$2-B2-1-04 CHARACTERIZATION OF THE SPECIFIC RECEPTOR FOR TISSUE-TYPE PLASMINOGEN ACTIVATOR EXPRESSED IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS IN CULTURE H. Fukao and O. Matsuo Dept. of Physiology, Kinki University School of Medicine, Osaka, Japan Vascular endothelilal cells (ECs) modulate fibrinolytic system in the blood by secreting tissue-type plasminogen activator (t-PA), urokinasetype plasminogen activator (u-PA) and their specific inhibitor, type-1 plasminogen activator inhibitor (PAI-1). Both t-PA and u-PA activates plasminogen to plasmin which digests fibrin and activates metalloproteinases, inducing thrombolysis or matrix degradation, u-PA can effectively express its proteolytic activity on the cell surface by binding to its specific binding protein, u-PA receptor (u-PAR). On the contrary, only a few reports have demonstrated the presence of t-PA receptor 0-PAR) on ECs or placenta, and thus the function of t-PAR still renrains to be elucidated. We previously demonstrated that t-PA specifically bound to human umbilical vein endothelial cells (HUVECs) in suspension culture which did not produce ECM and the binding between t-PA and PAI-1 was not involved. The binding constants was Kd of 7.56±2.30 (riM) and Bmax of (8.5 +_1.2) x 105 (sites/cell). The binding protein had M.W. of 20 kDa by SDS-PAGE analysis, and was identified as t-PAR which bound to only t-PA but not to plasminogen, forming a 90 kDa complex with I-PA (Fukao, H. et al., Biochem. Biophys. Res. Commun., 187, 956-962, 1992). In the present studies, we purified the 20 kDa t-PAR molecule from HUVECs and analyzed its functional properties in soluble or immobilized form. Lyzedfraction of HUVECs was subjected to molecular sieving by high performance liquid chromatography (HPLC) and the low molecular weight protein fraction (M.W. 10-40 kDa) was further purified by reverse phase separation in HPLC system. About 2.2 ~ g of purified t-PAR protein was obtained from 1 x 109 HLIVECs which revealed a single band on SDS-PAGE/silver staining. The purified t-PAR in immobilized form exhibited a 4-fold increase in plasminogen activation by t-PA. The t-PAR also enhanced ptasminogen activation by t-PA up to 2-fold in solution phase. Though the t-PAR isolated from human placenta by other group had a common binding site for t-PA and plasminogen, which is proposed as the membrane-associated, phospholipid binding protein, annexin II, our 20 kDa tPAR did not interact with either plasminogen or anli-annexin II antibody, indicating that 20 kDa t-PAR is quite diffemt from the placental annexin II. We concluded that our t-PAR from HUVECs is a novel and authentic t-PA receptor expressed in ECs which immobilizes t-PA molecule and enhances its proteolytic activity on the cell surface. These findings also suggest that the t-PAR expressed on the cell surface of ECs regulates one of the anti-thrombotic properties of ECs and that the modulation of t-PAR is related to the pathophysiologieal function at endothelium as cellular fibrinolysis.
$2-B2-1-05 R O L E OF U R O K I N A S E TYPE P L A S M I N O G E N A C T I V A T O R (U-PA) IN E P I T H E L I A L M I G R A T I O N A N D W O U N D H E A L I N G OF T H E C O R N E A T. N i s h i d a Dept. of O p h t h a l m o l o g y , Y a m a g u c h i Univ. School of Medicine, Ube, J a p a n Cell m i g r a t i o n is one of the f u n d a m e n t a l activities in v a r i o u s biological p h e n o m e n a , such as m o r p h o g e n e s i s , m e t a s t a s i s and w o u n d h e a l i n g . E p i t h e l i u m is a b a r r i e r w h i c h p r o t e c t s inside of the body f r o m the v a r y i n g e x t e r n a l e n v i r o n m e n t . To m a i n t a i n the u n i n t e r r u p t e d l a y e r of e p i t h e l i u m , active and c o n t i n u o u s r e g e n e r a t i o n a n d / o r r e p a i r of the d e f e c t s is m a n d a t o r y . We have been s t u d y i n g the p a t h o b i o l o g y of the p e r s i s t e n t c o r n e a l e p i t h e l i a l defects w