Integrins as dynamic regulators of vascular function - CiteSeerX

REVIEWS

Integrins

as dynamic

PRMCIS Vascular Boston,

ABSTRACT

W LUSGINSKAS

regulators AND JACK

Research

Division, Departments Massachusetts (12115, USA

The vascular

endothelium

endothelium

.

leukocytes

.

lines the entire

thrombospondin

selectins

INTERACTIONS IN THE cardiovascular system are dynamic processes that require precise regulation. Platelets circulate in a nonaggregated state despite the presence of 2 mg/ml of fibrinogen. Leukocytes must be recruited to sites of inflammation in the presence of blood flow. These processes involve a family of cell-surface, heterodimeric glycoproteins that have been designated integrins. Integrins are expressed by virtually all tissues in the human body. In the vasculature they are involved in maintaining normal structure and in remodeling associated with wound healing and angiogenesis. The term integrin was coined to indicate that they integrate the activities of the extracellular matrix and the cytoskeleton. The term goes beyond the molecular nature of this connection to indicate that these molecules integrate the cell’s activities in the context of its extracellular environment. Thus, integrins are important components of cellular signal transduction. The literature on integrins has been extensively reviewed in recent years. We have included five of the most recent and comprehensive reviews as references to the literature in general (1-5). In this review, we provide a brief summary of the common properties of the integrin family and summarize integrin expression on endothelial cells. We then give examples from vascular biology that illustrate these common functions. We will focus in particular on the dynamic aspect of vasculogenesis, angiogenesis, wound healing, and circulating blood monocyte recruitment to inflammatory sites under flow CELL-TO-CELL

0892-6638/94/0008-0929/$01

.50. © FASEB

function

LAWLEW of Pathology,

cardiovascular system and serves as a nonthrombogenic and selectively permeable boundary between the bloodstream and extravascular space. Endothelial cells are polar cells that are continuously subjected to fluid-generated forces on their luminal surface whereas their abluminal surface resides on basement membranes/extracellular matrix. The integrin family of cell-surface heterodimeric glycoproteins is located along both of these surfaces and participates in maintaining the normal endothelium and in the dynamic changes associated with the pathophysiology of the endothelium. Endothelial cell i3 i and f3 integrins function together with other families of adhesion molecules during vasculogenesis, angiogenesis, inflammation, and wound healing. Leukocyte /3 and (32 integrins, in conjunction with members of the Ig and selectin gene families expressed on endothelium, mediate leukocyte recruitment to sites of inflammation. The structural and functional properties of integrins make them uniquely suited to mediate these essential and complex processes in the vasculature.Luscinskas, F. W., Lawler, J. Integrins as dynamic regulators of vascular function. FASEB J. 8: 929-938; 1994. Key Words:

of vascular Brigham

and Women’s

Hospital

COMMON

FEATURES

OF

Integrins

are heterodimeric

and Harvard

Medical

INTEGRIN membrane

School,

FUNCTION glycoproteins

To date, 15 a subunits and 8 /3 subunits have been identified (Fig. 1) (1-5). The subunits are associated through noncovalent bonds and transported to the cell surface as a complex. Different cell types assemble and express different a/3 complexes. Some are restricted in their structure and tissue expression. For example, the integrins aLJ3s, aM/32, and ax/32 are unique in that they are expressed only on blood leukocytes and only as these complexes. By contrast, the a subunit is expressed by many different tissues and can associate with several different j3 subunits (Fig. 1B). Additional molecular heterogeneity in some subunits is generated by proteolytic processing and by alternative splicing of the cytoplasmic domains. The cell modulates its interaction with extracellular matrix and other cells by modifying the structure and function of the integrins. By specifically synthesizing certain subsets of a and /3 subunits, the cell can select for recognition of specific ligands. By modulating the reactivity of the integrins through activation, the cell can control ligand binding temporally (see section on inside-out signal transduction). Some cells can rapidly increase membrane expression of integrins by mobilizing specific storage pools. Neutrophils and monocytes transport /32 integrins from peroxidasenegative granules to the cell surface in response to inflammatory mediators. Integrins participate interactions

in cell-to-matrix

and

cell-to-cell

The combination of a and 3 subunits determines the ligand specificity (1-5). However, there is a great deal of redundancy in that most integrins are capable of binding several different adhesive glycoproteins and most of the adhesive glycoproteins bind to multiple integrins. This is also true for

tTo whom correspondence and requests for reprints should be addressed, at: Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Ave., LMRC Rm. 416, Boston, MA 02115, USA. 2Abbreviations: TGF, transforming growth factor; VCAM-1, vascular cell adhesion molecule-i; ICAM-l, intercellular adhesion molecule-i; CR3, CDllb, iC3b receptor, activated complement factor 3 receptor; CEA, carcinoembryonic antigen; lAP, integrinassociated protein; RGD, arg-gly-asp; PMA, phorbol-myristate acetate; VEGF, vascular endothelial growth factor; EA cells, endothelial-like cell line E Ahy 926; SCID, severe combined immunodeficient; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemotactic factor-i; PAF, platelet activating factor; bFGF, basic fibroblast growth factor; mAb, monoclonal antibody; ES, embryonic stem.

