The phosphatase PTP-PEST/PTPN12 regulates endothelial cell migration and adhesion, but not permeability, and controls vascular development and embryonic viability

Results: PTP-PEST is critical for adhesion and of endothelial cells. Its absence in cells results in embryonic Conclusion: The embryonic viability seen in

positive regulator of T cell activation, as a result of its capacity to promote homotypic adhesion between T cells (11). Lastly, it was recently uncovered that PTP-PEST is a tumor suppressor in human breast cancer and lung cancer, seemingly as a result of its capacity to control receptor PTK signaling (18).
Endothelial cells form the inner lining of blood vessels (19). They play an essential role during mammalian development, being the first fully functional system in the embryo (19,20). Endothelial cells are also critical for many processes in post-natal life, including exchange of nutrients, blood clotting and migration of immune cells towards inflamed tissues. Blood vessel development occurs through two consecutive processes, vasculogenesis and angiogenesis. Vasculogenesis involves blood vessel formation from pluripotent mesenchymal cells, whereas angiogenesis implicates blood vessel formation from existing vessels. Both require extensive endothelial cell proliferation and migration.
Here, we examined the role of PTP-PEST in endothelial cells. Using primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is required for migration and adhesion of endothelial cells, but not for proliferation, differentiation and modulation of permeability to macromolecules by these cells. This function correlates with the capacity of PTP-PEST to dephosphorylate Cas, paxillin and Pyk2. Studies of a mouse in which PTP-PEST was specifically eliminated in endothelial cells provided evidence that the function of PTP-PEST in endothelial cells is critical for normal vascular development and embryonic viability.

EXPERIMENTAL PROCEDURES
Mice. Mice expressing a tamoxifen-inducible Cre (Cre-ERT2) under the control of the ubiquitin C promoter (UBC-CreERT2) or the Cre recombinase under the control of the Tie2 promoter (Tie2-Cre), and ROSA26EYFP mice were obtained from The Jackson Laboratories (Bar Harbor, ME) (33)(34)(35). They were then bred with a conditionally-deleted allele of the PTP-PEST-encoding gene, Ptpn12 fl , back-crossed for at least 15 generations to the C57BL/6 background (11). A constitutive PTP-PEST-deficient mouse (Ptpn12 -/-), in which PTP-PEST expression was constitutively lacking in all cells, was generated by breeding Ptpn12 fl/+ mice with mice expressing a ubiquitous Cre transgene (11). Timed matings were set up between male Ptpn12 fl/+ ;Tie2-Cre + and female Ptpn12 fl/fl mice. Day 0.5 of embryonic development (E0.5) was defined as the morning when the vaginal plug was observed. All mice used in this study were bred under specific pathogen-free conditions. Animal experimentation was approved by the IRCM Animal Care Committee in accordance with the regulations of the Canadian Council for Animal Care. Inducible deletion of PTP-PEST in adult mice. Female Ptpn12 fl/fl mice were bred with male Ptpn12 fl/+ ;UBC-CreERT2 + mice. For tamoxifen-induced deletion of PTP-PEST, mice were fed for 5 consecutives days with tamoxifen (200 mg per g of weight per day, in corn oil; Sigma-Aldrich, St. Louis, MO). After 10 days, mice were sacrificed and used for endothelial cell isolation.

Isolation of lung-derived endothelial cells.
Primary lung-derived endothelial cells were extracted from lung tissue of 8-10 week-old mice, as described for the embryonic endothelial cells. Cells were then cultured in low-glucose DMEM:F12 medium (1:1 mix; Invitrogen), supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, USA), endothelial growth supplement (5 µg/ml; BD Biosciences) and heparin (100 µg/ml; Sigma-Aldrich), using gelatin-coated tissue culture dishes. Two successive rounds of endothelial cell purification were then performed using rat anti-mouse ICAM-2 antibodies (MAb 3C4; BD Biosciences) coupled to sheep anti-rat IgG-coated magnetic beads (Dynabeads; Invitrogen). The purity of endothelial cells was confirmed by flow cytometry analysis, using antibodies directed against the endothelial cell markers CD31 (rat anti-mouse CD31 MAb 390, eBioscience, San Diego, CA) and ICAM-2 (rat anti-mouse MAb 3C4, Biolegend, San Diego, CA). After 16 days in culture, cells were used for experimentation. At that time, >95% of cells expressed the endothelial cell-specific markers. Microscopy analyses of mouse embryos. After several washes, embryos were incubated overnight at 4 o C in the presence of a 1:500 dilution of Alexa Fluor 488-coupled goat anti-rat IgG (Invitrogen, Oregon, USA). After several washes, images were obtained by confocal microscopy using a LSM710 Microscope (Zeiss). For cross-section analyses, fixed embryos were embedded in Epon, sectioned (0.5 µm thickness) and stained with toluidine blue. They were then visualized by microscopy (Axiophot; Zeiss), as described above.

