1 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; 2 Johns Hopkins Asthma and Allergy Center, Department of Pulmonary and Critical Care, Baltimore, Maryland 21224; 3 Pulmonary Division, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242; 4 Department of Medicine, Columbia University, New York, New York 10019; and 5 Department of Pharmacology, College of Medicine, University of Illinois-Chicago, Chicago, Illinois 60612
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ABSTRACT |
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Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.
gp60; myosin light chain kinase; vascular endothelial-cadherin; adenyl cyclase; calcium
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INTRODUCTION |
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IN THE INTACT BLOOD VESSEL, the endothelium forms a continuous, semipermeable barrier. Barrier integrity differs between organs, and it has also become apparent that barrier integrity even differs within vascular segments of the same organ (1, 5, 14, 22, 27). For example, the lung endothelium in vessels < 30 µm in diameter forms a more restrictive barrier than either arterial or venular endothelium (1, 5, 22). In response to inflammatory stimuli, the endothelial barrier becomes less restrictive, resulting in increased water and protein permeability. Two mechanisms can account for such an increase in permeability: paracellular (i.e., between cell) pathway and transcytotic (i.e., through cell) transport. Paracellular transport of molecules was first reported in 1961 by Majno and Palade (17), who suggested that histamine induced the formation of inter-endothelial cell gaps at inflammatory sites. The concept that intercellular gaps represent the sites of small- and large-"pore" pathways has been the general model of endothelial permeability. Recently, it has become apparent that the endothelial barrier does not simply behave as a physical sieve (23-25, 28, 38). Transcellular albumin transport may also occur secondary to albumin binding to a specific docking protein (gp60) that induces vesicular transport across the endothelium (9, 28-30, 33, 37). This discovery prompted speculation that the transcytotic pathway may also account for the large-pore pathways (24), leading to a flurry of experiments to determine whether gp60-activated signaling represents a mechanism of site-directed protein delivery and increased transendothelial permeability. These advances were reviewed at the 2000 Experimental Biology Conference held in San Diego, CA.
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TRANSCELLULAR PATHWAYS IN ENDOTHELIAL CELLS1 |
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In the past five years, the presence of a gp60-mediated albumin
transport pathway has been well established (9, 23-26,
28-30, 33, 37, 38), although physiological function and
modes of regulation by specific signal transduction pathways are still poorly understood. New data indicate that gp60 activation induces the
transport of albumin without a concurrent increase in hydraulic conductivity across either endothelial cell monolayers in vitro or
isolated perfused rat lungs. These data suggest that one mechanism of
uncoupling protein permeability from water permeability involves the
gp60-mediated transport pathway. Whether inflammatory mediators induce
albumin transport and whether the gp60-mediated pathway is capable of
bulk albumin transport in physiological and pathophysiological conditions remain important future challenges. Localization of gp60
within caveolae suggests that its presence is associated with
endocytotic vesicles (10, 11, 34). Identification that albumin binding to gp60 induces tyrosine phosphorylation, activation of
Src tyrosine kinases, pp60c-Src, and
Fyn implicates receptor tyrosine kinase activity in albumin transcytosis (38). Interestingly, new studies demonstrate
that selective inhibition of Gi prevented albumin from
activating tyrosine kinase activity and from increasing albumin
transport, suggesting that gp60 is linked to activation of tyrosine
kinase through heterotrimeric G proteins (18). These
observations generally support the findings that genistein and
herbimycin A prevent gp60-mediated albumin transport (38).
Thus while considerable progress has been made in identifying certain
components of the signaling pathways activated on albumin binding to
the endothelium, key molecular events remain to be described.
Similarly, how these signaling events initiate transcytosis and the
molecular sequelae required for directed apical to basal vesicular
movement remain important future challenges.
