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. 2010 May 12;151(7):3432–3444. doi: 10.1210/en.2009-1305

Deciphering Mechanisms Controlling Placental Artery Endothelial Cell Migration Stimulated by Vascular Endothelial Growth Factor

Wu-xiang Liao 1, Lin Feng 1, Jing Zheng 1, Dong-bao Chen 1
PMCID: PMC2903938  PMID: 20463056

Abstract

Vascular endothelial growth factor (VEGF) stimulated fetoplacental artery endothelial (oFPAE) cell migration and activated multiple signaling pathways including ERK2/1, p38MAPK, Jun N-terminal kinase (JNK1/2), v-Akt murine thymoma viral oncogene homolog 1 (Akt1), and c-Src in oFPAE cells. VEGF-induced cell migration was blocked by specific kinase inhibitors of JNK1/2 (SP600125), c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and phosphatidylinositol 3-kinase/Akt (wortmannin) but not ERK2/1 (U0126) and p38MAPK (SB203580). VEGF-induced cell migration was associated with dynamic actin reorganization and focal adhesion as evidenced by increased stress fiber formation and phosphorylation of cofilin-1 and focal adhesion kinase (FAK) and paxillin. Inhibition of JNK1/2, c-Src, and phosphatidylinositol 3-kinase/Akt suppressed VEGF-induced stress fiber formation and cofilin-1 phosphorylation. c-Src inhibition suppressed VEGF-induced phosphorylation of focal adhesion kinase, paxillin, and focal adhesion. VEGF-induced cell migration requires endogenous nitric oxide (NO) as: 1) VEGF-stimulated phosphorylation of endothelial NO synthase (eNOS) via activation of Akt, JNK1/2, and Src; 2) a NO donor diethylenetriamine-NO-stimulated cell migration; and 3) NO synthase inhibition blocked VEGF-induced cell migration. Targeted down-regulation and overexpression of caveolin-1 both inhibited VEGF-induced cell migration. Caveolin-1 down-regulation suppressed VEGF-stimulated phosphorylation of Akt, JNK, eNOS, c-Src, and FAK; however, basal activities of c-Src and FAK were elevated in parallel with increased stress fiber formation and focal adhesion. Caveolin-1 overexpression also inhibited VEGF-induced phosphorylation of Akt, JNK, c-Src, FAK, and eNOS. Thus, VEGF-induced placental endothelial cell migration requires activation of complex pathways that are paradoxically regulated by caveolin-1.

VEGF stimulation of placental endothelial cell migration requires activation of JNK1/2, phosphoinositol-3 kinase/Akt, c-Src, and endogenous nitric oxide (NO) via endothelial NO synthase; these mechanisms are regulated by caveolin-1.

New vessel formation via angiogenesis is a pivotal mechanism to cause placental blood flow to rise such that adequate nutrients and oxygen can be delivered to the fetus and also for the fetus to exhaust its metabolic wastes during pregnancy. Angiogenesis denotes vascular growth from preexisting capillaries. It is initiated by increased production of local or systemic angiogenic factors, followed by endothelial cell (EC) activation to degrade extracellular matrix. ECs then migrate, proliferate, and differentiate to form tube-like structures with the recruitment of smooth muscle cells and pericytes (1). Angiogenesis is regulated by an elaborate balance between multiple proangiogenic and antiangiogenic factors. Angiogenesis in the placenta is greatest in late pregnancy, essentially keeping pace with the fast-growing fetus (2,3) and highly correlating with maternal and fetal placental production of vascular endothelial growth factor (VEGF). VEGF is essential for placental vascular development and trophoblast differentiation and invasion of the spiral arteries (4,5). In the sheep placenta, fetal cotyledon and maternal caruncle as well as placental membranes produce large quantities of VEGF during late gestation (6,7,8), coinciding with placental vascular density (7) and nitric oxide (NO) production (9). Thus, VEGF is critical for both angiogenesis and vasodilation at the maternal, fetal, and placental interface, which are two key routes governing the dramatic rises in local blood flows during pregnancy (2,3).

VEGF plays a crucial role in physiological and pathological angiogenesis prenatally and postnatally (10). VEGF elicits its functions via binding to specific receptors including fms-related tyrosine kinase 1 (Flt1)/VEGF receptor (VEGFR)-1 and kinase insert domain receptor (KDR)/VEGFR2 (10). Despite extensive studies showing the importance of VEGF and its receptors in placental vascular development, the mechanism underlying VEGF-regulated placental angiogenesis is still obscure. VEGF regulates many steps of angiogenesis such as EC proliferation, migration, and differentiation. We have shown that VEGF promotes placental EC proliferation via ERK2/1 and phosphatidylinositol 3 kinase (PI3K)/v-Akt marine thyme viral ontogeny homolog 1 (PI3K/Akt1) pathways (11,12,13) and differentiation (tube formation) via ERK2/1 pathway (14). However, how VEGF induces placental EC migration remains unknown. EC migration is a key step of angiogenesis. It involves sequential cellular changes in cytoskeleton remodeling and focal adhesion turnover (15) and is regulated spatiotemporally by multiple pathways including MAPKs (16), Src-family kinases (17), and PI3K/Akt (15). VEGF activates these pathways in placental ECs (12,13); however, it is currently unknown whether they participate in the VEGF-induced placental EC migration.