929

REVIEWS

A a i

cation binding sites

domain (7), which also has been referred to as the I domain (1, 2). It has been demonstrated recently for aM/32 (CR3, CDlIb, or activated complement factor 3 receptor) that this A domain (-200 amino acids) contains a fourth metal binding site that is essential for recognition of one of its substrates, iC3b, by the receptor (7). Similar calcium-binding motifs are present in the integrin /3 subunits. A point mutation in this region of the /33 integrin has been identified in the human population (3). Platelets from these individuals express aItb/33 with defective adhesive glycoprotein binding activity. These platelets also have abnormal binding of a monoclonal antibody that is capable of detecting

B 13 2 L

1/6

a

/-aUb

137

1

a47

a5/

Integrins a8

7

13

\

134

Figure 1. Schematic diagram of integrin structure (A) and subunit pairing (B). The locations of various structural motifs are indicated. The sites for ligand binding have been identified by chemical crosslinking studies and mapping loss of function mutations. Not all a subunits are proteolytically cleaved to form two disulfide-linked subunits. Some a subunits contain only three divalent cation binding sites. The pairing of aE with (3 has been reported recently (89). the integrins that participate in cell-to-cell interactions by binding to the members of the immunoglobulin superfamily. Both the a4j31 and cr4/37 integrins can bind to vascular cell adhesion molecule-I (VCAMl),2 and both a1j32 and aM/32 can bind to intercellular adhesion molecule-l (ICAM-l). The least selective integrins are the arIb/33 on platelets and av/33 on many cell types, including endothelial cells and smooth muscle cells. These integrins bind to adhesive glycoproteins in the vasculature including fibrinogen, fibronectin, vitronectin, thrombospondin, von Willebrand factor, and collagen.

Integrin structure dependent

and function

is divalent

cation

The presence of Ca2, Mg2, and Mn2 affects the structure and function of integrins. Three or four highly conserved divalent cation binding sites have been identified in the a subunits (Fig. 1A) (1-5). Calcium binding studies with a recombinant peptide that includes the divalent cation binding sites of ai1 indicate that two binding sites have a higher affinity for calcium than the other two (120 iM compared with 30 ISM) (6). The maximum binding to fibrinogen is observed when all four sites are occupied (6). Certain a subunits, aMf3s, ai132, a1/31, and cr2/31, contain an A-type

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Vol. 8

September

1994

calcium-dependent

conformational

changes.

The calcium binding sites of both the a and /3 subunits are in close proximity to the ligand binding site (1-5). Crosslinking studies with arg-gly-asp (RGD) peptide have identified the calcium-binding region of the /33 subunit of the allb/33 integrin as part of the ligand binding site (1-3). This observation, as well as characterization of /33 mutants, has led to the hypothesis that the bound cations interact directly with the ligand (3). This hypothesis is supported by the observation that differing effects of cations are observed on different ligands. Although binding of a3/31 to fibronectin and laminin is strongest in the presence of Mn, intermediate in the presence of Mg2, and weakest in the presence of Ca2, the binding of a3/31 to collagen is equivalent in the presence of any of these cations (8). interact

with

the cytoskeleton

Both the a and /3 subunits are transmembrane proteins with cytoplasmic tails at their carboxyl terminal (1-5). Except for /34, the cytoplasmic domains are relatively short. A specific interaction between two cytoskeletal components (a -actinin and talin) with /3 integrins has been demonstrated (9, 10). The integrins colocalize with vinculin and other focal contact proteins when cells spread on adhesive glycoprotein substrates. The composition of the extracellular matrix determines which integrins are included in focal contacts. The integrins provide a link for transmission of mechanical forces between the cytoskeleton and the extracellular matrix (11). Deletion of the cytoplasmic domain of $ results in a defect in the incorporation of integrins that contain this subunit into focal contacts (12, 13). Similarly, deletion of the cytoplasmic domain of a5 resulted in defective spreading and stress fiber formation when transfected cells were plated on fibronectin (14).

Integrin

function

is modulated

by cellular

environment

The ligand specificity and activation state of the integrins are modulated in a cell type-specific manner (1-5). The integrin a2/31 on endothelial cells functions as a receptor for collagen and laminin (15, 16). By contrast, platelet a2/31 binds to collagen but not to laminin (15, 16). When the receptors are isolated from the two cell types, this difference in ligand binding is preserved. A possible explanation for these findings is that integrin function is modulated in a cell type-specific fashion by ancillary molecules that copurify with the integrins. Lipids, glycoconjugates, and proteins have been reported to associate closely with integrins and to modulate their function (1-5). When purified a/33 is incorporated into lipid vesicles, the reactivity with vitronectin, fibronectin, and von Willebrand factor is influenced by the lipid composition of the vesicles (17). In vesicles composed of phosphatidylcholine only, binding to vitronectin but not to fibronectin or von Willebrand factor was observed. When phosphatidylethanolamine

The FASEB Journal

LUSCINSKAS AND

LAWLER

REVIEWS is included, binding to all three adhesive glycoproteins is observed. In neutrophils, integrin modulating factor-i is an unsaturated fatty acid that has been reported to modulate /32 integrin function (18). Integrin function is also supported by accessory membrane proteins. Three members of the Ig superfamily, carcinoembryonic antigen (CEA), embigin, and integrinassociated protein (lAP), have been reported to increase integrin-dependent cellular attachment to adhesive glycoproteins (19-21). lAP has been reported to function as an integrin-dependent calcium channel (22).

with an anti-$3 antibody, designated LIBS6. Treatment of platelets with LIBS6 alone did not cause a change in the pattern of tyrosine phosphorylation (27). In the presence of LIBS6 and fibrinogen, an increase in tyrosine phosphorylation of proteins of 50-60 kDa and 140 kDa is observed. Inhibition of fibrinogen binding with a function blocking monoclonal antibody inhibited tyrosine phosphorylation of these proteins (27). Thus, integrin occupation by adhesive glycoproteins serves to augment the signals that are transduced by soluble agonist.

