’ Endothelial
cells: adhesion
and tight junctions
lee 1. Rubin University
College
This article reviews recent cell adhesion molecules formation. Observations junctions between
Current
London,
Opinion
in Cell
Biology
is involved in many key aspects of normal physiology and is the source of and target for a variety of potentially therapeutic agents. The obvious function of the endothelium is in promoting the exchange of metabolites and secretory products between blood and tissues. Depending on the degree of exchange required, the ways in which adjoining endothelial cells are connected can vary from discontinuous or fenestrated (highly permeable) to continuous (less permeable). In the specific case of brain capillaries, neighbouring endothelial cells are coupled by tight junctions of high electrical resistance that severely limit molecular transfer between blood and brain. Serious diI%culties, such as tissue oedema, arise when endothelial cells are unable to regulate molecular exchange properly.
More recently, .tibelda and colleagues [6**] expressed PECAMI in COS cells where it became concentrated at borders between adjacent cells, but only if both cells expressed the protein. Thus, whatever cytoskeletal elements (if any) that are required for junctional localisation must be present in COS cells. L-cells, which do not normally adhere to one another in suspension, did so when transfected with PECAMl cDNA. Surprisingly, the binding was Cal+ -dependent, which is not characteristic of immunoglobulin superfamily members (although it is of cadherins). This leaves open the identity of the molecules to which PECAM-1 binds. Observations on the transfected cells seemingly suggested homophilic binding, but the authors also presented the possibility that heterophilic binding to integrins or cell surface proteoglycans might be taking place.
One of the most interesting aspects of endothelial cell biology is determining how endothelial cells respond to their environment by changing their structure and permeability properties. This will be covered in this review, and particular attention will be paid to the ways in which endothelial cells adhere to one another and form junctional complexes, including the high-resistance tight junctions at the blood-brain barrier. molecules
It is quite clear that the understanding of cell adhesion and the way in which adhesion triggers further structural changes is not nearly as advanced for endothelial cells
830
@
Current
Abbreviations adhesion Biology
4:83&833
Much of the definitive work concerning endothelial cell adhesion molecules has been directed at those involved in leukocyte binding to endothelial cells, but in the past few years several molecules involved in adhesion between endothelial cells have been discovered. One such cell adhesion molecule (CAM), endoCAM or PECAM1 (formerly referred to as CD31), has been studied by several groups who generated monoclonal antibodies that prevented endothelial cells from adhering to one another [ 2,3]. The antigen was shown to be a member of the immunoglobulin superfamily by the use of molecular cloning [4]. It is present on endothelial cells of large and small vessels, including those from brain in t&-o [5*-l, and is concentrated at interceuular junctions.
A less obvious function of endothelial cells is to serve as the point of entry for leukocytes into normal or i&lamed tissue. This property is absolutely vital to the functioning of the immune system, and defects are physiologicall) significant. Moreover, metastatic cells use similar mechanisms to bind to and enter target tissues. Understanding interactions between immune or metastatic cells and endothelial cells is therefore important, but is beyond the scope of this article. Brief mention will be made of the manner in which bound cells acquire the ability to traverse the endothelium.
CAM-cell
1992,
as it is for epithelial cells. However, it seems likely that the two cell types behave in a fundamentally similar fashion. For epithelial cells, homophilic interactions between Ca’+ -dependent E-cadherin molecules lead to adhesion followed by tight junction formation [ 11. How this occurs is not certain, but it is clear that a network of cytoplasmic elements linking the adherens and tight junctions is constructed following binding of the extracellular domains of E-cadherin molecules and then transmitted by their cytoplasmic domains. This suggests that it will be necessary to ident@ endothelial ceU adhesion molecules and their associated proteins.
The endothelium
cell adhesion
UK
discoveries concerning the identity of endothelial and their participation in intercellular junction relating to the formation of high-resistance tight brain endothelial cells are emphasized.
Introduction
Endothelial
London,
Ltd
molecule. ISSN
0995-0674
Endothelial
Some previous work had also pointed to the existence of cadherins in endothelial cells. Heimark et al. [7] generated a monoclonal antibody against a surface protein and found that it had the ability to block Ca*+ -dependent endothelial cell adhesion. Based on its characteristic sensitivity to trypsin treatment of intact cells, this 130kD protein appeared to be a member of the cadherin family. The protein was present on various types of endothelial cells, including those derived from brain [5**], and was relatively concentrated at intercellular junctions. It has not yet been completely characterized at the molecular level, although Suzuki et al. [8*] suggested that it might correspond to one of the cadherin-like proteins (cadherin 5) that they cloned using cDNAs derived from polymerase chain reactions. Liaw et al. [9] also cloned bovine endothelial cell cadherin cDNAs using oligomers derived from the highly conserved cytoplasmic domain that is common to many, but not all, cadherins. They found two cadherins present in all endothelial cells. One of these was homologous to mouse and chicken N-cadherin, while the other was homologous to mouse P-cadherin. Neither was restricted to endothelial cells. More recently, Rubin et al. [5**,10] presented evidence that a protein recognised by an Ecadherin antibody is present on rodent brain endothelial cells. Interestingly, this protein appeared to localize to intercellular junctions in cultures of bovine brain endothelial cells that had high resistance, but was diffusely distributed in those of low resistance. Neither PECAM-1 nor the vascular cadherin studied by Heimark et al. [7] had markedly different distributions in low- and high-resistance cells [ 5**]. Iampugnani and co-workers [ 11.1 studied integrins appearing in endothelial cells. The integrins a2pl and as& appeared to be relatively concentrated at the junctional region of human umbilical vein endothelial cells, but the amounts of these proteins seemed to be distinctly less than the amount of PECAM-1 in those cells, at least judging by the intensity of fluorescent antibody staining. An antibody against the as& integrin also seemed able to disrupt the monolayer partially and increase its permeability. It is still not clear whether the integrins subserve endothelial cell-cell or ceU-substrate interaction. Junction-associated cells
proteins
in endothelial
Information concerning molecules present in association with adherens and tight junctions in epithelial cells is becoming increasingly available. Proteins known to be located beneath adherens junctions in epithelial cells include actin, the catenins and a 220kD ankyrin-related protein [ 121. Molecules known to be present beneath the tight junction include ZO-1 [ 131 and cinguilin [ 141. ZO-1 has been shown to be present in peripheral [13] and brain endothelial cells in situ [ 151. Furthermore, ZO-1 is localized at intercellular regions of both low- and high resistance cultured brain endothelial cells [ 5**], which is not surprising as it is clearly present between low-resistance peripheral endothelial cells.