Caveolae are Ω-shaped plasma membrane microdomains mostly abundant in terminally differentiated cells including ECs (18). They are enriched in neutral lipids (sphingolipids and cholesterol) and participate in a plethora of physiological events, including endocytosis (19), apoptosis (20), and calcium signaling (21). Caveolae serve as a platform for organizing various ligated receptors initiated signaling pathways including MAPK (14), Src (22), PI3K/Akt (23), and endothelial NO synthase (eNOS) (24). Caveolae disruption by cholesterol depletion abolishes VEGF-stimulated ERK2/1 activation and placental EC proliferation and tube formation (14), suggesting that integral caveolae is critical for the VEGF-induced placental angiogenesis. Caveolin-1 (cav-1) is the major structural protein of caveolae, which functions as a scaffolding protein (25). We have recently shown that cav-1 and caveolae plays a paradoxical role in VEGF-induced placental EC proliferation and differentiation via ERK2/1 activation (14). However, the role of cav-1 and caveolae in VEGF-induced other signaling pathways and migration in placental ECs has yet to be determined.

The objectives of this study were to determine: 1) the role of various signaling pathways activated by VEGF in placental EC migration; 2) whether caveolae/cav-1 participates in the regulation of VEGF-stimulated placental EC migration; and 3) whether cav-1 and caveolae regulates VEGF stimulation of signaling pathways other than ERK2/1.

Materials and Methods

Antibodies and chemicals

These items were described in the Supplemental Materials and Methods published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Cell isolation and culture, experimental conditions, and preparation of total cell extracts

Primary ovine fetoplacental artery endothelial (oFPAE) cells were isolated and validated as described (11,14). The animal use protocol was approved by the Animal Subjects Committees from University of California San Diego and University of Wisconsin-Madison, and we followed the National Research Council’s Guide for the Care and Use of Laboratory Animals. oFPAE cells used in this study were at passages 8–11.

oFPAE cells were cultured in complete growth media [MCDB131 containing 10% fetal calf serum (FCS), 2 mm l-glutamine, and 1% antibiotics] and serum starved overnight in serum-free culture medium (M199 containing 0.1% BSA, 1% FCS, 25 mm HEPES) as described (14). After treatment with VEGF, cell lysates were prepared in a nondenaturing buffer (10 mm Tris-HCl, pH 7.4; 100 mm NaCl; 1 mm EDTA; 1 mm EGTA; 1% Triton X-100; 0.5% Nonidet P-40; 50 mm NaF; 1 mm Na3VO4; 1 mm phenylmethylsulfonyl fluoride; 1% proteinase inhibitor cocktail) (26). When pharmacological inhibitors [U0126, SB203580, SP600125, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimide (PP2), and wortmannin] were used, they were added 1 h before VEGF treatment. Caveolin-scaffolding domain (Cav-SD) and its control (Cav-SD-X) peptides were used at 5 μm for 2 h before VEGF treatment.

Stable cav-1 knockdown oFPAE cell line

Stable cav-1 knockdown oFPAE cell line was established as described (14). The cell line was established by transfection with the SureSilencing short hairpin RNA (shRNA) plasmid (SuperArray Bioscience Corp., Washington, DC) carrying ovine cav-1 RNA interference sequences: 5′-GGCAGTTGTACCATGCATTAA-3′ or a negative control sequence: 5′-GGAATCTCATTCGATG CATAC-3′. Stable oFPAE cells harboring shRNA plasmids were selected in complete MCDB-131 medium containing 600 μg/ml of Geneticin (G418; Invitrogen, Carlsbad, CA). The cell lines were confirmed by down-regulation of cav-1. The cells were maintained in complete MCDB131 medium containing 200 μg/ml of G418.

Adenoviral overexpression of cav-1

Adenoviruses carrying human cav-1 or green fluorescence protein genes were purchased from Vector Biolabs (Philadelphia, PA). Viral infections of oFPAE cells were performed and overexpression of cav-1 was confirmed by immunoblotting as described (14).

Immunoprecipitation, SDS-PAGE, and immunoblotting analysis

The cells were lysed with a nondenaturing lysis buffer as described (26). Equal amounts of proteins (1 mg/sample) were immunoprecipitated (IP) with pY99 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to pull down tyrosine-phosphorylated proteins as described previously (27). IP samples or total protein extracts were analyzed by Western blotting as described (26). Where indicated, integrated relative densities of individual bands were quantified by multiplying the absorbance of the surface areas using the ImageJ software (National Institutes of Health, Bethesda, MD).

Cell migration assay

Cell migration assay was carried out using transwell migration and scratch wound assays as described (14,28) and modified as below. For wound-healing assay, oFPAE cells were grown on fibronectin-coated culture plates to confluence, and the confluent cell monolayer was serum starved in M199 containing 0.1% BSA, 1% FCS, and 25 mm HEPES overnight and scratch by using a sterilized 200-μl pipette tip. After wounding, the cells were washed with serum-free M199 medium and cultured in M199 containing 1% FCS. The cells were then treated with or without VEGF (10 ng/ml) in the presence or absence of various kinase inhibitors for 12–24 h. Cell migration was then examined under a microscope with a ×10 objective, and digitalized images were captured. The distances of the cells of wounding edges moved toward the center of the wound were determined by using the SimplePCI image analysis software (Compix Inc., Cranberry Township, PA).

Double-immunofluorescence staining of stress fiber and focal adhesion

oFPAE cells undergone scratch wound assay were stained as described (26). The cells were incubated with antivinculin fluorescein isothiocyanate (FITC)-conjugated antibody (3.6 μg/ml) and tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (1 μg/ml) for 45 min, mounted with 4′,6-diamidino-2-phenylindole (Invitrogen), and examined under an inverted fluorescence microscopy (Leica MicroSystems Inc., Bannockburn, IL) for image acquisition by a charge-coupled device camera (Hamamatsu, Bridgewater, NJ) using SimplePCI software (Compix). The same concentrations of rabbit and mouse IgGs served as nonspecific binding controls (data not shown). Quantification of the relative immunofluorescence intensities were carried out using SimplePCI software. The mean fluorescence intensities of 100 migrating cells at the forefront edges of each group were averaged for statistic comparison among different treatment groups.

Isolation of caveolae membranes

Caveolae membranes were isolated by nondetergent sucrose gradient cell fractionation as previously described (14).