Integrins are components transduction

INTEGRINS

Cellular

modulation

of inside-out

of ligand

affinity

signal

has been

reported

for

l3, /32, and /33 integrins

(1-5). Various /32 integrins on monocytes and neutrophils are stimulated by inflammatory mediators to bind iC3b, fibrinogen, factor X, ICAM-i, or ICAM-2. These processes involve classical signal transduction pathways that lead to up-regulation of affinity for extracellular ligands from inside the cell (inside-out signaling). Thrombin-induced activation of platelets involves Gproteins and protein kinase C. Whereas the cytoplasmic domain of /33 is phosphorylated after thrombin treatment, only a small percentage of the activated atIbI33 complexes are phosphorylated. Involvement of the cytoplasmic domains is implied from mutagenesis experiments. Deletion of the cytoplasmic domain of the alib subunit results in a constitutively active complex that binds fibrinogen with an affinity equivalent to the wild-type complex (23). Involvement of the cytoplasmic domain is implied by the identification of a variant of Glanzmann’s thrombasthenia in which a serine residue in the cytoplasmic tail of /33 is replaced by proline (24). The platelets from these individuals have impaired coupling between activation and fibrinogen binding. In contrast to the aIIb/33 complex, a1j32 is constitutively active when it is expressed in COS cells, but requires activation in leukocytes (1, 2). These observations have led to the hypothesis that integrin activation is dependent on cellular components that act to repress or stimulate activation (25).

/33

Integrins are components transduction

of outside-in

AND VASCULAR

FUNCTION

THE

expression

VASCULAR

on endothelial

ENDOTHELIUM cells

The expression of many proteins by endothelial cells as well as their behavior has been shown to depend on the species and vascular bed from which they originate. In addition, variations have been observed with culture conditions and passage number. The integrin composition of human and bovine large-vessel and microvascular endothelial cells has been characterized by several groups (Table 1). Large-vessel endothelial cells express a2/31, a3/3j, a5/31, and a/33 (refs 28-33 and references therein). In addition, human foreskin and bovine adrenal cortex microvascular endothelial cells have been reported to express a1$1, a6/31, a6/34, and a/35 (32, 33). Maintenance of the endothelium involves attachments to adhesive glycoproteins and the basement membrane. Endothelial cell integrins function as receptors for collagen, laminin, fibronectin, and thrombospondin. In addition, a direct interaction of integrins with the proteoglycan perlecan has been reported to be involved in the interaction of endothelial cells with basement membrane (34). Perlecan is a heparin sulfate proteoglycan constituent of basement membrane that contains an RGD sequence in the core protein. Adhesion of endothelial cells to the perlecan core protein is inhibited by a mixture of antibodies directed to /3 and a/33. Expression of the a1 subunit on human umbilical vein (large vessel) endothelial cells is induced by tumor necrosis factor a (TNFa), retinoic acid, or phorbol-myristate acetate

signal

The binding of adhesive glycoproteins or antibodies to integrins has been shown to affect cellular differentiation, activation, gene expression, and proliferation (1-5). Intracellular pH and calcium levels, tyrosine phosphorylation, and inositol lipid turnover serve to transduce the signal that results from integrin occupancy. When human endothelial cells attach and spread on fibronectin or vitronectin, an increase in intracellular calcium concentration is observed (26). These effects can be mimicked by antibodies to (3i or a/33 integrins that are adsorbed to plastic. However, treatment of endothelial cells with the antibodies in solution did not increase intracellular calcium. Similarly, unactivated aIIb/33 will bind solid-phase fibrinogen but not fibrinogen in solution. These results indicate that in certain contexts, the adhesive glycoproteins can induce a conformational change, and thus permit binding and signal transduction. This conclusion is further supported by the observation that purified inactive aub/33 can be activated to bind fibrinogen by pretreatment with RGD or fibrinogen y-chain carboxyl-terminal peptides. Platelet activation results in an increase in the number of proteins that are phosphorylated on tyrosine residues (27). Fibrinogen binding to unstimulated platelets can be induced

INTEGRINS

Integrin

AND

TABLE

1. Expression of integrins on endothelial cells Large Resting

vessel

TGF-3

Microvessel TNF-cs

Resting

,b,

aj3

cx2/31 a3(31

+

a5f31

+

a6131

+

a/31

?

a/33

+

a6/34 afls

? ?

+

I

n.c. n.c. n.c. g

+

TGF-

bFGF

n.c.’

n.c.’

1

t

+ +

I J,d

I d

J, C’ C I

f

+

I

n.c!’

#{247} +

+

n.c.

I I I

‘The results in both bFGF columns (under Microvessel heading) were obtained from refs 32 and 33, respectively. 1The symbols used are: +, expressed; , not expressed; ?, not reported; I, increased expression; I, decreased expression; nc., no change in expression. ‘Also induced by retinoic acid and PMA, but not by IL-1f3 (30). dCited as preliminary studies (32). ‘a,fl1 was not detected on bovine microvascular cells before bFGF (33). Reported to be expressed by microvascular cell in culture but not on capillaries from heart, lung, and lymph nodes (28). A1so decreased by IL-’lfl (31). 5Unchanged by TNFa or IFN7 alone, but decreased when these proteins are used together (31).