The blood-brain
cells:
adhesion
barrier
and
tight
junctions
Rubin
in cell culture
AS mentioned in the introduction, one of the many interesting aspects of endothelial ceU biology is the ability of the endothelial cells to change phenotypes in different tissue environments. This is seen especially weU in the case of brain capillary endothelial cells. These cells differ from those that make up most large and smaU peripheral vessels in two major ways: they are coupled by tight junctions of high electrical resistance (greater than 1000 ncm*;[ I6,17]), and they have relatively low rates of fluid-phase endocytosis. These properties combine to dramatically reduce bidirectional exchange between blood and brain. Stewart and Wiley [18] discovered that when peripheral endothelial cells were made to vascularize brain, they were induced to resemble brain endothelial cells as they could then restrict the flow of certain tracers. Subsequently, Janzer and RaIf [ 191 showed that gIiaI cells (type 1 astrocytes) that form endfeet on brain endothelial cells induced this transformation in the virtual absence of other ceU types. While these studies demonstrated the influence of tissue environment on endothelial ceU phenotype, they did not provide clear information conceming the full extent of the changes in endothelial cells. For example, did peripheral endothelial cells transplanted to brain form high-resistance tight junctions? Electrical resistance measurements were not made in these studies, and high-resistance junctions would not be required to explain the limited permeability that was observed (see
[lOI >. Several groups have tried to establish ceU culture models of the blood-brain barrier, often by taking advantage of information provided by the transplant experiments described above, although it must be kept in mind that the full extent of the changes produced in endothelial cells by brain tissue or astrocytes in those experiments is still not clear. Raub et al. [21**] showed that bovine brain endothelial cells co-cultured with CG-gUoma cells had increased tight junction resistance and a slightly decreased rate of fluid-phase endocytosis. Nonetheless, the resulting cells had lower electrical resistance and greater leakiness than brain endothelial cells in vivo. Rubin and collaborators [5==] grew bovine brain endothelial cells in the presence of conditioned medium derived from rat brain type 1 astrocytes. They found that, on average, these cells had only a slightly increased electrical resistance compared with those grown without conditioned medium. However, treatment of those endotheiial cells with agents that elevate cyclic AMP levels produced a larger increase in resistance (on occasion reaching lOOO-15000 cm*) and provided cells that were much less leaky to molecular tracers. Increased cyclic GMP exerted an opposite effect: the resistance decreased significantly. It was also observed that cells treated with a combination of astrocyte-conditioned medium and cyclic AMP analogues underwent a noticeable morphological change, leading to an enrichment in the junctional region of Iilamentous actin and of a molecule immunologically related to E-cadherin.
831
832
Cell-to-cell
contact
Regulation resistance
and
extracellular
of tight junction
matrix
formation
and
Observations on brain endothelial cells implicate second messenger-mediated events as being important in determining the resistance of endothelial cell tight junctions. While the relevance of these results to in zYl)ophysiology remains to be determined, the simplest explanation for the in vitro results is that increased electrical resistance is obtained when cyclic AMP dependent protein kinase phosphorylates one or more proteins associated with the tight junction. As structural changes occur in the region of the adherens junction following cyclic AMP elevation in endothelial cells, there may be additional substrates for this kinase at that site. From this perspective, it is important to understand what is known about the role of phosphoxylation in tight and adherens junction formation in endothelial cells. As information pertaining to epithelial cells has accumulated more rapidly, it will be summarized briefly. A role for phosphorylation in tight junction maintenance was implied by Ojakian [22], who determined that phorbol esters decreased epithelial tight junction resistance and had disruptive effects on the junctions, as viewed ultrastructurally. Citi et al. [23**] suggested the participation of protein kinases by showing that the ability of EGTA to decrease tight junction resistance (presumably by interfering with cadherin binding) is reduced in the presence of certain protein kinase inhibitors. Thus, both studies suggested an inverse correlation between phosphovlation and resistance. There has also been a great deal of interest in the role of tyrosine kinases in junction formation. Geiger and colleagues [24-l have shown that inhibition of tyrosine phosphatases leads to a large increase in tyrosine phosphorylation and changes in the organisation of vinculin and actin at the adherens junction, with less effect on tight junctions and desmosomes. Tsukita and collaborators (25-l have also observed an accumulation of proteins phosphorylated on tyrosine residues at adherens junctions. It will aIs0 be important to determine which junction components are phosphorylated under the different conditions. Candidate phosphoproteins include vinculin, Ecadherin and ZO-1. The identities of other proteins that make up the tight junction are unknown at present, but might be substrates for kinases that atfect resistance. Thus, multiple junctional proteins are likely to be phosphorylated, and phosphovlation could well serve as a modulator of multiple aspects of epithelial and endothelial cell-cell interactions.