Experimental replication and statistical analysis

All experiments were repeated at least four times. Data were presented as mean ± sd. Statistic analysis was performed by one-way ANOVA, followed by Student-Newman-Keuls test for multiple comparisons using SigmaStat 3.5 (Systat Software Inc., San Jose, CA). Significant difference was defined as P < 0.05.

Results

Jun N-terminal kinase (JNK) 1/2, c-Src, and PI3K/Akt are important in VEGF-induced oFPAE cell migration

VEGF activates multiple intracellular signaling pathways, including ERK2/1, p38MAPK, JNK1/2, PI3K/Akt, and the nonreceptor tyrosine kinase Src in placental ECs. Activation of these pathways is important in VEGF-induced in vitro angiogenesis, e.g. proliferation (9,12), tube formation (14), and stimulation of eNOS and NO production (12,13). Herein we used specific kinase inhibitors, i.e. U0126 [MAPK kinase (MEK)-1/2] (29) and SB203580 (p38MAPK) (30), SP600125 (JNK1/2) (31), PP2 (c-Src) (32), and wortmannin (PI3K/Akt) (33), to investigate which signaling pathway(s) are critical for VEGF-induced oFPAE cell migration. We first determined the optimal does of these inhibitors in suppressing their respective pathways by measuring activation of their substrates (ERK2/1, heat shock protein 27, and Akt1) or kinases (c-Src and JNK1/2). As shown in Supplemental Fig. 1, VEGF stimulated phosphorylation of ERK2/1, heat shock protein 27, JNK1/2, c-Src and Akt1, which was does-dependently inhibited by U0126, SB203580, SP600125, PP2, and wortmannin, respectively (Supplemental Fig. 1). These data further confirmed the specificity and toxicity of these inhibitors as we have previously validated in oFPAE cells (13,14), which aided the decision on the concentrations of each inhibitor used for the current study.

We previously used a transwell migration assay by which we showed that VEGF promotes oFPAE cell migration (14). In this study, we used the scratch wound assay to verify the VEGF-induced oFPAE cell migration, which also generated data on directional migration (Fig. 1A and Supplemental Fig. 2). Treatment with VEGF significantly promoted oFPAE cell migration. Consistent with our previous report (14), U0126 (10 μm) did not suppress VEGF-induced cell migration. SB203580 (20 μm) also did not affect VEGF-induced cell migration. However, SP600125 (20 μm), PP2 (20 μm), and wortmannin (100 nm) attenuated VEGF-induced cell migration. These findings show that VEGF stimulation of placental EC migration requires activation of the JNK1/2, c-Src, and PI3K/Akt but not ERK2/1 and p38MAPK pathways.

Figure 1.

Figure 1

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Role of different signaling pathways in VEGF stimulation of oFPAE cell migration (A) and stress fiber (B) and focal adhesion formation (C). A, oFPAE cells were subjected to scratch wound cell migration assay as described in Materials and Methods in the presence or absence of VEGF (10 ng/ml) and/or various kinase inhibitors at the optimal doses determined in Supplemental Fig. 1. The concentrations of various kinase inhibitors were: U0126, 10 μm; SB203580, SP600125, and PP2, 20 μm; wortmannin, 100 nm. Cells were allowed to migrate for 16 h. Experiments were repeated four times independently and statistical analyses performed using Student’s t test. Bars (mean ± sd) with different letters differ significantly (P < 0.05). B and C, oFPAE cells were subjected to scratch wound migration assay described in Supplemental Fig. 3. At the end of cell migration, cells were labeled with TRITC-phalloidin (1 μg/ml) for stress fiber and antivinculin FITC-conjugated antibody (3.6 μg/ml) for focal adhesion. Relative immunofluorescence intensities of stress fiber and focal adhesion were measured using SimplePCI software as described in Materials and Methods. The mean relative fluorescence of 100 migrating cells at the forefront edges of each group was obtained. Bar graphs (mean ± sd) summarize four independent assays and bars with different letters differ significantly (P < 0.05).

JNK1/2, c-Src, and PI3K/Akt are important in VEGF-induced cytoskeleton reorganization

During cell migration, focal adhesion assembly and stress fiber formation are critical steps in growth factor-triggered cell migration (15). We accessed the effects of VEGF on stress fiber formation and focal adhesion and the involvement of various VEGF-initiated signaling pathways in these responses in oFPAE cells. Cells after directional migration in scratch wound assay were labeled with TRITC-labeled phalloidin and FITC-vinculin for measuring stress fiber and focal adhesion formation of the migrating cells at the forefront edges. As shown in Supplemental Fig. 3 (fluorescence images) and summarized in Fig. 1, B and C, VEGF significantly stimulated oFPAE cell stress fiber formation and focal adhesion assembly. U0126 and SB203580 did not inhibit these events induced by VEGF, consistent with cell migration results (Fig. 1A). SP600125, PP2, and wortmannin significantly suppressed VEGF-induced stress fiber formation. PP2, but not SP600125 and wortmannin, significantly inhibited VEGF-induced focal adhesion (Supplemental Fig. 3 and Fig. 1, B and C). Thus, JNK1/2, c-Src, and PI3K/Akt are important in VEGF-stimulated placental EC migration that involves cytoskeleton reorganization; c-Src is also important for VEGF-induced focal adhesion assembly. ERK2/1 and p38MAPK are not involved in these events induced by VEGF.