Isolation of embryonic endothelial cells. Male
Ptpn12 fl/+ ;Rosa26EYFP + ;Tie2-Cre + were bred with females Ptpn12 fl/fl ;ROSA26EYFP + mice. E10.5 embryos were harvested from timed pregnancies, washed, minced and digested at 37 o C for 1 hour with collagenase A (1 mg/ml; Roche, Indianapolis, IN). To remove undigested material, the cell suspension was washed through a cell strainer (BD Biosciences). Isolated cells were then incubated on ice for 1 hour with the antibodies described below. Cells were washed and EYFP + cells were analysed by flow cytometry (FACSCalibur, BD Biosciences). Flow cytometry. Embryonic cells or primary lung-derived endothelial cells were stained and analysed by flow cytometry, using the following antibodies (from Biolegend, BD Biosciences or eBioscience): Alexa 647-coupled rat anti-mouse CD31 (MAb 390), Alexa 647-coupled rat anti-mouse ICAM-1 (MAb YN1/1.7.4), Alexa 647-coupled rat anti-mouse ICAM-2 (MAb 3C4), Alexa 647-coupled rat anti-mouse VE-Cadherin (MAb VECD1), phycoerythrin (PE)-coupled hamster anti-mouse β1 integrin (CD29; MAb HMb1-1), FITC-coupled rat anti-mouse β2 integrin (CD18; MAb M18/2), and rat anti-mouse isotype control (MAb RTK2758). Proliferation assays. Primary lung-derived endothelial cells (4x10 4 cells) were seeded in 24-well, gelatin-coated tissue culture dishes. At the desired time point, cells were harvested by trypsin (Invitrogen) and counted. Adhesion assays. 96-well flat-bottom Immuno plates (Nunc, Thermoscientific, Rochester, NY) were coated overnight with fibronectin (10 µg/ml, BD Biosciences), type I collagen (10 µg/ml, BD Biosciences) or bovine serum albumin (BSA; 2 mg/ml, Sigma-Aldrich). After washing, plates were blocked with BSA, and 10 4 endothelial cells were allowed to adhere for 30 minutes at 37 o C. After washing away non-adherent cells, adherent cells were fixed and stained with crystal violet (Fisher Scientific, Ottawa, Ontario, Canada). Cell adhesion was estimated by measuring the absorbance at 562 in a Microplate Scanning Spectrometer (Power Wave X, Bio-Tek Instruments, Inc., Winooski, VT). Photographs were also obtained using an inverted microscope (Model Axiovert S100TV; Zeiss) with a QImaging camera and the Velocity image analysis software (PerkinElmer, Waltham, MA). Spreading assays. Coverslips were coated at 4 o C overnight with fibronectin or collagen, as above. Then, coverslips were washed with PBS and placed in 6-well dishes. Endothelial cells were harvested and seeded (1×10 5 cells) on the coverslips. After 90 minutes at 37 o C, unbound cells were washed and remaining cells were fixed with 2% paraformaldehyde, and mounted on a glass slide for examination by contrast microscopy. Data from 10 independent fields were acquired. Migration assays. For Transwell migration assays, endothelial cells were deprived of growth supplement and FBS overnight. Then, 10 5 cells were loaded in the upper chamber of a Transwell insert (8 µm pores, Costar, Corning, Lowell, MA), previously coated with fibronectin (5 µg/ml). The medium in the lower chamber was then replaced by medium containing 20% FBS, endothelial growth supplement (5 µg/ml; BD Biosciences) and heparin (100 µg/ml; Sigma-Aldrich). Cells were allowed to migrate for 24 hours. Cells having migrated in the lower chamber were counted using Velocity image analysis software.