931

REVIEWS (PMA), but not by interleukin-1/3 (IL-1/3) (30). Treated cells become more adhesive to the a /3 ligands, including collagen and laminin. The expression of integrins on large- and small-vessel endothelial cells is also modulated by transforming growth factor-/31 (TGF-/31) (Table 1) (29, 32). With bovine aortic endothelial cells, both subunits of the a5/31 complexes and /3 are up-regulated in response to ‘IGF-f31. The modulation of integrin expression has been shown to occur by regulation at both transcription and translation. For microvascular endothelial cells, a2, a5, a6, and /3 subunits are up-regulated by TGF-/31; however, no consistent increase in a/33 expression is observed (32). When these cells are treated with basic fibroblast growth factor (bFGF), increased surface expression of a2/31, a5/31, a6j31, a6/34, and a/3 has been observed (Table 1; 32, 33). Variable results have been obtained for the effect of bFGF on microvascular endothelial cell expression of a1/31, a3/31, a6/31, and a$3 integrins (32, 33). These differences in expression may depend on the species as well as tissue of origin. Whereas a/3 is sometimes referred to as the vitronectin receptor, on endothelium it serves as a receptor for von Willebrand factor, thrombospondin, fibrinogen, and thrombin as well as vitronectin. Initial attachment of human umbilical vein endothelial cells to von Willebrand factor and fibrinogen through a/33 is followed by synthesis and secretion of fibronectin, and by recruitment of integrins to focal contacts (35). When human umbilical vein endothelial cells are plated on fibronectin, the integrins are clustered but the /33 integrins are not (36). By contrast, plating foreskin microvascular endothelial cells on fibronectin results in the recruitment of aJ33 and a5/31, suggesting that the a/33 functions as a fibronectin receptor on these cells (37). a/33 is expressed on both the apical and luminal surfaces of the endothelium (38). On the luminal surface, aj33 may sequester adhesive glycoproteins along the surface of the endothelium. In addition to functioning at the apical and basal surfaces, it has been reported that af3j, a2/31, and a5/31 are located at the endothelial cell-to-cell contact borders (39). The

role of integrins

in vasculogenesis

During its development, the cell takes environmental cues from various factors including growth factors, cytokines, proteoglycans, and extracellular adhesive glycoproteins. Matrix can act directly on the cell through interactions with receptors, including integrins. In addition, the matrix can present various growth modulatory factors to the cell. The vasculature is formed by endothelial cell precursors, designated angioblasts, that differentiate from the mesoderm (refs 40-43, and references therein). The formation of new vessels from free angioblasts is referred to as vasculogenesis, and the subsequent neovascularization by sprouting from existing vessels is referred to as angiogenesis. In the mouse, vessels are formed by migration of angioblasts in the embryo and in the blood islands of the yolk sac. The appearance of angioblasts coincides with the expression of the flk-1 receptor tyrosine kinase (43). Flk-1 has been shown to function as a receptor for vascular endothelial growth factor (VEGF). The role of integrins in vasculogenesis has been approached by the injection of function blocking antibodies into the developing quail embryo and by “knocking” out the a5 gene by homologous recombination in the mouse. In the quail embryo, the formation of the dorsal aorta occurs over a period of approximately 7 h after the first appearance of clusters and linear arrays of angioblasts in the appropriate position (44). Injection of anti-integrin antibody CSAT inhibits the latter steps in the formation of the dorsal aorta.

932

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1994

Whereas the initial phases of angioblast commitment and cord formation proceed normally, the angioblasts are more spindle shaped and the developing vessels are without lumens. Yang et al. (45) have recently reported the results of deletion of the a5 gene from the mouse genome. The homozygote embryos die in utero after approximately 10-11 days of development with numerous morphological defects. At day 9.5, a defect in blood vessel formation is observed and many blood cells are found in the exocoelomic space. At the same time, a decrease in blood cells within the heart and embryonic circulation is observed. The authors concluded that the failure to form a competent vascular system may be the cause of the developmental arrest beyond day 9.5 of gestation (45). Because of the importance of the vascular system to the developing embryo, similar results may be observed with mice that are deficient in other proteins involved in vasculogenesis. Fibronectin-deficient mice also develop defects in the yolk sac vasculature and have blood cells in the exocoelomic space (46). These mice also have a defect in the structure of the dorsal aorta and die somewhat earlier than the a5deficient mice. These data are consistent with immunolocalization data indicating that vasculogenesis and angiogenesis occur in fibronectin-rich matrices initially, with laminin expression occurring later in mature vessel basement membrane (41). Taken together, these results establish the importance of the a5/31-fibronectin ligand-receptor pair in vasculogenesis. The in vitro differentiation of embryonic stem (ES) cells provides a useful tool for the study of vasculogenesis, particularly in the case of embryonic lethal gene deletions. In vitro, ES cells differentiate to cystic embryoid bodies that contain blood islands and vascular channels that contain hematopoietic cells (47, 48). Endothelial cells in embryoid bodies have been identified on the basis of uptake of acetylated low-density lipoprotein and von Willebrand factor expression. Integrin

function

during

angiogenesis

Endothelial cells adopt a migratory phenotype in response to angiogenic stimuli. This phenotypic change involves alterations in the level of expression of many proteins including protease and protease inhibitors, growth factors, adhesive glycoproteins and integrins, and other cell-surface receptors. By altering the profile of proteins that are secreted, the endothelial cell modifies the extracellular environment to support cellular migration. In vitro attachment and migration assays, as well as capillary tube formation, have been used to identify some molecules that are important to this process. These systems depend on the specific experimental protocols that are used. Human umbilical vein or neonatal foreskin endothelial cells will form networks of capillary tubelike structures when they are grown on collagen I or fibrin gels in the presence of PMA (49). An increase in the number and size of tubes is observed in the presence of antibodies to a2/31 or aV/33 on collagen I and fibrin gels, respectively. Whereas the lumen is formed by a single cell in the absence of the antibody, multiple cells could be identified surrounding the lumen in the presence of the antibodies. In this system, the anti-integrin antibodies seem to facilitate tube formation by shifting the balance of attachment forces away from a strong interaction with extracellular matrix and toward cell-to-cell interaction. The shift in the balance of forces has been proposed to represent a signal transduction pathway that may be important in the modulation of cellular phenotype during angiogenesis (11, 50). Consistent with this hypothesis, it has been shown that endothelial cells interact with two distinct sites on the laminin