Perspectives The ability to regulate endothelial cell tight junction permeability may have several applications. For instance, it would be useful to have a method for manipulating the permeability of the blood-brain barrier to permit the entry of therapeutic agents into the central nervous system. Conversely, it would be advantageous to be able to reverse the permeability increases that culminate in tissue
oedema in the periphery or the brain following certain types of insult. Another problem to be solved is determining how leukocytes and metastatic cells cross endothelial barriers. It is generally agreed that trans-endothelial migration occurs at the junctional region between cells (but see [ 261 1. A great deal is currently understood about the wa)’ in which leukocytes adhere to endothelial cells - weakly at lirst and then more tightly [27*] - but how bound cells actually move through the junction is less clear. It might be that the leukocytes secrete agents that open tight junctions. However, acti\ration of endothelial cell receptors b!T bound leukocytes may a&rate a signal transduction gstem that causes transient junction opening. Conclusion The formation of tight junctions in epithelial and endothelial cells is complex and still not well understood. Nonetheless, it is likely that the process is initiated b? cell surface adhesion proteins on adjacent cells that bind to one another. This trans.cellular ‘dimerization ma!’ cause a structural change in the c?Toplasmic domain of these membrane proteins that permits an association with other cytoplasmic molecules that somehow cause the tight junction to form. The resistance of the tight junction is subject to further regulation. Roth adherens and tight junctions appear to be modified b!r phosphoq%ition, with ditferent kinases potentially having different effects. This may retlect \rarying physiological demands on different cell populations. One major dificulty is to determine which endothelial cell molecules are important in adhesion and junction formation. Endothelial cells have many adhesion molecules (e.g. immunoglobulin superfamily, cadherins, integrins), but the ways in which they all function are as yet unknown. How phosl~,honlatioli modifies junction formation is also unknown. In the next few years. the ways in which adhesion molecules, kinases and phosphatases interact and regulate one another should become increasingly evident. Acknowledgements
References
and recommended
Papers of particular interest. published \ie~. have been hghlightd a: . of special interest .. of outstanding inwrest
reading aithin
the annual
paid
of re
1.
GlM3INl3l B, %l!\lIN5ON 13. GKIMUDI A: The Role Adhesion Molecule Uvomorulin in the Formation tenance of the Epithelial Junctional Complex. 1988, 107:1575-1587.
of the CeU and Main./ Cc// Bid
2.
M~LL!S WA, RA1-n CM. MCDONNELL 51, CWN LA: A Human Endothelial Cell-restricted Externally Disposed Plasmalemmal Protein Enriched in Intercellular Junctions. ./ iY.1~ Ib/e~/ 1989. 170:399-i1-1.
Endothelial
3.
Aweu% SM, O~TR PD, ROMER LH, BUCK CA EndoCAhl: a Novel EndotheliaI Cell-Cell Adhesion Molecule. / Cell Viol 19%. 110:1227-1237.
4.
I’J. BEKNDT MC, G~KSKY J. W~rni GC, Puxxxx LS. ML’UH WA: PECAhI-1 (CDjI): Cloning and Relation to Adhesion Molecules of the lmmunoglobulin Gene Superfamily. Science 1990. 247:1219-1222.
RUBIN LL, HA& DE, POR~K 5, B,UIBL’ K. CANNON C. HO~EH HC. JANATI~OL~R M. LL\w. CW. MANNING K. MOILUES J. ET ..u.: A Cell Culture Model of the Blood-Brain Barrier. ./ CeN Rio/ 1991. 115:172%1735. The authors describe a method of inducing brain endothelial cells to form tight junctions with high electrical resistance. They also discuss the distributions of cell adhesion molecules and qtoplasmic junctional components in low- and high-resistance cells. Finally, thq describe the role of phosphorylation in modulating both tight and adherens junc tions. 6. ..
AIJXIA SM. MIIUPR WA BUICK CA, NNhW PJ: Molecular and Cellular Properties of PECAM-1 (endoCAM/CD31) a Novel Vascular Cell-Cell Adhesion Molecule. / CeN Rid 1991, 114:105~1068 This paper demonstrates the adhesive htnction of PECAMI \ia tr;lns. fection of COS and L-cells. Perhaps the most surprising result is that transfected L-cell adhesion is CaL+ -dependent, mising the possibili~ that PECAM 1 binding is heterophilic. 7.
of the Human 1991, 1296-10.
HEwlARK RL, DEGNER M. Sc~w~ri! SM: Identification of a Ca2+ -dependent Cell-Cell Adhesion Molecule in EndotheIiaI Cells. ./ Cell Rio/ 1990, 1 IO:l’+i-I756
8. .
S~!ZIIKI 5, Swo K. T~WIHARA H. Diversity of the Cadherin Family: Evidence for Eight New Cadherins in Nervous Tissue. Cell Ke@cl 1991, 2:261-270. These investigators used polymerAse chain rr;lction techniques to iso. late cDNAs for eight potentially new cadherins in nervous tissue. The! suggest that one of these. cadherin-5. is present in endothelial cells and may correspond to the \zscular cadherin of Heimark e/ a/. 171. The cy toplasmic domain of this cadherin is not highly homologous to those of other cadherins and might not have been revealed in the cloning studies of Liaw Cl al. [9]. 9.
hw CW, CANNON C. POWER MD, KIHONEKA PK, R[IHIw IdentiIication and Cloning of Two Species of Cadherins Bovine EndotheIiaI Cells. T;IIBO J 1990, 9:2X-2708.
IL in
10.
LIAW CW. TOhlASEUJ KJ, C.&~NON C, DAVIS K, R~II~IN L: Disruption of MDCK and Bovine EndotheUaI Cell Tight Junctions with Cadherin Synthetic Peptides. / &/I Hiol 1990, 11 l:-tOX.