JNK1/2, c-Src, and PI3K/Akt are important in VEGF-induced cofilin-1 phosphorylation

Cofilin-1 is a member of a family of small actin-binding proteins that are essential for actin reorganization via depolymerizing and severing actin filaments during cell migration (34). The severing activity of cofilin-1 is reversibly regulated by Ser3 phosphorylation/dephosphorylation, with the phosphorylated form being inactive. Actin disassembly via cofilin-1 phosphorylation is important in VEGF-induced cell migration (35). As shown in Fig. 2A, treatment with VEGF stimulated cofilin-1 phosphorylation in oFPAE cells, which was inhibited by SP600125, PP2, and wortmannin. PP2 also inhibited the basal level of phosphorylated cofilin-1. These data indicate that cofilin-1 is phosphorylated by VEGF via multiple signaling pathways in placental ECs. These data further strengthened the role of JNK1/2, Src, and PI3K/Akt pathways in VEGF-induced cytoskeleton reorganization in placental ECs. U0126 and SB203580 also did not suppress VEGF-induced cofilin-1 phosphorylation (Supplemental Fig. 4A), consistently excluding the involvement of ERK2/1 and p38MAPK in VEGF-induced cell migration.

Figure 2.

Figure 2

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VEGF stimulates cofilin-1, FAK, and paxillin phosphorylations via JNK1/2, c-Src, PI3K/Akt, and c-Src, respectively. Serum-starved oFPAE cells were treated with or without SP600125 (20 μm), PP2 (20 μm), or wortmannin (100 nm) for 1 h before VEGF (10 ng/ml) treatment for 5 min. Cell extracts were prepared and subjected to Western blot analysis using specific antibodies. Bar graphs (mean ± sd) summarize four independent experiments, and bars with different letters differ significantly (P < 0.05). p, Phosphorylated.

VEGF induces focal adhesion kinase (FAK) and paxillin phosphorylation: role of c-Src

FAK is a widely expressed cytoplasmic nonreceptor protein tyrosine kinase that is found in focal adhesions of cultured cells (36). It is involved in receptor-proximal link between growth factor receptors and integrin signaling pathways (37). It plays an important role in the control of several biological processes, including cell spreading and migration (38). Src-mediated FAK phosphorylation on Tyr576/577 is important for cell motility (39). Furthermore, activated FAK stimulates Tyr118 phosphorylation of paxillin, the adaptor protein of FAK -mediated assembly of growth factor receptor signaling (40), which is important in cytoskeleton remodeling (41). We then further tested whether VEGF stimulates phosphorylation of FAK and paxillin and, if so, by which signaling pathways in placental ECs. As shown in Fig. 2B, VEGF stimulated Try576/577 phosphorylation of FAK. VEGF-induced FAK phosphorylation was inhibited only by the c-Src inhibitor, PP2, not by any other kinase inhibitors (Fig. 2 and Supplemental Fig. 4B). VEGF also stimulated paxillin phosphorylation, which was also inhibited only by PP2 (Figs 2C and Supplemental Fig. 4C).

Because VEGF may activate other tyrosine residues in FAK (42) important in cell migration, we carried out IP experiments using the pY99 antibody to pull down total tyrosine-phosphorylated proteins. As shown in Supplemental Fig. 5, VEGF induced FAK tyrosine phosphorylation. This activation was suppressed only by PP2, but not by SP600125 and wortmannin (Supplemental Fig. 5). These results suggest that phosphorylation on Tyr576/577 is likely to be the major mechanism for VEGF-induced FAK activation in oFPAE cells. These data are also consistent with focal adhesion turnover (Supplemental Fig. 3).

Role of eNOS-NO pathway in VEGF-induced oFPAE cell migration

VEGF activates eNOS via VEGFR2/PI3K/Akt pathway in ECs (43,44). Mice carrying mutant eNOS show impaired angiogenesis in response to ischemia (45) and VEGF (46). Furthermore, NO directly promotes or inhibits EC migration, depending on its concentrations (43,47). We investigated whether eNOS/NO are involved in VEGF-stimulated placental EC migration. As expected, VEGF stimulated eNOS Ser1179 phosphorylation in oFPAE cells (Fig. 3B), critical for its activation (43). In the presence of the NOS inhibitor N-omega-nitro-l-arginine methyl ester (l-NAME; 5 μm), VEGF-induced oFPAE cell migration was inhibited. Treatment with a NO donor diethylenetriamine (DETA)-NO at both 100 and 300 μm significantly stimulated oFPAE cell migration (Fig. 3A and Supplemental Fig. 6). However, high-dose DETA-NO (1 mm) inhibited oFPAE cell migration and was toxic to the cells (data no shown). These results indicated that the eNOS-NO pathway is important for VEGF-stimulated oFPAE cell migration and the effects of NO on placental EC migration is concentration dependent.

Figure 3.

Figure 3

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VEGF promotes oFPAE cell migration via eNOS-NO pathway. A, oFPAE cells were subjected to scratch wound cell migration assay in the presence or absence of VEGF (10 ng/ml) and/or n-omega-nitro-d-arginine methyl ester (d-NAME) l-NAME (both at 5 μm), or a NO donor DETA-NO at 100 or 300 μm, respectively. Cells were allowed to migrate for 24 h. B, VEGF-activated eNOS via JNK1/2, c-Src, and PI3K/Akt pathways. Serum-starved oFPAE cells were treated with or without SP600125 (20 μm), PP2 (20 μm), or wortmannin (100 nm) for 1 h before VEGF (10 ng/ml) treatment for 5 min. Cell extracts were prepared and subjected to Western blot analysis using specific antibodies. Bar graphs (mean ± sd) summarize four independent experiments, and bars with different letters differ significantly (P < 0.05). p, Phosphorylated.

We further tested the mechanism of VEGF-induced eNOS activation in oFPAE cells. VEGF-induced eNOS phosphorylation on Ser1179 was attenuated by the PI3K/Akt inhibitor wortmannin, consistent with previous reports (43,44). Interestingly, inhibition of JNK1/2 and c-Src also suppressed VEGF-induced eNOS phosphorylation (Fig. 3B). These findings indicate that VEGF-induced eNOS activation is via PI3K/AKT, JNK1/2, and c-Src pathways in placental ECs.