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KINASE REGULATION OF BARRIER FUNCTION2 |
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Considerable progress has been made in understanding how inflammatory agonists act to promote formation of intercellular gaps. The discovery that endothelial cells possess the molecular machinery needed to initiate and sustain a "contraction" had lead to the idea that inflammatory agonists increase constitutive actomyosin interaction sufficient to increase inwardly directed tension and pull cells apart at their sites of adhesion (16, 42). Several reports (7, 20, 31) showing that inhibition of nonmuscle myosin light chain kinase (MLCK), an enzyme required to initiate actomyosin interaction, prevented Gq-linked agonists from decreasing both cell-cell adhesion and endothelial barrier integrity are consistent with this idea . However, reports that inhibition of MLCK does not prevent direct intracellular Ca2+ concentration ([Ca2+]i) elevating agents from increasing permeability do not support this concept (16, 19). This evidence suggests that disruption of cell-cell adhesion is sufficient to increase endothelial cell permeability without an increase in tension per se (2). Cloning and expression of multiple endothelial cell-specific MLCK isoforms (and splice variants) by Garcia and colleagues (6, 8, 15, 32, 39, 40) provide an exciting advancement that may underlie these disparate findings. MLCK isoforms each possess unique regulatory sites. MLCK1 possesses a 922-amino acid NH2-terminal sequence that is not present in smooth muscle MLCK. Of particular interest is the presence of both SH2 and SH3 binding domains and a tyrosine residue (Tyr485) that is sufficient to activate kinase activity on phosphorylation without involvement of Ca2+/calmodulin. Indeed, Garcia has shown that stimulation of tyrosine kinase activity results in activation of the "contractile complex" that includes MLCK, Src, and cortactin. Completion of future key studies will require that these observations be placed in the context of endothelial cell barrier function in vivo. In addition, site-specific localization of MLCK isoforms and control of their discrete regulatory sites will also need to be considered. Finally, it will be important to determine whether MLCK isoforms control both intercellular gap formation and the molecular motor important for gp60-mediated vesicular trafficking.
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TENSION, ADHESION, AND CONTROL OF ENDOTHELIAL PERMEABILITY3 |
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That disruption of cell-cell adhesion mediated by vascular endothelial (VE)-cadherin is sufficient to induce inter-endothelial cell gap formation provides compelling evidence that tethering forces regulate barrier function (2). Although cell-cell adhesion in endothelial cells may be due to both tight and adherens junctions, VE-cadherins play a prominent role in cell tethering (2, 4). Disruption of VE-cadherin function resulted in interstitial edema and accumulation of inflammatory cells in the heart and lung microcirculation (2). Shasby and colleagues (21, 41) have recently shown that inhibition of VE-cadherin function decreases cell-cell but not cell-matrix resistance, suggesting that cadherins mediate cell-cell adhesion important for the control of barrier integrity. Moreover, they demonstrated that agonists such as histamine that raise [Ca2+]i activate signal transduction cascades that decrease the VE-cadherin-dependent sites of adhesion without increasing cell tension. These findings have begun to uncouple the Gq signaling events regulating cell tension from the Gq-activated signaling events that control cell adhesion. Future efforts will be required to establish the key signal transduction pathways that link inflammatory agonists to the sites of cell adhesion.
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CALCIUM REGULATION OF ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE: CONTROL OF ENDOTHELIAL PERMEABILITY4 |
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Endothelial cell biologists have recognized that a rise in [Ca2+]i is sufficient to induce inter-endothelial cell gap formation and increase permeability to macromolecules. Generally, however, agonists that elevate [Ca2+]i only increase permeability in the absence of a rise in cAMP (19). Stevens presented his work addressing the link between [Ca2+]i and cAMP. The observation that endothelial cells express the Ca2+-inhibited isoform of adenylyl cyclase, the enzyme responsible for cAMP synthesis, provides a compelling mechanism through which physiological increases in [Ca2+]i decrease cAMP and thereby control endothelial cell barrier function (36). However, direct [Ca2+]i elevating agents (i.e., agonists that increase [Ca2+]i without concurrent activation of heterotrimeric G proteins) only increase lung macrovascular endothelial cell permeability and not microvascular permeability. Although microvascular endothelial cells express the Ca2+-inhibited isoform of adenylyl cyclase, agonists elevating [Ca2+]i do not decrease cAMP, suggesting that maintenance of cAMP is an important homeostatic mechanism contributing to enhanced barrier properties of the microvascular endothelium (35). Further support for this idea has recently come from a study (3) showing that the physiological rise in [Ca2+]i induced microvascular endothelial cell gap formation only under experimental conditions in which Ca2+ inhibited adenylyl cyclase. These findings strongly support the involvement of this enzyme in control of endothelial cell barrier function through the integration of [Ca2+]i and cAMP signaling events. Perhaps most importantly, the findings support the developing idea that endothelial cells derived from conduit vessels and microvessels are phenotypically distinct. Thus unique signaling circuitry in these cell populations may underlie site-specific vascular responses to inflammation. Systematic exploration of cell-specific signaling cascades and their link to segment-specific function represent an important future challenge.