11. .
L~PIGNANI MG, RESNATI M. DEJANA E, MARCHISIO PC: The Role of Integrins in the Maintenance of EndotheIiaI Monolayer Integrity. J Cell Rio/ 1991, 112:-1?&i90. Thtl authors discuss the potential role of integrins in maintaining interactions between endothelial cells. While this paper concentrates on the effects of antibodies on endothelial cell monolayer integriv, consideration must also be given to the possibility that the primary erect of thy antibodies m on endothelial cell-substmte adhesion. 12.
ITOI-1 M, YONEMLIRA S, NI\GAFLICHI A. TSLIKIT.~ S. Ts[wr~ 220 kD Undercoat-constitutive Protein: Its Specific ization at Cadherin-based Cell-Cell Adhesion Sites. t?iol 1991. 115:144+1462.
13.
STEVENSON BR, Sicuwo JD, ,M~~SEKER MS. G~ODENOUGH DA: Identification of ZO-1: A High Molecular Weight Polypeptide Associated with the Tight Junction (Zonula Occludens) in a Variety of EpitheIia .I Cell Rid 1986, 103:755-766.
14.
Cm S, SAHANAY H, JAKES R, GEIGER B. KENIXUCKJONES J: Cinguilin: a New Peripheral Component of Tight Junctions. Nalrrre 1988, 333~272-276.
15.
WATSON PM, ANDERSON The Tight-junction++&&
TM, VANITAIUE C Protein ZO-1
and
Rat
and
tight
Blood-Brain
junctions
Barriers.
Rubin
iVeztro.cci
SP: Electrical Resistance of Brain Brain Res 1982, 241:4+55.
Len
CRONE C, Ou%II: cular EndotheIium.
17.
131:1-r AM, JONES IHC, AHHUIT NJ: Electrical the Blood-Brain Barrier in Anaesthetized mental Study. / Plqsiol 1990, 429:t7-62.
18.
Sr~%~,wt’ PA WILEY MJ: Developing Nervous Tissue Induces Formation of Blood-Brain Barrier Characteristics in Invading EndotheliaI Cells: a Study Using Quail-chick Transplantation Chimeras. Der, Rio1 1981, 84:18.3-192.
19.
J&~%EH RC, RV‘F MC: Astrocytes Properties in EndotheliaI CeUs.
20.
~LWAR~ JL. D~WRI\ISA’~-IAPHOR K: Occluding Structure-Function Relationships in a Cultured Monolayer. ,I Cell Biol 1985, 101:212&2133.
Induce IVatttre
Microvas-
Resistance Across Rats: a Develop-
Blood-Brain Barrier 1987. 325:253-257. Junction EpitheliaI
‘1. ..
RAOH TJ. KllElur/.t% Sl SAU’ADA GA: Permeability of Bovine Brain Microvessel Endothelial Cells In Vitro Barrier Tightening by a Factor Released from Astroglioma CeUs. Eq Cell Kes 1992, 199:33&340. This article describes another cell culture model of the blood-brain barrier in which endothelial cells are induced by factors secreted from C6-glioma cells. The absolute resistance of the endothelial cell monolayers ~3s not vev high, but the system may be quite useful in identifying glia-derived lactors that inIluence blood-brain barrier permeability. 22.
O!,wuclm meability
GK: Tumor Promoter-induced of Epithelial Tight Junctions.
Changes in the PerGel/ 1981, 23:9%103.
‘3. ..
ClTl S: Protein Kinase Inhibitors Prevent Junction Dissociation Induced by Low Extracellular Calcium in MDCK Epithelial Cells. J Cell Biol 1992. 117:16+1X This article is relcant to understanding regulation of tight junction resistance, % it implicates one or more kinases in the process of EGTAinduced changes in epithelial adherens and tight junctions. 211. .
VOI&KC T. ZICK Y. DROR R. S&LUAY I, GILON C. LEvrrw A. GEIGER B: The Effect of Tyrosine-specific Protein Phosphorylation on the Assembly of Adherens-type Junctions. EIIHO .I 1992, 1 1:1733-1742. The authors discuss the role of vrosine phosphorylation in the maintenance of adherens and tight junctions. 25. .
Tsl’Krr.4 5, OlSHl K, AKlYMM T, Y&w%UstlI Y, Y.&\lUlOTO T, TS~.KIT.A S: Specific Proto-oncogenic Tyrosine Kinases of src Family Are Enriched in Cell-to-Cell Adherens Junctions Where the Level of Tyrosine Phosphorylation is Elevated. .I Cell Bid 1991. 113:86?-879. This paper and [ 24* ] illustrate a striking increase in the level of tyrosine phosphoqkJtion on one or more adherens junction proteins when epithelial cells are treated nith an inhibitor of tyrosine phosphatases. The authors aIlso describe three proto-oncogene tyrosine kinases associated with adherens junctions. 26.
S: A Local./ Cell
M, Docmow SR: is a Component
adhesion
16.
NEwhlAN
5. ..
cells:
IOSSINSh?’ AS. PLL’TA R, SONG MJ, BmhtiJm’ V, MOW RC. WISNIEW,SKI HM: Mechanisms of Inflammatory CeU Attachment in Chronic Relapsing Experimental Allergic Encephalomyelitis: a Scanning and High-voltage Electron Microscopic Study of the Injured Mouse Blood-Brain Barrier. Microcvm Res 1991, 41:299-310.
‘7. .
B~ITCHER EC: Leukocyte-EndotheUaI CeU Recognition: Three (or More) Steps to Specificity and Diversity. Cell 1991, 67:1033-1036. An estremely provocative review illustrating the complexity of leukoqle-endothelial cell interactions and the advantages that this complexiv proffers.