Cav-1 is important in VEGF-stimulated placental EC migration

Cav-1 is a scaffolding protein that facilitates interactions among various signaling molecules, thereby compartmentalizing various signaling pathways in the caveolae. However, different and sometimes even opposite functions of caveolin-1 in cell migration have been reported in different cells surveyed (48,49,50). We investigated whether cav-1 participates in the VEGF-induced placental EC migration and, if so, how this is achieved. We found that cav-1 was evenly distributed in nonmigrating cells and was redistributed to the rear cell membrane in migrating cells (Fig. 4A), suggesting polarized redistribution of cav-1 in cell migration. We then investigated the effects of down-regulation or overexpression of cav-1 in VEGF-stimulated placental EC migration. To this end, we used oFPAE cells stably transfected with cav-1 shRNA for down-regulation and cells infected with adenovirus carrying cav-1 or addition of Cav-SD peptide for overexpression as we reported recently (14). Cav-1 knockdown resulted in significant reduction of basal directional migration and suppressed VEGF-induced cell migration. Control shRNA stably transfected cells displayed similar stress fiber and focal adhesion to nontransfected cells; however, cav-1 knockdown cells displayed greater basal levels of stress fiber formation and focal adhesion (Fig. 4B and Supplemental Fig. 7), Furthermore, cav-1 knockdown suppressed VEGF augmentation of stress fiber formation and focal adhesion (Fig. 5), indicating that cav-1 knockdown resulted in substantial changes in cytoskeleton remodeling during cell migration in the absence and presence of VEGF.

Figure 4.

Figure 4

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Caveolin-1 regulates oFPAE cell migration. A, Double-immunofluorescence labeling of caveolin-1 and stress fiber in no-migrating and migrating oFPAE cells. Cells were subjected to scratch wound cell migration assay as described in Materials and Methods. Cells were labeled wih anti-cav-1 antibody (red) and FITC-labeled phalloidin (green, 1 μg/ml) as described in Materials and Methods. Cells at the edge of wound were migrating cells, whereas confluent cells were no-migrating cells. Arrow indicates the direction of cell migration. Arrows indicates the extension of the leading edge where cav-1 level was reduced. B, Knockdown (KD) of cav-1 suppresses basal and VEGF-induced oFPAE cell migration. oFPAE cells stably transfected with cav-1 shRNA (Cav-shRNA) or control shRNA (Ctl-shRNA) as established previously (14) were subjected to scratch wound and transwell cell migration assays as described in Materials and Methods. Cells were allowed to migrate for 24 h. C and D, Overexpression of cav-1 inhibits VEGF-induced oFPAE cell migration. Cells were subjected to scratch wound and transwell cell migration assays as described in Materials and Methods in the presence or absence of cav-1 or green fluorescence protein (GFP) adenovirus (C, 20 multiplicity of infection) or Cav-SD (D) or its negative control (D; Cav-SD-X, both at 5 μm) or VEGF (10 ng/ml). Cells were allowed to migrate for 24 h. Bar graphs (mean ± sd) summarize four independent experiments, and bars with different letters differ significantly (P < 0.05).

Figure 5.

Figure 5

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Knockdown of cav-1 regulates basal and VEGF-induced oFPAE cell cytoskeleton remodeling. oFPAE cells stably transfected with cav-1 shRNA (Cav-shRNA) or control shRNA (Ctl-shRNA) were subjected to scratch wound cell migration assay as described in Fig. 4B. Cells were labeled with TRITC-labeled phalloidin (1 μg/ml) and antivinculin FITC-conjugated (3.6 μg/ml) antibodies as described in Fig. 1, B and C. The cells were mounted in Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole to label the nuclear. Fluorescence images (upper panels) of cells at the edges of wound were captured by a Hamamatsu charge-coupled device camera captured under an inverted Leica fluorescence microscopy using the SimplePCI image analysis software. The relative fluorescence intensities of F-actin and vaculin were measured using SimplePCI software as described in Materials and Methods. The mean fluorescence intensities of 100 migrating cells at the forefront edges of each group were obtained. Lower bar graphs (mean ± sd) summarizes four independent assays, and bars with different letters differ significantly (P < 0.05).

Moreover, adenoviral cav-1 overexpression inhibited VEGF-induced oFPAE cell migration (Fig. 4C). Similarly, Cav-SD peptide also inhibited VEGF-stimulated cell migration (Fig. 4D and Supplemental Fig. 8). These data indicate that too little or too much cav-1 both inhibited VEGF-induced placental EC migration.

Cav-1 regulates VEGF-activated signaling pathways in placental EC migration

As a scaffolding protein, cav-1 regulates cell function by compartmentalizing signaling pathways in the caveolae. We then accessed the role of cav-1 in various VEGF-activated signaling pathways important for oFPAE cell migration. As shown in Fig. 6, A–E, cav-1 knockdown suppressed VEGF-induced phosphorylation of Akt, JNK1/2, and eNOS. However, cav-1 knockdown increased basal levels of, but not VEGF-induced, phosphorylated c-Src and FAK. Increased basal levels of phosphorylated c-Src and FAK may account for the increased basal levels of stress fiber formation and focal adhesion as seen in the cav-1 knockdown cells (Fig. 5).

Figure 6.

Figure 6

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Knockdown of cav-1 regulates basal or VEGF-activated signaling pathways in oFPAE cells. Serum-starved oFPAE cells stably transfected with caveolin-1 shRNA (Cav-shRNA) or control shRNA (Ctl-shRNA) were treated with or without VEGF (10 ng/ml) for 5 min. Cell extracts were prepared and subjected to Western blot analysis of Akt (A), JNK1/2 (B), c-Src (C), FAK (D), and eNOS (E) phosphorylations using specific antibodies. Bar graphs (mean ± sd) summarize four independent experiments, and bars with different letters differ significantly (P < 0.05). p, Phosphorylated.

