t h e c o n t r a s t b e t w e e n t h em i c r o t u b u l e s a n d M i t r a t h a n e R , t h e s e s p e c i m e n s w e r e i n -ve r t ed t o a l l ow the U V l i gh t t o exc i t e t he ce l l s be fo re en -c o u n t e r i n g t h e p o l y u r e t h a n e m e m b r a n e .

Morphometric analysis . Q u a n t i t a t i v e d a t a w e r e o b -t a i n e d b y d i g i ti z i n g c el l m o r p h o l o g y f o r s p e c i fi c a r e a s o ft h e c e l l m o n o l a y e r s ( a s o b t a i n e d f r o m t h e v i d e o t a p e a n dp h o t o g r a p h i c d a t a ) u s i n g a Z e i s s V i d e o p l a n s y s t e m .

RESULTSB ov ine ao r t i c endo the l i a l c e l l s a l i gned pe rpend i cu l a r

t o t h e d i re c t i o n o f m e m b r a n e d e f o r m a t i o n i n f o u r o f f o u rexpe r im en t s i n w h ich t he ce l l s w e re cyc l i ca l l y s t r e t chedf o r 6 6 h ( F i g . 2 ). H U V E C a l i g n e d s i m i la r l y ( F i g . 3 ) i nt h r e e o u t o f t h r e e e x p e r i m e n t s u n d e r t h e s a m e c o n d i t i o n sa s t h e B A E C e x p e r i m e n t s . T h e a l i g n m e n t r e s p o n s e c a n b eq u a n t i t a t e d b y m o r p h o m e t r i c a n a l y s i s ( F ig . 4 ).

A q u a l i t a t i v e t r e n d t o w a r d a l i g n m e n t c o u l d c o n s i s t e n t l yb e o b s e r v e d i n c e ll s o f b o t h t y p e s a f t e r b e t w e e n 2 4 a n d 4 8h o f cyc l ic s t r e t ch ing . C on f luence d id i n f l uence a l i gn -m en t r a t e ; c e l l s i n l e s s con f luen t po r t i ons o f t hem e m b r a n e a l i g n e d m o r e r a p i d l y . A l i g n m e n t r a t e w a s n o td e p e n d e n t o n i n i t i a l c e l l s h a p e ; h o w e v e r , t h e r e s p o n s ew a s l e s s o b v i o u s i n m o r e p o l y g o n a l c e l l s ( u s u a l l y B A E C )t h a t d i d n o t h a v e a d i s t in c t l o n g i t u d i n a l a x i s .A, , , , . l O ] a 8"~ 6"O" 4 "00L. 2"U.

9 0 J0 9 0 ~ 1 8 0 ~A n g l e

g

0 0 90 100

A n g l eFie. 6. Distribution of an gl es made by cells' maximumdimensions in step stretch experiment a, corresponds to Fig. 5 a,the unstretehed membrane and is an essentially random pattern;b, corresponds to Fig. 5 b, stretched membrane. The cells

elongate in the direction o| mem brane elongation (0 to ]8 0~ axis).


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MECHA NICAL EFFECTS ON CELLS 505

Subject ing BAEC to a s ingle s tretch or s tep relaxationresulted in deformation of the individual cel ls paral le l tothe direction of axial strain, i.e. the change in celldimensions fol lowed the change in Mitrathane dimen-s ions |or shor t deformation t imes {Figs . 5 and 6) .

Morphometr ic analys is of s tep s tretched cel ls revealedan increase of cel l dimensions of 17.7% in the direct ion inwhich the membra ne was s tretched (n ~ 100 cellsmeasured). A decrease of 11..3% along the axis of stretchwas observed in cel ls that were grown on membranes inthe stretched positio n (n = 69 cells count ed) when themembr ane was r e laxed .

W hen BAEC wer e cu l tu r ed on Mi t r a thane backed wi thglass slides and subjected to a shear stress of 50d y n e s / c m ~, al ignment paral le l to the direct ion of f low wasobserved (Fig. 7) within 24 h in two of two exper iments .