h e r e e p i t h e l i a l m i g r a t i o n is d i s t u r b e d . Since the c o r n e a has a s i m p l e a n a t o m i c a l s t r u c t u r e and is a v a s c u l a r , the c o r n e a seems to be a s u i t a b l e t i s s u e to i n v e s t i g a t e the f a c t o r s w h i c h may affect the epithelial movements. Previous studies revealed that fibronectin-integrin (fibronectin receptors) system plays an i m p o r t a n t role f o r the c o r n e a l e p i t h e l i a l m i g r a t i o n . In the n o r m a l c o r n e a , i n t e g r i n ctS[~l is e x p r e s s e d only in the b a s a l l a y e r of the c o r n e a l e p i t h e l i u m ; f i b r o n e c t i n is located at the s t r o m a w h i c h is a u n d e r l y i n g s u b s t r a t e f o r the e p i t h e l i u m . W h e n the c o r n e a l e p i t h e l i u m is w o u n d e d , i n t e g r i n ctS[~l is e x p r e s s e d at the s u r f a c e of all the actively m i g r a t i n g e p i t h e l i a l cells. C o n c o m i t a n t l y m o r e f i b r o n e c t i n a p p e a r e d at the c o r r e s p o n d i n g c o r n e a l s t r o m a s e r v i n g as a p r o v i s i o n a l e x t r a c e l l u l a r m a t r i x . E i t h e r exogenous f i b r o n e c t i n o r s t i m u l a t o r of i n t e g r i n e x p r e s s i o n , such as E G F o r i n t e r l e u k i n 6, f a c i l i t a t e s c o r n e a l e p i t h e l i a l m i g r a t i o n in the o r g a n c u l t u r e of the c o r n e a and in vivo. T h e s e s t u d i e s show t h a t f i b r o n e c t i n - i n t e g r i n s y s t e m is p r i m a r i l y i m p o r t a n t f o r the e p i t h e l i a l m i g r a t i o n , t h u s f o r e p i t h e l i a l w o u n d h e a l i n g . On the b a s i s of these s t u d i e s , we have been t r e a t i n g the p a t i e n t s with p e r s i s t e n t c o r n e a l e p i t h e l i a l defects by f i b r o n e c t i n eye d r o p s s u c c e s s f u l l y . A t t a c h m e n t of e p i t h e l i a l cells to the u n d e r l y i n g s t r o m a t h r o u g h f i b r o n e c t i n - i n t e g r i n s y s t e m is i m p o r t a n t . H o w e v e r , the cells m i g h t not be able to move actively, if the cells a t t a c h to e x t r a c e l l u l a r m a t r i x so f i r m l y . W h e n we c o n s i d e r the p a t h o p h y s i o l o g y of e p i t h e l i a l w o u n d h e a l i n g , t h a t is the active m i g r a t i o n of cells as a l a y e r ( e p i b o l y ) , the p r o c e s s of the d e t a c h m e n t of cells f r o m e x t r a c e l l u l a r m a t r i x m i g h t be r e q u i r e d as well as t h e i r a t t a c h m e n t . In t h i s r e g a r d , the d e s t r u c t i o n of f i b r o n e c t i n by p r o t e o l y t i c e n z y m e s would be r e q u i r e d . To test t h i s h y p o t h e s i s , the c o r n e a s w e r e c u l t u r e d and the c h a n g e s in the e p i t h e l i a l m i g r a t i o n and u-PA activity w e r e m e a s u r e d . A l i n e a r c o r r e l a t i o n b e t w e e n u-PA activity in the c u l t u r e m e d i u m and the l e n g t h of e p i t h e l i a l m i g r a t i o n was o b s e r v e d . By a d d i n g s t i m u l a t o r y a g e n t s , such as f i b r o n e c t i n or E G F , o r i n h i b i t o r y a g e n t s , c y t o c h a l a s i n B, the l e n g t h of e p i t h e l i a l p a t h d e m o n s t r a t e d the l i n e r r e l a t i o n s h i p to u-PA activity. I f p r o t e i n a s e i n h i b i t o r , such as a p r o t i n i n and o v o - ~ 2 - m a c r o globulin w e r e added to the c u l t u r e m e d i u m , e p i t h e l i a l m i g r a t i o n was s i g n i f i c a n t l y i n h i b i t e d in a dose d e p e n d e n t f a s h i o n . In s u m m a r y , b o t h the a t t a c h m e n t and the d e t a c h m e n t s y s t e m s a r e m a j o r r e g u l a t o r y s t e p s f o r the cells to move actively; f i b r o n e c t i n - i n t e g r i n s e r v e s as a t t a c h m e n t s y s t e m and u-PA as d e t a c h m e n t system.
32