LL Rubin. Eisai London Research laboratories, Darwin sity College London, London WClE 6BT, UK.
Building,
Univer-
833
cells: adhesion
and tight junctions
lee 1. Rubin University
College
This article reviews recent cell adhesion molecules formation. Observations junctions between
Current
London,
Opinion
in Cell
Biology
is involved in many key aspects of normal physiology and is the source of and target for a variety of potentially therapeutic agents. The obvious function of the endothelium is in promoting the exchange of metabolites and secretory products between blood and tissues. Depending on the degree of exchange required, the ways in which adjoining endothelial cells are connected can vary from discontinuous or fenestrated (highly permeable) to continuous (less permeable). In the specific case of brain capillaries, neighbouring endothelial cells are coupled by tight junctions of high electrical resistance that severely limit molecular transfer between blood and brain. Serious diI%culties, such as tissue oedema, arise when endothelial cells are unable to regulate molecular exchange properly.
More recently, .tibelda and colleagues [6**] expressed PECAMI in COS cells where it became concentrated at borders between adjacent cells, but only if both cells expressed the protein. Thus, whatever cytoskeletal elements (if any) that are required for junctional localisation must be present in COS cells. L-cells, which do not normally adhere to one another in suspension, did so when transfected with PECAMl cDNA. Surprisingly, the binding was Cal+ -dependent, which is not characteristic of immunoglobulin superfamily members (although it is of cadherins). This leaves open the identity of the molecules to which PECAM-1 binds. Observations on the transfected cells seemingly suggested homophilic binding, but the authors also presented the possibility that heterophilic binding to integrins or cell surface proteoglycans might be taking place.
One of the most interesting aspects of endothelial cell biology is determining how endothelial cells respond to their environment by changing their structure and permeability properties. This will be covered in this review, and particular attention will be paid to the ways in which endothelial cells adhere to one another and form junctional complexes, including the high-resistance tight junctions at the blood-brain barrier. molecules
It is quite clear that the understanding of cell adhesion and the way in which adhesion triggers further structural changes is not nearly as advanced for endothelial cells
830
@
Current
Abbreviations adhesion Biology
4:83&833
Much of the definitive work concerning endothelial cell adhesion molecules has been directed at those involved in leukocyte binding to endothelial cells, but in the past few years several molecules involved in adhesion between endothelial cells have been discovered. One such cell adhesion molecule (CAM), endoCAM or PECAM1 (formerly referred to as CD31), has been studied by several groups who generated monoclonal antibodies that prevented endothelial cells from adhering to one another [ 2,3]. The antigen was shown to be a member of the immunoglobulin superfamily by the use of molecular cloning [4]. It is present on endothelial cells of large and small vessels, including those from brain in t&-o [5*-l, and is concentrated at interceuular junctions.
A less obvious function of endothelial cells is to serve as the point of entry for leukocytes into normal or i&lamed tissue. This property is absolutely vital to the functioning of the immune system, and defects are physiologicall) significant. Moreover, metastatic cells use similar mechanisms to bind to and enter target tissues. Understanding interactions between immune or metastatic cells and endothelial cells is therefore important, but is beyond the scope of this article. Brief mention will be made of the manner in which bound cells acquire the ability to traverse the endothelium.
CAM-cell
1992,
as it is for epithelial cells. However, it seems likely that the two cell types behave in a fundamentally similar fashion. For epithelial cells, homophilic interactions between Ca’+ -dependent E-cadherin molecules lead to adhesion followed by tight junction formation [ 11. How this occurs is not certain, but it is clear that a network of cytoplasmic elements linking the adherens and tight junctions is constructed following binding of the extracellular domains of E-cadherin molecules and then transmitted by their cytoplasmic domains. This suggests that it will be necessary to ident@ endothelial ceU adhesion molecules and their associated proteins.
The endothelium
cell adhesion
UK
discoveries concerning the identity of endothelial and their participation in intercellular junction relating to the formation of high-resistance tight brain endothelial cells are emphasized.
Introduction
Endothelial
London,
Ltd
molecule. ISSN
0995-0674
Endothelial
Some previous work had also pointed to the existence of cadherins in endothelial cells. Heimark et al. [7] generated a monoclonal antibody against a surface protein and found that it had the ability to block Ca*+ -dependent endothelial cell adhesion. Based on its characteristic sensitivity to trypsin treatment of intact cells, this 130kD protein appeared to be a member of the cadherin family. The protein was present on various types of endothelial cells, including those derived from brain [5**], and was relatively concentrated at intercellular junctions. It has not yet been completely characterized at the molecular level, although Suzuki et al. [8*] suggested that it might correspond to one of the cadherin-like proteins (cadherin 5) that they cloned using cDNAs derived from polymerase chain reactions. Liaw et al. [9] also cloned bovine endothelial cell cadherin cDNAs using oligomers derived from the highly conserved cytoplasmic domain that is common to many, but not all, cadherins. They found two cadherins present in all endothelial cells. One of these was homologous to mouse and chicken N-cadherin, while the other was homologous to mouse P-cadherin. Neither was restricted to endothelial cells. More recently, Rubin et al. [5**,10] presented evidence that a protein recognised by an Ecadherin antibody is present on rodent brain endothelial cells. Interestingly, this protein appeared to localize to intercellular junctions in cultures of bovine brain endothelial cells that had high resistance, but was diffusely distributed in those of low resistance. Neither PECAM-1 nor the vascular cadherin studied by Heimark et al. [7] had markedly different distributions in low- and high-resistance cells [ 5**]. Iampugnani and co-workers [ 11.1 studied integrins appearing in endothelial cells. The integrins a2pl and as& appeared to be relatively concentrated at the junctional region of human umbilical vein endothelial cells, but the amounts of these proteins seemed to be distinctly less than the amount of PECAM-1 in those cells, at least judging by the intensity of fluorescent antibody staining. An antibody against the as& integrin also seemed able to disrupt the monolayer partially and increase its permeability. It is still not clear whether the integrins subserve endothelial cell-cell or ceU-substrate interaction. Junction-associated cells
proteins
in endothelial
Information concerning molecules present in association with adherens and tight junctions in epithelial cells is becoming increasingly available. Proteins known to be located beneath adherens junctions in epithelial cells include actin, the catenins and a 220kD ankyrin-related protein [ 121. Molecules known to be present beneath the tight junction include ZO-1 [ 131 and cinguilin [ 141. ZO-1 has been shown to be present in peripheral [13] and brain endothelial cells in situ [ 151. Furthermore, ZO-1 is localized at intercellular regions of both low- and high resistance cultured brain endothelial cells [ 5**], which is not surprising as it is clearly present between low-resistance peripheral endothelial cells.