Us ing the an t i tubul in an t ibod y , the cy top lasmicm i c r o t u b u l e c o m p l e x e s o f t h e c o n t r o l c y c l i c a l l ys tretched, and shear s tressed BAEC monolayers werevisualized. In all cases, the cytoskeleton was a diffusematr ix of tubules with the organizing center c lear lyvis ible and the microtubules forming a nuclear band.Consis tent with the elongated nature of the exper imentalcel l monolayers there was a greater densi ty of microtu-bules running paral le l to the major axis of the cel l af tershear s tress or s tre tch- induced al ignment (Fig. 8) . S imilarf indings wer e ob ta ined f or HU VE C ( da ta no t shown) .

D ISCUSSIONBoth BAEC and HUVEC cul tur ed on Mi t r a thane a l ign

perpendicular to the axis of s tre tch when they are

cyclical ly s tretched at 1 Hz. This a l ignment response mayseem at f i r st to be 90 ~ dif ferent f rom the cel ls' response toshear stress. The parallel ali gnm ent of endothe lial cells inresponse to shear s tress can be rat ionalized as anadaptat ion (act ive or pass ive) to reduce their f luid draginteract ion. The perpendicular a l ignment of cel ls tocyclic s train may again be an at tem pt to reduce the s tressor s train energy exer ted on the cel l , a l though how this iseifected is not c lear ly apparent . Cells grown on anunstretcbed membrane, which is then s tretched, regaintheir polygonal shape within 24 h (data no t shown) . Theresponse t ime may be related to the t ime scale ofcytoskeletal component rear rangement. The reason forthe al ignment under cyclic deformations may be tominimize s tretching of microtubule or other cytoskeletalnetworks dur ing pulsat ion.

Alignment of endothelia l cel ls paral le l to the direct ionof f low in response to shear s tress has been observed inother s tudies (5,6,11,12) , and perpendicular a l ignment ofcells to the axis of stretch has been noted in f ibroblastssubjected to cyclic stretchin g (1). Cyclic stretching ofvascular smooth muscle cel ls has produced increasedprotein synthesis (13) and fine structural changes (21).Elongation of these cel ls paral le l to the direct ion ofs tretching has been noted {21) . In s tudies to be repor tedelsewhere we have observed perpendicular a l ignment ofcanine smooth muscle cells in response to cyclic strain,s imilar to the endothelia l cel l response descr ibed herein.

These responses are consis tent with a general izedconcept of vessel structure {Fig. 9). In a vessel thepr incipal direct ion of s train due to wall compliance iscircumferentia l . Thus al ignment perpendicular to this

FIG. 7. Bovine aortic endothelial c ell s (P S) cultured o n M itrathane backed with g la ss and subjected to 50dynes/cm2 for 20 h. Direction o f flow is left to right. X 488.


7/8

506 IVES ET AL.

woul d be i n t he ax i a l o r f l ow d i r ec t i on o f t he ves se l . T hef l a t m e m b r a n e , m o u n t e d i n th e s t r e tc h i n g a p p a r a t u s ,cou l d be cons i de red a ves se l cu t a l ong i t s ax is andf l a t t ened . I n t h i s way , bo t h f l u i d shea r s t r e s s and wa l ls t r a i n ac t t oge t he r t o cause a l i gnment o f t he endo t he l i a lcel l s par al le l to the f low ax is of the vessel .

. . . . . ~ ~ FcowMOSCLE STRESS. - ' '~ 'S ~ AT WALL

~ O O U G E D BYmENDOTHELIAL LINING PRESSURE, MAINLY CIRCUMFERENTIAL

THE CIRCUMFERENTIAL STRAIN IN THE VESSEL WALL CAN B [ MOOELLEDBY UNIAXIAL STRAIN IN A FLAT SHEET

FIG. 9. Diagram matic represe ntation of forces on the vesselwall. The principal direction of stretch due to wall compliance iscircumferential. N o t e that both the flow- and wall compliance-induced movement during the pressure pulse will tend to alignendothelial cells along the vessel axis.

FIG. 8. a, microtubule visualization of control BAEC monolay-er using antituhulin antibody; b, BAEC monolayer grown undercyclic strain of ] Hz and 7% maximum stretch for 48 h. A r r o windicates the principal axis of stretch. N o t e t h e alignment of themicrotubules perpend icular to the direction of stretch, c, BA ECmonolayer subjected to 50 dynes/cm2 wall shear stress for 48h--arrow indicates the direction of flow. Note the alignment of themierotubules with the flow direction.