The FASEB Journal

LUSCINSKAS AND LAWLER

REVIEWS molecule through different receptor systems (51). Although integrin receptors support cell attachment, a 67,000-kDa protein that recognizes the YIGSR sequence supports cellto-cell interaction and tube formation. Antibodies to laminin inhibit tube formation. By contrast, antibodies to thrombospondin-i have been reported to increase tube formation (52). In addition, the level of thrombospondin-1 synthesis is decreased during tube formation. The antiangiogenic effect has been mapped to two distinct regions of the thrombospondin-1 molecule (53). Endothelial cells attach to thrombospondin-1 through several different receptor systems including CD36 binding to the CSVTCG sequence in the type 1 repeats and aJ33 binding to the RGD sequence in the type 3 repeats (54). Peptides from the type 1 repeats inhibit angiogenesis (53), suggesting that the binding of thrombospondin-l to CD36 may be involved in the antiangiogenic response to thrombospondin-i. The effect of anti-integrin antibodies on in vitro tube formation has also been studied using the endothelial-like cell line EAhy 926 (EA cells) (55). When these cells are plated on matrigel, they form tubes within 12-16 h. This tube formation is dependent on new RNA and protein synthesis, and is inhibited by pertussis toxin, which suggests the involvement of a G-protein-mediated signal transduction pathway. Tube formation is also specifically inhibited by anti-a6 and anti-/31 integrin subunit antibodies. The amount of antibody required to inhibit in vitro angiogenesis is sufficient to bind 2-6% of the a6 and /3 integrin subunit expressed on the EA cell surface (55). This quantity of antibody is not sufficient to block EA cell adhesion or migration. These results suggest that antibody binding to the integrin sends a signal that inhibits tube formation. Role

of integrins

in wound

healing

Wound healing is an orchestrated series of processes that occur in the affected area including the inflammatory response, parenchymal cell proliferation and migration, neovascularization, and synthesis of extracellular matrix proteins (56, 57). Soon after cutaneous skin wounding, deposition of fibronectin, fibrinogen, and fibrin occurs, which forms a provisional matrix for migration of epidermal cells, principally keratinocytes, to cover the wound area. Simultaneously, changes in the composition of extracellular matrix proteins and expression of integrins can be detected, and release of growth factors and secretion of migration-inducing cytokines occurs. Granulation tissue forms below the provisional matrix, and within a few days neovascularization or angiogenesis begins. Ultimately, these events subside, and are followed by an increase in collagenization via synthesis of collagens by fibroblasts and an increase in the tensile strength of the repaired tissues. In cutaneous wound healing, an essential event is keratinocyte migration to cover the wound area. Under normal conditions in skin, keratinocytes reside on basement membrane composed primarily of laminin and type IV collagen, and express a2/31, a3f31, a6f34, and a integrins along their lateral borders (58-60). During wound healing, their phenotype becomes activated and migratory (ref 61 and references therein). Activation events include modulation or induction of integrins and their distribution in a suprabasal manner, induction of plasminogen activators, and an increase in ICAM-1 and IL-i receptors. Ultimately, the epidermal integrity is re-established and keratinocytes resume their sessile phenotype. A recent approach to study the changes in integrins on migrating keratinocytes during the wound healing process

INTEGRINS

AND VASCULAR

FUNCTION

has been the human skin/severe combined immunodeficient mouse model (60). In this model, full-thickness human skin (neonatal foreskin) transplanted onto severe combined immunodeficient (SCID) mice retains the human phenotype for at least 3 months. After deep excisional wounding of the transplanted tissue, immunohistochemical analysis demonstrated loss of laminin and collagen IV and an increase in expression of tenascin and fibronectin in the wound area. The exact role of tenascin in wound tissue is unknown, but may contribute to cell migration. On migrating keratinocytes, an increase in the expression of a subunits and induced expression of the fibronectin receptor, a5/31, was detected. By day 4 or 5 after wounding, epithelial integrity was achieved and a new basement membrane, composed of laminin and type IV collagen, was observed. At this time, a5$1 expression disappeared, whereas a expression persisted until 7-10 days before resuming to baseline expression. Likewise, detection of fibronectin also was markedly reduced, returning toward baseline patterns. Tenascin expression peaked at day 2 and declined thereafter. Results from this experimental model and others suggest that several mechanisms account for the observed changes in integrin expression and matrix protein synthesis during the wound healing process. As noted earlier (see section on vasculogenesis), cells can take cues from various environmental components including growth factors, cytokines, proteoglycans, and extracellular matrix proteins, as well as from mechanical signals (4, 11, 50). In this instance, cells are exposed to blood platelet-, leukocyte-, and serum-derived growth factors (PDGF, TGF-/3, bFGF, EGF, TNF, ILs, chemotactic factors, fibronectin, thrombospondin), any or all of which may modulate integrin expression or function and extraceliular matrix protein synthesis (1, 4, 50). Likewise, alterations, either loss or de novo synthesis in local extracellular matrix proteins, may directly influence integrins distribution, state of activation, and level of expression. LEUKOCYTE INFLAMMATION