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CELL SIGNALING IN THE PULMONARY MICROCIRCULATION5 |
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The proinflammatory effects of increased endothelial cell
[Ca2+]i in situ have been identified by
Bhattacharya and co-workers (12, 13), providing critical
integration between studies performed in culture and in the intact
organ. An increase in vascular pressure was shown to activate
mechanogated cation channels, resulting in Ca2+ entry that
stimulated the expression of endothelial cell P-selectin. More
recently, this group has demonstrated that direct instillation of tumor necrosis factor- into alveoli increased
[Ca2+]i not only in alveolar epithelium but
also, remarkably, in adjacent microvascular endothelium
(12). This rise in endothelial cell [Ca2+]i also resulted in an increase in
P-selectin expression. These data are provocative because they indicate
first that increased [Ca2+]i may direct an
inflammatory response to appropriate vascular segments through the
upregulation of endothelial adhesion molecules, perhaps without
inter-endothelial cell gap formation, and second that communications
between alveolar and vascular microcirculatory compartments may direct
leukocyte recruitment to sites of alveolitis. Future studies will be
critical to assess how these signaling events proceed and to determine
how a rise in microvascular versus macrovascular endothelial cell
[Ca2+]i coordinates with other signaling
events to induce a site-specific inflammatory response.
In conclusion, considerable progress has been made in the understanding of molecular events that regulate endothelial cell shape and signaling processes that initiate transcytotic and paracellular transport of macromolecules. Discriminating between the key events that regulate the site-specific endothelial cell response to inflammation and the unique signaling events activated to coordinate these processes represent important future challenges in microvascular biology.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Stevens, Dept. of Pharmacology, Univ. of South Alabama College of Medicine-MSB 3364, Mobile, AL 36688-0002 (E-mail: tstevens{at}jaguar1.usouthal.edu).
2 Presented by Joe G. N. Garcia.
3 Presented by D. Michael Shasby.
4 Presented by Troy Stevens.
5 Presented by Jahar Bhattacharya.
1 Presented by Asrar B. Malik.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albert, RK,
Kirk W,
Pitts C,
and
Butler J.
Extra-alveolar vessel fluid filtration coefficients in excised and in situ canine lobes.
J Appl Physiol
59:
1555-1559,
1985
2.
Corada, M,
Mariotti M,
Thurston G,
Smith K,
Kunkel R,
Brockhaus M,
Lampugnani MG,
Martin-Padura I,
Stoppacciaro A,
Ruco L,
McDonald DM,
Ward PA,
and
Dejana E.
Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo.
Proc Natl Acad Sci USA
96:
9815-9820,
1999
3.
Creighton, J,
and
Stevens T.
cAMP accumulation dictates calcium inhibition of adenylyl cyclase 6 necessary to increase gap formation in lung microvascular endothelial cells (Abstract).
FASEB J
14:
A693,
2000[ISI].
4.
Dejana, E,
Corada M,
and
Lampugnani MG.
Endothelial cell-to-cell junctions.
FASEB J
9:
910-918,
1995
5.
Effros, RM,
Schapira R,
Presberg K,
Ozker K,
and
Jacobs ER.
Stop-flow studies of solute uptake in rat lungs.
J Appl Physiol
85:
986-992,
1998
6.
Garcia, JG,
Lazar V,
Gilbert-McClain LI,
Gallagher PJ,
and
Verin AD.