For wound-healing assays, endothelial cells were cultured in gelatin-coated 6-well tissue culture plates (Nunc). Once the monolayer was confluent, a scratch was performed using a 200 µl pipet tip. Wound closure was monitored with a time-lapse microscope (DM IRE2; Leica), capturing images every 10 minutes. Speed of migration and persistence index, which is calculated as the ratio of the distance between the first and the last points of the tracked cells over the total distance traveled, were assessed after labeling cells with Hoechst and using the Velocity image analysis software. Image analyses and measurements were performed using the Matlab software (MathWorks Inc., Torrance, CA). Permeability assays.
Permeability was measured using type I collagen-coated Transwell units (0.4 µm pores; Costar). The indicated numbers of endothelial cells were seeded in the upper chamber and allowed to form a monolayer. Once a monolayer was formed, cells were deprived of serum and endothelial growth supplement for 1 or, in some cases, 4 hours. After deprivation, FITC-dextran (molecular weight = 40 kilodaltons; 1 mg/ml; Invitrogen) was added to the upper chamber and allowed to diffuse into the lower chamber for 30 or 60 minutes at 37 o C, in the absence or in the presence of vascular endothelial growth factor (VEGF; 75 ng/ml) (36). Endothelial permeability was determined by measuring the fluorescence in the lower chamber at 520 nm with an excitation wavelength of 492 nm. Triplicates measurements were taken. Immunoprecipitation and immunoblots. Cells were lysed directly on the plate with TNE buffer (50 mM Tris pH 8.0, 1% NP-40, 2 mM EDTA) supplemented with protease and phosphatase inhibitors, as described (37). Immunoprecipitation was accomplished by incubating 400 µg of total cell lysates with a protein-specific antibody for 45 minutes at 4 o C. To collect immune complexes, Pansorbin (Calbiochem, EMD Inc., Mississauga, Ontario, Canada) was added for an additional 45 minutes. Unbound proteins were washed away and the precipitated protein was loaded on 8% SDS-PAGE. Immunoblots were performed as outlined elsewhere (37). The following antibodies were used for immunoprecipitation: Cas (sc-860; Santa Cruz, Santa Cruz, CA), paxillin (610272, BD Biosciences) and FAK (sc-558; Santa Cruz). Phospho-specific antibodies against FAK (pY397; 3483 and pY576/7; 3281), and antibodies recognizing phospho-Erk (MAb E10), were from Cell Signaling, Danvers, MA. The following antibodies were used for reprobing the membrane: Cas (610272, BD Biosciences), paxillin (610272, BD Biosciences), FAK (61088; BD Biosciences) and Erk (06-182; Millipore, Burlington, Ontario, Canada). Rabbit antibodies directed against Pyk2, PTP-PEST and Csk were described elsewhere (12,38). To evaluate VEGF-induced signals, endothelial cells were deprived of serum and endothelial growth supplement for 2-4 hours. They were then stimulated with VEGF (50 ng/ml) for the indicated periods of time, and total cell lysates were probed by immunoblotting with antibodies recognizing phospho-Erk. Statistical analyses. t-Test analyses was performed using GraphPad version 5.00 for Mac (GraphPad Software; San Diego, CA).

PTP-PEST is not required for endothelial cell proliferation and differentiation in vitro-
The mid-gestation embryonic lethality previously observed in a constitutive PTP-PEST-deficient mouse prevented from establishing primary cultures of PTP-PEST-deficient endothelial cells in order to address the possible role of PTP-PEST in endothelial cells (13). To resolve this issue, we bred mice carrying a conditional allele of the PTP-PEST-encoding gene (Ptpn12 fl/fl ) with mice expressing an estrogen responsive-Cre (Cre-ERT2), under the control of the ubiquitin C (UBC) promoter ( Figure 1A) (34). In the absence of tamoxifen, Cre-ERT2 is cytosolic and not functional. However, when mice are fed tamoxifen, Cre-ERT2 translocates to the nucleus, where it can mediate deletion of the conditional allele. This enables deletion of the Ptpn12 gene in adult mouse tissues.