RECRUITMENT

TO

SITES

OF

Localized leukocyte accumulation is the cellular hallmark of inflammation. Early in vitro studies of leukocyte adhesion to cultured human umbilical vein endothelial cells typically were performed under static conditions, and indicated that basal adhesion of blood monocytes was high compared with neutrophils or lymphocytes. Activation of the endothelium with cytokines TNF-a, IL-i, or endotoxin from certain gram negative bacteria (LPS) resulted in a 2- to 5-fold increase in monocyte and lymphocyte adhesion and a 10- to 50-fold increase in neutrophil adhesion. This enhanced attachment reflects the induction of multiple adhesion molecules on the endothelial cell surface (for review, see refs 62-64) including E-selectin and P-selectin, which interact with sialyl-Lewis’ and similar carbohydrate ligands on leukocytes; VCAM-1, which interacts with a4/35 integrins; ICAM-1, which interacts with 132 integrins (ajft2 and aM/32); and an inducible endothelial ligand that interacts with L-selectin (65, 66). In addition, activated endothelial cells also synthesize leukocyte chemoattractants, including MCP-1, PAF, and IL-8 (for review, see refs 63, 64). That multiple adhesion molecules and leukocyte chemoattractants are induced suggests redundancy or overlap in their function. However, recent experiments performed in vivo (or in vitro under defined laminar flow conditions) have revealed that multiple receptor-ligand pairs can function in an sequential and overlapping fashion to mediate leukocyte attachment (63, 65, 67-70). The next

933

REVIEWS sections will discuss current models (see refs 63, 67, 68 for review) and present new information regarding blood monocyte adhesive interactions with activated endothelium under defined laminar flow conditions (65). In particular, these recent findings provide a clear indication that fl and /32 integrins have distinct functions in monocyte adhesive interactions with the vascular endothelium.

Molecular

under

basis

of leukocyte-endotheliai

cell adhesion

flow

In the molecular models that have been proposed (67, 68), the initial attachment of blood leukocytes to inflamed endothelial cells is mediated by members of the selectin gene family. The selectins (L-selectin, P-selectin, and E-selectin) interact with their carbohydrate ligands to mediate rolling (65-72) as wall as tethering (73) adhesive interactions between leukocytes and the vascular endothelium. Recent studies suggest that the ability of selectin molecules to mediate the experimentally observed rolling and tethering adhesive interactions, which require fast on-off kinetics, is related to their rates of bond formation and breakage (for review, see refs 74, 75). Future studies will be necessary to elucidate the role of each selectin during recruitment of the various circulating leukocyte types to sites of acute and chronic inflammation. These initial rolling or tethering interactions are reversible unless leukocytes are activated to undergo firm adhesion (arrest). Activation may be mediated through locally derived endothelial chemoattractants IL-8, PAF, or MCP-I, which are well-characterized mediators of leukocyte activation (63, 64), or possibly via ligation of specific adhesion molecules. Activation dramatically increases the adhesiveness of leukocytes for endothelium primarily by upregulated affinity and surface expression of /32 integrins.

These models are based primarily on observations with neutrophils, and until recently the extent to which blood monocytes and lymphocytes conform to this paradigm had not been tested. In addition to expressing /32 integrins and Lselectin, monocytes and lymphocytes also express multiple

13

integrins, of which a4f35 (VLA-4) has been shown to mediate attachment to extracellular matrix proteins and to function as a ligand for the endothelial-expressed adhesion molecule, VCAM-1. Using an in vitro flow chamber that can simulate flow conditions likely to occur in postcapillary yenules and function blocking monoclonal antibody (mAb), studies from our laboratory have extended this model and identified the cellular and molecular events necessary for monocyte arrest and transmigration across the endothelium, which has been selectively activated with IL-4 (65) (see Fig. 2). Unlike IL-i, TNF-a, and lipopolysaccharide (LPS), which induce E-selectin, P-selectin, and up-regulated VCAM-1 and ICAM-1 expression, IL-4 induces VCAM-1 expression without altering ICAM-1 or inducing E-selectin (76, 77).

Molecular basis of monocyte-endothelial under flow

interactions

Monocytes decelerate and roll on the IL-4-activated endothelial surface via L-selectin interacting with its inducible ligand (or ligands) (65; Fig. 2, step I). This step is reversible. In the presence of monoclonal antibodies that block Lselectin function (LAM1-3 mAb), few if any rolling monocytes were observed, whereas control nonblocking anti-Lselectin monoclonal antibody (LAMI-14) had no inhibitory effect on monocyte rolling or adhesion. These data are consistent with reports using blood neutrophils that demonstrate that L-selectin is crucial for rolling in vivo (71, 72) and for accumulation at sites of inflammation (78, 79). Most monocytes in contact with the activated endothelium rolled at

Figure 2. Model of monocyte attachment to IL-4-activated HUVEC monolayers under fluid shear at 1.8 dynes/cm2 (65). This figure depicts four sequential and overlapping cellular events (phases I-IV) observed during blood monocyte adhesive interactions with IL-4-activated human vascular endothelial cell monolayers under laminar flow at 1.8 dynes/cm2. L-selectin mediates monocyte rolling (I) on the endothelial monolayer and also facilitates cr4131-dependent arrest (II); (32 integrins are involved in monocyte spreading (III), and in conjunction with CD3I (80), mediate monocyte diapedesis (IV) under flow.