Myosin light chain kinase in endothelium: molecular cloning and regulation.
Am J Respir Cell Mol Biol
16:
489-494,
1997[Abstract].
7.
Garcia, JG,
Verin AD,
and
Schaphorst KL.
Regulation of thrombin-mediated endothelial cell contraction and permeability.
Semin Thromb Hemost
22:
309-315,
1996[Medline].
8.
Garcia, JG,
Verin AD,
Schaphorst K,
Siddiqui R,
Patterson CE,
Csortos C,
and
Natarajan V.
Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src.
Am J Physiol Lung Cell Mol Physiol
276:
L989-L998,
1999
9.
Ghinea, N,
Eskenasy M,
Simionescu M,
and
Simionescu N.
Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface.
J Biol Chem
264:
4755-4758,
1989
10.
Ghitescu, LD,
Crine P,
and
Jacobson BS.
Antibodies specific to the plasma membrane of rat lung microvascular endothelium.
Exp Cell Res
232:
47-55,
1997[ISI][Medline].
11.
Ghitescu, L,
Jacobson BS,
and
Crine P.
A novel, 85 kDa endothelial antigen differentiates plasma membrane macrodomains in lung alveolar capillaries.
Endothelium
6:
241-250,
1999[Medline].
12.
Kuebler, WM,
Parthasarathi K,
Wang PM,
and
Bhattacharya J.
A novel signaling mechanism between gas and blood compartments of the lung.
J Clin Invest
105:
905-913,
2000
13.
Kuebler, WM,
Ying X,
Singh B,
Issekutz AC,
and
Bhattacharya J.
Pressure is proinflammatory in lung venular capillaries.
J Clin Invest
104:
495-502,
1999
14.
Lamm, WJ,
Luchtel D,
and
Albert RK.
Sites of leakage in three models of acute lung injury.
J Appl Physiol
64:
1079-1083,
1988
15.
Lazar, V,
and
Garcia JG.
A single human myosin light chain kinase gene (MLCK; MYLK).
Genomics
57:
256-267,
1999[ISI][Medline].
16.
Lum, H,
and
Malik AB.
Regulation of vascular endothelial barrier function.
Am J Physiol Lung Cell Mol Physiol
267:
L223-L241,
1994
17.
Majno, G,
and
Palade GE.
Studies on inflammation. I. The effect of histamine and serotonin on vascular permeability: an electron microscopic study.
J Biophys Biochem Cytol
11:
571-605,
1961
18.
Minshall, RD,
Niles WD,
Tiruppathi C,
Gilchrist A,
Hamm HE,
Vogel SM,
and
Malik AB.
Association of endothelial cell surface gp60 with caveolin-1 mediates vesicle formation and trafficking by activation of Gi-coupled Src kinase pathway (Abstract).
FASEB J
14:
A412,
2000[ISI].
19.
Moore, TM,
Chetham PM,
Kelly JJ,
and
Stevens T.
Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP.
Am J Physiol Lung Cell Mol Physiol
275:
L203-L222,
1998
20.
Moy, AB,
Shasby SS,
Scott BD,
and
Shasby DM.
The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells.
J Clin Invest
92:
1198-1206,
1993[ISI][Medline].
21.
Moy, AB,
Winter M,
Kamath A,
Blackwell K,
Reyes G,
Giaever I,
Keese C,
and
Shasby DM.
Histamine alters endothelial barrier function at cell-cell and cell-matrix sites.
Am J Physiol Lung Cell Mol Physiol
278:
L888-L898,
2000
22.
Parker, JC,
Stevens T,
and
Martin SL.
Vascular segmental permeability after high peak inflation pressure (PIP) injury in isolated rat lungs (Abstract).
FASEB J
14:
A604,
2000.
23.
Predescu, D,
Horvat R,
Predescu S,
and
Palade GE.
Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide.
Proc Natl Acad Sci USA
91:
3014-3018,
1994[Abstract].
24.
Predescu, D,
and
Palade GE.
Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium.
Am J Physiol Heart Circ Physiol
265:
H725-H733,
1993
25.