Primary cultures of endothelial cells were established from the lungs of TAM-fed Ptpn12 fl/fl ;UBC-Cre-ERT2 + mice or, as control, TAM-fed Ptpn12 +/+ ;UBC-Cre-ERT2 + mice ( Figure 1A). Immunoblot analyses of total cell lysates with anti-PTP-PEST antibodies showed that cells from TAM-fed Ptpn12 fl/fl ;UBC-Cre-ERT2 + mice completely lacked expression of PTP-PEST ( Figure 1B). However, they exhibited normal expression of the endothelial cell markers CD31, VE-cadherin, ICAM-1, ICAM-2 and β1 integrin, suggesting that their differentiation was normal ( Figure 1C). In all experiments reported herein, >95% of cells expressed these markers. In the presence of endothelial cell growth supplement, these cells also proliferated at a normal rate in culture ( Figure  1D).
Hence, PTP-PEST was not needed for endothelial cell proliferation and differentiation in vitro.
PTP-PEST regulates integrin-mediated adhesion and spreading in endothelial cells-Integrins are adhesion molecules involved in cell-cell and cell-substratum interactions (23,29,32). Compared to control cells, endothelial cells lacking PTP-PEST had more irregular sizes and shapes ( Figure 2A). To address the possibility that this alteration was the result of abnormal integrin function, the ability of endothelial cells to adhere in response to the integrin ligands, fibronectin and collagen, was tested ( Figure 2B). Fibronectin binds α5β1 and αvβ3 integrins, whereas collagen recognizes α1β1 and α2β1 integrins, on endothelial cells. After 30 minutes of adhesion to integrin ligand-coated surfaces, cells were washed and adherent cells were detected by staining with crystal violet. In comparison to cells expressing PTP-PEST, cells lacking PTP-PEST had a decrease in adhesion to fibronectin and collagen ( Figure  2C).
Once cells adhere to an integrin ligand-coated surface, they undergo integrin-dependent signaling events that lead to cell spreading. In the light of this, the ability of endothelial cells to spread in response to integrin ligands was evaluated ( Figure 2C). Cells were added to fibronectin-or collagen-coated glass cover slips. After 90 minutes, non-adherent cells were washed and spreading of the remaining adherent cells was assessed. Whereas PTP-PEST-deficient cells exhibited less frequent adhesion than control cells, they underwent more frequent spreading ( Figure 2C). Therefore, PTP-PEST was critical for integrin-mediated adhesion of endothelial cells. It also regulated endothelial cell spreading.
PTP-PEST is not required for integrity of endothelial cell-cell junctions-Endothelial wall integrity in the embryo and post-natal life is maintained by close contacts between endothelial cells (20-22). This is mediated in large part by VE-cadherin, a self-binding endothelial cell-specific member of the cadherin family of adhesion molecules. Given the impact of PTP-PEST on integrin-mediated adhesion noted above, we also examined the role of PTP-PEST in endothelial cell permeability. To this end, we first stained primary endothelial cells expressing, or not expressing, PTP-PEST with antibodies against VE-cadherin, which identifies cell-cell junctions ( Figure 3A). Staining was then detected by confocal microscopy. As was the case for control cells, PTP-PEST-deficient cells exhibited staining with anti-VE-cadherin antibodies at the cell surface. Although cell sizes and shapes were distinct between the two cell populations, the anti-VE-cadherin antibody-decorated intercellular junctions did not seem to be appreciably affected by PTP-PEST deficiency.
Second, endothelial cell integrity was examined using a vascular permeability assay ( Figure 3B,C). In this assay, we monitored the ability of a small molecule, fluorescent isothiocyanate (FITC)-dextran, to diffuse across endothelial cells plated on a membrane with small pores (0.4 µm pores). First, we assessed basal permeability ( Figure 3B). By plating progressively higher numbers of endothelial cells, we found that lack of PTP-PEST had no appreciable effect on the ability of endothelial cells to block transfer of FITC-dextran. And second, we examined the ability of the growth factor, VEGF, to induce an increase in vascular permeability ( Figure  3C). In three independent experiments, absence of PTP-PEST had no impact on the capacity to augment transfer of FITC-dextran.
These findings indicated that PTP-PEST did not influence basal or VEGF-induced permeability of endothelial cells. They also implied that PTP-PEST had no effect on VE-cadherin function.