The blood-brain
cells:
adhesion
barrier
and
tight
junctions
Rubin
in cell culture
AS mentioned in the introduction, one of the many interesting aspects of endothelial ceU biology is the ability of the endothelial cells to change phenotypes in different tissue environments. This is seen especially weU in the case of brain capillary endothelial cells. These cells differ from those that make up most large and smaU peripheral vessels in two major ways: they are coupled by tight junctions of high electrical resistance (greater than 1000 ncm*;[ I6,17]), and they have relatively low rates of fluid-phase endocytosis. These properties combine to dramatically reduce bidirectional exchange between blood and brain. Stewart and Wiley [18] discovered that when peripheral endothelial cells were made to vascularize brain, they were induced to resemble brain endothelial cells as they could then restrict the flow of certain tracers. Subsequently, Janzer and RaIf [ 191 showed that gIiaI cells (type 1 astrocytes) that form endfeet on brain endothelial cells induced this transformation in the virtual absence of other ceU types. While these studies demonstrated the influence of tissue environment on endothelial ceU phenotype, they did not provide clear information conceming the full extent of the changes in endothelial cells. For example, did peripheral endothelial cells transplanted to brain form high-resistance tight junctions? Electrical resistance measurements were not made in these studies, and high-resistance junctions would not be required to explain the limited permeability that was observed (see
[lOI >. Several groups have tried to establish ceU culture models of the blood-brain barrier, often by taking advantage of information provided by the transplant experiments described above, although it must be kept in mind that the full extent of the changes produced in endothelial cells by brain tissue or astrocytes in those experiments is still not clear. Raub et al. [21**] showed that bovine brain endothelial cells co-cultured with CG-gUoma cells had increased tight junction resistance and a slightly decreased rate of fluid-phase endocytosis. Nonetheless, the resulting cells had lower electrical resistance and greater leakiness than brain endothelial cells in vivo. Rubin and collaborators [5==] grew bovine brain endothelial cells in the presence of conditioned medium derived from rat brain type 1 astrocytes. They found that, on average, these cells had only a slightly increased electrical resistance compared with those grown without conditioned medium. However, treatment of those endotheiial cells with agents that elevate cyclic AMP levels produced a larger increase in resistance (on occasion reaching lOOO-15000 cm*) and provided cells that were much less leaky to molecular tracers. Increased cyclic GMP exerted an opposite effect: the resistance decreased significantly. It was also observed that cells treated with a combination of astrocyte-conditioned medium and cyclic AMP analogues underwent a noticeable morphological change, leading to an enrichment in the junctional region of Iilamentous actin and of a molecule immunologically related to E-cadherin.
831
832
Cell-to-cell
contact
Regulation resistance
and
extracellular
of tight junction
matrix
formation
and
Observations on brain endothelial cells implicate second messenger-mediated events as being important in determining the resistance of endothelial cell tight junctions. While the relevance of these results to in zYl)ophysiology remains to be determined, the simplest explanation for the in vitro results is that increased electrical resistance is obtained when cyclic AMP dependent protein kinase phosphorylates one or more proteins associated with the tight junction. As structural changes occur in the region of the adherens junction following cyclic AMP elevation in endothelial cells, there may be additional substrates for this kinase at that site. From this perspective, it is important to understand what is known about the role of phosphoxylation in tight and adherens junction formation in endothelial cells. As information pertaining to epithelial cells has accumulated more rapidly, it will be summarized briefly. A role for phosphorylation in tight junction maintenance was implied by Ojakian [22], who determined that phorbol esters decreased epithelial tight junction resistance and had disruptive effects on the junctions, as viewed ultrastructurally. Citi et al. [23**] suggested the participation of protein kinases by showing that the ability of EGTA to decrease tight junction resistance (presumably by interfering with cadherin binding) is reduced in the presence of certain protein kinase inhibitors. Thus, both studies suggested an inverse correlation between phosphovlation and resistance. There has also been a great deal of interest in the role of tyrosine kinases in junction formation. Geiger and colleagues [24-l have shown that inhibition of tyrosine phosphatases leads to a large increase in tyrosine phosphorylation and changes in the organisation of vinculin and actin at the adherens junction, with less effect on tight junctions and desmosomes. Tsukita and collaborators (25-l have also observed an accumulation of proteins phosphorylated on tyrosine residues at adherens junctions. It will aIs0 be important to determine which junction components are phosphorylated under the different conditions. Candidate phosphoproteins include vinculin, Ecadherin and ZO-1. The identities of other proteins that make up the tight junction are unknown at present, but might be substrates for kinases that atfect resistance. Thus, multiple junctional proteins are likely to be phosphorylated, and phosphovlation could well serve as a modulator of multiple aspects of epithelial and endothelial cell-cell interactions.