Overexpression of cav-1 by addition of Cav-SD domain peptide inhibited VEGF-induced activation of Akt, JNK1/2, c-Src, FAK, and eNOS (Fig. 7, A–E). Furthermore, we found that Src, FAK, paxillin, eNOS, JNK1/2, and VEGFR1 were present in the caveolae membranes (Supplemental Fig. 9). These results indicate that cav-1 regulates VEGF-induced placental EC migration via a paradoxical regulation of multiple signaling pathways activated by VEGF.

Figure 7.

Figure 7

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Overexpression of cav-1 suppresses VEGF-induced phosphorylation of Akt, JNK1/2, c-Src, FAK, and eNOS. Serum-starved oFPAE cells were treated with or without Cav-SD or its negative control (Cav-SD-X; both at 5 μm) for 2 h before VEGF (10 ng/ml) treatment for 5 min. Cell extracts were prepared and subjected to Western blot analysis of Akt (A), JNK1/2 (B), c-Src (C), FAK (D), and eNOS (E) phosphorylations using specific antibodies. Bar graphs (mean ± sd) summarize four independent experiments, and bars with different letters differ significantly (P < 0.05). p, Phosphorylated.

Discussion

The current study follows up our recent mechanistic report on the role of cav-1 and caveolae in the VEGF regulation of placental EC proliferation and tube formation (14). Herein we focused on the mechanism of placental EC migration. EC migration is a major component of angiogenesis (1). The mechanism by which VEGF regulates placental EC migration is currently unknown. In this study, we first studied the intracellular signaling pathways involved in VEGF-triggered oFPAE cell migration and investigated whether and how cav-1/caveolae participated in these events. We have shown that activation of JNK1/2, c-Src, and PI3K/Akt are important in VEGF- induced migration in placental ECs, whereas ERK2/1 and p38MAPK are not required. We also studied VEGF-induced stress fiber formation and focal adhesion, two major events of cytoskeleton remodeling during cell migration. We found that VEGF-activated JNK1/2, c-Src, and PI3K/Akt are important in the stress fiber formation, and c-Src is also important in focal adhesion formation, whereas ERK2/1 and p38MAPK are not involved in these processes. Moreover, we found that cav-1 plays a paradoxical role in VEGF-induced oFPAE cell migration. These findings further strengthen our recent study (14), showing a paradoxical role of cav-1/caveolae that is important for VEGF-induced placental angiogenesis.

Our current results further affirm our recent report that ERK2/1 is not important in VEGF-induced placental EC migration (14). These results consistently agree with the literature. For example, inhibition of ERK2/1 signaling pathway by UO126 did not suppress VEGF-mediated bovine pulmonary arterial EC (BPAEC) and human umbilical vein endothelial cell (HUVEC) migration (51,52), and another MEK1/2 inhibitor, PD98059, also did not suppress HUVEC and keratocyte migration (53,54), although one report suggests that inhibition of ERK partially attenuated HUVEC migration (55),

Both p38MAPK and JNK1/2 are MAPKs that are also recognized as stress-activated protein kinase. Although originally thought to be important in inflammation and apoptosis (56), they also participate in growth factor-induced angiogenic responses, including cell migration (10). Disruption of p38αMAPK results in severe defects in placental development, particularly placental vasculature formation via vasculogenesis and angiogenesis (57). Activation of p38MAPK by VEGF has been reported, which was proposed to be important for VEGF stimulation of migration of various ECs (53) including BPAECs (51). p38MAPK inhibition down-regulates VEGF-stimulated HUVEC migration (58), whereas constitutive p38MAPK activation suffices to induce HUVEC cell migration in association with cytoskeleton remodeling characterized by enhanced lamellipodia (59). In contrast to these findings, we have shown that VEGF-induced cytoskeleton remodeling and cell migration was not suppressed by p38MAPK inhibition in oFPAE cells. The reason for this discrepancy is currently unknown. However, a recent report has shown that VEGF-stimulated migration was not constrained by SB203580 in HUVECs overexpressing urokinase plasminogen activator (uPA) and was inhibited by agents disrupting uPA-uPA receptor interaction (58), implicating a role of p38MAPK in VEGF-induced EC migration via uPA and its receptor interaction. It is possible that the nonessential role of p38MAPK in VEGF-stimulated oFPAE cell migration might be due to the absence of uPA and/or uPA receptor expressions or disruption of their interaction in oFPAE cells. However, this needs to be verified in further studies.

A few reports suggested that JNK signaling is important in growth factor-promoted cell migration (54,60,61); however, the underlying mechanism is largely unknown. VEGF-activated JNK signaling is important in VEGF-stimulated paxillin phosphorylation that is required for cell migration (54). We found that inhibition of JNK by SP600125 suppresses VEGF-promoted placental EC migration. However, suppression of JNK1/2 did not inhibit VEGF-induced paxillin phosphorylation and focal adhesion (Fig. 2C and Supplemental Fig. 3). On the other hand, VEGF-induced cofilin-1 phosphorylation and stress fiber formation were blocked by JNK inhibition. Because stress fiber formation via dephosphorylation of cofilin-1 is critical for VEGF-induced cell migration (62), our results suggest that VEGF-activated JNK1/2 signaling is important in placental EC migration, possibly via cofilin-1-mediated cytoskeleton remodeling. To our knowledge, this is the first report on VEGF-induced cell migration via JNK-cofilin-1 pathway in any ECs.