T h a t cyc l ic s t r e t ch i ng p rov i des an ap prop r i a t e mode lfor ef fects of pressure on endothel ia l cel l s can beexplained as fol lows. Physiological levels of s ta t icpres sure exe r t ed on ce l l s cu l t u r ed on a r i g i d , i mperm ea-b l e subs t r a t e shou l d have l i t t l e e f f ec t on ce l l morphol ogy .Hydros t a t i c p r es sures o f up t o 250 mmHg have no e f f ec ton endot he l i a l ce l l morphol ogy when ce l l s a r e cu l t u r edon po l ys t y r ene ( Ives, e t a l . , unpu bl i shed d a t a ) . T h i s i sbecause ce l l s cons i s t mos t l y o f wa t e r , and because wa t e ri s an i ncom press i b l e med i um, p r es sure ac t i ng on t he ce llw i l l be r ap i d l y equ i l i b r a t ed ac ros s t he ce l l membrane .S evera l a t mospheres o f p r es sure woul d be r equ i r ed t ocompress ce l l membranes s i gn i f i can t l y ( change f r eevo l ume) . Howeve r , t he pu l sa t i l e p r es sure waveformac t i ng i n t he na t u r a l a r t e ry i s ano t he r ma t t e r , because t hea r t e ry i s compl i an t . T he con t i nuous l y va ry i ng p res sures t retches the vessel wal l t i ssue and thus the cel l s 'env i ronment , sub j ec t i ng bo t h ce l l -t o - ce ll j unc t i ons andt he ce ll mem bran e an d cy t op l asm t o mecha n i ca l s t r a i n .

M orp hom et r i c measure ment o f ce ll s g rown on t hes t r e t ched o r uns t r e t ehed mem brane and t hen s t eps t r e t ched o r r e l axed demons t r a t e t h a t t he ce l ls r emai nadheren t and fo l low t he membr ane d i m ens i ona l changes ,t he change i n ce l l morph ol ogy be i ng p rop or t i ona l t o t hesubs t r a t e d i s t o r t ion fo r shor t de forma t i on t i me sca l es .T h i s r e sponse may be cons i de red a t endency t owardpara l l e l a l i gnment w i t h t he p r i nc i pa l ax i s o f s t r a i n , whi chi nd i ca t es t ha t t he pe rpend i cu l a r a l i gnment due t o cyc l i cs t r e t ch i ng i s an ac t i ve r e sponse . T he a l i gnment o fmi c ro t ubu l es pa ra l l e l t o t he l ong ax i s o f t he ce l l s sugges t st ha t t he i r ac t i ve r ea r r angement occur s dur i ng t hea l i gnment r e sponse , pe rhaps t o mi n i mi ze ce l l movementdur i ng each s t r e t ch cyc le and t hu s r educe mec hni ca lforces on the cel l s .


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MECHANICAL EFFECTS ON CELLS 507

In vivo studies of cardiovascular disease haveindicated that fluid mechanical forces may be importantin determining endothelial cell morphology and a factorin atherogenesis (12,15,16,19,23). High shear stress acrossthe vessel has been implicated as important by someworkers, whereas low shear stress regions (stag nan orrecirculating flow) have been identified as leading toatherosclerotic lesions by others (2,18,20). If thesignificance of mechanical factors as modulating agentsin the control of endothelial cell morphology andfunction is to be quantitatively determined, thenwell-controlled in vitro models need to he established.The results of the studies presented above suggest thatcell morphology is directly affected by both the shearstress and strain components produced by fluid mechani-cal forces in compliant vessels. The microtubule networkalso exhibits alignment in response to both of thesemechani cal forces.