Perspectives The ability to regulate endothelial cell tight junction permeability may have several applications. For instance, it would be useful to have a method for manipulating the permeability of the blood-brain barrier to permit the entry of therapeutic agents into the central nervous system. Conversely, it would be advantageous to be able to reverse the permeability increases that culminate in tissue
oedema in the periphery or the brain following certain types of insult. Another problem to be solved is determining how leukocytes and metastatic cells cross endothelial barriers. It is generally agreed that trans-endothelial migration occurs at the junctional region between cells (but see [ 261 1. A great deal is currently understood about the wa)’ in which leukocytes adhere to endothelial cells - weakly at lirst and then more tightly [27*] - but how bound cells actually move through the junction is less clear. It might be that the leukocytes secrete agents that open tight junctions. However, acti\ration of endothelial cell receptors b!T bound leukocytes may a&rate a signal transduction gstem that causes transient junction opening. Conclusion The formation of tight junctions in epithelial and endothelial cells is complex and still not well understood. Nonetheless, it is likely that the process is initiated b? cell surface adhesion proteins on adjacent cells that bind to one another. This trans.cellular ‘dimerization ma!’ cause a structural change in the c?Toplasmic domain of these membrane proteins that permits an association with other cytoplasmic molecules that somehow cause the tight junction to form. The resistance of the tight junction is subject to further regulation. Roth adherens and tight junctions appear to be modified b!r phosphoq%ition, with ditferent kinases potentially having different effects. This may retlect \rarying physiological demands on different cell populations. One major dificulty is to determine which endothelial cell molecules are important in adhesion and junction formation. Endothelial cells have many adhesion molecules (e.g. immunoglobulin superfamily, cadherins, integrins), but the ways in which they all function are as yet unknown. How phosl~,honlatioli modifies junction formation is also unknown. In the next few years. the ways in which adhesion molecules, kinases and phosphatases interact and regulate one another should become increasingly evident. Acknowledgements
References
and recommended
Papers of particular interest. published \ie~. have been hghlightd a: . of special interest .. of outstanding inwrest
reading aithin
the annual
paid
of re
1.
GlM3INl3l B, %l!\lIN5ON 13. GKIMUDI A: The Role Adhesion Molecule Uvomorulin in the Formation tenance of the Epithelial Junctional Complex. 1988, 107:1575-1587.
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M~LL!S WA, RA1-n CM. MCDONNELL 51, CWN LA: A Human Endothelial Cell-restricted Externally Disposed Plasmalemmal Protein Enriched in Intercellular Junctions. ./ iY.1~ Ib/e~/ 1989. 170:399-i1-1.
Endothelial
3.
Aweu% SM, O~TR PD, ROMER LH, BUCK CA EndoCAhl: a Novel EndotheliaI Cell-Cell Adhesion Molecule. / Cell Viol 19%. 110:1227-1237.
4.
I’J. BEKNDT MC, G~KSKY J. W~rni GC, Puxxxx LS. ML’UH WA: PECAhI-1 (CDjI): Cloning and Relation to Adhesion Molecules of the lmmunoglobulin Gene Superfamily. Science 1990. 247:1219-1222.
RUBIN LL, HA& DE, POR~K 5, B,UIBL’ K. CANNON C. HO~EH HC. JANATI~OL~R M. LL\w. CW. MANNING K. MOILUES J. ET ..u.: A Cell Culture Model of the Blood-Brain Barrier. ./ CeN Rio/ 1991. 115:172%1735. The authors describe a method of inducing brain endothelial cells to form tight junctions with high electrical resistance. They also discuss the distributions of cell adhesion molecules and qtoplasmic junctional components in low- and high-resistance cells. Finally, thq describe the role of phosphorylation in modulating both tight and adherens junc tions. 6. ..
AIJXIA SM. MIIUPR WA BUICK CA, NNhW PJ: Molecular and Cellular Properties of PECAM-1 (endoCAM/CD31) a Novel Vascular Cell-Cell Adhesion Molecule. / CeN Rid 1991, 114:105~1068 This paper demonstrates the adhesive htnction of PECAMI \ia tr;lns. fection of COS and L-cells. Perhaps the most surprising result is that transfected L-cell adhesion is CaL+ -dependent, mising the possibili~ that PECAM 1 binding is heterophilic. 7.
of the Human 1991, 1296-10.
HEwlARK RL, DEGNER M. Sc~w~ri! SM: Identification of a Ca2+ -dependent Cell-Cell Adhesion Molecule in EndotheIiaI Cells. ./ Cell Rio/ 1990, 1 IO:l’+i-I756
8. .
S~!ZIIKI 5, Swo K. T~WIHARA H. Diversity of the Cadherin Family: Evidence for Eight New Cadherins in Nervous Tissue. Cell Ke@cl 1991, 2:261-270. These investigators used polymerAse chain rr;lction techniques to iso. late cDNAs for eight potentially new cadherins in nervous tissue. The! suggest that one of these. cadherin-5. is present in endothelial cells and may correspond to the \zscular cadherin of Heimark e/ a/. 171. The cy toplasmic domain of this cadherin is not highly homologous to those of other cadherins and might not have been revealed in the cloning studies of Liaw Cl al. [9]. 9.
hw CW, CANNON C. POWER MD, KIHONEKA PK, R[IHIw IdentiIication and Cloning of Two Species of Cadherins Bovine EndotheIiaI Cells. T;IIBO J 1990, 9:2X-2708.
IL in
10.
LIAW CW. TOhlASEUJ KJ, C.&~NON C, DAVIS K, R~II~IN L: Disruption of MDCK and Bovine EndotheUaI Cell Tight Junctions with Cadherin Synthetic Peptides. / &/I Hiol 1990, 11 l:-tOX.