The role of PI3K/Akt signaling in VEGF-induced EC migration remains somewhat controversial. Our study agrees with previous studies showing that Akt is important in VEGF-stimulated actin reorganization and EC migration in microvascular ECs, both in vitro (63,64) and in vivo (64). PI3K/Akt is also important in VEGF-induced BPAEC (51) and HUVEC (65) migration. In other studies; however, PI3K was not required for VEGF-induced HUVEC migration (52). We have shown that PI3K/Akt activation by VEGF is essential for stress fiber formation, possibly linked to cofilin-1 phosphorylation. The latter agrees with a report in HUVECs that inhibition of PI3K attenuates VEGF-induced phosphorylation of Lin-11, Isl-1, and Mec-3 kinase responsible for phosphorylating cofilin-1 (65). We also have shown that PI3K/Akt is not involved in VEGF-induced focal adhesion in oFPAE cells, contrasting a previous report showing that VEGF induces BPAEC migration via PI3K/Akt pathway leading to focal adhesion (66).

It has been shown that c-Src is important in VEGF-induced cell migration, which regulates FAK activation and subsequent focal adhesion (67). c-Src is important in VEGF-induced BPAEC (51) and HUVEC (68) migration. Our data are well in line with these findings because c-Src inhibition significantly suppresses both basal and VEGF-induced FAK and paxillin phosphorylation as well as basal and VEGF-induced placental EC migration. Interestingly, we also observed that c-Src is involved in VEGF-induced stress fiber formation, possibly linked to cofilin-1 phosphorylation. The importance of c-Src in cofilin-1 phosphorylation has begun to be recognized recently. Activation of c-Src is important in epidermal growth factor stimulation of cofilin-1 phosphorylation and subsequent tight junction formation in epithelial cells (69). In addition, integrin-mediated cell adhesion also requires c-Src-mediated cofilin-1 phosphorylation in epithelial cells (70). To the best of our knowledge, this is the first report on VEGF-induced stress fiber formation and cofilin-1 phosphorylation via c-Src pathway.

We also have demonstrated a critical role of eNOS-NO in VEGF-induced placental EC migration because of the following findings. First, VEGF stimulates eNOS phosphorylation on ser1179 in oFPAE cells (Fig. 3B). Second, NOS inhibition with lr-NAME significantly suppressed VEGF-stimulated cell migration. Third, a NO donor DETA-NO significantly promotes oFPAE cell migration (Fig. 3). These results, combined with our previous reports showing that VEGF stimulates oFPAE cell NO production that is attenuated by lr-NAME (43,44), highlight a critical role of endogenous NO production via eNOS activation in the VEGF-induced placental EC migration. It is well established that Akt-dependent eNOS phosphorylation on ser1179 is critical for VEGF stimulation of NO production (43,44). Consistently, we have shown that VEGF-induced eNOS activation via PI3K/Akt pathway in placental ECs (Fig. 3B). Furthermore, VEGF-induced eNOS activation is also constrained by c-Src inhibition, similar to a previous report in bovine aortic ECs (BAECs) (71). Importantly, we have first shown that JNK1/2 is important in VEGF-induced eNOS phosphorylation in ECs, although a recent study has shown that JNK pathway is important for eNOS phosphorylation by insulin In HUVECs (72).

Because both VEGF receptors (Flt-1 and KDR) are present and activated by VEGF in oFPAE cells (14), we evaluated which VEGF receptor is important in VEGF-induced placental EC migration. As shown in Supplemental Fig. 10, inhibition of both VEGFR1 and VEGFR2 with their specific neutralizing antibodies significantly suppressed VEGF-induced oFPAE cell migration. These results suggest that both VEGFR1/Flt1 and VEGFR2/KDR are required for VEGF-induced placental EC migration. VEGFR2 is indispensable in VEGF-induced EC angiogenesis, including cell migration (73), whereas the role of VEGFR1 in this process is less conclusive. We have shown recently that activation of VEGFR1 by placental growth factor (PlGF) activates the PI3K/AKT pathway (14). It is possible that inhibition of Flt-1 suppresses VEGF-induced PI3K/AKT activation, leading to the attenuation of VEGF-induced stress fiber formation and oFPAE cell migration, similarly to a report showing that PlGF and PlGF/VEGF heterodimer induce primary microvascular EC tube formation via PI3K/AKT pathway (74). We also observed that neutralizing antibody of KDR, but not Flt-1, inhibits VEGF-stimulated c-Src activation (data not shown). In keeping with the critical role of Src in VEGF-induced focal adhesion turnover and stress fiber formation, it is possible that signaling via KDR is important in VEGF-induced oFPAE cell migration via these processes.

The role that cav-1/caveolae play in cell migration is an active area of research (75). Cav-1-null mice show abruption of multiple signaling pathways, resulting in a wide range of phenotypes including impaired angiogenesis most likely linked to decreased EC migration to exogenous stimuli (76). Cav-1 is important in regulating directional cell migration in vitro (22). When a cell migrates, cav-1 is redistributed and polarized at the rear of the cell (75) (Fig. 4A). However, the mechanism of cav-1 in regulating cell migration still remains a mystery. Several models have been proposed for cav-1 regulation of cell migration, e.g. controlling cell membrane composition and membrane surface expansion, polarization of signaling molecules and matrix proteolysis, and/or cytoskeleton remodeling (75). In this study, we used cav-1 knockdown and overexpression approaches to elucidate the role that cav-1 plays in VEGF-induced placental EC migration. We have shown that knockdown of cav-1 suppresses basal and VEGF-induced oFPAE cell migration (Fig. 4B). Of note, cav-1 knockout mice also display suppressed basal and stimulated angiogenic activities, including cell migration (22). Adenoviral cav-1 overexpression and addition of Cav-SD peptide also inhibit VEGF-induced placental EC migration. This paradox is consistent with our recent report that caveolae/cav-1 play a similar paradoxical role in regulating VEGF-induced oFPAE cell proliferation and tube formation via ERK2/1 (14). Indeed, both cav-1 knockdown and overexpression suppress VEGF-activated signaling pathways that are important in VEGF-induced placental EC migration (Figs. 6 and 7).