Predescu, D,
Predescu S,
McQuistan T,
and
Palade GE.
Transcytosis of alpha-acidic glycoprotein in the continuous microvascular endothelium.
Proc Natl Acad Sci USA
95:
6175-6180,
1998
26.
Predescu, SA,
Predescu DN,
and
Palade GE.
Plasmalemmal vesicles function as transcytotic carriers for small proteins in the continuous endothelium.
Am J Physiol Heart Circ Physiol
272:
H937-H949,
1997
27.
Qiao, RL,
and
Bhattacharya J.
Segmental barrier properties of the pulmonary microvascular bed.
J Appl Physiol
71:
2152-2159,
1991
28.
Schnitzer, JE.
gp60 is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis.
Am J Physiol Heart Circ Physiol
262:
H246-H254,
1992
29.
Schnitzer, JE,
Carley WW,
and
Palade GE.
Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein.
Proc Natl Acad Sci USA
85:
6773-6777,
1988[Abstract].
30.
Schnitzer, JE,
Ulmer JB,
and
Palade GE.
A major endothelial plasmalemmal sialoglycoprotein, gp60, is immunologically related to glycophorin.
Proc Natl Acad Sci USA
87:
6843-6847,
1990[Abstract].
31.
Sheldon, R,
Moy A,
Lindsley K,
Shasby S,
and
Shasby DM.
Role of myosin light-chain phosphorylation in endothelial cell retraction.
Am J Physiol Lung Cell Mol Physiol
265:
L606-L612,
1993
32.
Shi, S,
Verin AD,
Schaphorst KL,
Gilbert-McClain LI,
Patterson CE,
Irwin RP,
Natarajan V,
and
Garcia JG.
Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function.
Endothelium
6:
153-171,
1998[Medline].
33.
Siflinger-Birnboim, A,
Schnitzer J,
Lum H,
Blumenstock FA,
Shen CP,
Del Vecchio PJ,
and
Malik AB.
Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers.
J Cell Physiol
149:
575-584,
1991[ISI][Medline]. (Corrigenda. J Cell Physiol 151: June 1992, p. 642.)
34.
Stan, RV,
Ghitescu L,
Jacobson BS,
and
Palade GE.
Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein.
J Cell Biol
145:
1189-1198,
1999
35.
Stevens, T,
Creighton J,
and
Thompson WJ.
Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function.
Am J Physiol Lung Cell Mol Physiol
277:
L119-L126,
1999
36.
Stevens, T,
Nakahashi Y,
Cornfield DN,
McMurtry IF,
Cooper DM,
and
Rodman DM.
Ca2+-inhibitable adenylyl cyclase modulates pulmonary artery endothelial cell cAMP content and barrier function.
Proc Natl Acad Sci USA
92:
2696-2700,
1995[Abstract].
37.
Tiruppathi, C,
Finnegan A,
and
Malik AB.
Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.
Proc Natl Acad Sci USA
93:
250-254,
1996
38.
Tiruppathi, C,
Song W,
Bergenfeldt M,
Sass P,
and
Malik AB.
Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway.
J Biol Chem
272:
25968-25975,
1997
39.
Verin, AD,
Gilbert-McClain LI,
Patterson CE,
and
Garcia JG.
Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium.
Am J Respir Cell Mol Biol
19:
767-776,
1998
40.
Verin, AD,
Lazar V,
Torry RJ,
Labarrere CA,
Patterson CE,
and
Garcia JG.
Expression of a novel high molecular-weight myosin light chain kinase in endothelium.
Am J Respir Cell Mol Biol
19:
758-766,
1998
41.
Winter, MC,
Kamath AM,
Ries DR,
Shasby SS,
Chen YT,
and
Shasby DM.
Histamine alters cadherin-mediated sites of endothelial adhesion.
Am J Physiol Lung Cell Mol Physiol
277:
L988-L995,
1999
42.
Wysolmerski, RB,
and
Lagunoff D.
Involvement of myosin light-chain kinase in endothelial cell retraction.
Proc Natl Acad Sci USA
87:
16-20,
1990[Abstract].