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J. Biol. Chem., Vol. 279, Issue 18, 19230-19238, April 30, 2004
Intercellular Adhesion Molecule-1 (ICAM-1) Regulates Endothelial Cell Motility through a Nitric Oxide-dependent Pathway*![]() ¶ ||![]() ![]() ||
From the
Received for publication, November 3, 2003 , and in revised form, February 23, 2004.
Coordinated regulation of endothelial cell migration is an integral process during angiogenesis. However, molecular mechanisms regulating endothelial cell migration remain largely unknown. Increased expression of cell adhesion molecules has been implicated during angiogenesis, yet the precise role of these molecules is unclear. Here, we examined the hypothesis that intercellular adhesion molecule-1 (ICAM-1) is important for endothelial cell migration. Total cell displacement and directional migration were significantly attenuated in ICAM-1-deficient endothelium. Closer examination of ICAM-1-deficient cells revealed decreased Akt Thr308 and endothelial nitric-oxide synthase Ser1177 phosphorylation and NO bioavailability, increased actin stress fiber formation, and a lack of distinct cell polarity compared with wild-type endothelium. Supplementation of ICAM-1 mutant cells with the NO donor DETA NONOate (0.1 µM) corrected the migration defect, diminished stress fiber formation, and enhanced pseudopod and uropod formation. These data demonstrate that ICAM-1 facilitates the development of cell polarity and modulates endothelial cell migration through a pathway regulating endothelial nitric-oxide synthase activation and organization of the actin cytoskeleton.
Blood vessel growth and development are complex processes involving endothelial cell proliferation, invasion, and directional migration. All of these features are important characteristics of neovascularization; however, precise molecular mechanisms governing these events still remain undefined. Several lines of evidence suggest that endothelial cell adhesion molecules may be involved in angiogenesis. Diseases such as atherosclerosis, hypercholesterolemia, and diabetic retinopathy display increased adhesion molecule expression with concomitant neovascularization (1, 2). Several reports have demonstrated that increased adhesion molecule expression facilitates leukocyte-mediated angiogenesis (3-5). Moreover, soluble forms of E-selectin, vascular cell adhesion molecule-1 (VCAM-1),1 and intercellular adhesion molecule-1 (ICAM-1) have all been reported to stimulate angiogenic activity through unknown mechanisms (6, 7). Despite the active role adhesion molecules play during angiogenesis, little mechanistic information exists describing how these molecules facilitate neovascularization.
Tissue remodeling such as that seen during angiogenesis involves the transition of cells from a quiescent to a mitogenic and pro-migratory phenotype. Numerous environmental signals such as growth factors, extracellular matrix proteins, and mechanical forces are integrated in the cell to generate specific cellular responses. Migration occurs through coupling of actin-based protrusion and contraction with the dynamic formation and disassembly of cell-extracellular matrix adhesions and is highly adhesion-dependent. Both the formation of new matrix contacts and the generation of tractional forces are mediated through integrins (8-10). Altering the adhesive state of the cell induces a biphasic effect on cell migration, with low levels of adhesion insufficient to support tractional forces and high levels of adhesion preventing cell displacement (11, 12).
ICAM-1 is a member of the immunoglobulin protein super-family that mediates cell-cell adhesion and may participate in lamellipodium formation (13, 14). Several lines of experimental evidence implicate ICAM-1 in endothelial cell migration. For example, in atherosclerosis patients, increased endothelial cell ICAM-1 expression was observed within the neovasculature of coronary plaques, but not in endothelial cells directly overlying the plaque (15). ICAM-1 clustering has been shown to stimulate actin stress fiber formation in endothelial cells through a RhoA- and p38 MAPK-dependent pathway (16). In addition, ICAM-1 itself can associate with the actin cytoskeleton through direct binding with
NO production appears to be a key target in angiogenic signaling since known pro-angiogenic factors such as vascular endothelial growth factor, transforming growth factor- We show here that loss of ICAM-1 expression in endothelial cells significantly attenuates cell motility through a unique NO-dependent pathway. These data demonstrate that constitutive expression of ICAM-1 is important for basal Akt and eNOS phosphorylation. Loss of basal eNOS activity in ICAM-1-deficient cells induces the formation of actin stress fibers and significantly reduces the ability of these cells to migrate. Thus, our findings suggest a novel role of ICAM-1 during endothelial cell migration that may be important for neovascularization.