Our results are consistent with previous findings that cav-1 knockdown suppresses VEGF-induced Akt (77) and eNOS (77,78) phosphorylation in ECs in vivo. Overexpression of cav-1 also inhibits eNOS activation (79). Knockdown of cav-1 by small interfering RNA suppresses IL-6-induced JNK activation in human gingival fibroblasts (80), and adenoviral overexpression of cav-1 inhibits TGFβ1-induced JNK activation (81). These data also are in agreement with our findings showing that VEGF-induced JNK1/2 phosphorylation was inhibited by both knockdown and overexpression of cav-1 in placental ECs. Of note, cav-1 knockdown increases basal levels of stress fiber and focal adhesion in oFPAE cells (Fig. 5). The increase in basal cytoskeleton remodeling might be resulted from increased basal levels of c-Src and FAK activation (Fig. 6, C and D). However, these changes do not result in increased, but rather decreased, basal cell migration. This finding, although unexpected, is consistent with decreased basal cell migration of fibroblasts derived from cav-1 null mouse embryos (22). Basal c-Src phosphorylation is also increased in cav-1 knockdown cells, which may result in the increased focal adhesion turnover (22).

Although the exact underlying mechanism for these paradoxes remains unknown, both knockdown and overexpression of cav-1 may have disrupted the polarization or gradient redistribution of cav-1, which is important for proper compartmentalizing cell signaling pathways in the caveolae. For example, we have shown recently that VEGFR2 directly interacts with cav-1 and the Raf/Ras/MEK/ERK2/1 signaling cascade is compartmentalized in oFPAE cell caveolae (14), consistent with a previous report (73) in HUVECs. In this study, we further show that VEGFR1 also resides in the caveolae (Supplemental Fig. 9). The physical localization and interaction of these signaling molecules allows cav-1/caveolae to regulate VEGF-activated ERK2/1 signaling module and angiogenic responses in a paradoxical manner. On the one hand, caveolae serve as a platform to facilitate and amplify signaling cascades by compartmentalizing the necessary signaling components for instant responses to environmental factors. On the other hand, cav-1, the major structural protein of caveolae, interacts with these signaling molecules via its Cav-SD (amino acids 82–101), which in essence inhibits the catalytic activities of these signaling cascades. These structural and functional features allow cav-1 to regulate cell signaling in a concentration-sensitive manner. In other words, the optimal level of this protein is important to the maximal outcome to external stimuli. We have shown that, consistent with the findings in BAECs (82), c-Src and eNOS are detected in the caveolae of oFPAE cells (Supplemental Fig. 9). The finding that JNK1/2 is also detected in the caveolae of oFPAE cells is consistent with a report showing JNK1/2 in the caveolae of human renal proximal tubule epithelial cells (83). Interestingly, we detected FAK and paxillin in the caveolae of oFPAE cells, which is different from a previous report in rat cardiac fibroblasts (84). Although Akt is barely detected in the caveolae of oFPAE cells, similar to that of BAECs (85), it is possible that cav-1 is able to regulate these signaling pathways at multiple levels by either regulating the upstream (i.e. VEGFR2) or downstream (i.e. eNOS, c-Src, JNK1/2, FAK, and paxillin) signaling molecules.

Given the complex interplays among multiple signaling pathways toward placental EC migration, how can we reconcile the role of cav-1/caveolae and activation of cell signaling pathways (JNK1/2, c-Src, PI3K/Akt, and eNOS) in VEGF-induced placental EC migration? Our data support the following scenario (Fig. 8). Ligated VEGFR2, and possibly VEGFR1, interacts with cav-1 to compartmentalize cell signaling pathways in the caveolae. On the one hand, VEGF activates multiple pathways (i.e. JNK1/2, c-Src, and PI3K/Akt) leading to the formation of stress fibers and focal adhesion via phosphorylation of cofilin-1 and/or FAK/paxillin; on the other hand, activation of these pathways results in eNOS activation and NO production, which is also a potent angiogenic factor that promotes cell migration. Of note, although we have clearly demonstrated these links, more studies are needed for deciphering the mechanism by which VEGF activates each signaling pathway and how activation of each pathway by VEGF is finely tuned by cav-1/caveolae toward angiogenesis.

Figure 8.

Figure 8

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Diagrammatic signaling control of VEGF-induced placental EC angiogenesis via cav-1 and caveolae. VEGF promotes placental endothelial angiogenic responses via the activations of multiple signaling pathways, which is regulated by cav-1 in the caveolae.

Supplementary Material

[Supplemental Data]
en.2009-1305_index.html (775B, html)

Footnotes

This work was supported in part by National Institutes of Health Grants HL64703 (to J.Z.) and HL74947 and HL70562 (to D.C.).

Disclosure Summary: All authors have nothing to disclose.

First Published Online May 12, 2010

Abbreviations: BAEC, Bovine aortic EC; BPAEC, bovine pulmonary arterial EC; cav-1, caveolin-1; Cav-SD, caveolin-scaffolding domain; Cav-SD-X, Cav-SD control; DETA, diethylenetriamine; EC, endothelial cell; eNOS, endothelial NO synthase; FAK, focal adhesion kinase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; Flt1, fms-related tyrosine kinase 1; HUVEC, human umbilical vein endothelial cell; IP, immunoprecipitated; JNK, Jun N-terminal kinase; KDR, kinase insert domain receptor; l-NAME, N-omega-nitro-l-arginine methyl ester; MEK, MAPK kinase; NO, nitric oxide; oFPAE, ovine fetoplacental artery endothelial; PI3K, phosphatidylinositol 3 kinase; PlGF, placental growth factor; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimide; shRNA, short hairpin RNA; TRITC, tetramethylrhodamine isothiocyanate; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

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