PTP-PEST is required for endothelial cell migration-Next, we ascertained the impact of PTP-PEST on the ability of endothelial cells to migrate, which is required for vascular development (Figure 4). Migration was first analyzed using a Transwell migration assay ( Figure 4A). Endothelial cells were put in the upper chamber of a Transwell apparatus (4 µm pores) and their migration towards the bottom chamber, which contained endothelial growth supplement and serum, was analyzed. Lack of PTP-PEST significantly reduced (by ~60%) the ability to endothelial cells to migrate in this assay.
Migration was also examined in a wound-healing assay ( Figure 4B-E). Primary endothelial cells were plated and, when confluent, inflicted a scratch wound. Cell migration was then monitored over 24 hours by time-lapse microscopy and still photography ( Figure 4B). Endothelial cells from control mice migrated towards the wound and enabled its closure after 18 hours. Whereas PTP-PEST-deficient endothelial cells also migrated towards the wound, they failed to close the wound after 24 hours. Quantitation of the speed of wound closure ( Figure 4C) and the area repaired ( Figure 4D) showed that the speed of closure by cells lacking PTP-PEST cells was ~50% of the one of control cells. Nonetheless, tracking of individual cells after staining nuclei with DAPI showed that PTP-PEST-deficient cells moved directionally towards the wound, as was the case for control cells ( Figure 4E). The "persistence index", a measurement of directed migration, was not affected by PTP-PEST deficiency ( Figure 4F). Hence, the inability of PTP-PEST-deficient cells to close the wound was not due to compromised directional movement, but rather was caused by reduced speed of migration.
PTP-PEST regulates Cas, paxillin and Pyk2 in endothelial cells-To ascertain the biochemical mechanism by which PTP-PEST influences endothelial cell functions, protein tyrosine phosphorylation was examined by probing total cell lysates with anti-phosphotyrosine immunoblotting ( Figure  5A). Lack of PTP-PEST selectively enhanced the tyrosine phosphorylation of polypeptides of ~115-130 kilodaltons (kDas) and, less prominently, 70 and 90 kDas. To identify these products, tyrosine phosphorylation of canonical PTP-PEST substrates was evaluated by immunoprecipitation ( Figure 5B) (1,2). PTP-PEST deficiency resulted in a marked enhancement (3-to 6-fold) of tyrosine phosphorylation of Cas (~130 kDa) and paxillin (~70 kDa). A smaller increase (~2-fold) in the phosphotyrosine content of Pyk2 (~115 kDa) was seen. No effect was observed on FAK (~120 kDa). Similar results were obtained when phospho-specific antibodies directed against known phosphorylation sites of FAK, tyrosine 397 and tyrosines 576/577, were used ( Figure 5C).
We also analyzed the impact of PTP-PEST deficiency on VEGF-induced signals ( Figure 5D). Cells were stimulated or not for various periods of time with VEGF, and activation of extracellular signal-regulated kinase (Erk) was monitored by immunoblotting of total cell lysates with antibodies recognizing activated phospho-Erk. Lack of PTP-PEST had little or no effect on VEGF-induced Erk activation, in keeping with the lack of effect on vascular permeability noted earlier.
Thus, lack of PTP-PEST in endothelial cells resulted in augmented tyrosine phosphorylation of Cas, paxillin and, to a lesser extent, Pyk2. All three substrates are known regulators or effectors of the integrin pathway. However, it had no obvious effect of VEGF-induced signals.
PTP-PEST expression in endothelial cells is necessary for vascular development and embryonic viability-Previous analyses of a constitutive PTP-PEST-deficient mouse, in which PTP-PEST was missing from all cells, showed that PTP-PEST is critical for embryonic development and viability (13). To examine if this was due to a role of PTP-PEST in endothelial cells, Ptpn12 fl/fl mice were crossed with mice expressing Cre under the control of the Tie2 (or Tek) promoter (Table 1; Figure 6) (29,32,33). Tie2-Cre is active in all endothelial cells and a subset of hematopoietic cells, and is widely used to delete conditional alleles in endothelial cells. Heterozygous Ptpn12 fl/+ ;Tie2-Cre + mice were then bred with Ptpn12 fl/fl mice. Strikingly, genotype analyses of a total of 339 mice born from this cross failed to identify any Ptpn12 fl/fl ;Tie2-Cre + pups (Table 1A). Normally, ~25% of live pups would be expected to be Ptpn12 fl/fl ;Tie2-Cre + . This finding suggested that, as reported for constitutive PTP-PEST-deficient mice, the embryos lacking PTP-PEST in cells targeted by Tie2-Cre were dying in utero. To evaluate this possibility, embryos from timed pregnancies were studied (Table 1B). Live Ptpn12 fl/fl ;Tie2-Cre + embryos were observed at E10.5, but not at E11.5. This implied that Ptpn12 fl/fl ;Tie2-Cre + embryos died between E10.5 and E11.5.