MaterialsCell culture materials were purchased from Sigma. Endothelial cell mitogen was purchased from Biomedical Technologies, Inc. (Stoughton, MA). DETA NONOate ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) and N -nitro-L-arginine (L-NNA) were purchased from Alexis Biochemicals (Carlsbad, CA). Goat anti-eNOS antibody was obtained from Pharmingen, and rabbit anti-phospho-eNOS Ser1177, anti-phospho-Akt Thr308, anti-phospho-Akt Ser473, and anti-Akt antibodies were purchased from Cell Signaling (Beverly, MA). Alexa 546-phalloidin was purchased from Molecular Probes, Inc. (Eugene, OR). Mouse Endothelial Cell CultureMouse aortic endothelial cells (MAECs) were isolated from either wild-type or ICAM-1 mutant C57BL/6J mice and cultured as reported previously (29). Aortas were collected and incubated in 10 ml of Hanks' balanced saline solution (HBSS)/collagenase solution at 37 °C and agitated every 10 min for a total of 30 min to remove the connective fascia and adventitia. Aortas were then collected on 100-µm nylon filters and washed twice with HBSS/penicillin/streptomycin solution. The aortas were incised and incubated in 5 ml of HBSS/collagenase solution containing 1 unit/ml dispase at 37 °C and agitated every 10 min for a total of 30 min to release the endothelial monolayer. The digests were then passed over a 40-µm nylon filter to isolate single cells. The single cell suspension was centrifuged at 375 x g for 15 min at 4 °C. The resulting pellet was resuspended in 2 ml of HBSS containing 0.1% bovine serum albumin (fraction V) and labeled with 5 µg/ml fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia lectin I (Sigma) for 30 min at 4 °C with constant agitation. The labeled cell suspension was pelleted, washed twice, and resuspended in 1 ml of HBSS and 0.1% bovine serum albumin, and positively stained cells were collected using fluorescence-activated cell sorting. Bovine aortic endothelial cells were labeled with fluorescein isothiocyanate-conjugated B. simplicifolia lectin I and used as a positive control. Positively sorted cells were plated onto 0.5% gelatin-coated 6-well plates in medium containing MCDB-131 (pH 7.2), 10% fetal bovine serum, 1 mg/ml penicillin/streptomycin, 1 µg/ml hydrocortisone, 10 units/ml heparin, and 50 µg/ml endothelial mitogen. Western Blot Analysis and NO DetectionWestern blotting was performed as reported previously (30). Briefly, 25 µg of total protein was separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk powder in Tris-buffered saline/Tween overnight at 4 °C. Membranes were washed and incubated for 2 h at room temperature with anti-phospho-Akt Thr308 or Ser473, total anti-Akt, anti-phospho-eNOS Ser1177, or total anti-eNOS antibody at 1:1000 dilution. Membranes were washed three times with Tris-buffered saline/Tween and then incubated with anti-rabbit horseradish peroxidase-linked secondary antibody at 1:3000 dilution for 2 h at room temperature. Membranes were washed and developed using ECL Western blot detection reagents.
Total NO production in cell-conditioned medium was determined by measuring the NO metabolites nitrite ( Measurement of Cell MigrationDunn cell migration chambers were purchased from Weber Scientific International Ltd. (Teddington, United Kingdom). Cell migration experiments were performed as reported previously (32-34). Briefly, glass coverslips were coated with 0.1% gelatin for 1 h, and endothelial cells were seeded at a density of 20,000 cells/cm2 and allowed to attach in serum and growth factor-free MCDB-131 medium for 3 h. Glass coverslips were then loaded onto the Dunn chamber containing serum and growth factor-free MCDB-131 medium, cell side down, and sealed to the chamber with an equal mixture of vacuum grease and Vaseline blotted to remove excess oil. Cells were imaged on an Axiovert 100 microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a CCD camera (Model 300T-RC, Dage-MTI, Inc., Michigan City, IN) and a computer-controlled stage (Prior Scientific, Rockland, MA) to enable viewing of multiple fields over the time course of the experiment. The temperature on the stage was kept constant at 37 °C, and images were captured at 2-min intervals for a total of 6 h. Time-lapse images were analyzed by Metamorph software (Universal Imaging Corp., Downington, PA). Slight movements in position resulting from the use of a movable stage were removed by simultaneous tracking of a stationary point and normalization of the resulting (x,y) coordinates generated by Metamorph. Tracks were analyzed using programs written by one of us (D. F. K.) for this purpose. Total track distance was determined as the sum of the incremental distances between successive (x,y) coordinates. Cell speed was determined as the total distance migrated divided by the time of the assay. Total cell displacement was calculated as the distance between the final (x,y) coordinate and the initial (x,y) coordinate. x displacement and y displacement were also calculated for each cell using this method and could be used as the final cell position given an initial cell position at the origin (0,0). Fifty cells from each Dunn chamber experiment were analyzed, with each experiment repeated three separate times. Data were statistically compared using Student's t test between experimental groups. Actin Staining of Endothelial Cell MonolayersActin staining of endothelial cell monolayers was performed as described previously (35). Briefly, endothelial cells were fixed with 1% paraformaldehyde in phosphate-buffered saline for 5 min at room temperature. Monolayers were permeabilized by incubation with 0.5% Triton X-100 in phosphate-buffered saline for 5 min. Cells were washed and incubated with 6 µM Alexa 546-phalloidin for 20 min at room temperature. Coverslips were washed and mounted using Vectashield containing the nuclear counterstain 4',6-diamidino-2-phenylindole. Cells were imaged using Simple PCI software (Compix, Inc., Cranberry Township, PA) connected to an Eclipse TE2000 inverted epifluorescent microscope (Nikon Inc., Melville, NY).