934

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1994

The FASEB Journal

LUSCINSKAS AND LAWLER

REVIEWS 70-80 jsmls, and a significant proportion had rolling velocities between 3 and 10 tm/s. The slowly rolling monocytes exhibited characteristics similar to that previously described for neutrophils interacting with activated endothelium under flow (69, 70, 79): transient adhesion to endothelium (< S s) followed by detachment and release, or initiation of slow rolling (< 10 gem/s) on endothelial cells downstream. That monocytes roll rather than slide or tumble under laminar flow was clearly distinguished under a 40 x objective; the large nucleus rotated as the cell traveled across the surface of the endothelial monolayer.

/3 integrins under

but not

132

integrins

mediate

monocyte

arrest

flow

The next step, arrest, involves monocyte a4j31 interacting with VCAM-i (step II), but not /32 integrmns. It is not known whether a4/31-VCAM-1 interaction requires cell activation. Once the f3 mntegrmns engage, monocytes attach stably under flow conditions up to 1.8 dynes/cm2. Adherent monocytes then spread onto the luminal endothelial surface via /32 integrins engaging their ligand (or ligands), presumably ICAM-1 (step III). This step most likely requires activation. Because IL-4-activated endothelium produces monocyte chemotactic factor-i (MCP-i), monocyte (3 and/or /32 integrin activation could be triggered via endothelial-derived MCP-1. It is not known whether IL-4-activated endothelium produces platelet activating factor (PAF)-like molecules. These data are the first clear indication that /31 and /32 integrins have distinct functions in monocyte adhesive interactions with the vascular endothelium. In this system, /3 integrmns stabilize the initial attachment (arrest) of monocytes to endothelial cells under flow, whereas /32 integrins are required for spreading and motility of monocytes adherent to the luminal surface of an endothelial cell. This is in contrast to neutrophils, which use /32 integrins to mediate firm attachment to endothelium and lack a4j31 (51, 55, 77). From these data, we infer that neutrophils may use distinct (3 integrins for arrest and spreading via ligation to ICAM-i and/or ICAM-2, thus overcoming the lack of a4/31. More studies will be necessary to provide further insight into the molecular mechanisms of the differential function of /3 and /32 integrins in monocyte, as well as lymphocyte, arrest under flow. integrins migration

/32

function

in monocyte

spreading

and

Once spread, monocytes migrate to intercellular junctions and diapedeses between endothelial cells to the abluminal surface (step IV). Recent studies suggest that this latter process involves homotypic adhesion of PECAM-i (CD31) expressed both on leukocytes and endothelium (80) as well as 132 mntegrins (80, 81). In addition, an important role for /32 integrins in migration is clearly demonstrated by the heritable disease termed LAD 1(81) in which patients lack phagocytic cells (primarily neutrophils) at inflammatory sites, show impaired complement-mediated phagocytosis, and suffer from frequent life-threatening bacterial infections. Using IL-4-activated HUVEC monolayers that selectively express VCAM-i and an L-selectin ligand but not E-selectin and appropriate function blocking mAb, we found that monocytes rolled on and stably bound to IL-4-activated endothelium under flow, and that L-selectin mediated monocyte rolling and facilitated a4/3s-integrin-dependent arrest under flow. Subsequent monocyte spreading was dependent on 132-integrins. These findings extend current models of leukocyte-endothelial recognition to reveal a more complex, sequential, and overlapping model for monocyte-endothelial INTEGRINS

AND VASCULAR

FUNCTION

interactions than was appreciated previously. Thus, the process of leukocyte attachment and transmigration across the endothelial lining may be dynamically regulated by multiple activation-dependent adhesion molecules as well as by locally (i.e., endothelialor leukocyte-derived) elaborated, leukocyte-directed chemotactic mediators.

FUTURE

DIRECTIONS

The data summarized in this review establish the importance of the interactions of integrins with their ligands in vascular biology. The advent of cloning and sequencing techniques has facilitated an understanding of the structural and biochemical properties and identification of the proteins that comprise this family. The nature of the molecular interactions of the integrins with the adhesive glycoproteins on the outside of the cell and the cytoskeleton on the inside of the cell are beginning to be understood. In addition, we are beginning to understand the nature of the integrin-dependent signal transduction pathways. A great deal of work remains to be done to complete our knowledge in these areas. Furthermore, we need to integrate the integrin-dependent mechanisms with other systems for cell-to-cell and cell-tomatrix association. As noted in the section on leukocyte recruitment to sites of inflammation, current models have proposed that members of the selectin gene family mediate leukocyte rolling interactions with activated vascular endothelium. Recent studies with P-selectin-deficient mice have reported defects in leukocyte recruitment (82). In particular, P-selectindeficient mice exhibited a nearly complete absence of spontaneous rolling leukocytes in exteriorized mesenteric venules (ca. 25-35 jsm diameter) and a delayed neutrophil influx into the peritoneum 2-6 h after thioglycollate injection. Inhibition of L-selectin function by L-selectin receptor chimera molecules or antibodies also has yielded similar levels of inhibition of both spontaneous leukocyte rolling in mesenteric venules (71) and neutrophil influx into the peritoneum between 2 and 6 h (78). In addition, recent studies by Kansas and co-workers (83) and by Bargatze and Butcher (84) suggest that L-selectin interacting with its ligand (or ligands) expressed on high endothelial venules can regulate lymphocyte rolling and stable adhesion via G-proteins and cytoskeletal interactions. These data suggest that P- and L-selectin, interacting with distinct ligands on the opposing cells, are both required to generate the proadhesive forces, i.e., rolling and tethering, and activation signals necessary to mediate adherence under fluid shear. Although it is not the only interpretation, these results provide the rationale for future studies to dissect the contributions of each selectin to the recruitment of different leukocyte types to inflammatory events or immune reactions. Certainly, the availability of mice deficient in E- or L-selectin, as well as mice with multiple selectin deficiencies (combinations of L-, E, and P-selectin), will help address some of these issues. Moreover, we need to gain a better understanding of how selectins and /3 and /32 integrins (interacting with their Ig counterreceptors) mediate the sequential and overlapping cellular events of leukocyte rolling and arrest. Molecular assemblies are occurring inside the cell, in the plane of the membrane, and in the extracellular compartment (Fig. 3). These assemblies may be driven by the multivalent nature of the adhesive glycoproteins and by the ability of the integrins to associate with other membrane glycoproteins. Fibronectin can interact with proteoglycans a5/31, and a31 concurrently. The inclusion of a4/31 in such 935