11. .
L~PIGNANI MG, RESNATI M. DEJANA E, MARCHISIO PC: The Role of Integrins in the Maintenance of EndotheIiaI Monolayer Integrity. J Cell Rio/ 1991, 112:-1?&i90. Thtl authors discuss the potential role of integrins in maintaining interactions between endothelial cells. While this paper concentrates on the effects of antibodies on endothelial cell monolayer integriv, consideration must also be given to the possibility that the primary erect of thy antibodies m on endothelial cell-substmte adhesion. 12.
ITOI-1 M, YONEMLIRA S, NI\GAFLICHI A. TSLIKIT.~ S. Ts[wr~ 220 kD Undercoat-constitutive Protein: Its Specific ization at Cadherin-based Cell-Cell Adhesion Sites. t?iol 1991. 115:144+1462.
13.
STEVENSON BR, Sicuwo JD, ,M~~SEKER MS. G~ODENOUGH DA: Identification of ZO-1: A High Molecular Weight Polypeptide Associated with the Tight Junction (Zonula Occludens) in a Variety of EpitheIia .I Cell Rid 1986, 103:755-766.
14.
Cm S, SAHANAY H, JAKES R, GEIGER B. KENIXUCKJONES J: Cinguilin: a New Peripheral Component of Tight Junctions. Nalrrre 1988, 333~272-276.
15.
WATSON PM, ANDERSON The Tight-junction++&&
TM, VANITAIUE C Protein ZO-1
and
Rat
and
tight
Blood-Brain
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Rubin
iVeztro.cci
SP: Electrical Resistance of Brain Brain Res 1982, 241:4+55.
Len
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17.
131:1-r AM, JONES IHC, AHHUIT NJ: Electrical the Blood-Brain Barrier in Anaesthetized mental Study. / Plqsiol 1990, 429:t7-62.
18.
Sr~%~,wt’ PA WILEY MJ: Developing Nervous Tissue Induces Formation of Blood-Brain Barrier Characteristics in Invading EndotheliaI Cells: a Study Using Quail-chick Transplantation Chimeras. Der, Rio1 1981, 84:18.3-192.
19.
J&~%EH RC, RV‘F MC: Astrocytes Properties in EndotheliaI CeUs.
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~LWAR~ JL. D~WRI\ISA’~-IAPHOR K: Occluding Structure-Function Relationships in a Cultured Monolayer. ,I Cell Biol 1985, 101:212&2133.
Induce IVatttre
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‘1. ..
RAOH TJ. KllElur/.t% Sl SAU’ADA GA: Permeability of Bovine Brain Microvessel Endothelial Cells In Vitro Barrier Tightening by a Factor Released from Astroglioma CeUs. Eq Cell Kes 1992, 199:33&340. This article describes another cell culture model of the blood-brain barrier in which endothelial cells are induced by factors secreted from C6-glioma cells. The absolute resistance of the endothelial cell monolayers ~3s not vev high, but the system may be quite useful in identifying glia-derived lactors that inIluence blood-brain barrier permeability. 22.
O!,wuclm meability
GK: Tumor Promoter-induced of Epithelial Tight Junctions.
Changes in the PerGel/ 1981, 23:9%103.
‘3. ..
ClTl S: Protein Kinase Inhibitors Prevent Junction Dissociation Induced by Low Extracellular Calcium in MDCK Epithelial Cells. J Cell Biol 1992. 117:16+1X This article is relcant to understanding regulation of tight junction resistance, % it implicates one or more kinases in the process of EGTAinduced changes in epithelial adherens and tight junctions. 211. .
VOI&KC T. ZICK Y. DROR R. S&LUAY I, GILON C. LEvrrw A. GEIGER B: The Effect of Tyrosine-specific Protein Phosphorylation on the Assembly of Adherens-type Junctions. EIIHO .I 1992, 1 1:1733-1742. The authors discuss the role of vrosine phosphorylation in the maintenance of adherens and tight junctions. 25. .
Tsl’Krr.4 5, OlSHl K, AKlYMM T, Y&w%UstlI Y, Y.&\lUlOTO T, TS~.KIT.A S: Specific Proto-oncogenic Tyrosine Kinases of src Family Are Enriched in Cell-to-Cell Adherens Junctions Where the Level of Tyrosine Phosphorylation is Elevated. .I Cell Bid 1991. 113:86?-879. This paper and [ 24* ] illustrate a striking increase in the level of tyrosine phosphoqkJtion on one or more adherens junction proteins when epithelial cells are treated nith an inhibitor of tyrosine phosphatases. The authors aIlso describe three proto-oncogene tyrosine kinases associated with adherens junctions. 26.
S: A Local./ Cell
M, Docmow SR: is a Component
adhesion
16.
NEwhlAN
5. ..
cells:
IOSSINSh?’ AS. PLL’TA R, SONG MJ, BmhtiJm’ V, MOW RC. WISNIEW,SKI HM: Mechanisms of Inflammatory CeU Attachment in Chronic Relapsing Experimental Allergic Encephalomyelitis: a Scanning and High-voltage Electron Microscopic Study of the Injured Mouse Blood-Brain Barrier. Microcvm Res 1991, 41:299-310.
‘7. .
B~ITCHER EC: Leukocyte-EndotheUaI CeU Recognition: Three (or More) Steps to Specificity and Diversity. Cell 1991, 67:1033-1036. An estremely provocative review illustrating the complexity of leukoqle-endothelial cell interactions and the advantages that this complexiv proffers.
LL Rubin. Eisai London Research laboratories, Darwin sity College London, London WClE 6BT, UK.
Building,
Univer-
833