Macroscopic examination of these embryos showed that E9.5 and E10.5 Ptpn12 fl/fl ;Tie2-Cre + embryos were smaller than control embryos (control embryos were either Ptpn12 fl/fl or Ptpn12 fl/+ ;Tie2-Cre + ) ( Figure 6A). This was especially true for E10.5 embryos. Nonetheless, both were still alive, as reflected by the presence of active heart contractions.
Whole-mount staining of embryos with an antibody against CD31, a marker of endothelial cells, revealed that Ptpn12 fl/fl ;Tie2-Cre + embryos exhibited formation of some large blood vessels, but had dramatically altered sprouting of smaller blood vessels ( Figure 6B). Many of the smaller blood vessels appeared dilated. Furthermore, toluidine blue-stained, semi-thin sections of E10.5 embryos showed that the dorsal aorta of PTP-PEST-deficient embryos had multiple hypersegmented lumens, instead of a single lumen as found in control embryos ( Figure  6C).
To assess if lack of PTP-PEST affected endothelial cell differentiation in vivo, Ptpn12 fl/fl ;Tie2-Cre + mice were bred with ROSA26-EYFP mice, in which a stop sequence flanked by loxP sites is inserted upstream of the EYFP gene, to prevent expression of enhanced yellow fluorescent protein (EYFP) ( Figure 6D). In the presence of Cre, however, the stop sequence is removed, thereby enabling expression of EYFP and detection of Ptpn12-deleted cells (35). Flow cytometry analyses of EYFP + cells from disaggregated embryos revealed that large proportions of cells from PTP-PEST-deficient mice expressed CD31, ICAM-1 and ICAM-2. However, the proportions of cells expressing higher levels of these markers were lower in PTP-PEST-deficient mice, compared to control mice. This alteration might be secondary to the poor general condition of the embryos, or be an effect of PTP-PEST deficiency on the Thus, PTP-PEST expression in endothelial cells was critical for embryonic viability and vascular development, although it did not seem required for emergence of the endothelial cell lineage.

DISCUSSION
To generate PTP-PEST-deficient endothelial cells, Ptpn12 fl/fl mice were bred with UBC-CreERT2 transgenic mice. Adult Ptpn12 fl/fl ;UBC-Cre-ERT2 + mice were then fed with tamoxifen and primary cultures of lung-derived endothelial cells were established. These cells exhibited a total absence of PTP-PEST expression. Despite this, they proliferated normally in vitro and displayed normal expression of endothelial cell markers. The lack of effect of PTP-PEST deficiency on proliferation or differentiation of endothelial cells was consistent with similar findings made in other cell types, namely fibroblasts and T cells, in which lack of PTP-PEST had no appreciable impact on these functions (7,11).
Nonetheless, PTP-PEST deficiency resulted in alterations of the morphology of endothelial cells. Moreover, although integrin expression was not altered, PTP-PEST-deficient endothelial cells had markedly reduced adhesion to fibronectin-or collagen-coated surfaces. Once adhered to these surfaces, they also exhibited enhanced spreading. In addition, PTP-PEST-deficient endothelial cells exhibited a pronounced defect in migration. This effect was noted in Transwell migration assays and wound healing assays. In the latter assay, lack of PTP-PEST reduced the speed of migration, but had no effect on directional movement. This observation inferred that PTP-PEST is not required for sensing environmental cues that trigger migration, by rather is needed for the migration process itself.
Although PTP-PEST is necessary for homotypic adhesion between activated T cells (11), it was not needed for homotypic interactions between endothelial cells (this report). In support of the latter, staining with anti-VE-cadherin antibodies showed that the intercellular junctions between endothelial cells were not detectably affected by lack of PTP-PEST. Additionally, in a vascular permeability assay using the small molecule FITC-dextran, endothelial cell integrity was not compromised, in the absence or in the presence of VEGF. Although this distinction between T cells and endothelial cells might seem surprising, it likely reflects differences in the molecular basis of the homotypic interactions between these cells. Interactions between T cells are principally mediated by integrins, whereas interactions between endothelial cells are primarily due to VE-cadherin.