REVIEWS

Figure 3. Schematic representation of matrix-driven molecular assemblies on the surface of endothelial cells. Vitronectin binding to integrins (a, /3) induces cell spreading, formation of focal contacts, and recruitment of focal adhesion kinase (FAK). The integrinassociated protein (lAP) has been proposed to function as a calcium channel. Endothelial cells do not spread on thrombospondin (TSP) -coated substrates. TSP may be able to cluster integrins, CD36, and proteoglycans to stimulate a motile phenotype. TSP also has the ability to activate 113F13. CD36 has been shown to associate with the protein-tyrosine kinases Yes, Lyn, and Fyn; however, the molecular mechanisms for this association is not known and other proteins may participate in these assemblies. complexes depends on the presence of the alternatively spliced V region. Similarly, the thrombospondin-1 molecule binds to endothelial cell-surface proteoglycans, CD36, integrins, and yet-to-be identified cell-surface proteins. Thrombospondin-i can also bind to other extracellular matrix components and has the ability to activate TGF-(3 that is secreted from endothelial cells in culture (85). In several cases, the interaction of thrombospondin with cell surfaces involves multiple receptors simultaneously. Thus, the thrombospondin-l molecule can bind aV/33 and CD36 and result in clustering of these receptors. CD36 has been shown to coimmunoprecipitate with the Fyn, Lyn, and Yes protein-tyrosine kinases and thus has been proposed to participate in phosphotyrosine-mediated cell signaling (86). The ligand specificity of CD36 has been reported to be modulated by a novel mechanism involving the dephosphorylation of an extracellular domain (87). Whereas the phosphorylated CD36 preferentially binds collagen, the dephosphorylated protein preferentially binds thrombospondin. The change in ligand specificity could result in a change in the integrins that cluster with CD36. The significance of these observations to endothelial cell biology remains to be explored. Integrin function is also modulated by association with other membrane components, including lipids and proteins. The lAP is a 50,000-kDa protein that copurifies with aI33 (21, 22); it contains an extracellular Ig-like domain, five membrane-spanning domains, and a short cytoplasmic tail. An antibody to TAP blocks a13s-dependent attachment to vitronectin. This antibody also inhibits the fibronectinstimulated rise in endothelial cell cytoplasmic calcium, suggesting that /3 integrin function is also modulated by lAP. lAP has been reported to regulate calcium entry into endothelial cells by functioning as a calcium channel. This integrin-associated calcium channel activity may represent an integral part of the outside-in signal transduction pathway. The data reviewed here indicate that aV/33 can form a trimolecular complex with a calcium channel (lAP) and a protein that binds considerable calcium (thrombospondin-I). This raises the possibility that the function of thrombospondin-i is to provide a ready source of free cal936

Vol. 8

September

1994

cium ions in a specific and restricted environment at the cell surface. This mechanism may help to raise intracellular calcium in localized membrane patches in response to growth and migratory stimuli. Thrombospondin-i has been reported to act synergistically with growth factors, reducing the threshold concentration required to stimulate proliferation of smooth muscle cells (88). Extracellular matrix proteins provide environmental cues that modulate cell proliferation, migration, and differentiation during tissue genesis and remodeling. The dynamic assembly of extraceliular proteins, membrane proteins, cytoskeletal proteins, and cytoplasmic signaling molecules provides for complex and subtle changes in the message the cell receives. The changes in these complexes that occur during inflammation, angiogenesis, metastasis, and wound healing provide the stimulus for changes in the proliferative and migratory state of the cell. This view suggests that it is very difficult, if not impossible, to interpret pathophysiological events based on the expression or activity of a single molecular species. In vivo and in vitro data for multiple constituents must be accumulated and correlated before rational molecular modeling is attempted. We wish to thank Drs. Michael Gimbrone, Mary Gerritsen, Elizabeth George, Richard Hynes, and Thomas F. Tedder for helpful discussions and suggestions during the preparation of this manuscript. We wish to thank Drs. David Cheresh, Peter Davies, Elisabetta Dejana, Elizabeth George, Alan Horwitz, Rudolph Juliano, Peter Libby, Joe Madri, C. Wayne Smith, andJoy Yang for providing preprints and reprints. We also wish to thank Pamela Caffey and Tracy Baker for expert secretarial support. This work was supported by National Institutes of Health grants HL36028 and HL47646 (to F. W. L.), and HL28749 (toJ. L.).

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