ICAM-1 Deficiency Decreases Endothelial Cell DisplacementPrevious studies have shown that ICAM-1 expression in endothelial cells is involved in inflammation-mediated angiogenesis and that soluble ICAM-1 itself can also be pro-angiogenic (3, 4, 7). To investigate the role of ICAM-1 in endothelial cell migration, we compared cell migration in wild-type and ICAM-1-deficient MAECs. Fig. 1 shows representative cell migration tracks for wild-type and ICAM-1-deficient MAECs. We found that loss of ICAM-1 substantially attenuated basal endothelial cell migration. Individual cell tracks were further analyzed to determine the total migration distance and speed of migration. Interestingly, we observed that loss of ICAM-1 decreased both the total migration distance and speed compared with wild-type cells, as determined by tracking successive positions of the cell centroid (Fig. 1). Since the cell tracking software used does not distinguish between changes in cell shape and cell translocation, the actual effect of ICAM-1 deficiency on endothelial cell migration distance and speed may be more significant. Measurement of total displacement and directionality (total displacement/total distance) revealed that loss of ICAM-1 significantly inhibited the directional displacement of endothelial cells (Fig. 1). Consistent with this, time-lapse images showed that ICAM-1-deficient cells did not display considerable translocation from their point of origin.
What was most striking was that ICAM-1 migration paths were more annular with little linear tracks that would enable lateral cell displacement. Furthermore, ICAM-1 cell migration was most irregular in that the nature of movement was random and non-directional (as indicated by the directionality ratio), resulting in decreased lateral displacement. Together, these findings suggest that ICAM-1 mutant cells are quite capable of moving, but that the development of cell polarity and directional migration are compromised. ICAM-1 Deficiency and Alterations in the Endothelial CytoskeletonICAM-1 has been reported to associate with the actin cytoskeleton and may influence lamellipodium formation (13, 36, 37). Subconfluent endothelial cells were labeled with Alexa 546-phalloidin to examine intracellular actin organization. Fig. 2 (A and B) shows that wild-type endothelial cells displayed several actin-rich lamellipodium regions and distinct migration morphology with prominent pseudopod and uropod formation. In contrast, cytoskeletal organization was substantially different in ICAM-1-deficient cells. There were numerous stress fibers with smaller, less organized lamellipodia and a distinct loss of cell polarity as determined by poorly defined pseudopod and uropod formation (Fig. 2, C and D). Overall, ICAM-1-deficient cells appeared to exhibit a contractile phenotype compared with wild-type endothelium. These data suggest that ICAM-1 is important in determining endothelial cell actin cytoskeletal organization and may be important for generation of proper cell polarity.
Regulation of eNOS in ICAM-1-deficient Endothelial CellsNitric oxide has been reported to be an important biochemical requirement for endothelial cell migration (23, 38, 39). eNOS is the predominant NO-generating enzyme within endothelial cells, and gene-targeted removal of eNOS results in attenuated endothelial cell migration (20, 40). Recent reports have also shown that eNOS enzymatic activity is greatly influenced by phosphorylation via Akt/protein kinase B, suggesting that production of bioavailable NO is closely regulated (41-43). We examined the phospho-Akt and total Akt and eNOS protein levels in wild-type and ICAM-1 mutant endothelial cells. Fig. 3A illustrates decreased phospho-Akt Thr308 in ICAM-1 mutant cells, whereas phospho-Akt Ser473 and total Akt remained unchanged. eNOS Ser1177 (Ser1177 in human and Ser1179 in bovine) has recently been reported to be critical for both basal and agonist-induced eNOS activities (44, 45). Fig. 3B shows that a genetic loss of ICAM-1 resulted in decreased phosphorylation of eNOS Ser1177. Importantly, Western blot analysis of total eNOS protein was not different between endothelial cell types. These observations suggest that ICAM-1-mediated defects in endothelial cell migration may be due to a decrease in NO bioavailability.