Seemingly, PTP-PEST influences the function of integrins, but not that of VE-cadherin.
It is probable that dysregulated tyrosine phosphorylation of Cas, paxillin and Pyk2 explained the adhesion and migration defects seen in PTP-PEST-deficient endothelial cells. First, all three molecules are known regulators of the integrin pathway (23,24). More importantly, previous data showed that Cas plays a critical role in integrin-mediated endothelial cell migration and vascular development (39,40). Likewise, Pyk2 was shown to be involved in the control of angiogenesis (41). At this time, though, we cannot rule out that additional, as yet unidentified, substrates also contributed to the adhesion and migration defects seen in endothelial cells lacking PTP-PEST.
To examine the role of PTP-PEST expression in endothelial cells in vivo, we crossed Ptpn12 fl/fl mice with Tie2-Cre mice. Genotype analyses of embryos and born pups showed that all Ptpn12 fl/fl ;Tie2-Cre + embryos died in utero between E10.5 and E11.5. Furthermore, prior to death, these embryos showed markedly retarded growth. By staining embryos for CD31, it was found that Ptpn12 deletion resulted in compromised vascular development, in particular angiogenesis. This was likely due to a defect in endothelial cell adhesion and migration, as both processes are critical for normal vascular development (20,24). The time at which embryonic lethality occurred was also consistent with a vascular defect. Indeed, several other gene mutations affecting the vasculature result in embryonic lethality at ~E9. 5-10.5 (20,25-27,29-32,39).
Of note, however, the embryonic lethality observed in Ptpn12 fl/fl ;Tie2-Cre + mice arose approximately one day later than the one previously noted in constitutive PTP-PEST-deficient mice (13). Moreover, a side-by-side comparison showed that Ptpn12 -/embryos showed more extensive loss of vascular sprouting, in comparison to Ptpn12 fl/fl ;Tie2-Cre + embryos. These disparities might be explained by the inducible nature of conditional Cre-mediated deletion. Although Tie2-Cre is expressed as early as E6.5, deletion of both copies of the conditional allele, and degradation of all PTP-PEST RNA and protein [the half-life of the PTP-PEST protein has been estimated to be at least 4 hours (42)], are needed for complete PTP-PEST deficiency. This might require a few days and likely enabled partial expression of PTP-PEST between E6.5 and E10.5.
Alternatively, it might reflect a role of PTP-PEST in additional cell types that are not targeted by Tie2-Cre.
Adhesion receptors play critical roles in vascular functions during embryogenesis and adult life. The VE-cadherin pathway is a key regulator of vascular permeability. Previous data showed that Tie2, a PTK, and VE-PTP, a PTP, regulate the activity of VE-cadherin, and that both are essential for vascular development and embryogenesis (20, [25][26][27]. By opposition, the integrin pathway is important for the control of endothelial cell adhesion and migration. Earlier results, in addition to the findings herein, revealed that another PTK, FAK, and another PTP, PTP-PEST, control the activity of integrins. Both also have pivotal roles in vascular and embryonic development (29)(30)(31)(32). In this manner, unique pairs of PTKs and PTPs regulate the two major adhesion receptor families controlling vascular functions.
Together, the findings reported herein show that PTP-PEST is a key regulator of integrin-mediated adhesion and migration of endothelial cells in vivo and in vitro. This function is likely mediated by the ability of PTP-PEST to dephosphorylate the cytoskeleton regulators, Cas, paxillin and Pyk2. Moreover, such an activity likely explains the critical role of PTP-PEST in vascular development and embryonic viability. Given the continuing importance of angiogenesis after birth, it is probable that PTP-PEST also plays a key function in post-natal processes involving endothelial cells. These processes may include wound healing and tissue repair, and pathological situations such as tumor formation, inflammation and diabetic retinopathy. The possible involvement of PTP-PEST in pathological angiogenesis raises the possibility that pharmacological inhibitors of PTP-PEST, or of PTP-PEST-regulated pathways, may have therapeutic benefit in these conditions. Acknowledgements-We thank the members of our laboratory for discussions. We also thank Dominic Filion for help with microscopy and image analyses.