REFERENCES1. Buck, R. C. Reorientation response of cells to repeated stretchand recoi l of the substratum. Exp. Cel l Res. 127:470-474;1980.2. Caro, C. G.; Fitzgerald, J. M.; Schroter, R. C. Atheroma andarterial wall shear. Proc. Soc. Lond. B 177:109-159; 1971.3. DeForrest, J. M.; Hollis, T. M. Shear stress and aortic histaminesynthesis. Am. J. Physiol. 234;H701-H705; 1978.4. Dewey, C. F. Effects of fluid flow on living vascular cells. J.Biomech. Eng. 106:31-35; 1984.5. Dewey, C. F.; Bussolari, S. R.; Gimbrone, M. A., et al. Thedynamic response of vascular endothelial cells to fluid shearstress. J. Biomech. Eng. 103:177-185; 1981.6. Eskins, S. G.; Ives, C. L.; Mclntire, L. V., et al. Response ofcultured endothelial cells to steady flow. Microvasc. Res.28:87-94; 1984.7. Frangos, J. A.; Eskin, S. G.; McIntire, L. V., et al. Flow effectson prostacyclin production by cultured hmnan endothelial cells.Science 227:1477-1479; 1985.

8. Franke, R. P.; Grafe, M.; Schnittler, H., et al. Induction ofhuman vascular endothelial stress fibers by fluid shear stress.Nature 307:648-649; 1984.9. Fry, D. L. Acute vascular endothelial changes associated withincreased blood velocity. Circ. Res. 22:165-197; 1968.10. Gimbrone, M. A. Culture of vascular endothelium. Prog.Hemost. Thromb. 3:1-28; 1976.11. Ives, C. L.; Eskin, S. G.; McInt ire, L. V., etal . The importanceof cell origin and substrate in the kinetics of endothelial cellalignment in response to steady flow. Trans. Am. Soc. Art. Int.Org. 29:209-274; 1983.12. Langille, B. L.; Adamson, S. L. Relationship between blood flowdirection and endothelial cell orientation at arterial branch sitesin rabbits and mice. Circ. Res. 48:48i-488; 1981.13. Leung, D. Y. M.; Glagov, S.; Mathews, M. B. A new in vitrosystem for studying cell response to mechanical stimulation.Exp. Cell Res. 109:285-298; !977.14. McIntire, L. V.; Eskin, S. G. Mechanical and biochemicalaspects of leukocyte interaction with model vessel walls. In:Metselman, H.; Lichtman, M.; LaCelle, P., eds. White cellmechanics. Alan R. Liss, Inc. New York, NY; 1984:209-219.15. Nerem, R. M.; Corhill, J. F. The role of fluid mechanics inatherogenesis. J. Biomech. Eng. 102:i81-189; 1980.16. Reidy, M. A.; LangiUe, B. L, The effect of local blood flowpatterns on endothelial cel l morphology. Exp. Mol. Pathol.32:276-289; 1980.17. Remuzzi, A.; Dewey, C. F.; Davies, P. F., et al. Orientation ofendothelial cells in shear fields in vitro. Biorheology21:617-630; 1984.18. Roach, M. R.; Smith, N. B. Does high shear stress induced byblood flow lead to atherosclerosis? Perspect. Biol. Med.26:287-303; 1983.19. Ross, R. Atherosclerosis: A problem of the biology of arterial wallcel ls and their interactions with blood components.Atherosclerosis 1:293-311; 1981.20. Ross , R.; Glomset, J. A. The pathogenesis of atherosclerosis. N.Engi. J. Med. 295:369-377, 420-425; 1976.21. Sottiurai, V. S.; Kollros, P.; Glagov, S., et al. Morphologicalteration of cultured arterial smooth muscle cells by cyclic

stretching. J. Surg. Res. 35:490-497; 1983.22. White, G. E.; Gimbrone, M. A.; Fujiwara, K. Factors in-fluencing the expression of stress fibers in vascular endothelialcells in situ. J. Cell Biol. 97:416-424; 1983.23. Wong, A. J.; Pollard, T. D.; Herman, I. M. Actin filament stressfibers in vascular endothelial cells n vivo. Science 217:867-869;1983.This work was partiall y supported by grants HL 17437, HL 18072, and HL 23016 from the Natio nal Institutes

of Health, Bethesda, MD, and gran t C-938 from the Robert A. Welch Foundation.

ives et al., 1986, mechanical effects on endothlial cell morphology_in vitro assessment

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