Chemiluminescent detection of total NO in cell-conditioned medium was performed to determine whether NO production in ICAM-1 mutant cells was decreased. Fig. 3C shows that NO production in ICAM-1-deficient MAECs was significantly attenuated compared with wild-type cells. Together, these data provide strong evidence linking constitutive ICAM-1 expression with basal NO production via modulation of eNOS phosphorylation.
NO Donors Enhance ICAM-1-deficient MAEC MotilityEndothelial cell migration experiments were performed in the presence of the NO donor DETA NONOate to examine whether restoring NO bioavailability could correct the cell migration defects of ICAM-1 mutant endothelial cells (Fig. 4). 0.1 µM DETA NONOate donor treatment of wild-type endothelial cells did not significantly alter the total displacement distance compared with untreated cells. In contrast, DETA NONOate treatment of ICAM-1-deficient endothelial cells significantly increased the total displacement distance compared with untreated cells. Using the reported half-life of DETA NONOate (20 h at pH 7.4 and 37 °C) at a reaction stoichiometry of 2 molecules of NO released per molecule of DETA, the calculated amount of NO release over the 6-h incubation period was
NO Donors Restore Normal Cytoskeletal Organization to ICAM-1-deficient Endothelial CellsRecent reports have shown that NO and actin can reciprocally regulate each other (36). A recent study by Su et al. (46) revealed that differential association of eNOS with either G- or F-actin can influence enzymatic activity. Therefore, we examined whether DETA NONOate could alter actin cytoskeletal organization in ICAM-1 mutant endothelial cells. Fig. 6 shows that administration of DETA NONOate did not significantly alter the wild-type endothelial cytoskeleton. In contrast, DETA NONOate treatment diminished stress fiber formation and enhanced the appearance of actin-rich lamellipodia in ICAM-1-deficient endothelial cells. Interestingly, NO donor treatment also appeared to facilitate ICAM-1 mutant cell polarity as seen by distinct pseudopod and uropod formation and attenuated the contractile phenotype of these cells. These data demonstrate that ICAM-1 may exert a unique influence on the actin cytoskeleton through modulation of NO bioavailability.
NOS Inhibition Increases Stress Fiber Formation and Decreases Polarity of Wild-type Aortic Endothelial CellsWild-type endothelial cells were treated with 200 µM L-NNA to determine whether inhibition of NOS stimulates altered actin cytoskeletal organization similar to ICAM-1 mutant MAECs. Vehicle-treated MAECs displayed prominent lamellipodia with few stress fibers (Fig. 7, A and B). Conversely, treatment with L-NNA significantly attenuated lamellipodium formation, increased stress fiber formation spanning throughout the cell, and significantly attenuated membrane ruffling (Fig. 7, C and D). Together, these data demonstrate that NOS activity is critical for maintaining endothelial cytoskeletal organization and corroborate previous studies demonstrating a role for eNOS in modulating actin polymerization (46).
Directional migration of endothelial cells is a key requirement for neovascularization (47). Although much is known regarding molecules that stimulate endothelial motility, specific mechanisms necessary for endothelial cell migration remain poorly defined. Adhesion molecule expression (e.g. integrins) has been shown to be important for endothelial cell motility and angiogenesis (48). However, recent evidence suggests that other adhesion molecules, such as ICAM-1, may also participate in governing endothelial cell migration. For example, a requirement for ICAM-1 expression has recently been demonstrated during vascular endothelial growth factor-mediated chemotactic migration (25). This observation suggests that ICAM-1 may be involved in the regulation of endothelial cell motility.
Our study demonstrates that ICAM-1 serves a unique role in governing endothelial cell migration by facilitating directional displacement through controlling endothelial cell polarity development. Time-lapse video microscopy showed that ICAM-1-deficient cells displayed significantly reduced net translocation of the cell body compared with wild-type endothelial cells. Our observations are consistent with a previous report showing ICAM-1 association with the actin cytoskeleton, which may influence the development of cell polarity and affect lamellipodium formation (13). Our findings indicate that basal ICAM-1 expression likely governs cell polarity through an eNOS-dependent pathway and is involved in establishing NO bioavailability within endothelial cells since supplementation of NO reconstituted migration in ICAM-1 mutant cells. Surprisingly, these effects were observed with relatively low levels of NO (
ICAM-1 association with the actin cytoskeleton occurs through interactions with cortactin, Endothelial cell migration is significantly influenced by eNOS and its subsequent product, NO (20, 38-40). Regulation of eNOS activity has recently been reported to be governed by phosphorylation through Akt/protein kinase B and other kinases (41, 42). eNOS is phosphorylated at several amino acid residues, including Ser116, Ser617, Ser635, and Ser1177 (Ser1179 in bovine), with Ser1177 serving as the dominant residue for both basal and agonist-mediated NO production (44, 45). Given the importance of eNOS in endothelial cell migration, we investigated whether the significant attenuation of endothelial cell motility in ICAM-1-deficient cells involves altered eNOS activity. Surprisingly, constitutive phosphorylation of Ser1177 was markedly decreased in ICAM-1-deficient endothelial cells. Furthermore, media NO levels were significantly lower in ICAM-1-deficient cells, consistent with a decrease in constitutive eNOS activity. These data identify a link between ICAM-1 expression, eNOS phosphorylation, and enzymatic activity, suggesting that ICAM-1 expression is necessary for optimal eNOS activation. Akt/protein kinase B-dependent phosphorylation of eNOS is critical for increasing enzymatic activity. As such, we observed that phosphorylation of Akt Thr308 was significantly reduced in ICAM-1 mutant cells, whereas phosphorylation of Ser473 was unchanged. Regulation of Akt activity is complex, and both Thr308 and Ser473 have been shown to be important for activation (52). Although it is still not exactly clear how these residues facilitate activation, experimental evidence suggests that phosphorylation of Ser473 can facilitate localization of Akt to the membrane, where PDK1 then phosphorylates Thr308 to generate the fully active kinase (53). If Akt activation occurs in such a manner, our results suggest that ICAM-1 may be important for PDK1 activity in endothelial cells. ICAM-1 could also influence phosphatidylinositol 3-kinase activity, which in turn affects PDK1 activation. However, a recent study by Scheid et al. (54) has shown Ser473 phosphorylation to be sensitive to phosphatidylinositol 3-kinase inhibition, whereas Thr308 phosphorylation was unchanged. Together, these studies and our data suggest that functional loss of ICAM-1 may more directly affect PDK1 versus phosphatidylinositol 3-kinase activity. Studies are currently underway to better understand how ICAM-1 may influence PDK1/phosphatidylinositol 3-kinase activity in endothelial cells. It is possible that cytoskeletal perturbations associated with a deficiency of ICAM-1 modulate eNOS activation. Recent studies have shown that the actin and microtubule cytoskeletons play important roles in regulating eNOS activity (46, 55, 56). Other eNOS-binding proteins such as NOSIP and NOSTRIN have also been implicated in regulating eNOS activity by controlling intracellular enzyme trafficking (57, 58). ICAM-1 could interact with these or other proteins and influence eNOS activity through altered cellular localization of this enzyme. Although the exact mechanism is not known, our data clearly show that ICAM-1 expression can influence eNOS activity, as indicated by changes in enzyme phosphorylation and the increased sensitivity of ICAM-1-deficient endothelial cells to exogenous nitric oxide donors.
The bioavailability of NO has a significant impact on intracellular cytoskeletal organization. A study by Baldwin et al. (59) reported that treatment of endothelial cells in vivo with the NOS inhibitors N Previous studies have shown that endothelial adhesion molecules such as VCAM-1 and E-selectin may be involved in embryonic vascular development and regulation of endothelial cell proliferation, respectively (61, 62). Here, we have identified a novel role of ICAM-1 in governing endothelial cell motility. Loss of ICAM-1 expression appears to primarily affect lateral cell displacement, resulting in decreased cell migration. Our data further suggest that ICAM-1 in endothelial cells is important for governing Akt and eNOS activities, cytoskeletal organization, and the development of proper cell polarity. ICAM-1 expression is known to be induced by several angiogenic factors and may be a functional requirement for neovascularization. Given the prominent role of ICAM-1 during inflammation and its ability to govern endothelial cell migration, this molecule may act as a common denominator for many endothelial cell functions observed during angiogenic and inflammatory processes.
* This work was supported by National Institutes of Health Grant HL-010312 and an American Heart Association beginning grant-in-aid (to C. G. K.), by National Institutes of Health Grants HL-07918 (to A. W. O.), HL-70146 (to R. P. P.), HL-44575 (to J. M.-U.), and RR-17009 (to D. C. B.) and by a Veterans Affairs Medical Center merit review award (to D. F. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Pathology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. Tel.: 318-675-4694; Fax: 318-675-7662; E-mail: ckevil{at}lsuhsc.edu.
1 The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MAPK, mitogen-activated protein kinase; eNOS, endothelial nitric-oxide synthase; ERK, extracellular signal-regulated kinase; MAECs, mouse aortic endothelial cells; L-NNA, N
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